A FUNCTIONAL ANALYSIS OF THE SMALL NUCLEAR RNP

IMPORT ADAPTOR, SNURPORTIN1

by

JASON KERR OSPINA

Submitted in partial fulfillment of the requirements

for the degree of Doctor of Philosophy

Dissertation Advisor: Dr. A. Gregory Matera

Department of Genetics

CASE WESTERN RESERVE UNIVERSITY

August, 2005 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the dissertation of

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candidate for the Ph.D. degree *.

(signed)______(chair of the committee)

______

______

______

______

______

(date) ______

*We also certify that written approval has been obtained for any proprietary material contained therein. TABLE OF CONTENTS

Table of contents...... 1

List of tables ...... 3

List of figures...... 4

Acknowledgments...... 8

Abbreviations ...... 9

Abstract...... 11

Chapter 1: Background, purpose of study and thesis outline

Introduction...... 13

Cellular transport basics ...... 14

Cellular transport and snRNP biogenesis ...... 24

The role of snurportin in snRNP biogenesis...... 38

Purpose of study and thesis outline ...... 44

Chapter 2: SMN, the , forms a preimport complex with snurportin1 and β...... 47

Abstract ...... 48

Introduction...... 49

Materials and methods ...... 54

Results...... 58

Discussion ...... 75

Acknowledgements...... 79

1 Chapter 3: Crosstalk between snurportin1 subdomains...... 80

Abstract ...... 81

Introduction...... 82

Materials and methods ...... 86

Results...... 92

Discussion ...... 115

Supplemental data...... 119

Acknowledgements...... 122

Chapter 4: Conclusions, prospectus and future directions ...... 123

Conclusion...... 124

Discussion ...... 131

Future directions ...... 140

Concluding remarks...... 152

Appendices...... 153

Bibliography ...... 199

2 LIST OF TABLES

CHAPTER TWO

Table 2-1: Effects of SPN overexpression on Cajal bodies...... 64

CHAPTER THREE

Table 3-1: SPN in vitro binding and in vivo localization studies ...... 97

3 LIST OF FIGURES

CHAPTER ONE

Figure 1-1: Structure of the complex...... 17

Figure 1-2: General schematic of snRNA export ...... 26

Figure 1-3: Overview of snRNP import...... 27

Figure 1-4: Detailed depiction of snRNA export...... 31

Figure 1-5: Detailed schematic of snRNP import...... 37

CHAPTER TWO

Figure 2-1: Schematic of snurportin1 (SPN) and characterization of an anti-SPN antibody, R89...... 59

Figure 2-2: SPN is actively imported into the nucleus ...... 61

Figure 2-3: Comparison of mutant and wildtype and their effects on Cajal bodies...... 63

Figure 2-4: Colocalization of overexpressed SPN and endogenous SMN...... 64

Figure 2-5: SPN interacts indirectly with SMN in the ...... 67

Figure 2-6: SPN and SMN do not interact directly ...... 69

Figure 2-7: SMN interacts directly with importin β ...... 71

Figure 2-8: SPN forms a cytoplasmic complex with ZPR1 ...... 74

Figure 2-9: Model of the U snRNP import complex ...... 78

4 CHAPTER THREE

Figure 3-1: Schematic of snurportin1 (SPN)...... 93

Figure 3-2: Alignment of SPN orthologs ...... 94

Figure 3-3: Recombinant SPN can distinguish between m7G- and TMG-capped ...... 95

Figure 3-4: SPN mutants defective in TMG-cap binding are inhibited in Xpo1 binding ...... 99

Figure 3-5: SPN point mutants that are reduced for binding to Xpo1 in vitro interact in vivo...... 101

Figure 3-6: SPN import does not require bound cargo ...... 103

Figure 3-7: SPN accumulation in Cajal bodies correlates with the ability to bind TMG caps...... 105

Figure 3-8: Disruption of importin β binding and an alignment of importin β binding domains ...... 108

Figure 3-9: Mutants incapable of binding importin β in vitro are efficiently imported in vivo ...... 109

Figure 3-10: The binding of SPN to a TMG cap does not interfere with cap-independent import ...... 111

Figure 3-11: The binding of SPN to importin β is enhanced upon or truncation of the C-terminus of SPN...... 112

Figure 3-12: SPN N- and C-terminal domains interact ...... 114

Supplemental Data...... 119

Figure 3S1: SPN truncation and substitution mutants are altered in importin β binding...... 119

Figure 3S2: SPN mutants (R27A) and (25-27A) display decreased affinity for importin β...... 120

Figure 3S3: The importin β binding of wildtype and mutant SPN is responsive to -GTP ...... 121

5 CHAPTER FOUR

Figure 4-1: Model of snRNP import regulation via an auto-inhibitory function of snurportin...... 134

APPENDICES

Figure A1-1: Xpo1 is depleted from Cajal bodies upon LMB treatment...... 155

Figure A2-A: Excess TMG caps inhibit the enrichment of SPN in Cajal bodies...... 159

Figure A2-B: The enrichment of SPN in Cajal bodies is noted upon the import of U2 but not U1 ...... 162

Figure A3-1: The enrichment of in the cytoplasm does not result in formation in this compartment...... 165

Figure A4-1: SMN deficient Cajal bodies are enriched in U snRNPs ...... 168

Figure A5-1: PHAX mutants are altered in migration and interaction with SMN...... 171

Figure A6-1: pSV2-8.32 is unstable when propagated at 37°C ...... 175

Figure A6-2: Structural conformation of pJO7 ...... 176

Figure A6-3: pJO7 and pJO11 contain the appropriate arrays...... 177

Figure A6-4: Tet-inducible U2 snRNA stable cells display discrete lac repressor foci...... 181

Figure A6-5: Structure and expression of an inducible U2 array ...... 183

Figure A6-6: Stable line 41 is inducible for exogenous U2 expression...... 184

Figure A6-7: Cajal bodies associate with exogenous, Tet-inducible, U2 snRNA gene arrays ...... 185

Figure A6-8: Association frequencies and mean Cajal body numbers of stable cell lines...... 187

6 Figure A6-9: Mean Cajal body numbers of stable cell lines...... 188

Figure A7-1: Maps of Bac vectors generated for live cell studies...... 195

7 ACKNOWLEDGEMENTS

I would like to thank my advisor, A. G. Matera, for supporting my scientific development while in graduate school. He has challenged me to view science from many new perspectives and has encouraged me to be a better critical thinker. I am indebted to the members of the Matera lab for providing discussion and debate of topics ranging from science to concepts only warped scientific minds could conjure. In particular, I would like to thank Michael Hebert and Mark

Frey who provided valuable support during my first 3 years of graduate school. I would like to thank other lab members including: Graydon Gonsalvez, Usha

Narayanan, T.K. Rajendra, Karl Shpargel, Karen Tucker and Michael Walker for creating a great work and learning environment. Thanks to my committee members, Jim Bruzik, Peter Harte and Matthew Warman for advise and support.

I would like to thank my mom (Ann), sisters (Tina and Kathy) and brother

(Richard) for their support over the years. I would also like to thank my wife’s family, especially Barb English and Luise Bertetto. They are always eager to provide unwavering support and abundant amounts of really good cake.

Special thanks to my wife, Sue who I met during our first year in graduate school. The completion of my Ph.D. would not have been possible without her support, understanding and love. I would have never predicted that the initial decision to study at a particular institution could lead to something so extraordinary.

8 LIST OF ABREVEATIONS

ATCC American type culture collection ATP Adenosine triphosphate BSA Bovine serum albumin CB Cajal body CP Creatine phosphate CPK Creatine phosphokinase DAPI 4',6-diamidino-2-phenylindole DMSO Dimethyl sulfoxide DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid GFP Green fluorescent protein GST Glutathione S-transferase GTP His Histidine IF Immunofluorescence IP Immunoprecipitation IPTG Isopropyl-beta-D-Thiogalactopyranoside kDa Kilodalton LMB B m7G 7-methylguanosine MDa Megadalton mRIPA Modified radioimmunoprecipitation assay mRNA messenger RNA mut Mutant Myc c-Myc epitope NES NLS Nuclear localization signal NP-40 Nonidet-40 NPC Nuclear pore complex PBS Phosphate buffered saline PCR Polymerase chain reaction PHAX Phosphorylated adaptor for snRNA export rRNA ribosomal RNA RNA Ribonucleic acid RNP Ribonucleoprotein RT Room temperature RT-PCR Reverse transcriptase-PCR SDS Sodium dodecyl sulfate PAGE Polyacrylamide gel electrophoresis SMN Survival of motor

9 snRNA Small nuclear RNA snRNP Small nuclear RNP SPN Snurportin1 TMG 2,2,7-trimethylguanosine tRNA transfer RNA UTP Uridine triphosphate WT Wildtype Xpo1 Exportin1/Crm1 ZPR1 Zinc finger protein1

10 A Functional Analysis of the Small Nuclear RNP Import Adaptor, Snurportin1

Abstract

by

Jason K Ospina

Cells are the fundamental units of life and are therefore intimately connected to life’s vitality. Crucial to cellular function is an ability to coordinate and regulate numerous intricate processes. The generation of spliceosomal uridine-rich small nuclear ribonucleoproteins (U snRNPs) is a fascinating example, as it requires both cytoplasmic and nuclear phases. The generation of snRNPs begins in the nucleus with the of snRNAs. Nascent snRNAs are then exported to the cytoplasm where maturation results in newly assembled snRNP particles. A subset of these cytoplasmic steps requires the survival of motor neurons (SMN) . Resulting particles are imported back into the nucleus, where they are further modified. Mature snRNPs are then incorporated into the spliceosome and play a vital role in pre-mRNA splicing.

Central to snRNP biogenesis is the proper targeting of precursor molecules. As snRNPs are processed in the cytoplasm, but function in the nucleus, import factors are required to transport these components across the . Import is directed by two independent signals found on snRNPs. One import signal consists of a 5′ RNA cap structure and is bound by

11 the transport adaptor, snurportin1 (SPN). The precise identity of the other transport adaptor remains elusive, however it’s activity lies within the SMN complex.

I have utilized both in vitro and in vivo approaches to study the process of

U snRNP import. This thesis has identified a pre-import complex containing

SPN, SMN and the transport receptor, importin β. This work also provides significant insight into the function of SPN-mediated snRNP import. Residues required for SPN function have been identified and validated by functional studies. The utilization of mutants deficient in function illustrates that cargo is not a prerequisite for SPN import. The binding of SPN to its export receptor exportin1, is disrupted by the drug leptomycin B, resulting in the accumulation of

SPN in Cajal bodies. Finally, a novel interaction between the N- and C-termini of

SPN was uncovered, indicating the possibility of an intramolecular regulation of snRNP import.

12 CHAPTER 1

Introduction and Objectives

INTRODUCTION

The compartmentalization of eukaryotic cells into and sub-domains enables greater regulatory control over cellular processes compared to that afforded to prokaryotic organisms. The nucleus and cytoplasm are perhaps the best known examples of compartments within the eukaryotic cell. Further division of eukaryotic cells yields cytoplasmic compartments such as mitochondria and lysosomes and nuclear sub-domains including nucleoli and

Cajal bodies. These compartments provide local environments whereby the communication with, and access to, other cellular regions can be controlled. This enables the regulation of many key processes, depending on the requirements of metabolic demand.

Many compartments in eukaryotic cells are divided from their environment by a bilayer membrane. For example, the nuclear envelope separates nuclear and cytoplasmic compartments, allowing for regulated communication between these two cellular domains. Intercompartmental communication between the nucleus and cytoplasm is conducted via the transport of molecules through nuclear pore complexes (NPCs). NPCs are aqueous channels embedded within the nuclear envelope that facilitate the bidirectional passage of macromolecules. Transport proteins ferry cellular components including RNAs, proteins and RNA/protein (RNP) complexes through NPCs. Snurportin1 (SPN), 13 one such transport protein, specifically mediates the nuclear import of uridine-rich small nuclear ribonucleoproteins (U snRNPs). These RNP factors are essential to cellular gene regulation and function as a part of the splicing machinery.

CELLULAR TRANSPORT BASICS

Eukaryotic cells are separated into nuclear and cytoplasmic compartments, with the genomic material residing in the nucleus. Due to the lack of compartmentalization in prokaryotic cells, access to the genomic material is unregulated. As a result, DNA, RNA and protein synthesis occur in a single cytosolic compartment. In eukaryotic cells however, the nuclear synthesis of

DNA and RNA is uncoupled from protein synthesis, which occurs in the cytoplasm. This separation enables a greater ability to regulate both transcription and the of resulting mRNAs. For example, transcriptional enhancers can be sequestered in the cytoplasm to reduce the expression of specific genes. Additionally, nascent mRNAs may be selectively degraded in the nucleus, thus precluding their export and translation in the cytoplasm.

Multiple levels of regulation, do however, have their costs. A great deal of energy must be expended to maintain the higher order resulting from a separation into nuclear and cytoplasmic compartments. Furthermore, communication between these compartments must be established and regulated.

This communication proceeds through NPCs, and consists of the passive and facilitated transport of a wide range of cargoes.

14 A number of factors are required to ensure the proper regulatory control of nucleocytoplasmic transport and are either directly or indirectly involved in this process. Components playing a direct role in transport, include the cargo, transport receptor and adaptor molecules as well as cofactors regulating the associations between these elements. An environment conducive to transport, an indirect component, is provided by the NPCs.

Nucleocytoplasmic transport is critical for numerous cellular processes including proper . Fundamentally, gene expression requires the nuclear export of messenger (m)RNA, transfer (t)RNA, ribosomal (r)RNA, small nuclear (sn)RNA and ribosomal subunits as well as the import of proteins and snRNPs. Some processes require a single translocation of the nuclear envelope, whereas others, including and U snRNP biogenesis, require multiple exchanges between nuclear and cytoplasmic compartments. For example, the biogenesis of U snRNPs, key components of the splicing machinery, requires an initial export of RNA to the cytoplasm, followed by its import back into the nucleus as an RNP complex. This bidirectional transport of snRNP precursors is highly regulated and transmitted through macromolecular NPCs.

Nuclear Pore Complexes

NPCs are aqueous channels that perforate the nuclear envelope (reviewed in

Rout and Aitchison, 2001; Suntharalingam and Wente, 2003) and are situated at the sites of fusion between inner and outer nuclear envelope layers (Fig. 1-1). As

15 all nucleocytoplasmic transport must shuttle through NPCs, it is not surprising that these macromolecular complexes can negotiate a tremendous array of cargoes. NPCs permit the passive of ions and small molecules, while accommodating the transport of large complexes, such as ribosomal subunits, having molecular weights in excess of 2.0x106 kDa. It is hypothesized that in a resting state, NPCs are permeable to only smaller molecules and that sequential dilation of the NPC accommodates larger cargoes. Clues to the functioning and regulation that enable this versatility were obtained through the identification and investigation of NPC components.

The overall architecture of the NPC is conserved from to mammals (Davis, 1995; Fabre and Hurt, 1997; Yang et al., 1998).

This architecture consists of three general substructures (reviewed in Allen et al.,

2000): the cytoplasmic filaments, a central core and a nuclear basket (Fig. 1-1).

The arrangement of the NPC results in an octagonal symmetry when viewed along the axis of its central core and an asymmetry with reference to its cytoplasmic and nuclear extensions. Each vertebrate NPC is approximately 125

MDa (Reichelt et al., 1990) and consists of a minimum of 400 total proteins termed (reviewed in Suntharalingam and Wente, 2003). Many of these proteins are present in copies of eight, or multiples of eight, and therefore account for its octagonal symmetry.

16

Figure 1-1 Structure of the nuclear pore complex. The nuclear pore complex (NPC) is embedded in the nuclear envelope and consists of three substructures: the nuclear basket, a central core and cytoplasmic filaments. The protein components of the NPC are termed nucleoporins or nups. Many nups contain FG repeats, which mediate interactions with the so-called HEAT repeats of transport receptor proteins.

One feature common to many of these nucleoporins is the presence of multiple - (FG), FxFG (x denotes any ) or GLFG (L denotes leucine) amino acid repeats (Cronshaw et al., 2002; Rout et al., 2000; reviewed in Ryan and Wente, 2000). These FG repeats serve as binding sites for receptor proteins called and exportins, collectively referred to as . Although, the compositions of both yeast (Rout et al., 2000) and vertebrate (Cronshaw et al., 2002) NPCs were determined, little is

17 known concerning the mechanisms by which these components mediate transport.

Investigations of NPC activity during cargo translocation are in their early stages. Studies suggest that NPCs are highly dynamic, however, the components contributing to this dynamic nature, and the extent of its behavior, are unknown (reviewed in Powers and Dasso, 2004). Recent studies have investigated the dynamics of NPC components by tagging nucleoporins with green fluorescent protein (GFP) markers (Rabut et al., 2004). Data illustrate that the central core is extremely stable, whereas the more peripheral proteins, especially those of the nuclear basket, are increasingly dynamic. More work is needed to identify the roles of individual proteins and to incorporate these data into a model of NPC function, as it pertains to cargo translocation. Nonetheless,

NPCs provide an environment, whereby direct transport components, such as importin and exportin proteins, are able to mediate the nucleocytoplasmic exchange of cargoes.

Transport receptors

Karyopherins facilitate transport by providing cargo with an ability to negotiate the

NPC. This group is divided into two categories, depending on the direction of their transport activities. As their names indicate, importins mediate import into the nucleus, whereas exportins facilitate export to the cytoplasm. Despite this distinction, most importin and exportin proteins belong to the importin β family of

18 transport receptors (reviewed in Harel and Forbes, 2004; Mosammaparast and

Pemberton, 2004). All importin β members contain an N-terminal Ran binding domain and a series of tandemly repeated sequences of approximately 40 amino acids called HEAT motifs (reviewed in Andrade et al., 2001). The term HEAT is derived from the names of the four original proteins found to possess these motifs and include: huntingtin, elongation factor 3, the PR65/A subunit of protein phosphatase 2A and the lipid kinase TOR (target of rapamycin). HEAT motifs interact with the FG repeats of nuceloporins and facilitate the passage of cargo through the NPC. It is hypothesized that import competent -cargo complexes initially dock at several sites along the cytoplasmic filaments. This event is followed by multiple docking and release steps along the central core, which are mediated by FG repeat and HEAT motif interactions (models of translocation are reviewed in Fried and Kutay, 2003; Weis, 2003; Peters, 2005).

Import events are concluded when transport complexes reach the nucleoplasmic basket and are subsequently released into the nucleus (Blobel, 1995).

Transport adaptors and signals

In addition to the importin β family of transport receptors, some cargoes require the assistance of yet another group of transport molecules. This group consists of protein adaptors, which facilitate the transport of cargoes not directly bound by transport receptors. Protein adaptors perform this task through two binding functions. Adaptors must bind to their cognate transport receptor, depending on

19 the destination of the cargo, as well as bind to the cargo itself. This transport is slightly more complicated than simple binding of cargo to actual transport receptors. With direct binding between cargo and receptor, the direction of transport is indicated by the nature of the receptor. However, when adaptors are utilized, an additional signal is often required to indicate the destination of cargo transport.

Many molecules, including cargo and transport machinery, undergo multiple transport events. As a result, the signals directing this transport must persist for the life of the molecule. These transport signals are highly varied and range from simple amino acid sequences of proteins to 5′ cap modifications of

RNAs. Such signals can be inherent in the actual cargo or transport machinery or can be added specifically to the cargo through maturation or processing steps.

Signals marking cargo for import into the nucleus are called nuclear localization signals (NLSs) and those indicating that cargo should be exported to the cytoplasm are nuclear export signals (NESs). It is important to note that these transport signals can be protein (next paragraph) or RNA (see below) in origin or a combination of both.

The most well known transport signals consist of stretches of amino acids, which indicate transport direction. NLSs typically consist of stretches rich in basic lysine and arginine residues (Kalderon et al., 1984; Lanford and Butel,

1984), whereas NESs consist of stretches rich in leucine residues (Fischer et al.,

1995; Wen et al., 1995; reviewed in Mattaj and Englmeier, 1998). The classical

20 lysine/arginine rich NLS is recognized by one of the most well known transport adaptors, importin α (Conti et al., 1998), whereas short leucine rich NESs are substrates for the transport receptor, exportin1 (Xpo1; Kutay and Guttinger,

2005).

Most transport adaptors possess an NES or NLS domain or are able to bind these signals. Importin α is one such adaptor as it binds to NLS motifs, linking transport receptor (Weis et al., 1996b) and cargo molecules. Additionally, a transport adaptor may bind to an inherent modification of the cargo, such as an

RNA modification. These signals provide vital information for the transport machinery by indicating their intended cellular localization. However, there still exists the issue of distinguishing between the nuclear and cytoplasmic compartments. This requirement is fulfilled by the Ran-GTP gradient, which provides vectorality to cellular transport.

The Ran gradient and cellular compartment identification

In addition to interacting with nucleoporins, karyopherins can also interact with a small GTP-ase called Ran, through a conserved N-terminal domain. Ran is distributed throughout the cell, whereby most nuclear Ran is in the GTP-bound form while most cytoplasmic Ran is GDP-bound (Smith et al., 2002; reviewed in

Kuersten et al., 2001). It is estimated that this asymmetric distribution results in a

Ran-GTP ratio (from nucleoplasmic to cytoplasmic) of approximately 500:1

(Smith et al., 2002). This drastic gradient is established and maintained by the

21 restricted localization and activity of several protein regulators of Ran. One such regulator, nuclear transport factor 2 (NTF2), imports Ran-GDP from the cytoplasm to the nucleus, thus maintaining a large pool of nuclear Ran (Ribbeck and Görlich, 2001; Ribbeck et al., 1998; Smith et al., 1998). Nuclear pools of

Ran exchange factor (RanGEF) and RCC1 promote the dissociation of GDP from

Ran-GDP, thus allowing the binding of GTP. This reaction plays a key role in ensuring that the majority of nuclear Ran is GTP-bound. Additional regulators consist of the Ran Binding Proteins 1 and 2 (RanBP1, RanBP2) and Ran

GTPase activating protein (RanGAP). These factors promote the opposing reaction, whereby GTP hydrolysis by Ran results in conversion of Ran-GTP to

Ran-GDP. The function of these Ran-GDP promoting proteins ensures that the majority of cytoplasmic Ran is in a GDP-bound state. This difference in Ran distribution provides vital positional information to the transport machinery, enabling a distinction between nuclear and cytoplasmic compartments

(Izaurralde et al., 1997).

Ran and transport complex assembly

Ran-GTP differentially influences transport complex formation depending on the nature of the transport machinery with which it binds. This differential influence on complex formation is central to cellular transport and ensures the proper targeting of cargo. Importins bind their cargo in the cytoplasm (low Ran-GTP) and release them upon binding Ran-GTP in the nucleus. Importins are

22 subsequently exported to the cytoplasm bound to Ran-GTP (Hieda et al., 1999;

Izaurralde et al., 1997). Exportins display higher cargo affinity upon binding Ran-

GTP. Thus, exportins bind their cargo in the nucleus (high Ran-GTP). Following export to the cytoplasm, hydrolysis of GTP promotes complex disassembly and cargo release (Matsuura and Stewart, 2004). Exportins are then imported back into the nucleus where they can mediate additional rounds of cargo export. In addition to the transport machinery and a means of establishing location and direction, cells require energy to drive the highly entropic process of cellular transport.

The energy independence of NPC translocation

As dictated by the second law of thermodynamics, the accumulation of cellular components against a gradient requires an input of energy. As translocation across the nuclear envelope appears to be a complex process, it was assumed to be an energy consuming step of transport. However, this does not seem to be the case. A variety of examples illustrate that NPC translocation is not directly linked to tri-phosphate (NTP) hydrolysis, at least for simple cargo- receptor complexes (e.g. NLS/importin α/β) (Englmeier et al., 1999; Ribbeck et al., 1999; Schwoebel et al., 1998). In fact single-round transport events are independent of NTP hydrolysis (Huber et al., 2002; Ribbeck et al., 1999).

Consistent with these data, is the fact that neither NTPases nor motor-like activities were obtained from purified NPCs (Cronshaw et al., 2002; Rout et al.,

23 2000). Interestingly, nuclear export processes display a similar independence of

NTP hydrolysis (reviewed in Komeili and O'Shea, 2001). However, it is quite clear that continued rounds of karyopherin-mediated transport do require energy.

Data indicate that the energy required for transport is provided by the Ran-

GTPase system (Weis et al., 1996a). This system establishes the Ran-GTP gradient across the nuclear envelope, which guides the loading and release of cargo. The Ran-GTP gradient is sensed by transport receptors through their

Ran-binding domains and mediates the affinity of importins and exportins for their respective cargo. At least two vital transport processes require a component of the Ran-GTPase cycle, namely Ran-GTP. Following the translocation of importin

α/β mediated cargo, Ran-GTP is required for the release of the import cargo from the nuclear basket (Görlich et al., 1996c; Rexach and Blobel, 1995). Additionally, the formation of an export complex requires exportin to bind to Ran-GTP

(Fornerod et al., 1997). Thus, transport steps other than the actual translocation of the NPC, require the presence of Ran-GTP. Taken together, it seems likely that the energy required for nucleocytoplasmic transport is solely consumed by the establishment and maintenance of the Ran-GTPase system.

CELLULAR TRANSPORT AND SNRNP BIOGENESIS

The maintenance and vitality of cells depend on the proper functioning and regulation of cellular transport. Karyopherins are a key component in cellular transport and play a critical role in the proper distribution of cellular RNAs and

24 protein subunits. The biogenesis of U snRNPs is an exquisite example of the importance of correct cellular targeting.

Spliceosomal (Sm) U snRNPs are cellular components consisting of RNA and protein molecules and require a unique biogenesis pathway. This pathway consists of nuclear and cytoplasmic phases, whereby protein and RNA modifications occur in both compartments (reviewed in Will and Lührmann,

2001). U snRNP biogenesis begins in the nucleus with the generation of an RNA molecule, which is later exported to the cytoplasm. This transport is mediated by the cooperative action of protein receptor and adaptor molecules (Fig. 1-2). In the cytoplasm, the RNAs are modified and proteins are assembled onto the

RNAs resulting in RNP particles. These RNP particles will eventually function in mRNA splicing and therefore require transport back to the nucleus. This step of biogenesis is facilitated by the formation of a complex containing importins and

RNP cargo. Following complex formation, the RNP cargo is imported into the nucleus (Fig. 1-3) where it undergoes further maturation and subsequently mediates pre-mRNA splicing. Recycling of the components that mediate RNA export, as well as RNP import, replenishes both the nuclear and cytoplasmic compartments with transport machinery, enabling additional cycles of snRNP biogenesis.

25

Figure 1-2 General schematic of snRNA export. Transcription in the nucleus results in nascent snRNAs, which are cotranscriptionally modified with an m7G cap at their 5′ ends. These snRNAs are then transported to the cytoplasm by an export complex consisting of cap binding proteins, adaptor molecules and receptor proteins. Upon reaching the cytoplasm, protein modifications result in complex disassembly and the release of snRNA cargo. Export proteins are then shuttled back to the nucleus to mediate additional rounds of snRNA transport, whereas the snRNAs will undergo cytoplasmic modification (see below).

26

Figure 1-3 Overview of snRNP import. Subsequent to their nuclear export, U snRNAs are modified in the cytoplasm, resulting in newly assembled snRNPs. These snRNPs are then bound by import proteins, resulting in an import competent snRNP complex. Following import, complex disassembly results in free import proteins and snRNPs. Import proteins are then shuttled back to the cytoplasm to mediate additional rounds of snRNP transport, whereas the newly imported snRNPs are further modified prior to their functioning in the spliceosome.

27 The transcription of snRNA

The biogenesis of snRNPs is a complex process requiring both nuclear and cytoplasmic phases. This biogenesis consists of RNA modifications as well as the addition of protein components to the snRNP particles. The RNA components are small nuclear (sn) RNAs, which are transcribed in the nucleus and differ in RNA sequence from other snRNAs. For example, U1 snRNA shares only the Sm site (see later) and extensive secondary structure with other snRNAs. Protein components consist of molecules common to all snRNPs as well as those specific to each snRNP.

The production of snRNAs and protein constituents of snRNPs begins with the activity of RNA polymerase ΙΙ (RNA pol ΙΙ). This polymerase is responsible for the generation of transcripts encoding all snRNA transcripts (reviewed in

Lobo, 1994 and references therein) with the exception of U6, which is transcribed by RNA pol ΙΙΙ (Kunkel et al., 1986; Reddy et al., 1987). Following the generation of transcripts encoding the protein components of snRNPs, splicing results in mRNAs that are exported to the cytoplasm. Subsequently, translation results in proteins that either remain in the cytoplasm or are imported into the nucleus.

This differential localization depends upon whether the protein will be assembled onto the snRNA in the nuclear or cytoplasic compartment.

As for the snRNAs, transcription results in nascent molecules that are immediately modified by the nuclear machinery. This modification consists of the co-transcriptional addition of a 7-methylguanosine (m7G) cap to the 5' end of the

28 transcribed RNA (Neuman de Vegvar and Dahlberg, 1990 and references therein; reviewed in Cougot et al., 2004) and the addition of snRNA specific 3' , which will be processed in the cytoplasm (Neuman de Vegvar and

Dahlberg, 1990 and references therein).

The transport of snRNA

Early studies of snRNA transport indicated that the m7G cap is required for export

(Hamm and Mattaj, 1990; Jarmolowski et al., 1994). The m7G cap is bound by cap binding proteins (CBPs) 20 and 80, which heterodimerize forming the cap binding complex (CBC) (Hamm and Mattaj, 1990; Izaurralde et al., 1995;

Izaurralde et al., 1994; Mazza et al., 2001; Ohno et al., 1990; Visa et al., 1996).

Direct binding of the m7G cap is mediated by CBP20 (Izaurralde et al., 1994;

Mazza et al., 2002). However, as CBP20 cannot bind the m7G cap alone

(Izaurralde et al., 1995), it is thought that CBP80 promotes the proper cap binding conformation of CBP20.

Following CBC binding, the phosphorylated adaptor for snRNA export

(PHAX) binds to the CBC and to the snRNA molecule (Ohno et al., 2000; Segref et al., 2001). Subsequently, the nuclear export receptor, a protein called Xpo1

(Fornerod et al., 1997) and Ran-GTP (Izaurralde et al., 1997) bind to the

CBC/snRNA complex (Fig. 1-4). Two signals are known to promote complex formation and export to the cytoplasm. First, the phosphorylation of PHAX promotes its incorporation into the export complex (Ohno et al., 2000). Second,

29 the binding of Ran-GTP is required for the incorporation of Xpo1 into the transport complex (Fornerod et al., 1997). Following export complex formation,

Xpo1 plays a vital role in the negotiation of the NPC. Translocation through the nuclear pore is mediated by domains present within Xpo1, termed HEAT repeats

(Petosa et al., 2004, and references therein). Interactions between these repeats and the FG repeats of NPC proteins facilitate passage of the export complex through the nuclear pore.

30

Figure 1-4 Detailed depiction of snRNA export. U snRNAs are transcribed and modified in the nucleus, resulting in m7G capped molecules. The 5′ cap is bound by cap binding proteins 20 and 80, which form the cap binding complex (CBC). Phosphorylated PHAX binds the CBC and RNA, whereas Xpo1 binds both PHAX and Ran-GTP. Subsequent to complex formation, U snRNAs are exported to the cytoplasm. In the cytoplasm, dephosphorylation of PHAX and Ran-GTP hydrolysis promote complex disassembly. Export complex components are then shuttled back to the nucleus, whereas U snRNAs are modified in the cytoplasm, resulting in import competent snRNP particles.

31 Cytoplasmic modifications of snRNA transcripts

Subsequent to each round of cargo export, the complexes mediating such transport events must be disassembled. Upon cargo arrival in the cytoplasm,

GTP hydrolysis by Ran and the dephosphorylation of PHAX promote complex disassembly. Export complex proteins are then imported back into the nucleus to function in additional rounds cargo transport.

Following their export to the cytoplasm, U snRNAs are modified with the addition of Sm proteins, which heterodimerize forming the Sm core (reviewed in

Yong et al., 2004). This core consists of seven Sm proteins: B/B', D1, D2, D3,

E, F and G. These proteins bind to a site on the U snRNA that is enriched in uridine residues and termed the Sm binding site. It is hypothesized that the Sm core increases the stability of spliceosomal snRNPs (Bordonne and Tarassov,

1996; Noble and Guthrie, 1996; Roy et al., 1995; Rymond, 1993). The Sm proteins are not assembled onto the U snRNA individually, rather they are incorporated as three heteromeric complexes consisting of D1-D2, E-F-G and B-

D3 or B'-D3 (Raker et al., 1996).

The assembly of the Sm core is facilitated by a complex containing the survival of motor neurons (SMN) protein (Pellizzoni et al., 2002b). This complex promotes the assembly of the Sm core on the appropriate cellular RNAs

(reviewed in Yong et al., 2004). Mutation or deletion of the SMN gene results in a progressive, debilitating neurodegenerative disease termed spinal muscular atrophy (SMA) (Lefebvre et al., 1995). As SMN may perform other vital cellular

32 activities, in addition to Sm core assembly, it is unknown if the SMA phenotype is solely due to a disruption is snRNP assembly and biogenesis. Nonetheless, the addition of proteins to newly exported U snRNAs results in an RNA/protein complex or snRNP.

Sm core assembly is required for subsequent maturation of RNP complexes, including 5' cap and 3' end RNA modifications (Mattaj, 1986; Plessel et al., 1994; Seipelt et al., 1999). First, the 5' m7G cap is hypermethylated resulting in a 2,2,7-trimethylguanosine (TMG) cap (Fischer and Lührmann, 1990;

Hamm and Mattaj, 1990; Plessel et al., 1994). This hypermethylation is performed by a methyltransferase called Trimethylguanosine Synthase (Tgs1)

(Mouaikel et al., 2003; Mouaikel et al., 2002; Verheggen et al., 2002).

Subsequently, the snRNA is modified at its 3' end with the excision of approximately 8-20 nucleotides (Neuman de Vegvar and Dahlberg, 1990; Seipelt et al., 1999). The removal of these nucleotides is mediated by a currently unidentified exoribonuclease complex. It is known however, that this 3' end modification precedes import of newly assembled snRNPs (Neuman de Vegvar and Dahlberg, 1990 and references therein). These cytoplasmic maturation steps (Sm core assembly, 5' cap hypermethylation and 3' end trimming) are required for, or significantly accelerate, subsequent import of newly assembled snRNPs into the nucleus (reviewed in Yong et al., 2004 and references therein;

Fischer et al., 1994).

33 The import of newly assembled snRNPs

Following cytoplasmic maturation, snRNPs are imported into the nucleus for additional modifications. Import is mediated by cytosolic factors consisting of both adaptor and receptor proteins. These factors recognize two independent

NLSs found within the snRNP cargo (Fischer and Lührmann, 1990; Fischer et al.,

1993; Hamm and Mattaj, 1990). One signal consists of the Sm core domain, whereas the other is provided by the TMG cap (Huber et al., 2002; Marshallsay and Lührmann, 1994; Narayanan et al., 2004). The candidates for the Sm core domain NLS include SMN, the Sm proteins and SMN complex components

Gemins 2-7 (Pellizzoni et al., 2002a; Pellizzoni et al., 2002b). As a result, there are several potential import mediators specific to the Sm core NLS. In contrast, the mediators of the TMG cap specific NLS have been elucidated and include both import receptor and adaptor proteins (discussed below).

The protein receptor mediating snRNP import, importin β, was identified through kinetic competition studies. Depletion of importin β with an excess of the importin β binding domain of importin α, blocked snRNP import (Palacios et al.,

1997). Import was rescued upon the addition of recombinant importin β, indicating its vital role in snRNP import (Palacios et al., 1997). Additionally, studies indicated that an excess of classical-NLS-containing cargoes could not competitively inhibit snRNP nuclear import (Fischer et al., 1991; Fischer et al.,

1993; Izaurralde et al., 1997; Michaud and Goldfarb, 1992; Michaud and

Goldfarb, 1991). This observation indicated the existence of an additional

34 factor(s) specific for snRNP import and not shared with classical-NLS-mediated import. This additional factor was later identified and named Snurportin1 (SPN)

(Huber et al., 1998).

SPN contains an N-terminal importin β binding domain (IBB) as well as a centrally located TMG binding domain (Huber et al., 1998; Strasser et al., 2004).

Importin β contains HEAT repeats, similar to those of other karyopherins (e.g.

Xpo1), which are vital for NPC translocation (Bayliss et al., 2000; Petosa et al.,

2004 and references therein). As a result, complex formation consisting of importin β, SPN and the TMG capped cargo, facilitates nuclear import of newly assembled snRNPs (Fig. 1-5) (Huber et al., 1998; Huber et al., 2002). Upon nuclear import, snRNPs undergo further maturation, with a subset of these modifications occurring in the Cajal body (CB) (Darzacq et al., 2002; Jady et al.,

2003; Kiss et al., 2002). Subsequent to these and possibly additional nuclear modifications, snRNPs function as components of the pre-mRNA splicing machinery.

Nuclear export of import factors

Upon the nuclear delivery of cargo, import complex components must be exported back to the cytoplasm to mediate additional rounds of snRNP transport.

As for SPN, this process is mediated by the export receptor Xpo1 (Paraskeva et al., 1999). Recall that this protein, in cooperation with PHAX, also mediates the export of nascent snRNA transcripts. Xpo1 targets these snRNAs for export by

35 binding to the small leucine rich NES of PHAX. In contrast, SPN lacks a discernable leucine rich NES and is hypothesized to bind Xpo1 through an ambiguous domain, consisting of approximately two-thirds of the SPN protein

(Paraskeva et al., 1999). Export complex assembly, consisting of Xpo1 and

SPN, is promoted by the binding of Ran-GTP (Paraskeva et al., 1999). This complex is then exported to the cytoplasm, enabling SPN to mediate additional rounds of snRNP import.

36

Figure 1-5 Detailed schematic of snRNP import. Subsequent to cytoplasmic modifications, complex formation containing snurportin (SPN), which binds the TMG cap, and importin β, mediates snRNP import. Following import, Ran-GTP hydrolysis and additional unknown factors mediate complex disassembly. SPN and importin β are then shuttled back to the cytoplasm to mediate additional rounds of snRNP import. Newly imported snRNPs then undergo additional Cajal body-specific and nucleoplasmic modifications prior to mediating pre-mRNA splicing.

37 THE ROLE OF SNURPORTIN IN SNRNP BIOGENESIS

Prior to the identification of SPN, importin β was known to be a vital component necessary for snRNP import (Palacios et al., 1997). Importin β plays a critical role in the translocation of the NPC by interacting with nucleopore components.

The identification of the role played by importin β was significant to the understanding of snRNP import. However, the identity of the import adaptor, hypothesized to play a role in cap-dependent snRNP import, remained elusive. A critical component of our understanding of snRNP biogenesis was later obtained, when the adaptor protein, SPN, was found to promote snRNP import by linking importin β and snRNP cargo.

The identification of SPN

The discovery of SPN added a very important piece to the puzzle of snRNP biogenesis. The identification of SPN relied on the fact that it functions by binding to the 5′ TMG cap of its cargo in the cytoplasm. Due to this fact, a chemically synthesized, radiolabeled TMG cap oligo was incubated with HeLa cytosolic extracts. Following incubation, UV-crosslinking and SDS-PAGE analyses were conducted, which yielded a prominent 45 kDa band (Huber et al.,

1998). Size exclusion chromatography of HeLa cytosolic extracts, followed by affinity chromatography using the TMG cap oligo, resulted in a purified suspect protein adaptor. This protein was then microsequenced, yielding several peptide sequences corresponding to expressed sequence tags (ESTs) of SPN.

38 Further analyses indicated that snRNP binding was via the TMG cap alone, suggesting that SPN does not bind to any other RNA or protein component of the snRNP cargo (Huber et al., 1998). This conclusion was supported by the fact that TMG cap recognition was equally competed with either TMG caps alone,

TMG capped U snRNAs or TMG capped U snRNPs. Additionally, snRNPs lacking a 5′ TMG cap, failed to inhibit the crosslinking of SPN to TMG cap oligos

(Huber et al., 1998). Huber et al. (1998) also showed that SPN interacts with importin β by way of an N-terminal importin β binding domain (IBB). Finally, functional analyses illustrated that SPN greatly accelerates the nuclear import of cy3-labeled U1 snRNPs in Xenopus laevis oocytes and permeabilized HeLa cells

(Huber et al., 1998).

Details of SPN-mediated import

The formation of a complex containing SPN, cargo and importin β facilitates nuclear import of newly assembled snRNPs. This activity requires SPN to perform two functions. Namely, a SPN molecule must simultaneously bind to both importin β and TMG capped cargo. The assembly of a complex containing these components promotes a single round of snRNP import (Huber et al., 2002).

The initiation of snRNP import

The process of snRNP import begins with the arrival of newly exported snRNAs from the nucleus. Once in the cytoplasm, disassembly of the export complex

39 results in an snRNA that is ready for modification. The precise steps of export complex disassembly are unknown, however, this process is in part mediated by the dephosphorylation of PHAX (Ohno et al., 2000) by an unknown phosphatase(s). Subsequent to the release of snRNA from the export complex, a snRNP import complex must be assembled. The precise order of import complex formation is currently unknown, however, some events have been determined. For example, the addition of the Sm core by the SMN complex is a prerequisite for TMG cap formation. Additionally, due to the requirement of cap hypermethylation for binding of SPN to cargo, TMG capping must precede SPN binding. Details concerning the remaining events preceding cargo import are unclear. For example, whether SPN binds to importin β prior to, or following, incorporation into the import complex has not been deduced. The role of the

SMN complex after Sm core assembly is also unknown. Nonetheless, the formation of a complex containing snRNP cargo, a means of negotiating the NPC

(importin β) and an adaptor to bridge the two (SPN), promotes import.

The requirements for translocation of the NPC

Requirements for the translocation of snRNP cargo through the NPC have only recently come to light and require further elucidation. Nonetheless, data illustrate that the import of U snRNP cargo is both Ran and energy independent, at least in regards to single import events (Huber et al., 2002). These data were obtained by placing the IBB of SPN, or that of importin α, upstream of a β−galactosidase

40 (β−gal) cassette. The import characteristics of these heterologous substrates were then determined using in vitro import assays. The import of the importin α

IBB/β−gal cassette was Ran and energy dependent. However, replacement with the SPN IBB rendered this import Ran and energy independent (Huber et al.,

2002). This observation not only defined the Ran and energy requirements of

SPN-mediated import, it also illustrated that the Ran and energy independence

(in the case of SPN) is due solely to the nature of binding between importin β and

SPN (Huber et al., 2002). These data, although important in our understanding of snRNP import, only scratch the surface of this highly complex process.

The nuclear arrival of snRNP cargo

Having translocated the NPC, snRNP cargo must then be released into the , where it undergoes further maturation. As the import of snRNP cargo is independent of Ran (Huber et al., 2002), it appears that neither translocation across the nuclear envelope, nor the release of importin β-bound cargo from the NPC, requires Ran. This is in contrast to the Ran requirement of importin α-mediated import events (Görlich et al., 1996c). Interestingly, the region of importin β, which is bound by SPN, differs from the region bound by importin α (Rollenhagen et al., 2003). This difference in adaptor binding may promote the differential Ran requirement of cargo import. In accord with these differential import parameters is the observation that the time-frame between

NPC docking and the nuclear appearance of SPN-mediated cargo is shorter than

41 that of importin α-mediated cargo (Rollenhagen et al., 2003). The biological implications of these differential import requirements are unknown, however, the fact that snRNPs undergo further nuclear modifications, as compared to importin

α-mediated cargo, may predispose the snRNP pathway to altered regulation.

Import complex disassembly

Upon arrival in the nucleoplasm, snRNPs must be released from the import complex. The factors mediating this step are currently unknown. No additional direct protein interactors of SPN have been identified, however both direct and indirect interactions may facilitate disassembly. Whether or not Ran plays a role in snRNP import complex disassembly is also unknown, however in vitro data illustrate that the affinity between SPN and importin β is reduced upon the binding of Ran-GTP to importin β (Paraskeva et al., 1999). This reduction in binding may directly or indirectly promote complex disassembly, by destabilizing the complex and allowing access to other proteins. A potential role of Xpo1 in complex disassembly is discussed in Chapter 4 and may mediate this event.

Binding assays illustrate that the association of Xpo1 and cargo with SPN may be mutually exclusive (Paraskeva et al., 1999). This suggests that SPN is incapable of simultaneously being bound to both Xpo1 and snRNP cargo. Whether this is the case, and what potential role it might play in import complex disassembly, are unknown. Additionally, nothing is known regarding whether complex

42 disassembly, regardless of the pathway, is a prerequisite for further snRNP modifications.

Nuclear modifications and snRNP function

Upon reaching the nucleoplasm, snRNPs are further modified, resulting in the generation of mature snRNPs (Yu et al., 2001). Components mediating a subset of these processes have been recently identified and named small CB-specific

RNAs (scaRNAs), due to their enrichment in this nuclear body (Darzacq et al.,

2002). ScaRNAs act as guide RNAs and direct the pseudouridinylation and 2' O- methylation of RNA pol ΙΙ specific Sm class snRNPs (Darzacq et al., 2002; Jady et al., 2003; Richard et al., 2003). Additional modifications required for the generation of functional snRNPs have yet to be identified, however, judging from the complexity of snRNP biogenesis, these steps are likely to exist. Subsequent to these nucleoplasmic modifications, mature snRNPs are incorporated into the spliceosome and play a vital role in pre-mRNA splicing.

The recycling of SPN back to the cytoplasm

Following the deposition of newly imported snRNPs in the nucleus, SPN must be recycled back to the cytoplasm to mediate additional rounds of cargo import. The protein responsible for this recycling was quickly identified following the discovery of SPN and its role in snRNP import. The protein providing this recycling function turned out to be a well known export receptor, Xpo1 (Paraskeva et al., 1999).

43 Xpo1 is also responsible for the export of proteins containing NESs. This activity is mediated by the direct binding of Xpo1 to a leucine-rich NES motif, followed by translocation through the NPC. Interestingly, SPN contains no discernable motif resembling the consensus NES. Consequently, the region mediating the interaction between SPN and Xpo1 may be more difficult to determine and is currently unknown.

In addition to Xpo1, one other protein is necessary for efficient export of

SPN to the cytoplasm. Export complexes must be able to identify the cellular compartment they are in to efficiently direct transport. This identification, as well as the directionality of transport, are provided by the incorporation of Ran-GTP into the export complex. Binding of Ran-GTP to Xpo1 greatly increases its affinity for SPN (Paraskeva et al., 1999). As a result, export complex formation is likely to occur only in the nucleoplasm where most Ran is in the GTP-bound form. Subsequent to nuclear export, the recycling of SPN back to the cytoplasm facilitates additional rounds of snRNP import.

PURPOSE OF STUDY AND THESIS OUTLINE

The critical role of SPN in snRNP import has been established, however many questions remain concerning how SPN functions. For example, nothing is known concerning the order of import complex assembly or disassembly or if levels of regulation exist to modulate these processes. Currently, three SPN binding functions (importin β, TMG-capped RNA and Xpo1), have been identified,

44 however, nothing is known regarding the regulation of the affinities for these substrates. In fact no specific amino acids or motifs mediating these functions are known. Deficiencies in our basic understanding of SPN severely inhibit our ability to design and conduct further experiments aimed at deciphering function.

The aim of this dissertation is to gain insight into the role of SPN in snRNP transport and biogenesis. More specifically, I wish to address the following questions: What protein components constitute an import competent snRNP complex? Do interactions, in addition to those between SPN and importin β, mediate snRNP import? What SPN motifs and specific amino acids mediate its function? Can the Sm core-specific NLS mediate the import of TMG cap bound

SPN molecules? Lastly, is bound cargo a prerequisite for SPN import? To achieve these goals, I have employed both in vitro and in vivo techniques. In the following chapters I will describe my work toward defining the import complex that mediates snRNP biogenesis. Using biochemical techniques, we determined that

SPN interacts with SMN and that this interaction is RNA-dependent. Size exclusion chromatography of HeLa lysate illustrates that SPN, SMN and importin

β co-sediment, indicating their existence in a pre-import complex. Further work to define the role of the SMN complex as the unidentified Sm core NLS was carried out by a fellow graduate student and co-first author of Chapter 2, Usha

Narayanan, whereas I directed my thesis work on gaining a better understanding of the role SPN plays in snRNP biogenesis.

45 Work aimed at defining critical regions necessary for SPN function are described. These regions were initially identified using in vitro binding assays and subsequently validated using permeabilized HeLa cells and in vivo techniques. These mutants were then utilized to ask specific questions concerning SPN mediated snRNP import. Data illustrate that SPN binding to an

RNA cargo is not a prerequisite for its nuclear import. Results indicate that the interaction between SPN and importin β is not required to stabilize binding of the

SMN complex to importin β. Additionally, the binding of an IBB mutant of SPN to the TMG cap of cargo does not interfere with the Sm core dependent snRNP import pathway. This pathway promotes the import of a SPN IBB mutant in the presence of importin β, snRNPs and the SMN complex. Interestingly, an interaction between the N- and C-termini of SPN has been uncovered. This interaction may enable additional levels of regulation currently unknown in regards to this import adaptor.

46 CHAPTER 2

SMN, the Spinal Muscular Atrophy protein, forms a pre-import snRNP

complex with Snurportin1 and Importin β

Usha Narayanan‡, Jason K. Ospina‡, Mark R. Frey, Michael D. Hebert and

A. Gregory Matera*

Department of Genetics, Center for Human Genetics and Program in Cell

Biology, Case Western Reserve University and University Hospitals of Cleveland,

Cleveland, OH 44106-4955, U.S.A.

Note: This chapter is a manuscript published in 2002 in the journal, Human

Molecular Genetics.

‡These authors contributed equally to this work.

47 ABSTRACT

The survival of motor neurons (SMN) protein is mutated in patients with Spinal

Muscular Atrophy (SMA). SMN is part of a multiprotein complex required for biogenesis of the Sm-class of small nuclear ribonucleoproteins (snRNPs).

Following assembly of the Sm core domain, snRNPs are transported to the nucleus via importin β. Sm snRNPs contain a nuclear localization signal (NLS) consisting of a 2,2,7-trimethylguanosine (TMG) cap and the Sm core.

Snurportin1 (SPN) is the adaptor protein that recognizes both the TMG cap and importin β. Here, we report that a mutant SPN construct lacking the importin β binding domain (IBB), but containing an intact TMG cap-binding domain, localizes primarily to the nucleus, whereas full-length SPN localizes to the cytoplasm. The nuclear localization of the mutant SPN was not a result of passive diffusion through the nuclear pores. Importantly, we found that SPN interacts with SMN, Gemin3, Sm snRNPs and importin β. In the presence of ribonucleases, the interactions with SMN and Sm proteins were abolished, indicating that snRNAs mediate this interplay. Cell fractionation studies showed that SPN binds preferentially to cytoplasmic SMN complexes. Notably, we found that SMN directly interacts with importin β in a GST-pulldown assay, suggesting that the SMN complex might represent the Sm core NLS receptor predicted by previous studies. Therefore, we conclude that, following Sm protein assembly, the SMN complex persists until the final stages of cytoplasmic snRNP maturation and may provide somatic cell RNPs with an alternative NLS.

48 INTRODUCTION

The nuclear periphery is a very busy place: more than a million macromolecules are thought to be actively transported between the nucleus and the cytoplasm each minute (Allen et al., 2000; Görlich and Mattaj, 1996a; Ribbeck and Görlich,

2001; Smith et al., 2002). This bidirectional traffic is routed through nuclear pore complexes (NPCs), large protein structures that span the nuclear envelope bilayer (Allen et al., 2000; Rout and Aitchison, 2001; Ryan and Wente, 2000).

The NPCs are active participants in nucleocytoplasmic transport, which is mediated by soluble transport receptors. These receptor proteins, collectively known as karyopherins, shuttle between the nucleus and cytoplasm carrying a wide variety of cargoes. Unidirectional transport of the cargo molecules is achieved by two families of karyopherins called importins and exportins

(Izaurralde et al., 1997). Directionality is imposed on nucleocytoplasmic traffic by a small GTPase called Ran. The concentration of Ran-GTP is higher in the nucleus than in the cytoplasm and this gradient is maintained by the restricted subcellular localization of regulators of Ran among which is the guanine nucleotide exchange factor (RCC1), which is anchored to the

(Izaurralde et al., 1997; Kalab et al., 2002). Cargoes identify themselves to the nucleocytoplasmic transport machinery by motifs called nuclear localization signals (NLSs) and nuclear export signals (NESs). These signals can be protein- or RNA-based or a composite of the two (Conti and Izaurralde, 2001; Dingwall and Laskey, 1991; Jullien et al., 1999; Palacios et al., 1997; Pasquinelli et al.,

49 1997). Many classes of cargo contain signals that bind directly to a cognate receptor, while others have signals that bind to the receptor via adaptor proteins.

Small nuclear RNAs (snRNAs) of the Sm class have a rather unique life- cycle (Will and Lührmann, 2001). Following transcription in the nucleus, these

RNA pol ΙΙ transcripts are exported to the cytoplasm by binding to an adaptor protein called PHAX (Ohno et al., 2000). Assembly into stable ribonucleoprotein particles (snRNPs) requires the activity of the survival of motor neurons (SMN) protein complex (Paushkin et al., 2002; Will and Lührmann, 2001). After assembly of the heteroheptameric Sm-ring, the 5′ methylguanosine cap structure is hypermethylated (Mattaj, 1986) to form a trimethylguanosine (TMG) cap by a protein called Tgs1 (Mouaikel et al., 2002). Cap hypermethylation is a signal that triggers nuclear import (Fischer et al., 1993; Hamm and Mattaj, 1990; Mattaj and

De Robertis, 1985; Palacios et al., 1997) via binding to a protein called snurportin1 (Huber et al., 1998). Snurportin1 (SPN) is thus an adaptor that links snRNP cargo to importin β for subsequent transport to the nucleus (Huber et al.,

1998). The N-terminal, importin β binding domain (IBB) of SPN (Fig. 2-1A pg 59) shares significant similarity with importin α family members (Huber et al., 1998).

However, unlike other importins, SPN contains neither an NLS-binding domain, nor a domain that recognizes Ran-GTP (Huber et al., 1998; Paraskeva et al.,

1999). In contrast to that of importin α, the IBB of SPN allows for snRNP cargo to be imported in a Ran- and energy-independent fashion (Huber et al., 2002). It is important to note that SPN binds only the TMG cap and not to the Sm-core

50 (Huber et al., 1998). Thus, the C-terminus of SPN contains a novel TMG-binding domain.

Microinjection studies in Xenopus laevis oocytes have shown that Sm snRNPs (U1, U2, U4 and U5) possess a complex NLS composed of both the Sm core domain and the TMG cap (Fischer and Lührmann, 1990; Fischer et al.,

1993; Hamm and Mattaj, 1990). Interestingly, not all the spliceosomal snRNAs display the same TMG cap requirements for import in oocytes. Whereas U1 and

U2 snRNA absolutely require an intact cap, U4 and U5 snRNAs can be imported as ApppG-capped derivatives, albeit with reduced transport kinetics (Fischer et al., 1993). In contrast, the TMG cap is not essential for snRNP import in somatic cells (Fischer et al., 1994), although it accelerates the rate of transport. The Sm core domain is therefore necessary and sufficient to mediate nuclear targeting of snRNPs in somatic cells.

We were interested in perturbing the rate of Sm snRNP import in order to assay the effect on a prominent nuclear subdomain called the Cajal body

(reviewed in Gall, 2000; Matera, 1999). Cajal bodies (CBs) are thought to be one of the first sites of nuclear accumulation of newly-imported Sm snRNPs (Sleeman and Lamond, 1999). Furthermore, CBs associate with specific chromosomal loci in cells (Frey and Matera, 1995; Jacobs et al., 1999; Smith et al.,

1995); colocalization with the U2 snRNA gene cluster requires not only transcriptionally competent U2 loci (Frey et al., 1999) but the presence of intact

U2 snRNPs (Frey and Matera, 2001). Since Huber et al. (1998) showed that an

51 N-terminal deletion of SPN (SPN∆N65) was dominant negative for snRNP import in Xenopus oocytes, we decided to test the efficacy of this mutant in the mammalian system. To our surprise, SPN∆N65 overexpression did not allow us to assay the interaction of CBs and snRNA genes since it failed to titrate newly assembled snRNPs in the cytoplasm. Instead, we found that SPN∆N65 was actively imported into the nucleus, consistent with the existence of a parallel, cap- independent snRNP import pathway in somatic cells (Fischer et al., 1994;

Palacios et al., 1997). Furthermore, SPN and SPN∆N65 overexpression induced disassembly of CBs and SMN gems.

Previous studies suggested that a cytoplasmic factor bound to the Sm core domain mediates snRNP import in the absence of a functional TMG cap receptor (Fischer and Lührmann, 1990; Fischer et al., 1993; Palacios et al.,

1997). We hypothesized that the SMN complex, which is required for assembly of the Sm-ring (Fischer et al., 1997; Will and Lührmann, 2001), might provide this alternative import signal. Significantly, we found that SPN and SMN interact in

GST-pulldown and coimmunoprecipitation assays. The interaction of SPN and

SMN is not direct, but mediated by RNA and is restricted to the cytoplasmic compartment. Size exclusion chromatography assays confirmed that SMN, SPN and importin β cofractionate. As expected, only a relatively small fraction of the total SMN protein comigrates with importin β. Intriguingly, a zinc finger protein called ZPR1, implicated in the nuclear targeting of SMN (Gangwani et al., 2001), was also found to interact with a complex that contains SPN. These results

52 demonstrate that SMN is present not only during early cytoplasmic steps of Sm assembly but remains in the snRNP complex through late steps as well.

Consistent with the idea that SMN might accompany newly-assembled snRNPs into the nucleus, we also found that recombinant SMN interacts directly with importin β. Taken together, these results establish the existence of an import- competent snRNP complex that contains SMN and importin β.

53 MATERIALS AND METHODS

DNA constructs

Snurportin1 (SPN) and SPN∆N65 were amplified from a PCR-ready human brain cDNA library (BD Biosciences, Clontech) and cloned into pCR 2.1 (Invitrogen).

Primers containing EcoR I and BamH I restriction sites were used to PCR amplify

SPN from pCR 2.1. SPN was subsequently cloned into EcoR I/BamH I cut pEGFP-C3 (BD Biosciences, Clontech). 2X EGFP was generated by PCR amplification of EGFP from pEGFP-C3 using primers containing Bgl II and Hind

III restriction sites followed by cloning into Bgl II/Hind III digested pEGFP-C3.

SPN and SPN∆N65 were then excised from pEGFP-C3-SPN and pEGFP-C3-

SPN∆N65, respectively, with EcoR I and BamH I and cloned into EcoR I/BamH I digested 2xEGFP-C3 resulting in 2xEGFP-SPN and 2xEGFP-SPN1-∆N65.

Glutathione S-transferase (GST) fusions of SPN and SPN∆N65 were generated by PCR amplifying SPN from 2xEGFP-C3 SPN with EcoR I forward and BamH I reverse primers. This fragment was subsequently cloned into a BamH I/EcoR I digested pGex 2T vector (Amersham).

Size exclusion chromatography

The S100 cytoplasmic fraction was generated from 500 µl of packed HeLa cells

(National Cell Culture Center) using the NE-PER extraction system (Pierce

Chemical). 500 µl of S100 containing 10.5 mg of total protein were passed over a Superose 6HR 10/30 column and fractionated (300 µl fractions) using fast

54 performance liquid chromatography (FPLC). 25 µl of each fraction were added to

5 µl of 5X SDS-loading buffer, boiled and subjected to SDS-PAGE. 30 µl of S100 fraction were loaded as a positive control. Following transfer, membranes were probed with the appropriate primary and secondary antibodies.

Pulldowns

Expression and isolation of recombinant proteins were performed using standard techniques (Dignam et al., 1983). GST pulldowns were conducted by incubating

HeLa cell lysate, prepared as described (Dignam et al., 1983), with glutathione bead captured GST tagged proteins for 1 hr in a buffer that contains 450 mM

Na+. Beads were then washed 6 times with 1 ml modified (m)RIPA (50 mM Tris-

Cl, pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA), resuspended in 15 µl of 5X

SDS loading buffer, boiled and proteins were resolved by SDS-PAGE. Following transfer to nitrocellulose, membranes were probed with appropriate primary and secondary antibodies.

Coimmunoprecipitation

HeLa cells were cultured in DMEM (Invitrogen, Gibco BRL) with 10% FBS

(Invitrogen, Gibco BRL), penicillin and streptomycin. pEGFP-SPN or pEGFP-

SPN∆N65 was transfected into HeLa cells using SuperFect (QIAGEN). As a control, untransfected HeLa cells were also utilized. Cells were harvested 24 hours after transfection, washed with PBS and resuspended in 1 ml of mRIPA

55 buffer plus protease inhibitor cocktail tablets (Roche) to lyse the cells.

Resuspended cells were incubated at 4°C for 30 minutes followed by centrifugation for 5 minutes to pellet cellular debris. Where indicated, 5 mg of total protein from the lysate were treated with RNase A (1 mg, Sigma) and RNase

T1 (5000 units, gift of H. Salz) for 1 hr at 30°C. 50 µl of polyclonal anti-GFP (BD

Biosciences, Clontech) were then added to 900 µl of lysate. After incubating for

1 hr, 60 µl of 50% protein A Sepharose beads (Amersham) were added and to the lysates and incubated overnight. Beads were then washed 6 times with 1 ml mRIPA, resuspended in 15 µl of 5X SDS loading buffer, boiled and proteins were resolved by SDS-PAGE. After transfer to nitrocellulose, membranes were probed with anti-SMN (7B10) followed by incubation with goat anti-mouse conjugated horseradish peroxidase (Pierce Chemical) and chemiluminescence detection

(Roche).

Antibodies

A polyclonal SPN antibody was generated (Sigma Genosys) using a synthesized peptide consisting of CGMESEEENKKDDEE residues corresponding to amino acids 73-85 of human SPN. Other antibodies utilized in this study include: mouse monoclonal anti-SMN (7B10; Bühler et al., 1999); mouse monoclonal anti-

ZPR (LG1; Gangwani et al., 2001); mouse monoclonal anti-Gemin 3 (11G9;

Charroux et al., 1999); mouse monoclonal anti-Sm (Y12; Lerner et al., 1981); rabbit polyclonal anti-importin beta (gift of K. Weis); mouse monoclonal anti-GST

56 (Santa Cruz); rabbit polyclonal and mouse monoclonal anti-GFP (Roche); mouse monoclonal anti-myc (9E10; Santa Cruz); mouse monoclonal anti-T7 (Novagen); rabbit polyclonal anti-coilin (R288; Andrade et al., 1991); mouse monoclonal anti-

U2B′′ (gift of H. Salz); and rabbit polyclonal anti-glutaredoxin (gift of J. Mieyal).

Secondary antibodies utilized were goat anti-mouse and goat anti-rabbit conjugated horseradish peroxidase (Pierce Chemical) as well as goat anti-rabbit conjugated fluorescein (Vector Laboratories).

Immunofluorescence

HeLa cells were grown on slides (Nunc) to 80% confluency. Slides were fixed in

4% paraformaldehyde for 10 min at room temperature followed by extraction with

0.5% Triton for 5 min. Incubation in 10% normal goat serum was followed by the addition of anti-SPN rabbit polyclonal antibody, R89 and detection with fluorescein-conjugated goat anti-rabbit (Molecular Probes). Incubations at 30°C for 30 min were followed by 3 washes with 1X PBS for 5 min each at room temperature.

57 RESULTS

SPN is primarily a cytoplasmic protein

SPN is a protein of approximately 45 kDa that is required for nuclear snRNP import in Xenopus laevis oocytes (Huber et al., 1998). In order to better characterize the complexes that carry out snRNP import in mammals, we developed polyclonal antibodies against human SPN and cloned the cDNA by

RT-PCR using the published sequence (Huber et al., 1998). Western blot analysis with anti-SPN serum R89 identified a single band of the expected size

(Fig. 2-1B). Immunofluorescence with this antiserum revealed that the protein is localized throughout the cytoplasm, concentrating near the nuclear periphery

(Fig. 2-1C). This localization pattern is completely consistent with SPN’s role as a nuclear import adaptor. Equivalent results were obtained upon expression of

GFP-SPN (Fig. 2-1C). Western blot analysis of extracts from cells transfected with GFP-SPN revealed the presence of an additional band of the appropriate size (data not shown). Thus SPN antiserum R89 specifically recognizes both the exogenous and endogenous SPNs.

58

Figure 2-1: Schematic of SPN and characterization of antibody R89. (A) A cartoon of SPN indicating the importin β binding domain (IBB), the CRM1/Xpo1 and trimethylguanosine (TMG) cap binding domains. (B) Western blot analysis of HeLa extract with SPN antibody R89. The left lane was probed with pre-immune serum and the right lane with R89. The antibody specifically detected a band of approximately 45 kDa. (C) The subcellular localization of SPN was studied by transient transfection of HeLa cells with GFP-SPN (top panels). In the lower panels, cells were immunostained with anti-SPN R89. Both the exogenous and endogenous SPN localized to the cytoplasm with enrichment at the nuclear periphery.

59 A SPN mutant is actively transported to the nucleus

Huber et al. (1998) showed that the N-terminal 65 amino acids of SPN contain a domain (IBB, Fig. 2-1A) required for binding to importin β. Overexpression of

SPN∆N65 in frog oocytes abolished import of snRNPs into the nucleus (Huber et al., 1998). Thus we were quite surprised to find that GFP-SPN∆N65 was primarily nuclear when expressed in human cells (Figs. 2-2 and 2-3). The molecular weight cutoff for proteins passively diffusing through the NPC is reportedly ~40-60 kDa (Feldherr and Akin, 1990) and depends to some extent on the shape of the macromolecule. That is, proteins larger than this size require assistance from importins and exportins. GFP-SPN∆N65 contains an intact TMG binding domain but cannot bind to importin β (Fig. 2-1A). Therefore, it was formally possible that the construct could simply diffuse into the nucleus and bind to snRNPs. In the case of wildtype SPN, cargo release is effected upon binding to the export factor, Xpo1 (Paraskeva et al., 1999). However, binding to Xpo1 and recycling to the cytoplasm are disrupted in SPN∆N65, as export depends on multiple sites throughout the length of the protein (Paraskeva et al., 1999). Only the C-terminal 75 amino acids are dispensable for this activity (Fig. 2-1A). Since it was conceivable that GFP-SPN∆N65 (calc. MW ~65 kDa) was small enough to pass through the nuclear pores unassisted, we cloned a second GFP domain in- frame with GFP-SPN and GFP-SPN∆N65 to ensure that these constructs exceeded the limit for passive diffusion. As shown in Figure 2-2, the localization patterns of the 2xGFP constructs were identical to those of their 1xGFP

60 counterparts. These results indicate that GFP-SPN∆N65 is actively imported into the nucleus by an alternative pathway that does not involve direct binding to importin β (Fischer et al., 1993; Huber et al., 1998; Marshallsay and Lührmann,

1994; Palacios et al., 1997).

Figure 2-2: SPN is actively imported into the nucleus. Transient transfections of HeLa cells with 1x- or 2x-GFP tags. SPN (left panels) and SPN∆N65 (right) are shown. Surprisingly, SPN∆N65, which lacks an IBB, localizes primarily to the nucleus.

61 Overexpression of SPN and SPN∆N65 disassembles Cajal bodies

In the course of our localization experiments, we noticed that GFP-SPN∆N65 occasionally displayed nuclear foci (Fig. 2-2). Immunofluorescence with CB marker proteins coilin and SMN revealed that these focal accumulations did not strictly correspond to CBs, although they sometimes colocalized (Fig. 2-3 and data not shown). Strikingly, we found that cells transfected with GFP-SPN∆N65 typically displayed fewer CBs than did neighboring untransfected cells (Fig. 2-3).

In fact, careful analysis revealed that even cells transfected with wildtype GFP-

SPN showed a reduction in CB number (Table 2-1). Whereas moderate levels of

GFP-SPN∆N65 expression typically resulted in CB disassembly, only high levels of GFP-SPN had this effect (Fig. 2-3). Similar results were obtained in parallel experiments when SMN was used as the CB marker (Table 2-1). This effect was specific to SPN, as cells transfected with GFP alone showed no difference in CB numbers when compared to untransfected cells (Table 2-1).

As mentioned above, the typical SPN localization pattern was diffuse throughout the cytoplasm with a relatively pronounced staining of the nuclear periphery. Thus the majority of cells did not show any obvious colocalization with the cytoplasmic fraction of SMN (data not shown). However, we sometimes observed local accumulations of both GFP-SPN and SMN in the cytoplasm (Fig.

2-4), suggesting a possible interaction between the snRNP assembly and import machineries.

62

Figure 2-3: Comparison of mutant and wildtype proteins and their effects on Cajal bodies (CBs). Transient overexpression of GFP-SPN or GFP-SPN∆N65 causes a reduction in the number of CBs (see Table 2-1). The larger foci that were occasionally observed upon transfection with GFP-SPN∆N65 (top panels, arrow) did not strictly correspond to CBs (arrowhead). The low magnification image in the second set of panels shows that cells expressing GFP-SPN∆N65 displayed fewer CBs (arrows) than did the untransfected cells (e.g. the cell marked by the arrowhead). Cells expressing lower levels of GFP-SPN (third panel, lower cell) typically displayed higher numbers of CBs than did those cells expressing higher levels of the construct (upper cell).

63

Figure 2-4: Colocalization of overexpressed SPN and endogenous SMN. Transient overexpression of GFP-SPN was followed by immunofluorescence detection of SMN. Cytoplasmic accumulations of SMN (arrows) could sometimes be detected in the GFP-SPN channel (arrows) and were not visible in the DAPI channel (not shown).

Table 2-1. Effects of SPN overexpression on CBs.

64 SPN forms a cytoplasmic complex with SMN and splicing snRNPs

The SMN complex is required for assembly of the Sm-ring which, in turn, is required for cap hypermethylation (Will and Lührmann, 2001). Precisely when

SMN leaves the snRNP complex following Sm core assembly has not yet been established. In order to determine whether SMN and SPN are capable of binding to the same RNPs, we performed GST-pulldown assays with HeLa cytoplasmic extracts. As shown in Figure 2-5A, SMN, Gemin3 and SmB/B′ proteins were recovered by GST-SPN bound to glutathione-sepharose beads. Control experiments with GST alone were negative. Thus the SMN complex remains bound to TMG-positive Sm snRNPs. In order to prove that SPN forms a native complex with SMN cells were transfected with GFP-tagged SPN constructs and analyzed by co-immunoprecipitation with anti-GFP antibodies. As shown in

Figure 2-5B, SMN was coprecipitated by both GFP-SPN and GFP-SPN∆N65.

Parallel northern blotting experiments (Figs. 2-5C,D) showed that U2 snRNA was also co-precipitated with the SPN constructs. Both wildtype SPN and the IBB mutant protein have intact TMG binding domains and, as expected, each bound equivalent amounts of U2 snRNA in the GST-pulldown experiment (Fig. 2-5C).

However, quantification of the blot in Figure 2-5D showed that GFP-SPN∆N65 brought down five times more U2 RNA than did GFP-SPN and ten times more than GFP alone. GFP-SPN brought down twice as much RNA as did the GFP control. Given the nuclear localization of both GFP-SPN∆N65 and the bulk of the

Sm snRNPs, this result is not surprising. Additionally, SPN binding to Xpo1 is

65 required for cargo release and cytoplasmic recycling. Thus the SPN∆N65 construct, which cannot bind to Xpo1, likely binds “irreversibly” to snRNPs, whereas the wildtype protein does not.

66

Figure 2-5: SPN interacts indirectly with SMN in the cytoplasm. (A) SPN interacts with the cytoplasmic SMN complex in vitro. Pulldown assays with GST-SPN or GST alone were performed using HeLa cytoplasmic extracts. The pulldowns were analyzed by western blotting with monoclonal antibodies against SMN (7B10), Gemin3 (11G9) and SmB/B′ (Y12). (B) SPN interacts with SMN in vivo. HeLa cells were transiently transfected with GFP-SPN or GFP- SPN∆N65; untransfected cells were used as a negative control. Immunoprecipitations (IPs) were performed from total HeLa cell lysates with polyclonal antibodies against GFP. The IPs were then analyzed by western blotting with 7B10. (C) SPN and SPN∆N65 interact with U2 snRNA with similar affinities in vitro. GST pulldown assays were performed using GST-SPN, GST- SPN∆N65 and GST (negative control) from total HeLa cell lysate. RNA was isolated from the pulldowns, a U2 snRNA specific radiolabeled probe was used for analysis by northern blotting. The lower panel shows the coomassie image showing approximately equal amounts of protein were used. (D) SPN∆N65 and SPN bind U2 snRNA in vivo. HeLa cells were transiently transfected with GFP- SPN, GFP-SPN∆N65 or GFP (negative control). IPs were performed from total HeLa cell lysates with monoclonal antibodies against GFP. RNA was isolated from the IP, and analyzed by northern blotting.

67 To test whether SMN and SPN might interact directly, we purified recombinant proteins expressed in bacteria and performed GST-pulldowns. No direct binding was detected (Fig. 2-6A). Therefore, the interaction between SPN and SMN is likely mediated by the snRNA. To test this hypothesis, we performed GST- pulldown assays from cell extracts incubated in the presence or absence of

RNases A and T1. Figure 2-6B reveals that SMN was efficiently recovered in the absence of added RNase and not detectable in its presence. The blots were reprobed with anti-GST to demonstrate that roughly equivalent amounts of protein were used. As an independent control for monitoring RNase activity, extracts were incubated in the presence or absence of RNase and then assayed for binding of U2B′′ (a U2-specific snRNP protein) to U2 RNA (data not shown).

In order to determine if SMN and SPN complexes were restricted to a particular subcellular compartment, nuclear and cytoplasmic HeLa cell extracts were prepared and GST-pulldown assays performed with GST-SPN and GST alone. Cell fractionation was monitored using control antibodies (data not shown) against proteins that localize to the cytoplasm (Eriksson and Mannervik, 1970) or nucleus (Chan et al., 1994). As shown in Figure 2-6C, SMN was only recovered from the cytoplasmic fractions. Taken together, these data establish that SPN and SMN co-exist in a cytoplasmic complex, bridged by snRNAs. Thus we conclude that SMN remains bound to Sm snRNPs after cap hypermethylation, forming a putative pre-import complex.

68

A

B C

Figure 2-6: SPN and SMN do not interact directly. (A) Pulldown assays were performed using GST-SPN and GST (negative control), along with His-T7-SmB′ or His-T7-SMN. The pulldowns were analyzed by western blotting with anti-T7. (B) RNA mediates the interaction of SPN with SMN. Pulldown assays were performed using GST-SPN or GST (negative control) from HeLa cell lysates in the absence or presence of ribonucleases. The pulldowns were analyzed by western blotting with 7B10. (C) SPN interacts with cytoplasmic SMN. Pulldown assays were performed using GST-SPN and GST (negative control) from HeLa cells (nuclear or cytoplasmic fractions). The pulldowns were analyzed by western blotting with 7B10. Anti-GST and anti-GFP were used as loading controls where indicated. Inputs show 12% of the total lysate used in the pulldowns.

69 SMN and SPN form a complex that contains importin β

The interaction of Sm snRNPs, SPN and importin β has been well established

(Huber et al., 1998). Our finding that SMN and SPN form a complex together prompted the question as to whether SMN is present in an import-competent snRNP complex with importin β. As a first step, we transfected cells with myc-

SMN, immunoprecipitated with anti-myc antibodies and probed a western blot with anti-importin β. Interestingly, Figure 2-7A shows that SMN exists in a complex with importin β in vivo. In order to show that SPN, SMN and importin β are together in the same complexes, size exclusion chromatography was performed using HeLa cytoplasmic extract, followed by western blot analysis.

SMN runs in two broad peaks, the first one between 600 and 1200 kDa and the other between 100 and 400 kDa (Fig. 2-7C). Notably, importin β, SMN and SPN comigrate in the 400 kDa region, suggesting that these proteins form a single complex.

As described above, several lines of evidence suggest the existence of an

Sm core-directed, TMG cap-independent import mechanism that also impinges upon the importin β pathway (Palacios et al., 1997). In light of our findings, the simplest explanation for these data is that the SMN complex is the factor that recognizes both the Sm core and importin β, thus acting as the adaptor for this cap-independent pathway. We decided to test this idea further by asking whether SMN and importin β could bind to each other directly using recombinant

70 proteins. As shown in Figure 2-7B, GST-SMN, but not GST alone, clearly interacted with His-tagged importin β.

Figure 2-7: SMN interacts directly with importin β. (A) HeLa cells were transfected with myc-SMN; untransfected cells were used as a negative control. IPs were performed from total HeLa cell lysates with monoclonal antibodies against myc. The IPs were then analyzed by western blotting with anti-importin β. (B) SMN and importin β interact directly using purified recombinant proteins. Pulldown assays were performed using GST-SMN or GST alone and His-myc-importin β. The pulldowns were analyzed by western blotting with anti-myc and anti-GST (loading control). Input shows approximately 50% of the total lysate used in the pulldowns. (C) Size exclusion chromatography of HeLa cytoplasm. Fractions were analyzed by western blotting with the indicated antibodies. SPN, SMN and importin β cofractionate in the ~400 kDa range, consistent with the existence of a complex containing all three proteins.

71 SPN, SMN and the zinc finger protein ZPR1 form a complex

Given that SPN, importin β and SMN form what appears to be an import- competent complex, this raised the question as to whether the SMN complex is imported into the nucleus along with the newly assembled snRNPs. A recent study found that the zinc finger protein, ZPR1, is required for the accumulation of

SMN in the nucleus (Gangwani et al., 2001). ZPR1 is an essential protein in yeast (Gangwani et al., 1998). In mammals, the protein is involved in relaying proliferative growth signals from the cell surface to the nucleus (Galcheva-

Gargova et al., 1998; Galcheva-Gargova et al., 1996). Although ZPR1 is localized to the cytoplasm in quiescent cells, the protein is translocated to the nucleus upon mitogen stimulation (Galcheva-Gargova et al., 1998; Galcheva-

Gargova et al., 1996; Gangwani et al., 1998). Truncation of the C-terminus of

ZPR1 abolished the interaction with SMN and was dominant negative for SMN localization to (Gangwani et al., 2001). Therefore, we wanted to determine whether ZPR1 might also be complexed with SPN. Toward that end, we performed GST-pulldowns and co-immunoprecipitations. HeLa cell lysate was passed over GST and GST-SPN beads and then probed with anti-ZPR1 antibodies (Fig. 2-8A), revealing an interaction between the two proteins.

Similarly, lysates from cells transfected with GFP-SPN were immunoprecipitated with anti-GFP antibodies and probed for the presence of ZPR1. As shown in

Figure 2-8B, GFP-SPN but not GFP alone brought down ZPR1 protein. The interaction between SPN and ZPR1 was sensitive to RNase digestion (Fig. 2-8C)

72 and was primarily restricted to the cytoplasm (Fig. 2-8D). Furthermore, size exclusion chromatography showed that ZPR1 also comigrated with SPN, SMN and importin β (data not shown), suggesting that these factors are present in the same complexes. Thus the fact that ZPR1, which is involved in regulating the accumulation of SMN in the nucleus (Gangwani et al., 2001), is also complexed with SPN and importin β suggests that SMN could indeed accompany newly assembled snRNPs across the NPC and perhaps helps to target them to CBs

(Hebert et al., 2001; Sleeman et al., 2001).

73

Figure 2-8: SPN forms a cytoplasmic complex with ZPR1. (A) SPN interacts with ZPR1 in vitro. Pulldown assays with GST-SPN and GST were performed using HeLa lysate. The pulldowns were analyzed by western blotting with monoclonal antibodies against ZPR1 (LG9). (B) SPN interacts with ZPR1 in vivo. HeLa cells were transiently transfected with GFP-SPN or GFP alone. IPs were performed from total HeLa cell lysates with polyclonal antibodies against GFP. The IPs were then analyzed by western blotting with LG9 and GFP (loading control). (C) RNA mediates the SPN-ZPR1 interaction. Pulldown assays were performed using GST-SPN; GST (negative control) from HeLa cell lysates in the presence or absence of ribonucleases. (D) SPN interacts with cytoplasmic ZPR1. Pulldown assays were performed using GST-SPN and GST (negative control) along with nuclear (N) or cytoplasmic (C) HeLa cell extracts.

74 DISCUSSION

One function of the SMN protein complex is to assemble the Sm core domain

(Fischer et al., 1997; Meister et al., 2001a). To accomplish this task, members of the complex must contact both the snRNA (Yong et al., 2002) and the Sm proteins (Bühler et al., 1999; Charroux et al., 1999; Charroux et al., 2000;

Pellizzoni et al., 1999). In vivo, a subset of the Sm proteins are targeted to the

SMN complex by symmetrical dimethylarginine residues that are posttranslationally added to their arginine and glycine rich tails (Brahms et al.,

2001; Brahms et al., 2000; Friesen et al., 2001a; Friesen et al., 2001b; Friesen et al., 2002; Meister et al., 2001b). The precise mechanism used by SMN to direct cytoplasmic assembly of the heptameric Sm-ring is unknown. Following assembly, the events whereby the newly assembled snRNP particle acquires its hypermethylated 5′ cap and is transported across the nuclear pore are poorly understood. Our results demonstrate that SMN does not fall off immediately following assembly, but remains associated with the snRNP throughout the downstream cytoplasmic maturation steps. Together with data from the literature, our results suggest the existence of at least three distinct cytoplasmic

SMN/snRNP subcomplexes: (i) a post-export, pre-Sm assembly particle (Yong et al., 2002), (ii) one that contains an Sm-ring but lacks a TMG cap (Pellizzoni et al., 1998) and (iii) a pre-import complex that contains SPN and importin β (this work).

75 Furthermore, the results implicate the SMN complex in TMG cap formation since SMN is present both before and after cap hypermethylation. It is therefore possible that the SMN complex also functions to recruit the requisite methyltransferase. The enzyme that performs this function has recently been identified in both yeast and mammals (Mouaikel et al., 2002; Verheggen et al.,

2002). Interestingly, the subcellular distribution of this protein, called Tgs1, mirrors that of SMN: it is diffusely localized throughout the cytoplasm and concentrated in nuclear Cajal bodies (Verheggen et al., 2002).

Finally, our findings that SMN directly interacts with importin β and that

SPN, SMN, ZPR1, importin β and TMG-capped snRNPs form a complex indicates that SMN may be imported into the nucleus along with the snRNPs.

The simplest explanation to emerge from these data is that the SMN complex is, in fact, the long-awaited Sm core NLS receptor (Fig. 2-9). Consistent with this interpretation, heterokaryon fusion experiments using FP-tagged Sm proteins showed that only SMN-positive CBs accumulated newly imported snRNPs

(Sleeman et al., 2001). Recent work from our laboratory has shown that coilin contains symmetrical dimethylarginine residues (Hebert et al., 2002) and can compete with Sm proteins for binding sites on SMN (Hebert et al., 2001). Thus it is possible that coilin may help to dissociate the SMN/snRNP complex following nuclear import (Hebert et al., 2001). However, direct evidence of SMN crossing the pore along with the snRNPs is currently lacking. Alternatively, SMN may be released from snRNPs just prior to, or concomitant with import. In that case,

76 nuclear SMN may be involved in other aspects of assembly or reassembly of snRNPs and snoRNPs (Jones et al., 2001; Pellizzoni et al., 2001), although these two functions need not be mutually exclusive.

In summary, we have shown that SMN is associated with snRNPs during the final steps in cytoplasmic Sm snRNP assembly and that it directly interacts with importin β. Thus the SMN complex is uniquely positioned to act as a quality control factor at multiple steps in the Sm snRNP maturation process. As such, the complex may act as a kind of “master assembler” or chaperone of RNP assembly (Gubitz et al., 2002; Terns and Terns, 2001). In the future, elucidating the molecular mechanisms behind these processes should help to better understand the phenotype of patients with Spinal Muscular Atrophy.

77

Figure 2-9: Model of the U snRNP import complex.

Adapted from Palacios et al. 1999. SPN binds to the TMG cap (m3G) of Sm class snRNPs and interacts with importin β via its IBB. The hypothetical snRNP core-binding factor (see text) is predicted to be the SMN complex. Since SMN lacks an IBB, direct binding to importin β (Fig. 2-7B) is therefore unlikely to proceed via the SPN/importin α binding pocket (α). Two importin β molecules are shown in the model, as predicted by (Palacios et al., 1997). Alternatively, binding of the Sm core by SMN might stabilize the binding of a single importin β. Additional experiments will be required to distinguish between these two possibilities.

78 ACKNOWLEDGEMENTS

We thank the members of our lab for helpful discussions of the project and Erica

Jacobs, a past lab member in particular for her technical assistance. Special thanks to the members of the Mieyal lab, particularly David Starke for all his help with size exclusion chromatography. We are also indebted to U. Fischer, K.

Weis, R. Davis, H. Salz, J. Steitz, G. Dreyfuss, for generously providing us with their reagents. We are thankful to Juan Valcarcel and Patrick Foersch at EMBL for their help with some techniques. We acknowledge the National Cell Culture

Center for providing HeLa S9 cells. This work was supported by NIH grants R01-

GM53034 and R01-NS41617 (to A.G.M.) and by a research grant from the

Muscular Dystrophy Association. J.K.O. was supported in part by an NIH predoctoral traineeship (T32-GM08613). MDH was supported in part by an NIH postdoctoral fellowship (T32-HD07518-04) and by a Development Grant from the

Muscular Dystrophy Association.

79 CHAPTER 3

Crosstalk between snurportin1 subdomains

Jason K. Ospina1, Graydon B. Gonsalvez1, Janna Bednenko2, Edward

Darzynkiewicz3, Larry Gerace2 and A. Gregory Matera1*

1Department of Genetics, Case Western Reserve University, School of Medicine,

Cleveland, OH 44106-4955 USA

2Department of Cell Biology and Department of Molecular Biology, The Scripps

Research Institute, La Jolla, CA 92037 USA

3Department of Biophysics, Institute of Experimental Physics, Warsaw

University, Warsaw, 02-089, Poland

Note: This chapter is a manuscript in press in the journal, Molecular Biology of the Cell.

All data were generated by Jason K. Ospina with the exception of Supplemental Figure 3S2, which was provided by Janna Bednenko.

80 ABSTRACT

The initial steps of spliceosomal small nuclear ribonucleoprotein (snRNP) maturation take place in the cytoplasm. Following assembly of an Sm-core and a trimethylguanosine (TMG) cap moiety, the RNPs are transported into the nucleus via the import adaptor, snurportin1 (SPN) and the import receptor, importin β. To better understand this process, we identified SPN residues that are required to mediate interactions with TMG caps, importin β and the export receptor, exportin1 (Xpo1/Crm1). Mutation of a single arginine residue within the importin

β binding domain (IBB) disrupted the interaction with importin β, but preserved the ability of SPN to bind Xpo1 or TMG caps. Nuclear transport assays showed that this IBB mutant is deficient for snRNP import but that both snRNP and SPN import can be rescued by addition of purified survival of motor neurons (SMN) protein complexes. Conserved tryptophan residues outside of the IBB are specifically important for TMG binding. However, SPN can be efficiently imported into the nucleus in the absence of cargo. SPN interacts with Xpo1 in a leptomycin B-sensitive fashion and relocalizes to Cajal bodies upon treatment with the drug. Finally, we uncovered an interaction between the N- and C- terminal domains of SPN, suggesting an autoregulatory function similar to that of importin α.

81 INTRODUCTION

A key feature of all eukaryotic cells is their ability to regulate the flow of macromolecules between various subcellular compartments. The nuclear envelope is perhaps the best example of this type of cellular partitioning, as the nuclear pore complexes (NPCs) embedded within this structure allow for the selective transport of specific RNA and protein cargoes (reviewed in Pante, 2004;

Rout and Aitchison, 2001; Suntharalingam and Wente, 2003). Individual cargoes contain nuclear localization signals (NLSs) and/or nuclear export signals (NESs), which are recognized by members of the karyopherin family (reviewed in Fried and Kutay, 2003). Karyopherins can be divided into two subfamilies, called importins and exportins, depending on the direction of their transport activities

(reviewed in Mosammaparast and Pemberton, 2004). Nucleocytoplasmic trafficking is thus a highly orchestrated process, mediated by transport receptor proteins.

Despite their opposing transport activities, most importins and exportins are structurally related to importin β (reviewed in Harel and Forbes, 2004).

Importin β family members are characterized by an N-terminal Ran binding domain and a series of HEAT repeats (reviewed in Andrade et al., 2001). The

HEAT repeats interact with the FG-rich motifs present in most nucleoporins and allow for passage of cargo through the NPC. The direction of cargo transport is regulated by a small GTPase called Ran (Izaurralde et al., 1997). In the nucleus,

Ran exists primarily in the GTP-bound state, whereas cytoplasmic Ran is

82 predominantly GDP-bound. Nuclear Ran-GTP promotes dissociation of importins from their cargoes and association of exportins with their substrates, thereby conferring directionality to the system (Görlich and Mattaj, 1996a).

An additional group of adaptor proteins mediates cellular transport in cooperation with the importin β superfamily. These adaptors facilitate transport of cargoes that cannot bind directly to a given receptor protein. For example, importin α forms the bridge between most “classical” NLS motifs and importin β

(Adam and Adam, 1994; Adam and Gerace, 1991; Moroianu et al., 1995; Weis et al., 1995). The N-terminal region of importin α contains an importin β binding motif (IBB), whereas the C-terminal domain mediates recognition of the NLS- containing cargoes (Conti et al., 1998; Görlich et al., 1996b). Interestingly, the N- terminal IBB also contains a weak NLS that is thought to perform an autoregulatory function (Conti et al., 1998; Kobe, 1999). Thus, adaptor proteins like importin α, must shuttle between the nucleus and the cytoplasm binding cargo in one compartment and dropping it off in the other. However, transport proteins are not the only factors known to shuttle.

Certain cargo proteins (e.g. cyclins, hnRNP proteins) are known to contain both NLSs and NESs (reviewed in Dreyfuss et al., 2002). Thus these factors can also shuttle between the nucleus and cytoplasm. Sm-class ribonucleoproteins

(RNPs) represent a unique category of cargoes, as they are one of the few factors known to make two “one way” trips, traveling from the nucleus to the cytoplasm and back again, albeit with significant remodeling on each leg of the

83 circuit (reviewed in Kiss, 2004; Will and Lührmann, 2001). Interestingly, the RNA component of the RNP forms an integral part of the signals used for these transport events. Export of small nuclear (sn)RNA transcripts from the nucleus to the cytoplasm is mediated by specific factors that recognize the RNA pol ΙΙ- encoded 7-methylguanosine (m7G) cap structure (Izaurralde et al., 1994;

Masuyama et al., 2004; Ohno et al., 2000). Once in the cytoplasm, snRNAs are assembled with core factors, called Sm proteins, forming a stable RNP. This process is mediated by the activity of the survival of motor neurons (SMN) protein complex (Meister et al., 2002; Yong et al., 2004). Following Sm-core formation, the m7G cap is hypermethylated by an enzyme called Tgs1 to create a 2,2,7- trimethylguanosine (TMG) cap (Mouaikel et al., 2003; Mouaikel et al., 2002;

Verheggen et al., 2002). The TMG cap and the Sm core collaborate to form two separable NLSs through which two independent import adaptors utilize the same import receptor, importin β (Fischer et al., 1993; Marshallsay and Lührmann,

1994; Palacios et al., 1997). Snurportin1 (SPN) is the adaptor protein for the

TMG cap-dependent pathway (Huber et al., 1998; Huber et al., 2002), whereas the SMN complex is required for the Sm-core pathway (Narayanan et al., 2004).

Subsequently, importin β exits the nucleus in a complex with Ran-GTP (Hieda et al., 1999; Izaurralde et al., 1997); whether or not components of the SMN complex are exported from the nucleus is unknown. Recycling of SPN is carried out by the export receptor exportin1 (Xpo1; Paraskeva et al., 1999) which is also known as Crm1 in yeast (Adachi and Yanagida, 1989; Stade et al., 1997).

84 Human SPN is a 45 kDa protein that contains three known functional domains, consisting of an N-terminal IBB, a centrally-located TMG cap binding domain and an ill-defined region responsible for binding to Xpo1 (Fig. 3-1 pg 93).

The SPN N-terminus shares significant similarity with the IBB of importin α, but the TMG-binding domain is completely novel, with no obvious similarity to other

RNA-binding proteins (Huber et al., 1998). Despite the fact that SPN binds to

Xpo1 with high affinity, the protein lacks a discernable leucine-rich NES

(Paraskeva et al., 1999). In order to better define the motifs within SPN that are important for its function, we undertook a mutational analysis of the protein.

Using a combination of in vivo localization, in vitro binding and nuclear transport assays, we have identified specific residues within both the IBB and TMG domains that are required for proper SPN function. Despite an extensive survey of internal deletion and substitution , we were unable to define a specific region of the protein that mediates Xpo1 binding, suggesting that Xpo1 recognizes a structural motif, rather than a linear amino acid sequence. Notably,

SPN rapidly accumulates in nuclear Cajal bodies upon treatment with leptomycin

B (LMB), a powerful Xpo1 inhibitor. Furthermore, we generated two mutations that uncover the existence of an interaction between the N- and C-terminal domains of SPN. In vivo and in vitro analyses demonstrate that SPN binding to an RNA cargo is not a prerequisite for its import. Finally, SPN binding to the

TMG cap does not interfere with Sm core-dependent snRNP import. Taken together, these studies provide important insight into role of SPN in the biogenesis of small nuclear RNPs. 85 MATERIALS AND METHODS

Plasmid construction and mutant generation

All deletions, single and block amino acid mutations as well as truncations were generated using the QuikChange Mutagenesis kit (Stratagene), primer sequences available on request. All block mutations consisted of the substitution of alanines for the wildtype amino acids (indicated as first amino acid-last amino acidA e.g. 48-52A; therefore amino acids 48-52 have all been changed to alanines in this example). Deletions involved removal of the indicated amino acids along with the insertion of an in-frame five-residue linker (5′-

ATCGTCGCAGGATCC-3′) that includes a novel BamH I restriction site used for identification of positive clones. All constructs were subsequently sequenced throughout the entire SPN open reading frame. Primers containing BamH I and

Not I restriction sites were used to PCR amplify human Xpo1 from Myc-Xpo1 and this fragment was subsequently cloned into pET 24b (Novagen).

Generation of radiolabeled RNA

A plasmid containing an Ascaris U2 snRNA gene driven by a T3 promoter (gift of

T. Nilsen) was linearized with Sma I. Linearized DNA was then purified by phenol/chloroform extraction, resuspended in TE buffer and utilized to generate single-stranded RNA. In vitro transcription using the Riboprobe system

(Promega) was then conducted in the presence of radiolabeled UTP, and m7G- or TMG-cap analogs (as directed) and resulting RNA was purified using Bio-Spin

86 Tris columns (Bio-Rad). One microgram of GST or GST-tagged protein was then incubated with 1.6 x 106 counts of RNA for 1 hr at 4°C. Beads were then washed four times with modified (m)RIPA (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% NP-

40, 1 mM EDTA) containing 2 mM DTT plus protease inhibitor cocktail tablets

(Roche) and bound counts determined by an LS6500 scintillation counter

(Beckman Coulter).

Protein purification

GST- and His-tagged proteins were expressed in the Escherichia coli strain BL-

21 Star (DE3) (Invitrogen). Cells were grown at 37°C to an optical density at 600 nm of 0.6, followed by induction with 1 mM IPTG (Sigma-Aldrich). Cells were induced at 30°C for 2 hr except for cells expressing RanQ69L (gift from K. Weis), which were induced at 25°C for 4 hr. GST- and His-tagged constructs were purified using either glutathione beads (Amersham) or Ni-NTA agarose beads

(QIAGEN) as per the manufacturer's instructions. RanQ69L was purified as described (Klebe et al., 1993; Nilsson et al., 2001) and loaded with GTP as described (Askjaer et al., 1998).

Antibodies

A rabbit polyclonal anti-coilin antibody (R124) was generated (Covance Research

Products) using a His-tagged fragment of coilin consisting of the C-terminal 214 amino acids of human coilin. Mouse monoclonal anti-Xpo1 (BD Biosciences),

87 rabbit polyclonal anti-Myc and mouse monoclonal anti-GST (Santa Cruz) were used at 1:5000 while (R124) was used at 1:600. A His-probe (Pierce Chemical) was used at 1:5000 to detect His-tagged proteins as per the manufacturer's instructions. Secondary antibodies used were goat anti-mouse and goat anti- rabbit conjugated horseradish peroxidase at 1:5000 (Pierce Chemical) and goat anti-rabbit conjugated Texas Red at 1:200 (Vector Laboratories).

GST-pulldown assays

E. coli lysates containing GST, GST-SPN or mutant SPN were incubated with glutathione beads for 1 hr at 4°C and washed two times with 1X PBS. All pulldowns utilized 1 µg of GST-SPN except for experiments involving Xpo1 which utilized 2 µg. Glutathione-bead captured GST, GST-SPN or mutant SPN was incubated with 1 µg of importin β for 1 hr at 4°C in 800 µl mRIPA buffer.

Pulldowns utilizing Xpo1 involved incubation of GST, GST-SPN or mutant SPN with 150 µl of E. coli lysate expressing Xpo1-His for 3 hr in the presence of 30 µg

RanQ69L-GTP. Leptomycin B (LMB; Calbiochem) was added at 20 nM to E. coli lysate 1 hr proceeding the addition to glutathione-bead captured GST-SPN.

Reactions were incubated with gentle inversion for 1 hr at 4°C and washed 4 times with 1 ml mRIPA, resuspended in 10 µl 5X SDS loading buffer, boiled and analyzed by SDS-PAGE. Following transfer to nitrocellulose, membranes were probed with the appropriate primary and secondary antibodies prior to chemiluminescence detection (Pierce Chemical). The assay shown in Figure 3-

88 4B was conducted as above except that a buffer described in Paraskeva et al.

(1999) was used. Namely, this reaction was incubated and washed in 50 mM

Hepes-KOH [pH 7.5], 200 mM NaCl, 5 mM Mg[OAc]2 and 0.005% digitonin.

Immunochemical methods

HeLa-ATCC cells were cultured in DMEM (Mediatech) supplemented with 10%

FBS and penicillin/streptomycin (Invitrogen) to 70% confluency. Cells were harvested and electroporated using a GenePulser Xcell electroporator (Bio-Rad) as directed using 2 µg of DNA. Cells were then seeded on slides (Nunc) for 16 hr, fixed in 4% paraformaldehyde, and permeabilized in 0.5% Triton X-100 as described (Frey and Matera, 1995). Incubation in 10% normal goat serum preceded antibody detection. LMB at 20 nM was added to cell culture media for

1 hr prior to cell fixation.

Solid phase binding assays

Solid phase binding assays were performed essentially as described in

Bednenko et al. (2003), with the following modifications. 200 ng of importin β were adsorbed to each well and GST-SPN binding reactions were performed in

PBS containing 0.2% NP-40 and 1% BSA. GST-SPN was detected using anti-

GST antibody (Amersham Biosciences).

89 Import assays

HeLa-ATCC cells were grown to 50% confluency on slides (Nunc) and washed once with 1 ml P buffer (50 mM HEPES-KOH [pH 7.5], 50 mM KOAc, 8 mM

Mg[OAc]2, 2 mM EGTA, 1 mM DTT, and 1 µg/ml each aprotinin, leupeptin, and pepstatin). Cells were permeabilized with digitonin in the presence of an ATP regenerating system (0.2 mg/ml BSA, 1 mM ATP, 10 mM creatine phosphate, 50

µg/ml creatine phosphokinase (Roche) and 0.2 mM GTP) for 5 min at 26°C.

Cells were then washed twice (1 ml each) and incubated in 100 µl P buffer for 15 min at 26°C to remove endogenous transport factors. Following two washes with

P buffer, cells were transferred to T buffer (1 ml each; 20 mM HEPES/KOH [pH

7.5], 80 mM KOAc, 4 mM Mg[OAc]2, 1 mM DTT, and 1 µg/ml each of aprotinin, leupeptin, and pepstatin) before performing the import assay. Import reactions

(100 µl total volume) were incubated at 26˚C for 30-40 min. Unless specified otherwise, each reaction contained 0.2 mg/ml tRNA, 0.2 mg/ml BSA, 1 mM ATP,

10 mM creatine phosphate, 50 µg/ml creatine phosphokinase (Roche) and 40 nM

Cy3-labeled U1 snRNPs. Purified SMN or control complexes (Narayanan et al.,

2004; Pellizzoni et al., 2002a) were a gift from G. Dreyfuss and were used at 400 ng per assay. Importin β and His-GFP-SPN were added at 700 ng each to the import reactions. After incubating, cells were washed in transport buffer, then fixed in 4% paraformaldehyde for 10 min at room temperature and permeabilized with 0.5% Triton for 5 min. Cells were visualized by a Zeiss Axioplan upright epifluorescence microscope (100X objective). Digital images were taken with a

90 Hamamstsu ORCA-ER C4742-95 CCD camera and Open Lab software

(Improvision).

91 RESULTS

Mutational analyses identify residues important for TMG binding

SPN contains a small N-terminal IBB and a large centrally-located TMG cap binding domain (Fig. 3-1). Although the central region of the protein is conserved among higher eukaryotes (Fig. 3-2), it does not share significant similarity with other known RNA binding domains. In order to gain insight into the process of snRNP import, we set out to define sequences that are critical for SPN function.

As a first step in our analysis, we tested wildtype GST-SPN for its ability to bind to TMG capped snRNA. Radiolabeled U2 snRNAs were transcribed in vitro in the presence of either m7G or TMG cap dinucleotide triphosphates. As shown in

Figure 3-3, GST-SPN specifically recovered TMG- but not m7G-capped RNA, whereas only background levels of U2 RNA bound to GST alone. We conclude that, at least by this criterion, recombinant wildtype GST-SPN is a functional protein.

92

Figure 3-1: Schematic of snurportin1 (SPN). A cartoon of SPN illustrating the trimethylguanosine (TMG) cap, exportin1 (Xpo1) and importin β binding domains (IBB). The IBB of SPN is defined as amino acid residues 26-65, based on similarity with the IBB of importin α (Huber et al., 1998). The region of SPN responsible for Xpo1 binding activity has not been mapped and may not be a modular domain (Huber et al., 1998; Paraskeva et al., 1999). Based on proteolytic cleavage of SPN and UV crosslinking studies, the TMG-binding domain is thought to span residues 79-301 (Strasser et al., 2004).

93

94

Figure 3-3: Recombinant SPN can distinguish between m7G- and TMG- capped RNA. GST pulldowns were conducted using GST or GST-SPN and radiolabeled m7G- or TMG-capped U2 snRNA. Following a 1 hr incubation at 4°C, complexes were washed and bound counts determined.

95 Previous studies utilized truncation mutants in attempts to map the various domains of SPN (Huber et al., 1998). Therefore, we generated a large battery of block substitution and internal deletion mutants in various conserved regions throughout the length of the SPN molecule and tested them for their abilities to bind to TMG caps in vitro. The entire data set is summarized in Table 3-1. For comparison, the sequence conservation of the mutated regions is illustrated in

Figure 3-2.

Since tryptophan and other aromatic residues are known to play important roles in binding to m7G caps (reviewed in Fechter and Brownlee, 2005; Quiocho et al., 2000), we paid special attention to conserved motifs containing such residues. Surprisingly, we found that nearly all of the deletion mutants abolished

TMG binding (Fig. 3-4A and Table 3-1). We therefore made a number of point and block substitution mutations in these conserved motifs (e.g. W107A, 104-

107A, 203-207A and W276A) and found that these also significantly reduced binding to TMG capped snRNAs (Fig. 3-4A and Table 3-1). Two mutations bordering the TMG domain (∆1-65 and P291L) disrupted TMG binding only slightly (Fig. 3-4A and Table 3-1). Together with previous findings (Huber et al.,

1998; Strasser et al., 2004), these results identify a minimal TMG binding domain, located between residues 100 and 280.

96 Table 3-1 In vitro binding and in vivo localization studies

SPN Construct Imp β TMG Xpo1 Localization Wildtype + + + Cytoplasmic (1-65) +++ n.d. –1 Nucleoplasmic (1-280) ++ n.d. +/–3 Nuc+Cyto R27A – +/– + Cytoplasmic 25-27A – +/– +/– Cytoplasmic 30-32A + n.d. n.d. n.d. 43-45A + n.d. n.d. n.d. 48-52A ++ n.d. – Nucleoplasmic 63-64A + n.d. n.d. n.d. 65-69A + n.d. n.d. n.d. 104-107A ++ – +/– Nucleoplasmic W107A +2 – +/– Cytoplasmic W107,276A +2 – +/– Nuc+Cyto 203-207A +1 n.d. –1 Nucleoplasmic W276A +2 – +/– Cytoplasmic P291L ++ +/– + Cytoplasmic Δ 1-65 – +3 –3 Nucleoplasmic Δ 39-52 +/– n.d. +/– Nuc+Cyto Δ 96-112 ++ – – Nucleoplasmic Δ 119-134 + – +/– Nucleoplasmic Δ 135-159 + n.d. +/– Nucleoplasmic Δ 170-187 + n.d. – Nucleoplasmic Δ 203-214 + – +/– Nucleoplasmic Δ 255-262 + n.d. – Nucleoplasmic Δ 266-279 + – – Nucleoplasmic

1Binding inferred from localization studies 2Not tested in vitro but relocalizes to nucleus upon LMB treatment 3See Huber et al. (1998) and Paraskeva et al. (1999) n.d. (Not Determined)

97 Point mutants in the TMG domain can interact with Xpo1 in vivo

Following import of newly assembled snRNPs, SPN must be recycled to the cytoplasm to facilitate additional rounds of snRNP import. Recycling depends on the ability of SPN to interact with its export receptor, Xpo1 (Paraskeva et al.,

1999). Despite the lack of a discernable NES, Xpo1 binds to SPN with 50-fold greater affinity than it does to leucine-rich NES-containing proteins such as HIV

Rev (Paraskeva et al., 1999). We therefore tested whether the Xpo1 interaction with SPN was sensitive to LMB (Fornerod et al., 1997; Kudo et al., 1998;

Ossareh-Nazari et al., 1997). Treatment with 20 nM LMB significantly reduced

Xpo1 binding to GST-SPN (Fig. 3-4B), suggesting that SPN binding utilizes the typical NES docking site on Xpo1. Using a similar pulldown assay, we also tested various mutant GST-SPN constructs for their ability to form a ternary export complex with Xpo1 and Ran-GTP in vitro. As shown in Figure 3-4B, the deletion mutants we tested were uniformly inhibited for binding to Xpo1. One of the point substitution mutants (W276A) bound Xpo1 to a lesser extent, whereas

W107A binding was drastically reduced (Fig. 3-4B and data not shown). Since neither of these constructs were functional in our TMG cap assay, the fact that

W276A can bind to Xpo1 suggested that this protein could recycle to the cytoplasm, whereas W107A might be inhibited in this regard.

98 A

B

Figure 3-4: SPN mutants defective in TMG-cap binding are inhibited in Xpo1 binding. (A) Mutation of residue W107 or residues 104-107 abolish TMG binding. GST pulldowns were conducted using GST alone, GST-SPN and the following GST- tagged SPN mutants: R27A, W107A, 104-107A, ∆119-134, ∆203-214 and P291L in the presence of radiolabeled, TMG-capped U2 snRNA. Following incubation and washes, bound counts were determined using a scintillation counter. (B) SPN binding to Xpo1 is extremely sensitive to mutation. GST pulldowns were conducted using GST, GST-SPN (+/- LMB) and the following GST-tagged SPN mutants: 25-27A, R27A, 104-107A, W107A, ∆119-134, ∆203-214, W276A and P291L in the presence of lysate expressing recombinant Xpo1-His and containing RanQ69L-GTP. Western blot analysis was conducted using anti-Xpo1 and anti-GST antibodies (loading control). Input shows 5% of the total lysate used in the pulldown.

99 As an initial step in characterizing the recycling capacities of the TMG domain point mutants in vivo, we analyzed the steady-state subcellular distributions of various GFP-tagged constructs in the presence or absence of

LMB. As expected, wildtype GFP-SPN localized to the cytoplasm and redistributed to the nucleoplasm upon LMB treatment (Fig. 3-5). Similarly, the

GFP-SPN(W276A) construct was primarily cytoplasmic and relocalized to the nucleus in the presence of LMB (Fig. 3-5). Despite the fact that the W107A was greatly reduced in Xpo1 binding in vitro, we found that it localized to the cytoplasm in untreated cells (Fig 3-5). Since this construct was responsive to

LMB (Fig. 3-5), we conclude that W107A can interact with Xpo1 in vivo.

Consistent with their inability to bind both TMG and Xpo1 in vitro, all of the

TMG domain deletion constructs were primarily nucleoplasmic (Fig. 3-5 and

Table 3-1). These results suggest that the TMG deletion constructs and the point substitution mutants described above can each bind to importin β, as they were either nuclear in untreated cells or they were relocalized to the nucleus after treatment with LMB (see Table 3-1).

100

Figure 3-5: SPN point mutants that are reduced for binding to Xpo1 in vitro interact in vivo. The subcellular localizations of GFP-SPN, W107A and W276A were studied following transient transfection of HeLa cells in the presence or absence of LMB. GFP-tagged constructs bearing deletions or block substitutions (104-107A, ∆119- 134 and ∆203-214) were found to be nuclear in the absence of LMB treatment. Bar, 10 µm.

101 Cargo binding is not a requirement for SPN nuclear import

The fact that the TMG domain deletion mutants were nuclear suggested that SPN can be imported into the nucleus in the absence of RNA cargo. We decided to test this hypothesis using the digitonin-permeabilized HeLa cell system (Adam et al., 1990). As shown in Figure 3-6, wildtype GFP-SPN and Cy3-labeled U1 snRNPs were efficiently imported into the nucleus when incubated with recombinant importin β at 26˚C. GFP-SPN was also imported in the absence of the labeled U1 snRNPs (Fig. 3-6), although the level of nucleoplasmic fluorescence was somewhat variable and fluorescence at the nuclear rim was more pronounced (see Discussion). Thus, SPN can be imported into the nucleus in the absence of exogenous cargo. Because we cannot exclude that the protein was imported along with endogenous factors present in the permeabilized HeLa cells, we tested a TMG domain mutant in this assay. GFP-SPN(104-107A) was used for these studies, as this construct can bind neither TMG caps (Fig. 3-4A) nor Xpo1 (Fig. 3-4B) and is nuclear upon transfection into HeLa cells (Fig. 3-5).

This substitution mutant was therefore used in a nuclear transport assay by incubating it in the presence of importin β and Cy3-U1 snRNPs at 26˚C. As shown in Figure 3-6, GFP-SPN(104-107A) was imported into the nucleus (albeit with a pronounced accumulation at the nuclear envelope), but the construct was completely defective in transporting snRNPs. These results demonstrate that

TMG domain mutants are incapable of importing snRNPs and reveal that SPN does not require an RNA cargo to access to the nucleus.

102

Figure 3-6: SPN import does not require bound cargo. The ability of GFP-SPN or -SPN(104-107A) to mediate U snRNP import was examined using an in vitro nuclear transport assay. Recombinant importin β and purified Cy3-labeled U1 snRNPs were incubated with either wildtype or mutant His-GFP-SPN constructs and digitonin-permeabilized HeLa cells. In the top panels, GFP-SPN and U1 snRNPs were efficiently imported. In the bottom panels GFP-SPN(104-107A) was imported into the nucleus (with pronounced staining of the nuclear rim). However, the mutant construct failed to mediate U1 import. In the middle panels, GFP-SPN was imported in the absence of added U1 snRNPs, showing variable degrees of nuclear accumulation; some cells displayed prominent rim staining, whereas others were more uniformly labeled. Import reactions were incubated at 26°C for 35 min. Bar, 10 µm.

103 SPN accumulates in Cajal bodies upon treatment with leptomycin B

As described above, we found that short treatments with LMB resulted in a dramatic relocalization of GFP-SPN from the cytoplasm to the nucleus (Fig. 3-5).

Although most of the protein was distributed throughout the nucleoplasm under these conditions, we were surprised to find that wildtype SPN also accumulated in nuclear foci (Figs. 3-5 and 3-7). Costaining with anti-coilin antibodies revealed that these foci were, indeed, Cajal bodies (CBs) (Fig. 3-7). Interestingly, SPN accumulation in CBs in the presence of LMB correlated with a given construct’s ability to bind TMG capped RNA. For example, the steady-state distribution of

GFP-SPN(W107A) is primarily cytoplasmic in untreated cells, but the protein relocalizes to the nucleus after LMB treatment (Figs. 3-5 and 3-7). However, this construct did not accumulate in the CBs of LMB treated-cells (Fig. 3-7).

Likewise, W276A and all of the TMG-domain deletion mutants we tested were negative for CB accumulation upon LMB treatment (Fig. 3-7 and Table 3-1).

Thus LMB does not induce localization to CBs independent of SPN’s ability to bind TMG caps.

104

Figure 3-7: SPN accumulation in CBs correlates with the ability to bind TMG caps. HeLa cells were transiently transfected with wildtype GFP-SPN, -SPN(W107A) or -SPN∆119-134 and treated with 20nM LMB for 1 hr. Immunofluorescence was then conducted with anti-coilin antibodies to localize CBs. Arrows mark CBs in the wildtype panel. Bar, 10 µm.

105 A conserved arginine residue is required for binding to importin β

As part of our mutational analysis, various SPN constructs were also tested for their ability to bind importin β. We found that neither the deletion nor the substitution mutations within the TMG binding domain had an effect on importin β binding, with the exception of SPN(104-107A) and SPN∆96-112, which bound slightly better than wildtype (see Supplemental data Fig. 3S1 and Table 3-1). We therefore concentrated our efforts within the SPN IBB and found that, as expected, deletion of the entire N-terminal domain (∆1-65) abolished the interaction with importin β (Fig. 3-8A). However, a smaller deletion in the IBB

(∆39-52) had only a modest effect (Table 3-1). Intriguingly, certain alanine- scanning mutations of conserved regions within the IBB disrupted binding to importin β (e.g. 25-27A), whereas others (e.g. 48-52A) enhanced the binding

(see Supplemental data Fig. 3S1, Fig. 3-8A and Table 3-1). The molecular implications of the SPN(48-52A) mutation will be discussed below. Given that

SPN(25-27A) failed to bind to importin β, the results suggest that this motif contains residue(s) necessary for the interaction.

Because the crystal structure of the IBB of importin α complexed with importin β has been solved (Cingolani et al., 1999), we compared the IBB of SPN and importin α (Fig. 3-8B). Notably, in the α/β co-crystal, three importin α residues that make direct contacts with importin β are conserved in human SPN

(asterisks in Fig. 3-8B). Mutation of only one of these regions (R27) disrupted binding; substitutions within motifs containing the other two residues (K32 and

106 R64) had no effect (Table 3-1 and Fig. 3-8A). We tested the GST-SPN(R27A) mutant and found that it fails to interact with importin β in vitro (Fig. 3-8A). In

order to measure the apparent binding affinities (i.e. the relative Kd’s) of the IBB mutants, we utilized a solid phase binding assay (Bednenko et al., 2003). In agreement with our qualitative analysis (Fig. 3-8A), we found that both the

(R27A) and (25-27A) mutations decreased the affinity of the IBB by roughly 20 fold (see Supplemental data Fig. 3S2). The apparent affinities of importin β for wildtype, (R27A) and (25-27A) SPN constructs were: 4.7±1.0 nM; 92.5±18.0 nM and 132.9±28.5 nM, respectively.

Importantly, the (R27A) mutation had little effect on SPN’s ability to bind either TMG caps (Fig. 3-4A) or Xpo1 (Fig. 3-4B), suggesting that these functions were unperturbed. We therefore analyzed the subcellular localization of the two

IBB mutants (25-27A and R27A) and found that they were similar to wildtype

(Fig. 3-9C). Likewise, treatment with LMB demonstrated that the constructs were imported into the nucleus (presumably by an SMN-mediated pathway, see below) and subsequently exported to the cytoplasm by Xpo1 in vivo (Fig. 3-9C). This latter finding was interesting because the 25-27A mutant bound poorly to Xpo1 in vitro (Fig. 3-4B). We also noted that each of the IBB mutant constructs accumulated in CBs upon LMB treatment (Fig. 3-9C), as predicted by their ability to bind TMG-capped RNAs in vitro (Fig. 3-4A and Table 3-1). Having successfully identified a mutant that interacts with Xpo1 but is incapable of binding to importin

β, we next tested the GFP-SPN(R27A) construct in a nuclear transport assay.

107

A

B

Figure 3-8: Disruption of importin β binding and an alignment of IBBs. (A) Mutation of residue R27 disrupts SPN binding to importin β. GST-pulldown assays were conducted using GST (negative control), GST-SPN, and the following SPN mutants: Δ1-65 (N-terminal deletion of 65 amino acids), R27A, 48- 52A or P291L, along with recombinant His-myc-importin β. Western analysis was conducted using anti-myc and anti-GST (loading control) antibodies. Input shows 10% of the total used in the pulldown. (B) Alignment of N-terminal regions of human importin α and various SPN orthologs (human, Xenopus and worm). Residues R27, K32 and R64 are marked with asterisks, regions 25-27, 30-32, 48-52 and 63-65 are overlined with bars.

108

Figure 3-9: Mutants incapable of binding importin β in vitro are efficiently imported in vivo. HeLa cells were transiently transfected with wildtype GFP-SPN, or GFP-tagged SPN mutants 25-27A and R27A and treated with 20 nM LMB for 1 hr. Immunofluorescence was then conducted with anti-coilin antibodies to localize CBs. Arrows indicate CBs in untreated cells. Bar, 10 µm.

109 SPN(R27A) is defective in snRNP import

Together with purified Cy3-labeled U1 snRNPs, GFP-tagged SPN constructs were assayed for import using recombinant importin β and digitonin- permeabilized HeLa cells. As shown in Figure 3-10, the import of both GFP-SPN and Cy3-U1 was robust when cells were incubated at 26˚C for 35 min. Strikingly,

GFP-SPN(R27A) was incapable of supporting U1 import (Fig. 3-10). Thus, despite the fact that SPN(R27A) can bind TMG-capped snRNAs, the mutant was defective for snRNP import in vitro. Significantly, we found that import of both snRNPs and SPN(R27A) could be rescued by addition of purified SMN complexes to the import reaction (Fig. 3-10). When control protein complexes were used (see materials and methods), neither SPN(R27A) nor snRNPs were imported. These studies provide an explanation for the nuclear localization of

SPN(R27A) upon LMB treatment in vivo (Fig. 3-9), demonstrating that SPN binding to the TMG cap does not interfere with the SMN-mediated, cap- independent snRNP import pathway (Narayanan et al., 2004). Furthermore, the results indicate that the interaction between SPN and importin β is not required to stabilize binding of the SMN complex to importin β.

110

Figure 3-10: The binding of SPN to a TMG cap does not interfere with cap- independent import. Digitonin-permeabilized HeLa cells were incubated at 26°C for 35 min with purified Cy3-labeled U1 snRNPs, recombinant importin β and either GFP-SPN or GFP-SPN(R27A) in the presence or absence of purified SMN complexes. Note that neither GFP-SPN(R27A) nor Cy3-U1 snRNPs are imported in the absence of added SMN complexes. Both are imported when 400 ng of purified SMN complexes were added to the reconstituted . Bar, 10 µm.

111 SPN subdomains form an intramolecular interaction

During the course of our importin β binding studies, we recovered two SPN mutations that actually increased importin β binding, relative to wildtype (Fig. 3-

8A). One such mutation was within the IBB (48-52A), whereas the other was in the C-terminus (P291L). One possibility suggested by these observations is that the C-terminus of the protein adopts a conformation that partially sequesters the

N-terminus. We therefore truncated the C-terminus of the protein and assayed binding to importin β relative to wildtype. We generated two constructs, one truncating the entire C-terminus SPN(1-65), whereas the other removed the last

80 amino acids, SPN(1-280). Notably, both constructs (Fig. 3-11 and

Supplemental data Fig. 3S1) bound importin β to a greater extent than either wildtype or the P291L mutant.

Figure 3-11: The binding of SPN to importin β is enhanced upon mutation or truncation of the C-terminus of SPN. Pulldown analyses were conducted using GST, GST-SPN, GST-SPN(1-65) or GST-SPN(P291L) and recombinant importin β. The pulldowns were analyzed by western blot and probed with anti-myc antibody or anti-GST as the loading control. Input shows 10% of the total used in the pulldown.

112 To directly assay for crosstalk between SPN subdomains, we generated differentially tagged N- and C-terminal SPN fragments. Recombinant His-

SPN(65-360) was incubated with GST-SPN(1-65). Western analysis demonstrated that these two fragments interacted in trans (Fig. 3-12, lane 3).

Control assays with GST-alone were negative (Fig. 3-12, lanes 1 and 2). We reasoned that in the context of the full-length protein, substitution mutations that increased binding to importin β in cis (i.e. 48-52A and P291L), did so by disrupting a putative intramolecular interaction between elements located in the

N- and C-termini. Such a disruption might allow the SPN IBB to adopt a more

“open” conformation. These substitution mutations should also disrupt the ability of isolated N- and C-termini to interact in trans. We therefore tested this prediction by introducing these substitution mutations within the N- and C- terminal fragment backbones and found that they abolished the interaction (Fig.

3-12, lanes 4 and 5). Thus mutations that stimulate importin β binding in the context of full-length SPN disrupted association between the N- and C-terminal domains of SPN supplied in trans. These data indicate that the C-terminus of the protein can attenuate the affinity of SPN for importin β by sequestering the SPN

IBB.

113

Figure 3-12: SPN N- and C-terminal domains interact. The N-terminal IBB and C-terminus of SPN interact. GST pulldown assays were conducted using GST (negative control), GST-SPN(1-65) or GST-SPN(1-65) containing the 48-52 mutation, referred to as GST(1-65,48-52A). Lysates containing recombinant His-SPN(65-360), referred to as SPN-Cter(wt), or His- SPN(65-360) containing the P291L mutation, referred to as SPN-Cter(P291L) were used. Western blot analysis was conducted and the blot probed with a His- probe or anti-GST antibody (loading control). Inputs show 1% of the total lysate used in the pulldown.

114 DISCUSSION

In vitro, Sm-class snRNPs can be imported into the nucleus via two separate importin β-dependent pathways (Fischer et al., 1993; Marshallsay and Lührmann,

1994; Palacios et al., 1997). One pathway depends on the presence of a TMG cap and is mediated by SPN (Huber et al., 1998; Huber et al., 2002), whereas the other is cap-independent and relies upon the SMN complex (Narayanan et al.,

2004). Previously, we showed that truncation of the entire IBB resulted in a protein that localizes primarily to the nucleus (Table 3-1 and Narayanan et al.,

2002). Thus, in vivo, the domain through which SPN is thought to be imported is actually dispensable for import. However, the N-terminus of SPN is also required for binding to Xpo1 in vitro (Table 3-1 and Paraskeva et al., 1999), suggesting that the IBB deletion construct (GFP-SPN∆1-65) is able to access an alternative import pathway. Since SPN∆1-65 retains the ability to bind TMG caps (Huber et al., 1998), we theorized that import of the mutant protein was facilitated by an import signal present on newly-assembled snRNPs (Narayanan et al., 2002). We tested this hypothesis directly, by creating a SPN mutant (R27A) that can bind to

TMG caps and Xpo1 (Fig 3-4), but not to importin β (Fig. 3-8A). Interestingly, this protein relocalizes from the cytoplasm to the nucleus upon LMB treatment (Fig.

3-9), suggesting that SPN(R27A) is imported together with snRNPs via the cap- independent, Sm-core import pathway. Consistent with this interpretation, in vitro transport assays showed that import of the R27A mutant depended upon addition

115 of the SMN complex (Fig. 3-10). Thus SPN is able to bind to the TMG cap and

‘piggyback’ into the nucleus via the cap-independent snRNP import pathway.

Cargo binding and the directionality of snRNP transport

Transport adaptors like SPN or importin α shuttle continuously between the nucleus and cytoplasm. In the case of importin α, the high nuclear concentration of Ran-GTP is critical for release of importin β from the nuclear side of the NPC, whereas import of SPN-bound complexes can be achieved in the absence of Ran

(Huber et al., 2002). Our data reveal that cargo binding is not a requirement for

SPN import (Figs. 3-5 and 3-6) and that internal deletion/substitution mutations disrupting TMG-cap binding also inhibited Xpo1 binding (albeit to varying degrees) (Fig. 3-4). Thus we conclude that SPN nucleocytoplasmic shuttling is relatively insensitive to the presence of cargo and that the mutually-exclusive nature of TMG versus Xpo1 binding (Paraskeva et al., 1999) provides a mechanism by which newly-imported snRNPs are prevented from being re- exported to the cytoplasm.

Upon arrival in the nucleus, newly-assembled snRNPs are thought to target to CBs before proceeding on to their final nucleoplasmic destinations

(reviewed in Kiss, 2004). Whether SPN accompanies the import complex to CBs or not is unclear, however, the protein accumulated in these structures when export was blocked with LMB (Figs. 3-7 to 3-9). Curiously, we found that under steady-state conditions, GFP-SPN(25-27A) localized in both the cytoplasm and

116 CBs (Fig. 3-9). Thus SPN can bind to TMG-capped RNAs while in the nucleus.

Given that SPN(25-27A) is slightly defective in binding to Xpo1, the results suggest that perturbation of recycling can result in SPN accumulation within CBs.

Huber et al. (2002) showed that Ran-GTP is not strictly required for SPN translocation across the nuclear pore. However, Ran could still play a role in cargo release or dissociation of the import complex. In this context, it is important to note that Ran(Q69L)GTP destabilizes complexes between importin β and either wildtype (Paraskeva et al., 1999) or mutant SPN constructs (see

Supplemental data Fig. 3S3). Whether or not the interaction with importin β plays a role in modulating SPN’s affinity for TMG cargo is unknown. Future experiments will be required to address this issue.

SPN autoregulation via an intramolecular interaction?

Access to the IBB of importin α is thought to be regulated by sequences within the C-terminal, NLS-binding domain (Kobe, 1999). Disruption of this so-called

“autoinhibitory” interaction was shown to have functional consequences in yeast

(Harreman et al., 2003a; Harreman et al., 2003b). Our discovery that the SPN N- and C-termini interact suggests that SPN may function in a similar manner. Thus the current perceived modular character of the SPN IBB must be re-evaluated.

We favor a snRNP import model wherein folding of the C-terminus regulates the availability of the N-terminal IBB. Consistent with this interpretation, we found that mutation or removal of the C-terminal domain increased the binding of SPN

117 to importin β (Fig. 3-11 and Supplemental data Fig. 3S1) and that interactions between isolated N- and C-terminal fragments of SPN were disrupted by mutations within either of these subdomains (Fig. 3-12). Thus we conclude that

SPN forms an intramolecular interaction and that crosstalk between subdomains may modulate the efficiency of nuclear import.

To facilitate snRNP import, SPN must form a complex with both snRNPs and importin β. The order of complex formation is unknown. Following export and release from Xpo1 in the cytoplasm, SPN is presumably free to bind to the receptor, the cargo or to itself via an intramolecular interaction. Because an intramolecular interaction would be kinetically favorable, we propose that sequestering of the SPN IBB might help prevent cargo-less SPN molecules from binding to importin β in the cytoplasm, thus reducing the number of futile import cycles.

A recent structure-function study of Xpo1 has also led to an autoinhibitory hypothesis regarding the Ran binding loop of this transport protein (Petosa et al.,

2004). Similarly-detailed structural studies, comparing the TMG bound and unbound states, will be required in order to demonstrate the existence of an intramolecular interaction within SPN. However, our finding that the SPN N- and

C-termini can interact reveals a common theme amongst nucleocytoplasmic transport proteins. In the future, it will be interesting to see if other transport factors utilize similar mechanisms.

118 Supplemental Data

Supplemental Figure 3S1

SPN truncation and substitution mutants are altered in importin β binding. SPN truncation (1-280) or the mutation of residues 104-107 increases importin β binding (left panels), whereas mutation of residues 25-27 inhibits this interaction (right panels). GST-pulldown assays were conducted using GST (negative control), GST-SPN and the following GST-tagged SPN mutants: 1-280, 104-107A and 25-27A along with recombinant His-myc-importin β. Western analyses were conducted using anti-myc and anti-GST (loading control) antibodies. Input shows 10% of the total used in the pulldown.

119 Supplemental Figure 3S2

SPN mutants (R27A) and (25-27A) display decreased affinity for importin β. Solid phase binding assays were conducted using GST-SPN, GST-SPN(R27A) and GST-SPN(25-27A) in the presence of importin β. GST-SPN was detected using an anti-GST antibody and HRP-coupled secondary antibody. The colorimetric reaction was stopped by the addition of 125 nM HCl and the signal was measured at 450 nm.

120 Supplemental Figure 3S3

The importin β binding of wildtype and mutant SPN is responsive to Ran- GTP. GST-pulldown assays were conducted using GST (negative control), GST-SPN and the following SPN mutants: P291L, 1-65 and 65-360;P291L (N-terminal deletion of 65 amino acids containing the P291L mutation) along with recombinant His-myc-importin β in the presence or absence of RanQ69L-GTP. Western analysis was conducted using anti-myc and anti-GST (loading control) antibodies. Input shows 10% of the total used in the pulldown.

121 ACKNOWLEDGMENTS

We thank U. Narayanan, K. Shpargel, T.K. Rajendra and M. Walker for scientific discussions. We are indebted to G. Dreyfuss, R. Lührmann, M. Fornerod, K.

Weis, and T. Nilsen for reagents. This work was supported by NIH grants R01-

GM53034 and R01-NS41617 to (A.G.M.). J.K.O. was supported in part by an

NIH predoctoral traineeship (T32-GM08613).

122 Chapter 4

Conclusions, Discussion and Future Directions

123 CONCLUSIONS

This thesis has addressed the role of SPN in snRNP biogenesis. It has identified a pre-import complex containing importin β, SPN and SMN. SMN is found to directly interact with importin β, implying a role for SMN in snRNP import. It has identified SPN mutants that are disrupted in one function, while preserving the capacity of additional activities. These deficiencies have been verified in functional studies and mutants were subsequently utilized to gain insight into the role of SPN in snRNP biogenesis. SPN binds to Xpo1 in an LMB-sensitive manner and relocalizes to CBs when Xpo1 binding is disrupted. Interestingly, a novel SPN interaction, which may facilitate the regulation of snRNP biogenesis, has been discovered.

Pre-import complex formation

The biogenesis of snRNPs requires both nuclear and cytoplasmic phases and therefore necessitates the bidirectional transport of precursor molecules.

Transcription in the nucleus results in snRNAs that are exported to the cytoplasm by an export complex containing PHAX and Xpo1. This export relies on the presence of NESs found within PHAX to interact with the export machinery. In the cytoplasm, snRNAs are assembled with an Sm core, hypermethylated and modified by exoribonuclease activity. Following cytoplasmic maturation, these newly assembled snRNPs are incorporated into an NLS-contatining import complex, which enables interaction with the cellular import machinery.

124 It is well established that snRNPs possess two such NLSs. One NLS is provided by the 5′ hypermethylated TMG cap (Huber et al., 1998; Huber et al.,

2002), whereas the other consists of the Sm core (Fischer and Lührmann, 1990;

Fischer et al., 1993; Görlich and Kutay, 1999; Hamm and Mattaj, 1990). Data from Chapters 2 and 3 illustrate the possibility that this elusive Sm core-specific

NLS may be the SMN protein.

In support of this hypothesis, we found that SPN interacts with SMN in vitro and in vivo. HeLa cell lysates were used to determine that SPN interacts with cytoplasmic SMN in an RNase sensitive manner. SPN was also found to interact with a subset of the SMN complex members. These data indirectly illustrated the existence of an SMN containing, pre-import complex. Further evidence for the existence of this complex was obtained when we performed size exclusion chromatography using HeLa cytoplasmic lysate. Utilizing this approach, we identified a pre-import complex containing SPN, importin β and

SMN.

The finding that SMN interacts with importin β, provided additional evidence that the SMN complex is the unidentified Sm core NLS. This interaction was detected using HeLa cell lysates as well as purified recombinant SMN and importin β proteins. This finding illustrates that SMN and importin β interact directly and highlights the possibility that the NLS activity of the Sm core is provided by SMN itself. Interestingly, the SMN complex, in the presence of recombinant importin β, rescued the import of snRNPs and the IBB-deficient SPN

125 mutant, R27A. A control experiment containing added importin β and a control complex failed to rescue import. These data demonstrate that the SMN complex, along with importin β, can provide NLS activity (presumably through the interaction between SMN and importin β).

The role of SPN in snRNP biogenesis

SPN harbors three known functional domains to facilitate U snRNP biogenesis.

These domains consist of an N-terminal importin β binding domain (IBB), a central TMG cap binding domain and Xpo1 binding domain. The identification of these domains has relied on protein sequence homologies, interaction studies using gross truncation mutants and cross-linking techniques. These studies are limited in that they provided very general data concerning regions necessary for binding function. Consequently, we possess a vague understanding of the interactions required for SPN to mediate snRNP biogenesis. The identification of specific amino acids or motifs of SPN that mediate function, would therefore provide greater insight into its role in this critical process.

To this end, I have conducted a study investigating SPN binding functions.

This study has yielded three main outcomes. First, we have a better understanding of the interactions between SPN and its binding partners.

Second, tools were generated that will allow greater control over experimental parameters when investigating SPN function. Finally, novel SPN activities were uncovered.

126 Identification of critical regions mediating SPN function

Importin β binding

Amino acids required for importin β binding were identified. Mutation of these residues results in either an inhibition (discussed here) or an enhancement

(discussed below) of importin β binding. Mutation of residues 25-27 disrupted the ability of SPN to bind to importin β. A more precise mutation within this motif

(R27A) also disrupted this interaction, indicating that this residue is vital for importin β binding. Solid phase binding analyses confirmed these in vitro qualitative data and illustrated that these IBB mutants (25-27A and R27A) display a reduced affinity for importin β. Additional analyses revealed that SPN(R27A) is specific in its functional disruption as this mutant retains an ability to bind both

TMG caps and Xpo1.

SPN(R27A) was initially identified using in vitro binding assays with importin β and was subsequently confirmed to be deficient in snRNP import using the HeLa permeabilized cell assay. However, transient transfection of HeLa cells indicated that, in vivo, R27A was imported into the nucleus in the absence of direct binding to importin β. Further analyses revealed that the in vitro import deficiency of R27A could be rescued by addition of the SMN complex and importin β. This observation suggested that R27A is imported in vivo by accessing the Sm core-specific NLS of snRNPs. As R27A can bind the TMG caps of snRNPs and the SMN complex (bound to the Sm core) can bind to

127 importin β (directly through SMN), R27A can indirectly access the import machinery through snRNPs.

TMG binding

Mutants disrupted in TMG cap binding, while retaining the ability to bind to importin β, were also generated. These TMG binding mutants were also primarily identified by in vitro binding assays and later confirmed to be functionally deficient, as they were incapable of importing U1 snRNPs in the permeabilized HeLa cell system. Transient transfections of HeLa cells illustrated that these SPN mutants were imported (in the absence of bound cargo) and failed to localize to CBs (see below), providing in vivo evidence for their inability to bind TMG caps.

Xpo1 binding

Mutational analyses illustrate that the Xpo1 binding property of SPN is highly sensitivite to mutation. In vitro analyses indicated that all block-deletion (e.g.

∆119-134) and substitution (e.g. 25-27A) mutants were disrupted for Xpo1 binding. Point mutant W276A displayed reduced in vitro Xpo1 binding, whereas

W107A was drastically inhibited in this regard. Interestingly, the level of residual binding displayed by these mutants (W276A and W107A) was sufficient to support Xpo1 binding and SPN export in vivo. As the affinity of SPN for Xpo1 is

50-fold greater than the affinity of HIV Rev (which contains a leucine rich NES)

128 for Xpo1 (Paraskeva et al., 1999), a large dynamic range of binding is expected and may account for these observations. Nonetheless, as this export was sensitive to LMB treatment (see below), we conclude that it is mediated by Xpo1 and not some unknown factor.

Two point mutants (R27A and W276A) retained the ability to bind to Xpo1 in vitro. Whereas R27A bound to Xpo1 at wildtype levels, W276A displayed reduced binding. Block deletion and substitution constructs were confirmed to be disrupted in Xpo1 binding as determined by their nucleoplasmic localization in transfected HeLa cells. In parallel experiments, I determined that LMB disrupts the interaction between SPN and Xpo1 using in vitro binding analyses and LMB treatment of transfected HeLa cells. When HeLa cells were transfected with wildtype SPN and treated with LMB, nucleoplasmic enrichment was observed.

This indicated that the nucleoplasmic enrichment of block deletion and substitution mutants is due to a disruption in Xpo1 binding.

Interestingly, while SPN(R27A) was able to bind Xpo1, mutation of two adjacent amino acids (resulting in SPN25-27A) disrupted this ability. The highly sensitive nature of Xpo1 binding may illustrate a direct interaction of wildtype amino acids and/or their indirect promotion of proper SPN folding and 3D conformation to mediate interaction. Additionally, most mutants disrupted in

TMG cap binding were also disrupted for Xpo1 binding. The exceptions include

W107A and W276A, which were deficient in TMG binding, but displayed Xpo1 binding in vivo. Interestingly, W107A was greatly reduced in Xpo1 binding in

129 vitro, whereas W276A displayed only reduced Xpo1 binding. These data illustrate the possibility that the binding to both TMG and Xpo1 may be mediated by similar regions or motifs of SPN, thus impeding the simultaneous binding of these molecules to the same molecule of SPN.

SPN localizes to CBs when its export is disrupted

SPN is relocalized to the nucleoplasm upon LMB treatment or mutation that disrupts its interaction with Xpo1. Wildtype SPN and mutants that retain an ability to bind to TMG caps, accumulate in CBs in addition to this diffuse nucleoplasmic enrichment. Importantly, the CB enrichment can be competed with the addition of excess TMG caps in the HeLa permeabilized cell assay (see

Appendix AΙΙ). Control studies using m7G caps failed to inhibit the CB localization, illustrating the specificity of the TMG cap effect.

Bound cargo is not a prerequisite for SPN import

Having identified a mutant deficient for TMG cap binding, we utilized this protein to address the question of whether SPN could be imported into the nucleus in the absence of bound cargo. Upon transient transfection of HeLa cells, we noted that SPN(104-107A) was enriched in the nucleoplasm and absent from CBs. As this mutant is unable to bind TMG caps in vitro, we conclude that bound cargo is not required for SPN to access the import machinery. Interestingly, upon transient transfection of HeLa cells, we found that SPN(W107A) could not only be

130 imported in the absence of cargo, but was subsequently exported back to the cytoplasm. This finding paralleled observations that wildtype SPN alone or SPN lacking an ability to bind TMG cargo was imported in permeabilized HeLa cells.

SPN N- and C-termini interact

During the course of analyzing SPN mutants for disruption in binding capacity, a handful of mutants differed from the rest. These mutants stood out as they showed increased binding activity. More specifically, SPN mutants 48-52A, 104-

107A and P291L all bound to importin β to a greater extent than wildtype. This led us to hypothesize that the increased affinity may be due to an alteration in

SPN folding that allows for greater access to importin β. Data supporting this hypothesis was obtained when we determined that isolated SPN N- and C- termini interact in trans. This interaction was found to be disrupted by the introduction of the 48-52A or P291L mutations in the N- or C-terminal SPN fragments, respectively.

DISCUSSION

Transcompartmental communication is crucial to the functioning and vitality of eukaryotic cells. It is for this reason that we should strive to better understand both the generalities and intricacies of this process. An emerging hypothesis in regard to transport receptors and adaptors is that their activities can be self- regulated. A recent crystallographic study led authors to hypothesize that the

131 Ran binding loop of Xpo1 mediated an autoinhibitory method of regulation

(Petosa et al., 2004). Additionally, crystallographic analysis of importin α revealed an intramolecular interaction between its N- and C-terminal domains

(Kobe, 1999). Situated in the N-terminal IBB of importin α is a weak NLS which can be bound by the C-terminal NLS-binding domain. Disruption of this autoinhibition has a functional consequence in yeast, as it results in a disruption of cargo release and subsequent recycling of importin α back to the cytoplasm

(Harreman et al., 2003a; Harreman et al., 2003b). It will be interesting to see if other transport proteins, in addition to Xpo1 and importin α, display intramolecular interactions that regulate their function.

Intramolecular interaction and snRNP biogenesis

Our finding that the N- and C-termini of SPN interact is suggestive of an intramolecular interaction and warrants further investigation. What is the significance of this interaction to SPN function and general snRNP biogenesis?

Currently nothing is known concerning the regulation of snRNP import complex assembly. An intramolecular interaction might play an “autoinhibitory” role in this process and provide for an additional level of regulation. For example, following an import event, SPN is exported back to the cytoplasm by a complex containing

Xpo1. Subsequent to reaching the cytoplasm and export complex disassembly,

SPN is free to bind to importin β, cargo or possibly itself through an intramolecular interaction. The kinetically favored outcome might be the latter.

This outcome would benefit snRNP biogenesis in several ways. First, it would 132 reduce the number of futile rounds of SPN import (i.e. cargoless import events).

The subset of SPN molecules not participating in cargo import would be sequestered via their intramolecular interactions, precluding undesired importin β or cargo binding. This would effectively remove “idle” SPN molecules from the transport pathway when demand for such transport is low (Fig. 4-1). Upon increased demand for snRNP biogenesis, the binding of importin β may disrupt the intramolecular interaction and increase access to snRNP cargo. Second, the binding of snRNP cargo may increase the effective access of importin β to SPN, again through a disruption of the intramolecular interaction (Fig. 4-1). These scenarios could reduce futile rounds of SPN import and provide regulation of import complex formation. See future directions for discussion concerning the testing and implications of an intramolecular interaction.

Additionally, regulation of snRNP biogenesis may be provided by post- translational modifications, which might attenuate an intramolecular interaction.

For example, phosphorylation of SPN may result in an alteration of the strength of the intramolecular interaction. An increase in this affinity may result in more molecules being sequestered from cargo import, whereas a decrease might enable a larger pool of SPN to participate in snRNP biogenesis. These modifications could quickly and efficiently address alterations in metabolic demand by modifying levels of snRNP biogenesis. Nevertheless, our data support a more complex picture of importin β binding regulation, whereby an

133 intramolecular interaction between the N- and C-termini of SPN may regulate access to importin β and/or cargo.

Figure 4-1: Model of snRNP import regulation via an auto-inhibitory function of snurportin. An intramolecular interaction might sequester SPN molecules when the demand for snRNP biogenesis is low. Upon increased demand, the binding of importin β or snRNP cargo might increase access to snRNP cargo or importin β, respectively. This would enable the regulation of snRNP biogenesis through the modulation of import complex formation. IBB indicates the importin β binding domain.

134 SPN localization to CBs upon nuclear enrichment

The fact that SPN accumulates in CBs upon enrichment in the nucleus is interesting as SPN is not known to have any specific function in this compartment. In fact the only known activity of nuclear SPN is binding to Xpo1 for transport back to the cytoplasm following snRNP import. As chapter 3 of this thesis contains the first description of any specific nuclear localization of SPN, the CB accumulation may be indicative of a novel function for this import adaptor.

The CB accumulation of SPN was originally noted upon LMB treatment of transiently transfected HeLa cells. Using both in vivo and in vitro analyses (see

Chapter 3 and appendices), I determined that the presence of TMG-capped snRNPs in CBs mediates this enrichment of SPN. Further investigation yielded additional conditions whereby SPN accumulated in CBs. CB enrichment was also noted in cells expressing SPN(25-27A) in the absence of LMB. Binding of this mutant to Xpo1 is barely detectable by western analysis, however this mutant can bind Xpo1 fairly well in vivo, as it displays a steady-state cytoplasmic localization with nuclear redistribution following LMB treatment. Therefore, this mutant appears to be reduced in its export kinetics due to impaired Xpo1 binding.

As a result, there is an increase in the nucleoplasmic pool of this protein and enrichment in CBs. Why might SPN, a snRNP import adaptor, accumulate in

CBs and is this indicative of a wildtype process? Under wildtype conditions, rapid export kinetics would result in rapid nuclear clearance of SPN. However, a reduction in Xpo1 affinity for SPN (by LMB treatment or mutation) would result in

135 slowed kinetics of SPN export and be observed as an accumulation of SPN in

CBs.

An additional hypothesis for the accumulation of SPN in CBs depends upon the fact that newly assembled snRNPs are further modified following nuclear import. Upon import, these snRNPs undergo additional modifications in

CBs (Darzacq et al., 2002; Jady et al., 2003; Richard et al., 2003). In fact, CBs that contain SMN are the first sites of enrichment of these newly imported snRNPs (Sleeman et al., 2001; Sleeman and Lamond, 1999). This thesis and other data (Massenet et al., 2002; Narayanan et al., 2004) indicate that these snRNPs are bound by the SMN complex in the cytoplasm and remain bound during their translocation through the NPC. Interestingly, coilin, the marker protein of the CB, functions in the recruitment of SMN to this nuclear subdomain

(Hebert et al., 2001). Therefore, it is conceivable that coilin may facilitate the CB localization of newly assembled snRNPs (for further modification by scaRNAs) by binding to the SMN protein of the SMN complex. Subsequent to reaching the

CB, disassembly of the remaining members of the import complex (including

SPN) would result in cargo release. A potential protein that might mediate this disassembly is Xpo1, as it is also found to accumulate in CBs (see appendices).

Based on these data, two scenarios come to mind that could describe the nature of SPN’s accumulation in CBs. One is that SPN stays bound to the snRNP as part of the import complex, until delivery to the CB. Release from the snRNP would require Xpo1, however in the presence of LMB, Xpo1 is not able to

136 bind SPN. Therefore, the outcome of LMB treatment or mutant SPN expression

(25-27A) would be a CB enrichment due to a lack of Xpo1 binding and displacement of SPN from the snRNP. Another possibility is that the dissociation of SPN from the snRNP is mediated by an unknown factor and occurs immediately following nuclear arrival. Due to the inability of SPN to bind Xpo1,

SPN export kinetics are greatly slowed, resulting in an enrichment in the nucleus.

Nucleoplasmic SPN could then rebind TMG capped RNAs, which are abundant in

CB-bound snRNPs. This scenario would, however, require an additional component or components (other than Xpo1) to mediate the release of SPN from the TMG cap of imported snRNPs.

Regulation of transport directionality

What regulates the functional timing of intracellular transport? This is a fundamental question that arises at many stages of the snRNP biogenesis pathway. For example, how does the snRNA export machinery know when to export newly generated RNA molecules? Additionally, what mechanism inhibits the transport machinery from exporting newly imported snRNP molecules? In terms of snRNA export, this event requires the incorporation of a phosphorylated version of PHAX and the binding of Ran-GTP to Xpo1. These events must occur subsequent to the binding of the CBC, which itself requires m7G cap formation on the snRNA. Regulation is also required in the cytoplasm, and provided by many snRNP modifications, which are prerequisites for downstream maturation events.

137 This ensures that alterations occur at the correct time and at the proper stage of snRNP maturation.

The concept of functional regulation is also important when examining the recycling of transport components. For example, following a transport event, adaptors and receptors must by recycled to the proper cellular compartment, in order to mediate additional rounds of cargo transport. What prevents these transport proteins from being recycled while they are still bound to their respective cargo? The data presented in Chapter 3 illustrate that, at least for

SPN, many of the same regions or motifs may be used to bind both cargo and an export receptor molecule. This explanation is supported by the observation that most mutations disrupting TMG cap binding also perturb Xpo1 binding. As a result, SPN molecules that are bound to TMG caps, would be unable to bind

Xpo1, because the regions required for this function would be involved in cargo binding. This scenario would inhibit the binding of Xpo1 to SPN while it is still bound to TMG cargo and prevent the export of newly imported snRNPs.

Following import, the release of SPN from cargo could then be mediated by unidentified factors, or could occur in a step-wise fashion, whereby regions of

SPN bind to Xpo1 immediately following their release from cargo. It seems likely that both scenarios would defy the principle of parsimony. The former would simply require additional participants to promote cargo release from SPN. The latter would depend on added regulation to shield the intermediate complex, containing snRNP, SPN and Xpo1 from export. Therefore, the most likely

138 scenario might be an extremely rapid exchange of SPN from cargo to Xpo1 and subsequent export of SPN to the cytoplasm.

139 FUTURE DIRECTIONS

In science, each answer potentially yields many new questions. Therefore, carefully evaluation of new data is crucial when choosing which avenues of research to pursue. This advice is applicable to most scientific endeavors and this thesis is no exception. To most effectively continue the study of snRNP biogenesis, we must critically evaluate the current knowledge base, including this work, and plan our investigations wisely. Several new questions arise from this thesis, a subset of which deserve further attention and warrant investigation.

140 I. Determine the functional significance of SPN’s intramolecular interaction

Having identified that isolated SPN N- and C-termini can interact, the next question to address is whether this interaction exists in the full-length protein.

This is a rather difficult question to address and may require X-ray crystallography (see future direction IV), nuclear magnetic resonance (NMR) spectroscopy (see future direction V) or the utilization of live cell video microscopy.

One approach to evaluate the existence of an intramolecular interaction would require fluorescence resonance energy transfer (FRET) analysis. FRET involves the non-radiant transfer of energy from a donor fluorophore to an acceptor fluorophore. This transfer occurs over a very short distance (1 to 10 nm), referred to as the Forster radius, and yields a subsequent fluorescence emission from the acceptor (Forster, 1948; reviewed in Day et al., 1999). This technique allows the investigator to determine the existence of an intermolecular interaction (between two of the same or different molecules) or intramolecular interaction (between two subdomains within a single molecule). To determine the existence of a SPN intramolecular interaction, donor and acceptor fluorophores would be tagged to the N- and C-termini of this protein. Based on an analysis comparing the effective Forster radii for GFP and its variants

(Patterson et al., 2000) the most appropriate combination would be to use a yellow donor and cyan acceptor as this pair has the shortest relative Forster radius.

141 In designing this line of research, extra care must be taken to properly control the experimental parameters. Heterologous proteins of approximately

360 amino acids should by used to obtain base-line FRET values for the absence of an intramolecular interaction. A protein known to display an α-helical (linear) three dimensional structure, should be included in these base-line FRET readings. A set of fluorophores, separated by 360 amino acids, could be incorporated into this linear protein and subsequently assayed by FRET. This would yield a FRET value for the absence of globular folding or interaction within the protein. Additionally, as importin α harbors an intramolecular interaction

(Kobe, 1999), it should be used to establish what to expect from a positive outcome. Again, this would involve the placement of fluorophores at the N- and

C-termini of this protein, and a determination of FRET values from both nuclear and cytoplasmic compartments.

One limitation to this line of investigation is the fact that a positive outcome

(obtaining a positive FRET signal) is informative, whereas a negative result tells very little. There are numerous caveats that might yield a negative result, in addition to the possibility that snurportin may not even display an intramolecular interaction. However, if the controls (e.g. importin α) yield a positive outcome (as would be expected due to the fact that this import adaptor displays an intramolecular interaction), this would lend support that a negative or positive result is due to the absence or presence of an intramolecular interaction, respectively.

142 II. Determine the order of import complex assembly and whether Xpo1 facilitates its disassembly upon arrival in the nucleus

Does the assembly of an import competent RNP represent a point of regulation for snRNP biogenesis? An ability to modulate assembly, via a SPN intramolecular interaction, might assist the general regulation of snRNP biogenesis. However, a direct assessment of this capacity would require one to determine whether the binding of SPN to cargo is increased subsequent to SPN binding importin β. Alternatively, the binding to importin β may increase binding of TMG cargo. I suggest using in vitro binding assays to address these possibilities.

The first question is to determine whether binding of SPN to cargo results in an increase in the binding of SPN to importin β. To address this question,

GST-SPN would be incubated with TMG- or m7G-capped cargo. Importin β would then be added to these reactions and its binding determined by glutathione bead pulldown analysis and western blotting. The use of m7G-capped RNA would control for any non-specific increase in importin β binding due to the presence of RNA molecules. There are a number of caveats to this line of investigation. For example, the binding of cargo to SPN may increase the binding to importin β in vivo due to additional snRNP components not present in this in vitro assay. To partially circumvent this problem, snRNPs could be used in the pulldown assay, which would more closely mimic an in vivo situation.

However, additional snRNP or non-snRNP components, found in vivo, might still

143 be lacking in vitro and thus potentially hinder the assay. To address this caveat, I would consider adding recombinant snRNP components to the in vitro pulldown analyses.

A related question would be to determine whether binding of SPN to importin β increases SPN binding to cargo. This would involve incubating SPN with importin β or an equivalent amount of BSA. Radiolabeled snRNA would then be added followed by further incubation. A series of centrifugation and wash steps would yield bound complexes containing SPN, importin β and radiolabeled snRNA. A comparison of bound counts would then indicate if the binding to importin β increases the binding of SPN to cargo. Again, similar caveats exist including the absence of proteins or factors present in vivo, which may be lacking in these in vitro assays.

To circumvent the inherent problems with in vitro analyses, in vivo alternatives might be utilized. These assays would, however, require the increased expression of importin β or the increased generation of snRNPs.

Overexpression of importin β is straightforward and could be directly achieved by transient expression in HeLa cells. Conversely, the increased generation of snRNPs would require the overexpression of a snRNP component, indirectly leading to increased snRNP biogenesis (Sleeman et al., 2001). This study reported that overexpression of Sm proteins yielded a downstream increase in snRNP generation (Sleeman et al., 2001). Following increased importin β or snRNP expression, a resulting increase in the binding of SPN to snRNPs or

144 importin β, respectively, could be determined by coimmunoprecipitation analyses and western blotting. These experiments would, of course, require the inclusion of controls in which heterologous proteins are overexpressed to ensure the specificity of any alteration in SPN binding. Analyses determining whether the overexpression of snRNP components actually results in an increase in snRNP biogenesis (as measured by steady-state snRNP levels) are also vital experimental controls.

III. Identify the SMN complex member(s) required for Sm core dependent import

The existence of a bipartite snRNP NLS (consisting of the TMG cap and Sm core) has been hypothesized for some time (Fischer and Lührmann, 1990;

Fischer et al., 1993; Görlich and Kutay, 1999; Hamm and Mattaj, 1990), however, data illustrating the nature of these NLSs have only recently come to light. The adaptor for the TMG pathway was identified with the discovery of SPN (Huber et al., 1998), whereas this thesis and other work (Narayanan et al., 2004), support the notion that the SMN complex is the Sm core import adaptor. Whether or not

SMN is the actual adaptor is unknown as these experiments utilized the entire

SMN complex to rescue import of either snRNPs (Narayanan et al., 2004) or snRNPs and SPN (Chapter 3). An important line of investigation would be to identify the actual import adaptor(s).

145 To date, the SMN complex consists of several “Gemin” proteins as well as a few additional proteins, a subset of which are unidentified (Narayanan et al.,

2004; Pellizzoni et al., 2002a; Pellizzoni et al., 2002b) To identify which SMN complex component(s) is acting as an import adaptor, I propose the use of the

HeLa permeabilized cell assay. The full set of recombinant Gemin proteins, along with importin β, SMN and snRNPs, would be incubated with permeabilized

HeLa cells. If snRNPs are imported, additional experiments would utilize only a subset of Gemin proteins to identify the necessary Gemin(s) required to mediate

Sm core dependent snRNP import. Other SMN complex components, in addition to Gemins, might also be necessary to support Sm core-dependent snRNP import. To address this possibility, these proteins could be recombinantly expressed and combined with Gemin proteins prior to conducting the in vitro import reactions. This line of investigation would address whether SMN complex proteins actually provide targeting signals to promote the import of newly assembled snRNPs.

IV. Directly determine residues mediating SPN binding functions

An important aspect of understanding how a protein functions is the identification of specific residues mediating such functions. The identification of functional motifs is generally straightforward when small NLS or NES regions are responsible for function. Identification and analysis is also greatly simplified when a consensus sequence exists. The deletion of a suspect motif, along with

146 its addition to an innocuous protein such as GFP, is a valuable approach to determine its functionality. However, these approaches are insufficient when no suspect region(s) can be identified due to the lack of a consensus motif or the use of a relatively large or several smaller motifs.

To determine specific residues mediating SPN function, I propose using X- ray crystallography and/or NMR spectroscopy. In light of this thesis and data regarding other transport proteins (Kobe, 1999; Petosa et al., 2004), I suggest using full-length SPN protein for these analyses. The structure of full-length SPN should be initially determined to identify the three dimensional structure of this protein in the absence of any binding partners. This would yield important information concerning the conformation adopted by SPN in the absence of cargo or importin β. Additionally, it would address the possibility of whether SPN possesses an intramolecular interaction.

Following the determination of the structure of full-length SPN, studies should identify amino acids critical for interaction with other molecules. Again X- ray crystallography and NMR spectroscopy would be a powerful techniques to address these questions. These studies would utilize either full-length SPN or fragments of the protein bound to cargo, importin β or Xpo1. However, this thesis indicates that using fragments of SPN to identify surfaces mediating Xpo1 binding may yield an incomplete story. Due to the possibility that SPN may bind

Xpo1 through many small motifs spanning the entire protein, full-length SPN might be the more appropriate protein to use. The caveat to this line of

147 investigation is, however, the requirement of generating co-crystals of SPN and its various binding partners. Additionally, current molecular size limitations of

NMR spectroscopy limit its applicability to proteins smaller than 40 kDa and may hinder the usefulness of this technique.

V. Characterize SPN structural modifications resulting from complex formation

Although the data obtained from simple one-to-one binding is important, it is restricted in its power. In addition to the importance of identifying amino acids mediating function, structural data would enable a comparison of the different conformations that mediate binding. Vital insight into SPN function could be gained with comparisons of structural modifications resulting from binding to importin β, cargo or Xpo1. The comparison of data obtained from X-ray crystallography and NMR studies may be valuable in this line of investigation.

As shown in Chapter 3, nucleoplasmic SPN can to bind to TMG caps.

What keeps SPN from rebinding cargo following the disassembly of the import complex? Additionally, what keeps SPN from binding Xpo1 while still bound to cargo following an import event? This thesis indicates that these events are prevented because similar regions or motifs of SPN may be required in interactions with both proteins. Additionally, the conformation that SPN assumes upon binding cargo may sequester the necessary motifs needed to bind Xpo1 and vise versa. A comparison of the three dimensional conformations adopted

148 when binding these substrates would provide insight into this question. Do these conformations sequester domains or motifs shown to be vital in the association with other partners?

VI. What are the phenotypic consequences of a loss of SPN

To date, studies investigating SPN function have utilized Xenopus oocyte and

HeLa cell model systems. Much power lies in a model system whereby one can eliminate SPN and determine the functional effect on snRNP biogenesis and the overall organismal consequences.

Three model systems come to mind. One system would require the knockdown of SPN protein in HeLa cells, using SPN RNA interference (RNAi) constructs. An additional approach would utilize the power of Drosophila genetics to generate a SPN null organism. Lastly the generation of a SPN null mouse may provide many insights into the complex process of snRNP biogenesis.

RNA interference

The permeabilized HeLa cell system enables control over the presence of most soluble components, including SPN. These components can be globally depleted by washes that effectively remove the cytoplasm or they can be selectively depleted and/or supplemented to assess the impact on snRNP biogenesis. However, much could be learned using an RNAi system, whereby

149 endogenous factors, other than SPN, are at normal levels. RNAi constructs specific for SPN are commercially available, and only require the validation of their efficacy prior to experimentation.

The RNAi system would allow the expression of mutant SPN proteins in a background whereby endogenous SPN had been targeted for disruption. This is made possible by using RNAi constructs specific to wildtype RNA, followed by the expression of mutants that differ from wildtype in their RNA sequence

(therefore not targeted for degradation), but encode either wildtype or mutant amino acids. This system would enable the investigation of many aspects of snRNP biogenesis. Are newly assembled snRNPs imported via the Sm core pathway in the presence of mutant SPN proteins? Do newly imported snRNPs require the SPN import pathway to be initially targeted to CBs? If snRNPs are targeted differently in this system, what are the ramifications to nuclear snRNP maturation?

Targeted disruption of SPN in Drosophila and mouse

Studies requiring the knockout of SPN in the Drosophila or mouse system must be carefully considered due to the time commitment of such approaches.

However, these techniques would be powerful in that they enable one to investigate the potential developmental consequences of a loss of SPN.

Currently, no Drosophila P-element insertions disrupting the SPN gene (RNUT1) exist. Additionally, there are no known RNUT1 null mice or cell lines lacking this

150 gene. Therefore, the generation of these systems would require one to start from the step of construct assembly. Is SPN required for life? If not, are there development consequences of its loss? What about tissue specific consequences? Does a heterozygous SPN null display a haploinsufficiency phenotype? Cell lines derived from mouse null organisms could be used to ask specific questions concerning the effects of a loss of SPN on snRNP biogenesis

(see RNAi knockdown above).

151 CONCLUDING REMARKS

A greater understanding of the intricacies of snRNP biogenesis will demand much work. This thesis has addressed a role of the SMN complex in the Sm core snRNP import pathway. Furthermore, it provides insight into the function of

SPN in snRNP biogenesis. The identification of mutations specific in functional disruption, provides new tools for the study of snRNP biogenesis. These reagents will hopefully enable experiments that were previously impossible.

Additionally, a novel interaction between the N- and C-termini of SPN was identified, highlighting the possibility of additional levels of snRNP import regulation. It is my hope that the work presented in this thesis will enhance our knowledge of the vital process of snRNP biogenesis and provide inspiration to anyone wanting to pickup where I have left off.

152 APPENDICES

During the course of my graduate career, I conducted some experiments that were legitimate, however not directly tied to my thesis objectives. Several of these studies yielded results that were either inconclusive or difficult to understand based on our current knowledge. Additionally, due to an alteration in my research objectives, I am leaving behind data generated from my original thesis project. I present these date here, due to an inability to incorporate said results into the main body of this thesis.

153 Appendix Ι

Question under investigation

Does Xpo1 mediate the LMB-induced accumulation of SPN in CBs?

Rationale

I have previously shown that Xpo1 is enriched in CBs (see Figure 5 in Boulon et al., 2004). As Xpo1 interacts with SPN, we wanted to test if Xpo1 could mediate the enrichment of SPN in CBs upon LMB treatment.

Materials and methods

HeLa-ATCC cells were cultured in DMEM (Mediatech) supplemented with 10%

FBS and penicillin/streptomycin (Invitrogen) to 70% confluency. Cells were harvested and electroporated using a GenePulser Xcell electroporator (Bio-Rad) as directed using 2 µg of Xpo1-GFP. Cells were then seeded on slides (Nunc) for 16 hr and treated with the addition of 20 nM LMB to the cell culture media 1 hr prior to cell fixation. Cells were then fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 as described (Frey and Matera, 1995).

Incubation in 10% normal goat serum for 30 min preceded antibody detection.

CBs were detected by immunofluorescence using anti-coilin (R124) at 1:600 and goat anti-rabbit conjugated Texas Red at 1:200 (Vector Laboratories).

Results

Xpo1 localizes to the nucleoplasm with enrichment in CBs and at the nuclear rim.

The nuclear rim enrichment of Xpo1 is reduced, and its accumulation in CBs undetectable, in the presence of LMB (Fig. A1-1).

154

Figure A1-1: Xpo1 is depleted from Cajal bodies (CBs) upon LMB treatment. HeLa cells were transiently transfected with Xpo1-GFP and treated with 20 nM LMB for 1 hr. Immunofluorescence was then conducted with anti-coilin antibodies to localize CBs. Xpo1-GFP is enriched in CBs and at the nuclear rim in the absence of LMB. A reduction in the nuclear rim concentration and a depletion from CBs are noted upon LMB treatment. Bar, 10 µm.

155 Discussion

The concentration of Xpo1 at the nuclear rim is anticipated based on its role in cellular transport (Fornerod et al., 1997). As LMB inhibits the ability of Xpo1 to bind its cargo (Fornerod et al., 1997; Kudo et al., 1998; Ossareh-Nazari et al.,

1997), it was anticipated that treatment would result in a reduction in this nuclear rim concentration. However, an inability to detect Xpo1 enrichment in CBs on

LMB treatment was surprising. I reasoned that if Xpo1 is playing a role in the enrichment of SPN in CBs, then Xpo1 should behave similarly to SPN on LMB treatment. However, Xpo1 was not detected in CBs upon treatment. The fact that SPN in enriched in CBs on LMB treatment, while Xpo1 is depleted, argues that Xpo1 is not mediating the enrichment of SPN in CBs.

These data also suggest that the binding capacity of Xpo1 is important for its accumulation in CBs. It is important to note that this binding capacity is LMB sensitive, as Xpo1 is depleted from CBs upon treatment with this drug. An excellent candidate for the CB targeting of Xpo1 is PHAX, as it is also enriched in

CBs and contains an NES motif. The binding of PHAX would be inhibited in the presence of LMB and if this binding is vital for the accumulation of Xpo1 in CBs, one would expect a depletion from these nuclear domains on treatment.

156 Appendix ΙΙA

Question under investigation

Do TMG caps mediate the accumulation of SPN in CBs?

Rationale

Data in Chapter 3 illustrate that the ability of SPN to concentrate in CBs depends on its capacity to bind TMG caps. Therefore, I wanted to test whether the CB enrichment of SPN could be competed with excess TMG caps.

Materials and methods

HeLa-ATCC cells were grown to 50% confluency on slides (Nunc), washed once with complete transport buffer (CTB; 20 mM HEPES-KOH [pH 7.5], 110 mM

KOAc, 2mM MgCl2, 5 mM EGTA, 2 mM DTT, and 1 µg/ml each aprotinin, leupeptin, and pepstatin) and permeabilized with digitonin for 5 min at 4°C. Cells were then washed twice with CTB to remove soluble endogenous transport factors. Import reactions were then carried out in the presence of an ATP regenerating system (0.2 mg/ml tRNA, 0.2 mg/ml BSA, 1 mM ATP, 10 mM creatine phosphate, 50 µg/ml creatine phosphokinase (Roche)) for 35 min at 4°C in the absence of added caps or in the presence of excess m7G or TMG caps.

After incubation, cells were washed in CTB, then fixed in 4% paraformaldehyde for 10 min at room temperature and permeabilized with 0.5% Trition for 5 min.

Cells were visualized by a Zeiss Axioplan upright epifluorescence microscope

(100X objective).

157 Results

I initially wanted to use the 4°C incubation of SPN in the digitonin permeabilized

HeLa cell assay as a negative control for import. However, nuclear enrichment of

SPN was noted under these conditions. Additionally, a small percentage of cells displayed an enrichment of SPN in CBs when incubated at 4°C (Fig. A2-A). This enrichment was competed with the addition of excess TMG caps, but not with added m7G caps.

158

Figure A2-A: Excess TMG caps inhibit the enrichment of SPN in CBs. Permeabilized HeLa cells were incubated for 35 min at 4°C in the presence of GFP-SPN and importin β. Reactions were carried at in the absence of added caps or in the presence of excess m7G or TMG caps. Bar, 10 µm.

159 Discussion

The conclusions that can be drawn from this experiment are limited. It is quite clear that excess TMG caps can compete the enrichment of SPN in CBs, while m7G caps cannot. This observation indicates that TMG caps (probably at the 5’ ends of snRNPs), mediate the enrichment of SPN in CBs. Aside from this observation, little can be concluded or speculated, as the experimental conditions were non-physiological (4°C incubation).

More experimentation is required to understand the import of SPN at 4°C.

This observation might however, actually be expected, as it appears that NPC translocation is independent of NTP hydrolysis. This coupled to the fact that SPN transport is Ran-GTP independent, could account for its import at 4°C. However, there is still the question of NPC dynamics and functioning at such low temperatures. Therefore, more experimentation will be necessary to draw any further conclusions from these observations.

160 Appendix ΙΙB

Question under investigation

Does the accumulation of SPN in CBs depend upon the type of snRNP it imports?

Rationale

I have shown that the accumulation of SPN in CBs requires the capacity of SPN to bind TMG caps. However, no SPN enrichment in CBs is noted when assaying

U1 snRNP import. Interestingly, these snRNPs are considered mature as they contain all known U1 specific components. Therefore, I wanted to test whether the import of an immature snRNP, such as the 12S U2 snRNP, would display an altered SPN localization when compared to that of U1 import. A differential SPN localization may be reflective of the fact that CBs are a site of maturation of RNA pol ΙΙ specific Sm class snRNPs (Darzacq et al., 2002; Jady et al., 2003; Richard et al., 2003).

Materials and methods

These experiments were conducted as above (Appendix ΙΙA) with the following exceptions: import reactions were incubated at 26°C in the absence of added caps. Additionally, reactions were conducted in the presence of importin β and

His-GFP-SPN at 700 ng each, and either 40 nM Cy3-labeled U1 or U2 snRNPs.

161 Results

The accumulation of SPN and snRNPs in CBs is noted upon the import of U2 but not U1 snRNPs (Fig. A2-B).

Figure A2-B: The enrichment of SPN and snRNPs in CBs is noted upon the import of U2 but not U1 snRNPs. Permeabilized HeLa cells were incubated for 35 min at 26°C in the presence of His-GFP-SPN, importin β and either cy3-labeled U1 or U2 snRNPs. Bar, 10 µm.

Discussion

The observation that the CB enrichment of SPN and snRNPs is dependent upon the type of snRNP being imported is interesting and warrants further investigation. The next set of experiments should determine the specific component(s) of the U2 snRNP that mediate this CB enrichment of SPN. This will require the reconstitution of U2 snRNPs with varying U2-specific components, to effectively mimic different stages of snRNP maturation.

Additionally, immature U1 snRNPs could be utilized to determine whether the import of these RNPs might also result in the accumulation of SPN in CBs.

162 Appendix ΙΙΙ

These data can be found in Shpargel et al. (2003) published on January 15,

2003 in the Journal of Cell Science.

Question under investigation

Do Cajal bodies form in the absence of transcription i.e. in the cytoplasm?

Rationale

Nothing is known concerning what promotes Cajal body nucleation. Therefore, I wanted to determine if transcription is required for the formation of these structures. To address this possibility, I generated GFP-tagged coilin mutants incapable of being imported into the nucleus.

Materials and methods

GFP-coilin(Δ106-234) was generated using QuikChange mutagenesis

(Stratagene). The mouse embryonic fibroblast (MEF) cell line was established from coilin knockout (-/-) mice as described previously (Tucker et al., 2001). The coilin-knockout MEF or HeLa-ATCC cells were grown in DMEM (GIBCO BRL), supplemented with 10% FBS (GIBCO BRL). Cells were grown to subconfluency on chambered slides (Nunc) and transfected for 24 hours. MEF cells were transfected using LippofectAMINE (GIBCO BRL); HeLa cells were transfected using SuperFect (Qiagen) as directed. Cells were fixed in 4% paraformaldehyde, extracted in 0.5% Triton X-100 and processed for microscopy as previously described (Frey and Matera, 1995). Immunofluorescence was conducted with

163 myc (1:40; Santa Cruz Biotechnology), R288 [1:100 (Andrade et al., 1993)] and

R508 [1:200 (Chan et al., 1994)] antibodies.

Results

Expression of GFP-coilin results in CB numbers similar to those found in untransfected cells, upon detection of endogenous coilin. When the GFP tag is generated at the C-terminus of coilin, there is an increase in CB numbers. This increase is also seen a truncation mutant, GFP-coilin(1-481), whereby the the C- terminal 95 amino acids have been removed.

No CBs were detected in the cytoplasm of HeLa or MEF cells transfected with the GFP-coilin(Δ106-234) construct, despite the relocalization of coilin to these compartments. Additionally, CBs were undetected in the nuclei of HeLa cells transfected with GFP-coilin(Δ106-234). However, when coilin-GFP(Δ106-

234) was transfected into HeLa cells, nuclear CBs were noted, indicating that coilin-GFP(Δ106-234) is imported at low levels. Interestingly, when MEF cells lacking endogenous coilin were transfected, neither coilin import nor CB formation was noted (Fig. A3-1). This suggests that the import of coilin-

GFP(Δ106-234) may be due to its association with endogenous coilin. This is consistent with the observation that coilin-GFP proteins display unregulated CB formation (probably due to increased coilin-coilin interactions, Hebert and Matera,

2000).

164

Figure A3-1: Coilin mutants display unregulated nuclear body formation. Upper panels, HeLa cells were transfected with GFP fused to the N-terminus of coilin (left), GFP fused to the C-terminus of coilin (middle) or a GFP-tagged truncation of coilin (1-481, right). The GFP-coilin(1-481) foci colocalized with other CB markers (SMN and fibrillarin) when transfected into HeLa cells (data not shown). Note that several out-of-focus dots appear blurred. In the lower panels, deletion of the nuclear localization signals (residues 106-234) in the GFP-coilin background results in exclusively cytoplasmic localization without foci in HeLa cells (left). Deletion of the NLSs in the coilin-GFP background results in a similar pattern, with the exception that nuclear foci are detected. Although the bulk of the fluorescence was cytoplasmic, two populations of cells were observed, some with faint nuclear foci and others with brighter foci (middle). When transfected into coilin knockout MEFs, this same coilin NLS-GFP construct localized solely to the cytoplasm (right).

165 Appendix ΙV

These data can be found in Hebert et al. (2002) published on September 3,

2002 in the Journal Developmental Cell.

Question under investigation

Does the localization of U snRNPs in Cajal bodies require the presence of SMN?

Rationale

Sleeman et al. (2001) showed that newly imported snRNPs (visualized by FP- tagged Sm proteins) do not accumulate in coilin foci lacking SMN. These analyses, in addition to others studying snRNP localization, have relied upon the monoclonal antibody Y12 for snRNP detection. Interestingly, Hebert et al. (2002) found that Y12 crossreacts with coilin, indicating that the foci lacking SMN in

Sleeman et al. (2001) might also be lacking coilin. This raised the possibility that both SMN and coilin are required for accumulation of snRNPs in CBs. To test this I utilized the HeLa-PV cell line, which displays a greater percentage of cells with a separated CB and Gem phenotype when compared to HeLa-ATCC cells

(Hebert et al., 2002).

Materials and methods

Cells were grown on chamber slides (Nunc) to 50%–80% confluency. Cells were fixed in 4% paraformaldehyde for 10 min and then permeabilized with ice-cold

0.5% Triton X-100 for 5 min. Immunofluorescence was conducted with the following antibodies: anticoilin rabbit serum (R508) and anti-SMN (mAb 2B1).

166 Hybridization to U2 snRNA was conducted with a biotinylated U2 peptide nucleic acid (PNA) probe (5′-Bio-OO-ACAGATACTACA-3′, where O represents an abasic linker). An antisense oligomer U7-(3-20), 5′-

CTAAAAGAGCUGUAACACBBBBdC-3′, was used for detection of U7 snRNA.

The residue preceded by a lowercase "d" represents deoxyribose (DNA), B marks positions of biotin phosphoramidites, and A, G, C, and U are 2′-O-methyl

RNA residues. Hybridization solution consisted of 4× SSC/10% dextran sulfate and 2 pmol/µl of U2 PNA or U7 oligo. Hybridization for 30 min at 37°C was followed by 3 washes in 4× SSC/0.1% Tween 20.

167 Results

The presence of SMN in CBs is not required for snRNP accumulation in these nuclear structures (Fig. A4-1).

Figure A4-1: SMN deficient Cajal bodies are enriched in U snRNPs. HeLa-PV cells were grown at 32°C to exacerbate the separation phenotype and then assayed for colocalization of coilin, SMN, and U snRNPs with antibody R508, mAb 2B1, and anti-sense in situ oligoprobes, respectively. U2 (top) and U7 (bottom) snRNAs were visualized by RNA FISH. In cells that display both gems and CBs, U snRNPs were enriched in the CBs lacking SMN. Arrows in the U2, U7, and coilin panels indicate the presence of U snRNPs in CBs. Arrows in the SMN panels indicate positions of the CBs that lack SMN, which is enriched in the cytoplasm and gems.

168 Appendix V

Question under investigation

Does mutation of PHAX, at predicted sites of phosphorylation, disrupt its localization or interaction with Xpo1?

Rationale

The transport of snRNAs to the cytoplasm requires the incorporation of the phosphorylated version of PHAX into the an export complex. The identity of these phosphorylated amino acids is currently unknown.

Materials and methods

A mouse PHAX cDNA was used in these experiments, however the nomenclature of the mutants reflects the amino acid positions of human PHAX.

The amino acid sequence of PHAX was entered into a phosphorylation prediction program. Residues predicted to be phosphorylated were examined for their conservation, resulting in two interesting sites. PHAX-GFP mutants (S14A,

S14D, T284A and T284E) were generated using QuikChange mutagenesis

(Stratagene). Cells were grown to subconfluency on chambered slides (Nunc) for fluorescence microscopy or in flasks (BD Biosciences) for coimmunoprecipitations and transfected for 24 hours using SuperFect (Qiagen) as directed. Cells were fixed in 4% paraformaldehyde, extracted with 0.5% Triton

X-100 and processed for microscopy as previously described (Frey and Matera,

1995). For coimmunoprecipitations, cells were harvested, washed with PBS and resuspended in 1 ml of mRIPA buffer (50mM Tris-Cl, pH 7.5, 150 mM NaCl, 1%

169 NP-40, 1mM EDTA) plus protease inhibitor cocktail tablets (Roche) to lyse cells.

Resuspended cells were incubated at 4°C for 30 minutes with gentle inversion, followed by centrifugation for 5 minutes to pellet cellular debris. 50 µl of polyclonal anti-GFP (BD Biosciences, Clontech) were then added to 900 µl of lysate. After incubation for 1 hr, 60 µl of 50% protein A Sepharose beads

(Amersham Pharmacia) were added and to the lysates and incubated overnight.

Beads were then washed 3 times with 1 ml mRIPA, resuspended in 15 µl of 5X

SDS loading buffer, boiled and proteins were resolved by SDS-PAGE. After transfer to nitrocellulose, membranes were probed with anti-SMN (7B10) followed by incubation with goat anti-mouse conjugated horseradish peroxidase (Pierce

Chemical) and chemiluminescence detection (Roche).

Results

The localization patterns of mutant PHAX-GFP proteins mimicked that of wildtype

PHAX-GFP (see discussion) with enrichment in the nucleoplasm and CBs (Frey and Matera, 2001). Two differences were noted when mutant PHAX proteins were used to coimmunoprecipitate SMN. PHAX(S14A) migrates faster than either wildtype or the other PHAX mutants by SDS-PAGE (Fig. A5-1). It was also noted that less SMN was coimmunoprecipatated by PHAX(T284A) (Fig. A5-1).

170

Figure A5-1: PHAX mutant (S14A) migrates faster and (T284A) coimmunoprecipitates less SMN compared to wildtype. HeLa cells were transfected with wildtype PHAX-GFP or the following PHAX mutants: S14A, S14D, T284A or T284E. Lysates were generated and immunoprecipitated with anti-GFP antibodies. SDS-PAGE analysis was then conducted, followed by western blotting with anti-GFP and anti-SMN antibodies. Note the difference in migration of S14A and the reduction of coimmunoprecipatated SMN with T284A.

Discussion

The incorporation of PHAX into an snRNA export complex requires that it is phosphorylated. Therefore, the mutation of PHAX, whereby the phosphorylation of critical residues is prevented (S14A or T284A), should inhibit its participation in snRNA export. This would however, be detected as a nucleoplasmic enrichment which mimics endogenous PHAX localization. On the other hand, if constitutive phosphorylation (S14D or T284D), resulted in a greater propensity for inclusion in the export complex, then more mutant PHAX would be exported. However, these molecules could then be imported back into the nucleus, presumably by importin

171 α (PHAX contains two signals which resemble classical NLSs (Ohno et al., 2000;

Segref et al., 2001). Both outcomes may be difficult to observe in transfected cells. Thus, the absence of an alteration in localization tells us nothing.

However, the difference in migration of (S14A) and the reduction in the ability of (T284A) to coimmunoprecipitate SMN do suggest functional alterations.

The migration difference of (S14A) is suggestive of a difference in the post- translational modification of this mutant, possibly due to an alteration in phosphorylation. In regards to the difference in SMN coimmunoprecipitation,

Massenet et al. (2002) found that SMN interacts with PHAX in the cytoplasm

(Massenet et al., 2002). It is hypothesized that this interaction occurs immediately following the arrival of the snRNA export complex in the cytoplasm.

A PHAX mutant that displays a hypophosphorylated state, might be less likely to participate in snRNA export and would therefore immunoprecipitate less SMN.

172 Appendix VΙ

The dynamics of Cajal bodies and snRNA gene loci

INTRODUCTION AND BACKGROUND

Cajal bodies associate with a subset of U snRNA gene loci

CBs associate with RNU1, RNU2, RNU3, RNU4, RNU11 and RNU12 gene loci in interphase human cells (Frey and Matera, 1995; Gao et al., 1997; Jacobs et al.,

1999; Smith et al., 1995). An association is observed when the immunofluorescence staining of a CB visibly contacts the FISH signal from a gene locus.

The biogenesis of snRNPs begins when snRNAs are transcribed in the nucleus and subsequently exported to the cytoplasm (reviewed in Mattaj and

Englmeier, 1998). In the cytoplasm, Sm core assembly, cap hypermethylation and 3′ end processing result in newly assembled snRNPs, which are imported into the nucleus (Mattaj et al., 1993). Newly assembled snRNPs primarily enrich in CBs upon nuclear import (Sleeman and Lamond, 1999) followed by localization to clusters (IGCs) and perichromatin fibrils.

Interestingly, mature snRNPs return to IGCs following (Sleeman and

Lamond, 1999). It is not known why CBs are the first recipients of newly assembled snRNPs or which CBs are the recipients of these snRNPs. These findings, coupled with the fact that CBs contain partially mature snRNPs, suggest

173 that snRNPs are returning to their sites of transcription, possibly as a feedback mechanism (Matera and Frey, 1998).

Recently, our lab showed that CBs colocalize with artificial U2 snRNA gene constructs (Frey et al., 1999; Frey and Matera, 2001). Transcription of arrays containing U2 promoter elements, a U2 coding region, 3′ box and CT microsatellite is sufficient for colocalization with CBs (Frey et al., 1999).

Additionally, CB colocalization with U2 snRNA genes was preserved upon replacement of the U2 promoter elements with a Rous Sarcoma Virus (RSV) promoter (Frey and Matera, 2001). These data illustrate that nascent U2 snRNA transcripts are required for colocalization with CBs. These data also support the idea that one may manipulate U2 snRNA promoter and 3′ noncoding regions, without disturbing the interaction with CBs. To date, studies investigating the association between CBs and U snRNA genes have utilized fixed cells. This approach restricts the investigator to a mere snapshot of a dynamic process. I proposed the use of live cell imaging to elucidate the dynamics and mechanics of the association between CBs and U snRNA genes.

Materials and methods

Generation of pJO7B pSV2-8.32 containing an array of 256 copies of lac repeats was a gift from A.

Belmont. The lac array was removed by digestion with BamH I and Sph I restriction enzymes and ligated into pBeloBAC11 (New England Biolabs) to

174 create pJO1. This was conducted for two reasons: 1. pSV2 is a multi-copy plasmid, which contributes to the instability of pSV2-8.32, (Fig. A6-1). 2. A vector capable of harboring large inserts, was required. pBeloBAC11, a single copy bacterial artificial chromosome (BAC) vector, can maintain such large inserts and is more stable when harboring repetitive sequences.

Figure A6-1: pSV2-8.32 is unstable when propagated at 37°C. Twenty colonies were cultured at 37°C from isolated colonies containing intact pSV2-8.32. The sizes of three plasmids (4, 15, 16) were determined by restriction digestion. Gel electrophoresis reveals that #4 and #16 contain a fully intact lac array of 10 Kb (arrows), while #15 contains a truncated lac array (arrowhead). C represents cut while U represents uncut DNA.

A neo-dhfr cassette was excised from pE1 (gift from A. Weiner) with BamH I and

Bgl II restriction enzymes and ligated into the BamH I site of pJO1 to generate pJO3. A tandem array containing 24 copies of U2 genes (driven by the endogenous U2 promoter) was excised from pVJ104 (32) with BamH I and Bgl II restriction enzymes and ligated into the BamH I site of pJO3 to create pJO7. An ampicillin/blasticidin resistance cassette was then generated as follows:

QuikChange mutagenesis (Stratagene), was utilized to introduce a BamH I site

175 into pGEX-3X (Invitrogen) at nucleotide 2356 yielding pGEX-3X Bam. A blasticidin resistance cassette was excised from pPAC4 (gift from H. Willard) with

EcoR I and ligated into pGEX-3X Bam yielding pGEX-3X Bam blast. The ampicillin/blasticidin cassette was then removed with BamH I from pGEX-3X Bam blast and ligated into pJO7 to create pJO7B. The introduction of an ampicillin/blasticidin resistance cassette was used to simplify the screening of potential positive inserts. All clones were checked by restriction digestion for the recovery of predicted fragments (Fig. A6-2). Digestion of pJO7 with EcoR I and

Sma I digests the lac array into ~300 bp fragments and the U2 gene array into

~1.7 Kb fragments, respectively further confirming the structure of pJO7 (Fig. A6-

3). The structure of pJO7B was confirmed to be correct by the following criteria: restriction digestion, ampilcillin and chloramphenicol resistance and the generation of a PCR band corresponding to U2 coding sequence.

1 2

U2 array ~ 41 Kb

12 Kb lac array ~10 Kb 9 Kb pBeloBAC 11 ~ 7 Kb

Figure A6-2: Structural conformation of pJO7. pJO7 was digested and analyzed by field inversion gel electrophoresis to determine the sizes of the U2 and lac arrays. All arrays were of the desired sizes.

176

Figure A6-3: pJO7 and pJO11 contain the appropriate arrays. pJO7 and 11 were digested leaving the U2 and lac arrays intact (left lanes) or cut into monomers (right lanes). Southern analysis with U2 and lac repeat probes reveals ~300 bp lac monomers and ~1.7 Kb U2 monomers.

Generation of pJO11B

Primers were used to introduce Bgl II and Sgf I sites flanking the Tet-responsive elements and minimal CMV promoter of pTRE2 (BD Biosciences, Clontech).

PCR was conducted with PfuTurbo (Stratagene) and the resulting fragment was

Zero Blunt TOPO cloned (Invitrogen). The Tet-CMV fragment was then cloned into pRc/RSV U2 and the insertion of the U2 gene at the +1 start site of transcription was confirmed by sequencing. The Tet-CMV U2 cassette was then excised with BamH I and Bgl II and ligated into the BamH I and Bgl II sites of pUC18 Bgl (gift from A. Weiner) to create pJO8.1. Multimerization of the Tet-

CMV U2 cassette was then conducted with serial digestion and ligation reactions using BamH I, Bgl II and Hind III. An 8 copy Tet-CMV U2 cassette was excised with BamH I and BsrB I and ligated into pVJ104 at BamH I and Swa I. One

177 round of multerimerization was conducted to yield a cassette containing 16 copies of Tet inducible U2 genes. This 16 copy array was then excised with

BamH I and Hpa I and ligated into pJO3 yielding pJO11. The ampicillin/blasticidin resistance cassette from pGEX-3X Bam blast was then ligated into pJO11 at BamH I yielding pJO11B. All constructs were confirmed by restriction digestion for recovery of the predicted fragments.

Establishment of stable cell lines pJO11B was digested with Spe I, purified by phenol/chloroform extraction and ethanol precipitated. Ligations using 25 µg of recovered linear arrays were then conducted using T4 DNA ligase for 15 minutes at 25°C. Resulting arrays were ethanol precipitated and transfected into HeLa-ATCC cells at 50% confluency using Calcium Phosphate Transfection reagent (Invitrogen). After 48 hours, cells were seeded to 150 mm dishes (Nunc) and selected with 4 µg/ml blasticidin

(Invitrogen). After 10 days of selection, isolated colonies were picked to 24 well dishes (Nunc). Cells were selected for an additional seven days, replica plated and frozen in DMEM containing 10% Dimethyl Sulfoxide (DMSO). Cell lines were then seeded to 8-well chamber slides (Nunc) and transiently transfected

(Superfect) with YFP-lac repressor. Following fixation, cell lines were examined by fluorescence microscopy to determine the presence of lac array sequences.

Positive insertion was scored by the presence of one or more nuclear YFP foci in addition to diffuse nuclear YFP fluorescence.

178 RT-PCR of stable cell lines

Stable cell lines were grown in T75 flasks and transfected (Superfect) with pTet-

ON. Cells were then treated with doxycycline (BD Biosciences, Clontech) at 2

µg/ml for 16 hours to induce transcription of exogenous U2 gene arrays. Total

RNA was then obtained using the Perfect Eukaryotic Mini Kit (Eppendorf) and

DNase treated. Reverse transcription using the SuperScript First-Strand

Synthesis System (Invitrogen) as per the manufacture’s instructions, then followed. PCR was then conducted using a forward primer recognizing all U2 transcripts and a reverse primer specific for the exogenous RNA.

Cell scoring and association analyses

Stable cells were seeded to chamber slides and transfected with YFP-lac repressor, CFP-SMN and pTet-ON. Cells were treated with doxycycline at 2

µg/ml for 16 hours and fixed in 4% paraformaldehyde. CBs were counted and association frequencies determined by counting the occurrences of overlap between a CB and U2 gene array, detected by CFP-SMN and YFP-lac repressor fluorescence, respectively.

Live cell imaging

Cells were seeded to dishes and transfected with YFP-lac repressor, CFP-SMN and pTet-ON. Cells were then treated with doxycycline for 16 hours. Prior to imaging, cells were incubated for 2 hours with DMEM lacking phenol red and

179 containing 10% FBS and HEPES. Cells were imaged for 100 minutes with images acquired at 5 minute intervals using both CFP and YFP filter sets at each time point.

Results

I hypothesize that CBs form at sites of active U2 snRNA gene transcription. Cell lines have been generated to address this question. In short, a vector containing

Tet-inducible U2 snRNA genes and the lac array was linearized and subsequently ligated to generate longer arrays. Tet-inducible genes were desired to enable control over both the timing and level of exogenous U2 gene expression. Using Tet-inducible arrays, a single cell can be imaged in the “off” state and upon induction, visualized for responses to transcription initiation from exogenous arrays. YFP-lac repressor was transiently expressed in U2 stable cells and localized to the nucleoplasm and bright nuclear foci in cells positive for lac array sequences (Fig. A6-4).

180

HeLa 45 60

HeLa 41 41

Figure A6-4: Tet-inducible U2 snRNA stable cells display discrete lac repressor foci. Cell lines were transfected with YFP-lac repressor and examined by fluorescence microscopy. HeLa panels illustrate the localization of YFP-lac repressor in cells prior to stable transfection. YFP-lac repressor localizes to foci, when transiently expressed in stable lac/U2-array cells (panels 45, 60 and 41).

181 Following the identification of stable cell lines, experiments determined whether the integrated arrays were inducible. Stable cells were transiently transfected with pTet-ON and treated with doxycycline. Expression of pTet-ON yields the reverse tetracycline-controlled transactivator which, in the presence of doxycycline, turns on transcription of tetracycline-regulated genes (Fig. A6-5A).

Cells were treated with doxycycline for 16 hours and total cellular RNA obtained.

Total RNA was also obtained from a control cell line, R51B6, containing non- inducible U2 genes (Frey and Matera, 2001). Recovered RNA was DNase treated and reverse transcribed using oligo-dT primers. PCR was then conducted using a forward primer annealing to the first stem loop of U2 and a reverse primer annealing to the bovine growth hormone poly A tail (Fig. A6-5B).

182 A CMV

TRE BGH polyA U2

Figure A6-5: Structure and expression of an inducible U2 array. (A) A single U2 tetracycline-inducible gene is shown: the tetracycline response elements (TRE), CMV promoter, U2 coding sequence and bovine growth hormone (BGH) poly A signal are illustrated. (B) RT-PCR illustrates the inducibility of stable cell lines. HeLa RNA served as a negative control while RNA and cDNA from the R51B6 cell line served as positive controls. R51B6 cDNA PCR products were loaded in both upper and lower wells as a size maker.

183 Figure A6-5B illustrates the generation of exogenous U2 transcripts in stable cell lines. However, the expression of these transcripts in the absence of induction had not been tested. Therefore, cell line 41 was investigated for the inducibility of exogenous U2 expression. Cell line 41 was transfected with pTet-ON and treated with doxycycline, transfected with pTet-ON, or treated with doxycycline in the absence of pTet-ON. HeLa cells served and a negative control and the previously obtained cell line 41 RT-PCR product as a size marker. Total RNA was isolated, DNase treated and subjected to reverse transcription using an oligo-dT primer, followed by PCR using U2 and BGH poly A specific primers (Fig.

A6-6).

Figure A6-6: Stable cell line 41 is inducible for exogenous U2 expression. Cell line 41 was transfected with pTet-ON and treated with doxycycline, (Dox) transfected with pTet-ON alone or only treated with Dox. U2 expression was noted with transfection of pTet-ON followed by Dox treatment. Very low expression was noted with pTet-ON transfection alone and no expression was seen in the absence of pTet-ON. Note that the lane labeled 41+ simply contains a previously obtained RT-PCR product from cell line 41 as a size marker.

184 Next, I wanted to determine if expression from exogenous U2 snRNA genes was sufficient to promote CB association. To address this question, cells were examined for association after induction of exogenous U2 gene arrays. Cells were transfected with pTet-ON, YFP-lac repressor and CFP-SMN, and subsequently treated with doxycycline for 16 hours. As illustrated in Figure A6-7, associations (arrows) were noted upon visualization of cells by fluorescence microscopy.

Figure A6-7: CBs associate with exogenous, Tet-inducible, U2 snRNA gene arrays. Cell line 41 was transiently transfected with pTet-ON, YFP-lac repressor and CFP-SMN. Cells were then treated with doxycycline for 16 hours and visualized by fluorescence microscopy. CBs are blue while exogenous U2 gene arrays appear green. Associations are indicated by the arrows and were also noted in cell lines 41A, 45 and 60.

185 Stable cell lines were then examined to determine the association frequencies of exogenous U2 snRNA gene arrays with CBs. Cells were transfected as described in Figure A6-7 and then examined following the merger of YFP and

CFP images. Stable cells lines displayed similar association frequencies as well as mean CB numbers (Fig. A6-8). Importantly, no associations were noted in the absence of transcription proving that transcription is required for CB association.

This is significant because comparisons of transcriptional states are made within a single cell line as compared to previous studies, which made comparisons between different cell lines (Frey et al., 1999; Frey and Matera, 2001).

Additionally, CB numbers were compared in induced and non-induced cell lines

(Fig. A6-9). Induced stable cell lines had similar CB numbers compared to parental HeLa cells, with the exception of higher CB numbers in cell line 41 (Fig.

A6-9). This is interesting due to the fact that around 60% of cells in line 41 have large clusters of integrated U2 genes (Fig. A6-4). This is in agreement with published data illustrating that an increase in gene number, from diploid to tetraploid HT1080 cells, also doubled CB numbers (Frey et al., 1999).

186

Figure A6-8: Association frequencies and mean CB numbers of stable cell lines. Stable cells were transfected with pTet-ON, YFP–lac repressor and CFP-SMN and treated with doxycycline, then scored for associations. An association is noted when the fluorescence of the lac focus overlaps with the fluorescence of a CB (see arrows in Fig. A6-7).

187

Figure A6-9: Mean Cajal body numbers of stable cell lines. CB numbers were determined in induced and non-induced stable cells as well as in parental HeLa cells. CB numbers are similar in all cell lines with the exception of greater CBs numbers in induced cell line 41 (see text).

Live cell analyses

In a collaboration with Judith Sleeman and Angus Lamond at the University of

Dundee, we have conducted live cell imaging with inducible cell lines. Live cell movies have been generated by transiently transfecting stable cells with pTet-

ON, YFP-lac repressor and CFP-SMN followed by doxycycline treatment.

Transfected cells were imaged for 100 minutes with images taken every 5 minutes. Preliminary analyses of resulting movies indicated that CB associations were quite stable and lasted on the order of 30-45 minutes. Additional associations appeared to be lost twice and then reestablished over the course of

188 the movie. Several instances of CB joining were noted, in agreement with published data (Platani et al., 2000).

Conclusions

I have obtained data illustrating that stable cell lines harboring tetracycline responsive, CMV promoter driven U2 snRNA arrays are inducible and that CBs associate with these induced arrays. Furthermore, preliminary live cell analyses illustrate the feasibility of additional studies investigating the dynamics of the association between CBs and U2 snRNA gene arrays.

Future Directions

Despite the current status of this project, studies investigating the dynamics of the association between CBs and U2 snRNA genes will require fulltime attention and entail a major research project. Below, I outline a manuscript indicating my current data and hypothesized experiments that may yield a complete story.

189 CB/U2 association dynamics manuscript outline

Figure 1: Describe the stable construct arrays (diagram)

-Illustrate genes and proteins used and why

-Show metaphase fluorescence in situ hybridization (FISH) data.

-Quantify integration copy number (not completed)

Figure 2: Show that inducible arrays behave as expected

-U2 gene array is present and inducible (completed)

-lac repressor accumulates in foci (completed)

-Foci vary between cell lines (most cell lines give single foci, whereas one

yields several foci with clusters) (completed)

-CBs associate, with X frequency and transcription is required for this

association (completed)

Figure 3: Describe the association dynamics on a general level

-What are the residence times of association? (requires more studies)

-How stable are the associations, are they strictly maintained or transient

i.e. on and off over time? (the initial experiments are complete but more

studies are required)

-How long do the associations last? (requires more studies)

-Is ATP required for association? (not completed)

-Is translation required association? (not completed)

Figure 4: Focus on the CB side of the association (not completed)

190 -Do active arrays nucleate CBs? If yes, then are snRNPs required in

nucleating CBs. Determine this by titrating snRNPs in the cytoplasm e.g.

introduce snurportin mutant that can not bind importin β (R27A).

-If active arrays do not nucleate CBs, then where do associating CBs

come from?

-If CBs come from other nuclear locations, then where?

-Look at the cell line that has greater than one gene array integration site.

-Where do CBs go after association? Use a coilin mutant that is found in

CBs and nucleoli to localize both domains. Determine the post-

association CB migration in relation to the nuclear periphery.

-Characterize CB mobility pre- and post-association. Are they the same,

do they differ, i.e. does CB mobility resemble simple diffusion pre-

association, then switch to a more directed mobility post-association?

Figure 5: Focus on the gene side of the association (not completed)

-Does an association alter the position of a gene array?

More transciptionally active genes are often found in the nuclear

interior. Therefore, image pre-induction, are gene arrays more often seen

at the nuclear periphery?

-If yes, do they migrate to the nuclear interior upon induction?

-Compare arrays that associate with arrays that do not? Do non-

associating arrays remain in the nuclear interior, while associating arrays

are repositioned at the nuclear periphery?

191 -If associating arrays are repositioned, does that repositioning require sustained or transient CB association?

-If arrays are repositioned to the nuclear periphery following association, this supports the hypothesis that CB association may regulate gene expression (by a negative feedback mechanism, discussed in Matera and

Frey, 1998).

192 Appendix VΙΙ

Descriptions and maps of Bac vectors generated for live cell studies

pBeloBAC 11 BAC vector New England BioLabs - Single copy plasmid whose “MCS” contains only three sites pJO1 pJO1 was generated by ligating a 256 copy lac operator array flanked with BamH I and Sph I into the BamH I and Sph I sites of pBeloBAC11. pJO3 pJO3 was generated by putting a DHFR-Neor cassette flanked by BamH I sites into the BamH I site of pJO1. The DHFR-Neor cassette was generated by the Alan Weiner lab. Neor cassette is from Stratagene’s pMC1NEO and the DHFR is from mouse, but I don’t know its sequence. pJO3 MCS pJO3 containing an expanded MCS with various 8-cutters pJO6 pRc/RSV with the RSV promoter replaced by a Tet-inducible promoter driving a single U2 gene ending with a Bovine Growth Hormone poly A signal and tail.

BGH poly A

Single Tet-inducible gene

193 pJO7 pJO3 containing 24 copies of U2 genes (each one being 1.7 kB mU2 +CT fragments) totaling ~41 kB pJO7 B pJO7 containing a blasticidin resistance cassette pJO11 pJO3 containing 16 copies of a Tet-inducible promoter driven U2 gene ending with a Bovine Growth Hormone poly A signal and tail cassette (see pJO6 vector) pJO11 B pJO11 containing a blasticidin resistance cassette

STABLE CELL LINE CLONES

#11 1 dot per cell #41 Many dots #41A 1 dot per cell #45 1 dot per cell #60 1 dot per cell

All are stable via blasticidin resistance in HeLa cells. Grow in DMEM + pen/strep + 10% FBS with 2 ug/ml blasticidin.

To induce transfect with pTet-ON and treat with 2 ug/ml doxycycline.

194 Figure A7-1: Maps of Bac vectors generated for live cell studies.

195

196

197

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