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SR proteins in microRNA/mRNA biogenesis by Han Wu Department of Cell Biology Duke University Date:_______________________ Approved: ___________________________ Jun Zhu, Supervisor ___________________________ Brigid L.M. Hogan, Chair ___________________________ Christopher B. Newgard ___________________________ Uwe Ohler ___________________________ Kenneth D. Poss ___________________________ Yuan Zhuang Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Cell Biology in the Graduate School of Duke University 2011 ABSTRACT SR proteins in microRNA/mRNA biogenesis by Han Wu Department of Cell Biology Duke University Date:_______________________ Approved: ___________________________ Jun Zhu, Supervisor ___________________________ Brigid L.M. Hogan, Chair ___________________________ Christopher B. Newgard ___________________________ Uwe Ohler ___________________________ Kenneth D. Poss ___________________________ Yuan Zhuang An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Cell Biology in the Graduate School of Duke University 2011 Copyright by Han Wu 2011 Abstract Serine/arginine‐rich proteins or SR proteins are well‐known protein factors involved in splicing regulation. Despite the tremendous efforts that have been made in characterizing their functions, several fundamental questions still remain: how are the expression levels of SR proteins regulated; what are the molecular mechanisms underlying SR protein‐mediated gene regulation; and what are the physiological targets of SR proteins in vivo. In my dissertation study, I employed a number of genomic and molecular approaches to study the functional involvement of two SR proteins, SF2/ASF and SRp20, in regulating microRNA/mRNA biogenesis. Negative feedback regulation has been shown as a common mechanism to maintain SR protein homeostasis (i.e. SC35 and SRp20). In the first part of my thesis, I set out to examine the potential involvement of miRNA‐mediated feedback regulation in maintaining the steady‐state level of SF2/ASF. MicroRNA deep sequencing was employed to identify differentially expressed miRNAs by elevating SF2/ASF level in an inducible cell line system. The sequencing data were further integrated with miRNA target predictions, which allowed me to identify a putative SF2/ASF‐miR‐7 negative feedback loop. A series of molecular and cellular techniques were then employed to validate the circuit structure. To our knowledge, this is the first negative feedback circuit iv reported between an SR protein (SF2/ASF) and a miRNA (miR‐7), and the paradigm may be broadly applicable in regulating the homeostasis of other SR proteins. This initial study was then extended to characterize the mechanism underlying SF2/ASF‐enhanced miR‐7 expression. Notably, one of the miR‐7 primary transcripts, miR‐7‐1, is embedded in an alternatively spliced intron of the hnRNPK gene. It provides a unique system to simultaneously study the involvement of SF2/ASF in alternative splicing and miRNA biogenesis. Through a series of mutagenesis assays in a minigene reporter system, I have uncovered a novel splicing‐independent function of SF2/ASF in regulating miRNA biogenesis. Direct interaction between SF2/ASF and primary‐miR‐7 (pri‐miR‐7) was demonstrated by cross‐linking and immunoprecipitation assay (CLIP) as well as by RNA affinity purification assay. Furthermore, I showed that this interaction is required for efficient miR‐7 processing in vivo. Finally, an in vitro pri‐miRNA processing assay was employed, and the results showed that SF2/ASF promotes the Drosha cleavage step of pri‐miR‐7 depending on the predicted binding site. Taken together, this was the first study showing the direct involvement of an SR protein in miRNA biogenesis. In order to study the global involvement of SR proteins in RNA biogenesis, one important stepping stone is to identify their targets in vivo. To this end, I focused on SRp20, another classic SR protein. PAR‐CLIP (Photoactivatable‐Ribonucleoside‐ Enhanced Cross‐linking and immunoprecipitation assay) combined with Illumina v sequencing was employed to systematically identify potential SRp20 binding targets, a subset of which were randomly selected and validated. As expected, our results showed that SRp20 primarily targets exonic regions for splicing regulation, and such interactions are likely to be mediated by a CWWCW motif. Surprisingly, extensive interactions between SRp20 and the 3’ UTRs of mRNAs were also observed. Such interactions presumably affect the choices between alternative polyadenylation sites. Thus, SRp20 may be one of the master regulators of both splicing and 3’ end processing. In summary, my thesis study was centered on functional characterization of two SR proteins, SF2/ASF and SRp20, in post‐transcriptional gene regulation. I have identified a negative feedback circuit between SF2/ASF and miR‐7: SF2/ASF directly binds to pri‐miR‐7 and promotes its maturation in a splicing‐independent manner; while mature miR‐7 negatively regulates SF2/ASF at the translational level. Furthermore, PAR‐ CLIP assay identified genome‐wide interactions between SRp20 and its potential targets, which may be linked to splicing regulation as well as alternative 3’ end processing. Altogether, this study has provided novel insights into how SR protein homeostasis can be achieved through negative feedback regulation, and the regulatory role of SR proteins in miRNA/mRNA biogenesis. vi Contents Abstract ......................................................................................................................................... iv List of Tables ................................................................................................................................. xi List of Figures .............................................................................................................................. xii List of Abbreviations .................................................................................................................xiv Acknowledgements ................................................................................................................... xix 1. Introduction ............................................................................................................................... 1 1.1 Pre‐mRNA splicing .......................................................................................................... 2 1.2 The SR family of splicing factors .................................................................................... 4 1.3 SR proteins in splicing regulation .................................................................................. 6 1.4 Other regulatory functions of SR proteins in gene expression .................................. 8 1.5 SR protein‐mediated gene regulation: functional significance ................................ 10 1.6 The substrate specificities of SR proteins .................................................................... 13 1.7 SR protein homeostasis .................................................................................................. 16 1.8 MicroRNA biogenesis .................................................................................................... 18 1.9 MicroRNA functions ...................................................................................................... 22 1.10 MicroRNA‐mediated gene regulatory networks ..................................................... 26 2. Methods .................................................................................................................................... 29 2.1 Cell culture and transfection ......................................................................................... 29 2.2 Plasmids ........................................................................................................................... 29 2.3 Profiling miRNA expression by deep sequencing ..................................................... 30 vii 2.3.1 Construction of miRNA sequencing library .......................................................... 30 2.3.2 Analysis of miRNA deep sequencing data ............................................................ 31 2.4 Dual‐Luciferase reporter assay ..................................................................................... 32 2.5 Semi‐quantitative PCR ................................................................................................... 33 2.6 Cross‐linking and Immunoprecipitation (CLIP) ........................................................ 34 2.6.1 CLIP‐PCR .................................................................................................................... 34 2.6.2 PAR‐CLIP .................................................................................................................... 35 2.6.3 Analysis of PAR‐CLIP sequencing data ................................................................. 36 2.7 Northern blotting ............................................................................................................ 37 2.8 Western blotting ............................................................................................................. 38 2.9 RNA affinity purification .............................................................................................. 39 2.10 RNA interference .........................................................................................................
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