Characterization of the Terminal Oligopyrimidine Mrna Binding Properties of La-Related Protein 1

Characterization of the Terminal Oligopyrimidine mRNA Binding Properties of La-Related Protein 1

By Jaclyn Hearnden

Department of Biochemistry McGill University Montreal, Quebec, Canada

April 2016

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Masters of Science. © Jaclyn Hearnden, 2016

Abstract The conservation of cellular energy during unfavorable conditions requires efficient deactivation of protein production. When environmental conditions cannot support basic cellular functions, various intracellular signaling modules respond, notably mammalian

Target of Rapamycin Complex 1 (mTORC1)- a master regulator of protein synthesis.

Although the bulk of mRNA translation is rapidly downregulated following nutrient and oxygen deprivation by mTORC1, a subset of mRNA transcripts featuring Terminal

OligoPyrimidine (TOP) sequences in their 5’ UnTranslated Region (5’UTR) are uniquely sensitive to mTORC1 regulation through an ill-defined mechanism. These mTORC1-sensitive transcripts include many ribosomal proteins and some general translation factors. La-

Related Protein 1 (LARP1) has been suggested as the missing link between TOP transcript translational regulation and mTORC1; LARP1 is an established mTORC1 phosphorylation target with multiple RNA binding domains. In this thesis, the direct binding of LARP1 to various TOP 5’UTR RNA sequences is shown to be higher affinity than binding to non-specific targets by incubating purified recombinant proteins with various radioactive RNA probes and visualizing complex formation through ElectroMobility Shift Assays (EMSAs). Using such an approach, we also define which domains within LARP1 contribute to TOP RNA binding, and assign a putative functional role to a non-TOP RNA binding domain. Based on homology alignments and structural predictions, we generated point mutations within the domain of interest and map residues relevant to maintaining TOP RNA binding activity. Several conserved features of TOP mRNAs necessary for LARP1 binding are also briefly examined.

All together, this thesis expands our understanding of the complex contribution of LARP1 as a regulator of TOP mRNA translation.

Résumé

“Caractérisation de l’intéraction entre LARP1 et les ARNm TOP”

La conservation de l’énergie cellulaire lors de conditions défavorables requiert la désactivation efficace de la production de protéines. Lorsque l’environnement ne permet pas le support des fonctions cellulaires de base, plusieurs cascades de signalisation intracellulaire réagissent, notamment mTORC1 (de l’anglais mammalian Target of

Rapamycin Complex 1), un régulateur important de la synthèse protéique. Bien que la majorité de la traduction d’ARNm soit arrêtée par mTORC1 dès la détection d’un manque d’oxygène ou de nutriments, un groupe d’ARNm comportant une séquence riche en pyrimidines ou TOP (de l’anglais Terminal OligoPyrimidine) dans leur région 5’ non-traduite ou 5’UTR (de l’anglais 5’ UnTranslated Region) sont particulièrement sensible à l’action de mTORC1, mais le mécanisme responsable de cet effet demeure mal compris. Ces transcrits mTORC1-sensibles en incluent de nombreux encodant des protéines ribosomal ainsi que des facteurs généraux d’initiation de la traduction. Il a été suggéré que LARP1 (de l’anglais La-

Related Protein 1) pourrait représenter le chaînon manquant entre la régulation traductionnelle des transcrits TOP et l’activité de mTORC1, LARP1 étant une cible bien établi de phosphorylation par mTORC1 qui possède plusieurs domaine d’interaction à l’ARN. Dans la présente thèse, il est démontré que LARP1 lie certaines séquences TOP avec une affinité supérieure aux séquences d’ARN non-spécifiques en incubant des proteins recombinantes purifiées avec différentes sondes d’ARN radioactives et en visualisant la formation d’un complexe par essai de décalage de mobilité électrophorétique (EMSA, de l’anglais electromobility shift assay). Grâce à cette approche, nous avons identifié les domaines de

LARP1 contribuant à l’interaction avec l’ARN TOP et nous assignons une fonction putative à un domaine liant l’ARN non-TOP. En nous basant sur l’alignement d’homologues de LARP1 et des prédictions sur sa structure, nous avons généré des mutations ponctuelles dans le domaine d’intérêt et cartographié les résidus maintenant la capacité de LARP1 à lier l’ARN

TOP. Un survol de plusieurs caractéristiques de LARP1 maintenues à travers les espèces nécessaires à cette interaction est aussi inclus. Prise dans son ensemble, cette thèse élargie notre compréhension de la complexité avec laquelle LARP1 contribue à la régulation de la traduction des ARNm TOP.

Acknowledgements Endless thanks to Dr. Edna Matta-Camacho, who was responsible for my training and guidance. Dr. Matta-Camacho made great contributions to the experimental design involved in this project and the execution of some experiments (ITC, NMR, crystallization screenings, certain computational analyses).

Thank you to Dr. Nahum Sonenberg for the opportunity.

Thank you to Nathaniel Robichaud for the translation of my abstract.

Thank you to Dr. Abba Malina and Dr. Edna Matta-Camacho for thesis corrections.

Thank you to Isabelle Harvey, Annamaria Kiss, Sandra Perreault, and Christine Sgherri for invaluable administrative assistance.

Thank you to all members of the Sonenberg lab but especially: Soroush Tahmasebi, Nathaniel Robichaud, Chadi Zakaria, Yuri Svitkin, Nadeem Siddiqui, Tommy Alain, and Bruno Fonseca.

Special thanks to Meena Vipparti.

Thank you to Défi Canderel and the Canadian Institute of Health Research (CIHR) for financial support.

Table of Contents

Abstract………………………………………………………………………………………………………………….…………………1

Resume……………………………………………………………………………………………………………………….…………….2

Acknowledgements……………………………………………………………………………………………………….………….4

Table of Contents……………………………………………………………………………………………………………….……..5

1. Introduction ...... 7

1.1 An overview of translation……………………………………………………………………………………….7

1.2 Characteristics of ribosomal protein transcripts……………………………………………………….8

1.3 mTORC1 and TOP mRNA translation ...... 10

1.4 The increasing interest in La-Related Protein 1 ...... ….11

1.5 The La-Related Family ...... 14

1.6 Functions for LARP1 in Model Organisms...... 16

1.7 LARP1 and TOP Transcripts ...... 19

1.8 LARP1 in Cancer………………………………………………………………………………………...... 23

1.9 Research Question, Hypothesis, Aims…………………………………………………………………….23

2. Results……………………………………………………………………………………………………………………..25

2.1 Recombinant LARP1……………………………………………………………………………………………….25

2.2 Direct binding of TOP 5’UTR by LARP1……………………………………………………………………29

2.3 LARP1 species and their RNA shift patterns…………………………………………………………….30

2.4 LARP1 TOP binding and sequence specificity………………………………………………………….33

2.5 TOP transcript binding by LARP1 domain fragments……………………………………………….37

2.6 The La domain of LARP1…………………………………………………………………………………………40

2.7 LARP1 C-terminal aromatic residues contribute to TOP binding by LARP1………………43

2.8 Truncation of a TOP sequence from the 3’ end abolishes binding by LARP1……………46

2.9 LARP1 transcript levels in human breast tumors…………………………………………………….48

3. Discussion ...... 51

4. Materials and Methods ...... 55

4.1 Cloning……………………………………………………………………………………………………………………55

4.2 Site-Directed Mutagenesis ...... 56

4.3 Protein Expression and Purification ...... 56

4.4 Electrophoretic Mobility Shift Assay ...... 57

4.5 Coomassie Protein Gels………………………………………………………………..………………………..57

4.6 Western Blot…………………………………………………………………………………..………………………57

4.7 RNA Sequences………………………………………………………………………………………………………58

4.8 Isothermal Titration Calorimetry…………………………………………………………………………….58

4.9 Nuclear Magnetic Resonance………………………………………………………………………………….58

4.10 Quantitative Real Time Polymerase Chain Reaction……………………………………………..59

5. References……………………………………………………………………………….………………………………….60

Introduction

1.1 An overview of translation

Translation concludes the conversion of genetic information into proteins capable of carrying out cellular functions. Each mRNA transcript generated from genomic DNA is soon modified and joined by various RNA-binding factors that work to ensure efficient translation into protein. Transcripts are typically modified at the 5’ end with a methyl 7’guanosine cap and at the 3’ end with the addition of a string of adenosine residues, referred to as the polyA tail. The 5’ cap forms the binding site for eukaryotic Initiation Factor 4E (eIF4E), and along with eIF4E, additional translation initiation factors eIF4A and eIF4G form the eIF4F complex, which acts to recruit the ribosomal pre-initiation complex to the 5’ end of the message.

Transcript circularization is facilitated by the scaffolding protein eIF4G, which also binds

Poly A Binding Protein (PABP), linking the 5’ cap to the 3’ tail [1]. The circularized transcript is bound by numerous ribosomes, each catalyzing peptide bond formation between amino acids to generate protein. In the event of conditions without sufficient nutrients and other essential factors, cell growth and proliferation can be efficiently reduced by targeting the protein translation machinery. Constraint of translation can occur via the modification of translation factors, or the activation of translation inhibitors, or by limiting the availability of translational components at their production. Lower cellular levels of ribosomal proteins and translation factors reduces overall protein translation, switching off an enormously energy-consuming process. It follows that dysregulated translation is a factor in enhanced proliferation, survival, and angiogenesis following tumorigenic mutation [2]. Impeding mRNA translation via the core machinery targets cancer cells with augmented growth and proliferation.

1.2 Characteristics of ribosomal protein transcripts

Control of protein production is exerted partially at the level of mRNA transcripts: translation may be activated or repressed and stability may be altered by trans-acting RNA- binding protein factors. The selection of a specific group of transcripts for regulation by a particular factor occurs via a “cis element”: a shared mRNA sequence. The 5’ untranslated regions (UTR) of ribosomal transcripts, as well as some translation factor and RNA binding protein transcripts, contain a terminal oligopyrimidine (TOP) motif, referring specifically to a stretch of 4-14 cytosine and uracil residues. The full consensus for transcripts belonging to the TOP category also includes an invariable 5’ C residue, followed by the TOP, and concluded by a GC-rich region of variable length [3]. These three features form the basis for recognition of these transcripts by TOP-sensitive RNA-binding regulatory factors, including Lupus autoantigen (La), zinc Finger Protein 9 (ZNF9), and T-cell intracellular antigen 1 (TIA-1) [4-

7]. TOP sequence-containing transcripts are present in all vertebrates and in Drosophila, though they are unreported in C. elegans and yeast [3]. In humans, TOP describes 79 ribosomal proteins, eight translation initiation and elongation factors, and a limited number of assorted RNA-binding proteins including PABP [8]. Most but not all TOP transcripts are united by this motif in synchronous transcript-level regulation, the rate of translation or stability of these transcripts might be modified in concert. 5’UTR sequence motifs, while necessary to define TOP transcripts, are not sufficient to classify them: only those transcripts that are exceptionally sensitive to regulation by mammalian Target of Rapamycin Complex

1 (mTORC1) in their translational regulation are considered “true” TOPs.

5’ 1C2CTTTCCGGTT3GCGGCGCCGCGCGG TG ‘3

Figure 1: Conserved features of a TOP motif. As demonstrated by the partial 5’ UTR of

RPS16: 1. 4-14 cytosine and uracil residues (red), their position relative to the transcription

and translation start sites varies. 2. Translation begins at a cytosine residue (bold red), a rare

feature present in only 19% of eukaryotic transcripts [9]. 3. GC-rich region (blue).The RPS16

TOP sequence is defined in Fonseca et al., 2015 [10]. eIF2S2 NM 003908 1 AGGCGCATTT CCAACGCTTG GAGGAGAGGG CGGGGTGTCG TTTCCTTTCG CTGATGCAAG 61 AGCCTAGTGC GGTGGTGGGA GAGGTATCGG CAGGGGCAGC GCTGCCGCCG GGGCCTGGGG 121 CTGACCCGTC TGACTTCCCG TCCGTGCCGA GCCCACTCGA GCCGCAGCCA TG RPS16 NM 001020

1 GAAAAGCGGC CAGGGTGGCC CCTAGCTTTC CTTTTCCGGT TGCGGCGCCG CGCGGTGAGG 61 TTGTCTAGTC CACGCTCGGA GCCATG RPL23A NM 000978 1 GGCCACGTGA GGAGGGTGGG CGGGGCGTTA AAGTTCATAT CCCAGTGTCC TTTGAATCGA 61 CTTCCTTTTT TCTTTTTTCC GGCGTTCAAG ATG

RPL32 NM 000994 1 AGGGGTTACG ACCCATCAGC CCTTGCGCGC CACCGTCCCT TCTCTCTTCC TCGGCGCTGC 61 CTACGGAGGT GGCAGCCATC TCCTTCTCGG CATCATG

Figure 2: The appearance of different TOP-containing 5’UTR varies due to flexibility in the

consensus elements. The amount of sequence between the cytosine cap site, occurring at the

9 beginning of the oligopyrimidine tract, and the translation start codon is highly variable with consequences for RNA secondary structure.

1.3 mTORC1 and TOP mRNA translation

In the early 1990’s studies by Jefferies et al. and Terada et al. linked TOP transcripts, then called the polypyrimidine tract family, to mammalian Target of Rapamycin (mTOR) [11, 12].

Following treatment with allosteric mTORC1 inhibitor rapamycin, the shift of TOP transcripts from monosomes to polysomes that typically follows serum stimulation was suppressed [11]. Though rapamycin causes an overall reduction in mRNA translation, TOP demonstrate the greatest sensitivity.

This result was expanded in genome-wide ribosome profiling studies by Thoreen et al. and

Hsieh et al. [13, 14] TOP transcripts and transcripts featuring similar motifs comprised a subset of transcripts demonstrating a significant reduction in translation efficiency upon treatment with mTORC1 active site inhibitor Torin1 [13]. Relating nutrient responsive growth and proliferation signalling by mTORC1 to the production of the protein translation machinery is a topic of great interest. Numerous mechanisms by which mTORC1 may regulate TOP mRNA have been proposed. The proposed models must allow regulation by mTORC1 activity, directly or indirectly, and interaction with TOP mRNA. As the knockdown of 4E-BP rendered TOP translation efficiency insensitive to Torin1, TOP mRNAs were initially thought to have a strong dependence on eIF4F complex formation [13]. The 5’ mRNA cap binding activity of eIF4E depends on the phosphorylation status of the 4E-BP inhibitors,

10 which are mTORC1 substrates. Unphosphorylated 4E-BPs bind eIF4E, preventing cap binding. Phosphorylation of the 4E-BPs by mTORC1 liberates eIF4E allowing translation initiation. However, this model is incomplete as overexpression of eIF4E cannot restore translation of TOP mRNA in the presence of mTORC1 inhibitors [15].

1.4 Increasing interest in La-Related Protein 1

Screening methods have recently been applied to identify proteins involved in mTORC1- dependent translational regulation [16, 17]. An RNA binding protein related to La protein and thought to be a target for mTORC1-dependent phosphorylation, La-Related Protein 1

(LARP1) would appear to be a promising hit [18, 19]. In one strategy, mass spectrometry was applied to the products of 7-methylguanosine cap pulldowns, changes in cap complex composition were measured following insulin stimulation or pharmacological inhibition of mTORC1 [17]. LARP1 was consistently found in the same fraction as PABP, both proteins could be collected from the cap predominantly during insulin stimulation. The activation of mTORC1 following treatment with insulin results in the phosphorylation of 4E-BP, uninhibited eIF4E is the basis of the eIF4F translation complex that indirectly links PABP to the cap. LARP1 was shown to associate with the translation machinery in an mTORC1- dependent manner. A concurrent study used mass spectrometry to identify proteins recovered from Regulatory Associated Protein of mTOR (RAPTOR) pulldowns [16]. RAPTOR is the subunit of mTORC1 that interacts with substrates, which are then phosphorylated by

TOR kinase. mTOR complexes 1 and 2 share components such as mammalian lethal with

SEC18 protein 8 (mLST8) and Disheveled, Egl-10, Pleckstrin (DEP)-domain-containing mTOR interacting protein (DEPTOR). RAPTOR and Proline-Rich Akt Substrate 40kDa

(PRAS40) are unique to mTORC1. Rapamycin Insensitive Companion of TOR (RICTOR), mammalian Stress-activated protein kinase Interacting protein 1 (mSIN1) and proline-rich protein 5/Protein Observed with RICTOR (PRR5/PROTOR) are only found in Complex 2.

Probing RAPTOR specifically eliminates the contributions of proteins phosphorylated indirectly following mTORC1 activation and proteins that interact with mTORC2. LARP1 was identified as a RAPTOR binding partner and therefore likely direct target of mTORC1, at some portion of its numerous putative phosphorylation sites (Figure 3). Demonstration of mTORC1-dependent activity by LARP1 in each of these screens was quickly linked to an earlier study of the protein that demonstrated its ability to bind TOP mRNA [20].

Figure 3: Translational regulation of TOP transcripts is impacted by mTORC1 in a yet-to-be- elucidated mechanism. Modified from Fonseca et al., 2015 [10]. Nutrient and growth factor stimulation precedes the activation of numerous kinases, LARP1 is a direct target of mTORC1.

1.5 The La-Related Family

La proteins are present in all eukaryotes and act as RNA chaperones in various contexts.

Human La (also known as LARP3) is the best-characterized member of the La-Related family.

LARP3 is an essential nuclear phosphoprotein implicated as an autoantigen in Systemic

Lupus Erythematosus, Neonatal Lupus, and Sjorgen’s Syndrome [21]. In its primary context,

LARP3 binds 3’ termini and primarily prevents the exonucleolytic degradation of RNA polymerase III transcripts [22, 23]. In these transcripts, which include precursor-tRNAs, transcription termination is signalled by a stretch of thymidine residues resulting in a polyuridine-containing mRNA (polyU mRNA). The affinity of LARP3 for polyU transcripts extends to some Pol II intermediates, such as precursor U1 snRNA in humans, which contain

UU 3’OH following post-transcriptional modification [24, 25]. However, the binding of

LARP3 is not limited to transcripts containing UUU 3’OH and this RNA-binding protein has been linked to numerous atypical translation initiation contexts, as in IRES-dependent viral translation and TOP translation [4, 26]. Roles exist for LARP3 in RNA metabolism beyond the simple protection of transcripts. In mRNA translation, the effect of LARP3 may be positive or negative and may depend on the transcript, the context, and LARP3 phosphorylation status and localization [4, 27, 28].

As determined by composition and the presence of a La Motif (LAM), LARP3 is one of seven

La-Related Proteins in humans [21]. In each LARP the LAM is off-set toward the protein N- terminus and followed by 1-2 RNA Recognition Motifs (RRM) (Figure 4). The LAM adopts a winged helix fold observed in other RNA-binding proteins and typical of DNA-binding transcription factors [29]. In LARP3, the LAM and downstream RRM1 collaborate toward the

14 binding of UUU 3’OH. This interaction, as described by a crystal structure, does not involve the expected interaction surfaces in each domain [30]. In this model highly conserved residues not shown to interact with UUU 3’OH are available for RNA binding in alternative contexts: the configuration of each motif is flexible such that its contribution may be directed by the target mRNA bridging isolated segments of protein sequence. Both LAM and RRM1 are essential for RNA binding by LARP3 [31].

La-related protein C-termini increase in size from yeast to higher organisms and allow divergent functions among human LARPs [32]. The seven LARPs are distributed in five families (LARP1/2-, LARP3-, LARP4-, LARP5/6-, or LARP7-like) that differ in the position and composition of their RRM, and their C-termini [21]. The RRM of LARP1 proteins, referred to as RRM-L5, are atypical and lack essential modules of the LARP3 RRM despite conservation of LAM residues used by LARP3 for UUU 3’OH binding [32]. LARP1/2 proteins are also defined by a C-terminal “DM15” domain, composed of 3 iterations of the same sequence [32]. Genome wide, the DM15 motif is only found in LARP1/2 [32]. Although LARPs may demonstrate a high degree of homology in their LAM, C-terminal determinants contribute to target selection. LARP7 is highly similar to LARP3 and similarly binds UUU

3’OH but effectively discriminates against pre-tRNAs in S. pombe pre-tRNA-mediated suppression assays that disregard subcellular localization [33]. Eliminating the C-terminus of LARP7 allowed it to behave as LARP3 does in this assay.

Figure 4: The La-Related Protein Family is comprised of 7 proteins. A DM15 motif and RRM-

L5 defines LARP1/1b.

1.6 Functions for LARP1 in model organisms

The first LARP1 functional studies involve the Drosophila protein, dLarp [34-36]. LARP1- type proteins, featuring a dual RNA binding activity at La motif and C-terminal DM15 motif, are evolutionarily conserved and a suggested function in development ascribed to dLarp was similarly suggested by transcript expression patterns in mice [34]. Further studies in

Drosophila established a requirement for dLarp in male meiosis and syncytial embryo development. PABP and dLarp interacted to form physical complexes in Drosophila and their

16 genetic ablation produced similar meiotic-type phenotypes, these phenotypes were enhanced by simultaneous ablation [35].

A function for the DM15 domain was first shown in C.elegans [37] (Figure 5). There are two

Larp proteins in C.elegans, one of which is homologous to human LARP1. In this model organism full length Larp1 bound polyU and polyG RNA though neither polyA nor polyC. The in vitro binding of polyU/polyG was replicated by the both the LAM/RRM-containing N- terminus of the protein and the DM15-containing C-terminus, though neither as strongly as the full length protein. A combinatorial mechanism resembling the action LAM and RRM in

LARP3 was then suggested. The physiological function of LARP1 in C.elegans again impacted the germline, as it was determined to be essential for proper oocyte development. The defects observed in Larp1 deletion mutants resembled the effects of phenotypes occurring from overactive Ras-MAPK signalling in C.elegans. It followed that Larp1 was discovered to attenuate Ras-MAPK signalling in C.elegans, through the reduction of transcripts encoding

Ras signalling pathway components. These Ras signalling transcripts were thought to be

LARP1 targets, degraded following accumulation of the protein in cytoplasmic Processing

(P) bodies in the germline.

Figure 5. Full length Drosophila LARP1 and DM15-containing fragment (C) bind polyU and polyG RNA while LAM-containing fragment (N) fails to capture polyU. From Nykamp, et al.,

2008 [37]

Indication that LARP1 might be involved in translational control appeared in a 2010 study performed in HeLa cells [38]. As in Drosophila, LARP1 was found in complex with PABP.

LARP1 was also isolated in 7-methyl GTP pulldowns, via translation complexes of eIF4E,

RNA, and PABP. An increase in subpolysomal components in LARP1-depleted HeLa cells seemed to link LARP1 to translation initiation. Within the same study LARP1 acquired additional functions in cell division, apoptosis, and cell migration. LARP1 was found to be associated with cytoskeletal proteins and defects observed following LARP1 knockdown all impacted cytoskeletal processes. These observations of cells lacking LARP1 do little to explain the role of LARP1 via RNA-binding. No cohesive, uniting theory for LARP1 targeting and physiological role justifying strong species conservation had been proposed.

1.7 LARP1 and TOP Transcripts

Requirements for the RNA targets of human LARP1 were first explored in a previously mentioned 2012 study by Aoki et al [20]. In pulldown experiments using Flag-conjugated in vitro transcribed RNA probes, polyA probes collected significant amounts of LARP1 protein from cell lysates, as identified by mass spectrometry. The addition of a 5’ methyl guanosine cap to the probe did little to improve or reduce polyA binding, though addition of excess 3’ sequence beyond the polyA abolished binding. PolyU/C/G did not bind LARP1, and binding to polyA was abolished with the addition of U, C, or G in the 3’ position of the probe (Figure

6). A length requirement was also established for polyA binding as a dramatic reduction in

LARP1 binding was observed as the polyA probe shrunk from 60 to 9 residues, with very little observable binding below 9 residues. The transcripts bound to endogenous LARP1 were assessed by immunoprecipitation in HEK293 cells followed by qRT-PCR of “numerous transcripts commonly used for reference” in an experiment that first defined TOP mRNAs as targets of LARP1. LARP1 was observed to prefer the mature TOP transcripts while excluding the unspliced transcripts. TOP transcript abundance was found to follow LARP1 abundance, in that a siRNA-mediated depletion of LARP1 protein resulted in lower levels of TOP transcripts with little effect on select non-TOP mRNAs. Northern blot analysis of TOP mRNAs destabilized by LARP1-depletion revealed no deadenylation suggesting a binding and stabilization mechanism separate from the polyA tail. This was the first indication of dual

RNA binding modes for LARP1.

Figure 6. PolyA RNA precipitates LARP1 from cell lysates and PolyU/G/C do not. PolyA probes of increasing length were more successful at LARP1 collection. Substituting a 3’ adenine residue reduces LARP1 binding. From Aoki et al., 2013 [20]

The connection between LARP1 and TOP was quickly noted by the Roux group following their screen for proteins associated with the mRNA cap complex in an mTORC1-dependent manner [17]. Similar cap binding responsiveness was observed for LARP1 and PABP: both proteins associated with the cap complex components during mTORC1 activation, which phosphorylates 4E-BPs allowing eIF4E to associate with the rest of eIF4F complex. However, the activation status of mTORC1 did not affect the interaction between LARP1 and PABP.

PABP was expected to bring LARP1 to the cap and to polysomes, as LARP1 does not bind to other components of the translation apparatus independently. LARP1 was found associated with polysomal subunits as well as with intact polysomes associated with active translation.

The inhibition of mTORC1 was shown to stimulate the re-distribution of LARP1 from polysomal to monosomal fractions. The direct interaction between mTORC1 subunit

RAPTOR and LARP1 was demonstrated, implicating LARP1 as a potential substrate for phosphorylation-mediated regulation by mTORC1. A limited reduction in total protein synthesis was observed upon LARP1 depletion by shRNA with an accumulation of cells in

G0/G1 and decreased proliferation. Considering TOP mRNAs specifically, polysome preparations revealed decreased loading in LARP1-depleted cells and long term knockdown reduced the abundance of TOP-encoded proteins. Thus, in this study it was concluded that

LARP1 positively regulates the translation of TOP mRNAs.

A 2015 study by Fonseca et al. concluded that LARP1 represses the translation of TOP transcripts but also contained a brief investigation of LARP1 as a factor regulating TOP mRNA stability [16]. Following the initial post screening identification of LARP1 as a

RAPTOR binding partner, the relevant domains in each protein were investigated. LARP1 binding activity was mapped to the C-terminal WD40 repeats, with contributions from the

RAPTOR Conserved N-terminal Domains (RNC). RAPTOR substrates share a TOS motif of five amino acids, deletion of a similar sequence in the C-terminus of LARP1 impaired RAPTOR binding. The LARP1 putative TOS motive, residues 889-893, did not function as the equivalent sequences in the 4E-BPs and S6K do as deletion of the initial phenylalanine did not completely eliminate RAPTOR binding. Whether or not the TOS tested is the key to

LARP1-RAPTOR interaction, the C-terminus of LARP1 was plainly implicated. LARP1-PABP interaction was declared RNA-independent and mapped to the N-terminus. Additionally,

LARP1-PABP binding was not impacted by Torin1 treatment, while even acute treatments abolished the interaction between LARP1 and RAPTOR or mTOR. The presence of RNA, or omission of RNAse A, also decreased LARP1-RAPTOR binding perhaps indicating shared

21 interaction surfaces. LARP1 knockdown shifted TOP transcripts into heavier polysomal fractions while steady state transcript levels were reduced. LARP1 was proposed to repress

TOP translation and to stabilize these transcripts. The ability of Torin1 to reduce TOP translation was impaired without LARP1, suggesting LARP1 is part of in the mTORC1-based regulation of these transcripts. LARP1 overexpression produced the expected results, less

TOP in heavy polysomes and increased stability.

Figure 7. Full length recombinant LARP1 binds directly to TOP 5’ UTR. The TOP sequence and 5’ invariable C residue of the TOP impact binding by LARP1. Fonseca et al., 2015 [16]

1.8 LARP1 in Cancer Translation, particularly of TOP transcripts, can be an integral point of dysregulation preceding tumor development [2]. LARP1 functions downstream of mTOR, one of the most prevalent dysregulated pathways in a variety of tumor types [39]. Whether it is a gas pedal or a brake LARP1 is applying, it appears to be helping to dictate cellular levels of proteins essential for cell growth and proliferation. It would follow that the clues to LARP1’s true status as a repressor or activator and the contribution of its stabilization activity might be revealed by analyzing LARP1 levels in cancer cells. Preliminary studies have begun concerning several different cancers. According to Oncomine, most epithelial malignancies have higher levels of LARP1 than adjacent tissues [40]. LARP1 protein levels were studied in ovarian cancer revealing increases that coincided with cancer progression [41]. In hepatocellular carcinoma, high levels of LARP1 protein were correlated with 35% increase in death after five years [42]. Expanded studies in human cancer cell lines seemed to link

LARP1 to migration, epithelial-to-mesenchymal transition, and invasion, with distinct phenotypes in malignant and non-malignant cells [40, 41].

Question: How do the various domains of LARP1 contribute to its function?

Hypothesis: An RNA-binding domain within LARP1 binds TOP mRNA, facilitating a role for

LARP1 in cancer progression

Aims:

- Calculate quantitative binding between full length LARP1 and TOP mRNA 5’UTR

- Determine which LARP1 RNA-binding domain (LA-HTH or DM15) binds TOP mRNA

- Identify residues within this domain that impact TOP mRNA binding

- Suggest a putative role for the non-TOP RNA binding domain within LARP1

- Investigate features of TOP 5’UTR sequences important for LARP1 binding

- Contribute to early suggestions of a role for LARP1

Results

2.1 Recombinant LARP1

Rationale: Producing LARP1 protein in bacteria enables the generation of large quantities of pure protein for biochemical assays.

LARP1 occurs as two isoforms, isoform1 with 1096 amino acids and isoform 2 with 1019 amino acids [38]. As indicated in secondary structure predictions by “PredictProtein”

(https://www.predictprotein.org/), a program which uses a variety of databases and methods to estimate regions of structure and solvent accessibility based on amino acid composition, LARP1 structure is limited to two small domains, one central region ascribed to the LAM RRM-L5, and the C-terminal DM15 domain (Figure 8b). These two structured regions, LAM RRM-L5 and DM15, are well-conserved among different species as demonstrated by sequence alignments produced using Geneious software (Figure 8c). Based on these secondary structure predictions and sequence alignments, full length LARP1 and small fragments encompassing the entire sequence of the protein were cloned into pET28a-

5xHisSUMO for bacterial expression and were purified by His-Tag affinity. In a preliminary construct design, the amino acid sequence of LARP1 was divided according to homology- based domain predictions (Figure 8a). Initially a eIF4G-like domain (59-192) was mapped in addition to a LAM, adjacent RRM (300-450), and DM15 (799-959) domains but it was later excluded as it is not widely agreed upon. A second preliminary fragmentation strategy divided the protein roughly for a total of 7 constructs. High expression levels were observed for 3/7 constructs (Figure 8d), two constructs representing the C-terminal DM15 (799-959,

750-1019) and full length LARP1. The boundaries of constructs representing the LAM RRM-

L5 were later varied such that a more stably expressing construct was also obtained for this region (321-441). The expression and purification of full length SUMO-LARP1 was optimized to produce high yields of pure protein without contamination with RNA from bacteria

(Figure 9).

LARP1

- + - + - + - + - + - + - + IPTG Induction LARP1 + - +

*Expression Figure 8: LARP1 Domain Structure, Homology, and Fragment Expression. (a) LARP1 is large mostly unstructured protein, the presence of structural elements and homologous sequence indicates two domains: the LAM/RRM-L5 and the DM15. (b) PredictProtein sequence-based

26 analysis indicates likely alpha helices and beta sheets (top), corresponding to regions of solvent inaccessibility (bottom), which again indicate two domains. (c) A sequence alignment among different species containing LARP1 proteins. LARP1 was fragmented according to two different strategies: first considering domains and predicted structure and second dividing the protein roughly into quarters. (d) Coomassie staining of the total bacterial protein production for each different fragment, uninduced (left) and induced

(right). Only constructs corresponding to the full length protein (1-1019) or C-terminal fragments (750-1019, 799-959) showed high yield expression. The LAM/RRM-L5 construct was later redesigned to produce a stably expressing fragment.

Figure 9: Expression and Purification of Full Length SUMO-LARP1. LARP1 expression was tested with 0.5 mM IPTG for 4 hours at 37°C or overnight at 18°C (a). Expression was also tested overnight at 18°C with 1 mM (b), 0.1 mM (b), 0.01 mM (c), or 0.001 mM IPTG (c). IPTG at 0.5-1 mM for 4 hours at 37°C or overnight at 18°C was used for future protein production.

LARP1 was purified using a standard His-Tag purification protocol and stepwise elution with

500 mM imidazole (d). The purification of full length LARP1 was modified to include the use of an imidazole gradient from 100-500 mM imidazole for elution, increases in pure eluted protein and reductions in contaminants were modest at best (e). The 14kDa SUMO tag was stably and completely cleaved from full length LARP1 using Ubiquitin-Like Protease 1

(ULP1), which was then removed from the protein preparation via its own 6 histidine tag (f).

LARP1 produces two peaks by size exclusion chromatography when prepared in 0.5 mM

TCEP, shown here as highly concentrated sample prepared for crystallization screens (g).

Peak 1 eluted at 46.8 minutes, corresponding roughly to a complex or aggregate >130kDa, and Peak 2 eluted at 58.3 minutes, indicating monomeric SUMO-LARP1 (h). Contaminants or degradation products were significantly reduced and eluted individually at multiple time points corresponding to smaller size proteins.

2.2 Direct binding of TOP 5’UTR by LARP1

Rationale: Analysing the proportion of TOP mRNA bound over a range of concentrations of

LARP1 protein indicates the strength of this interaction quantitatively.

An earlier study used Electrophoretic Mobility Shift Assay (EMSA) to demonstrate qualitatively the binding of recombinant LARP1 to the 5’ UTR of Ribosomal Protein L32

(RPL32), a known translationally-regulated TOP sequence [16, 43]. EMSA, in which the

“shift” of a radioactively labelled nucleic acid probe indicates protein binding, provides a simple and direct method of assessing protein-nucleotide binding in vitro. A range of LARP1 protein concentrations, from 10 nM to 20 µM, was used to assess the affinity of LARP1

29 binding to the 5’ UTR of RPL32. The density of signal for bound and unbound RNA was calculated and compared at each protein concentration. These measurements were used to calculate a constant of dissociation (Kd) of 110 nM for this interaction (Figure 10).

10nM 20µM

Binding Curve: LARP1 (FL)- RPL32 5’UTR

Figure 10: LARP1 binds the 5’ UTR of RPL32 with micromolar affinity. (a) Full length SUMO- tagged LARP1 was combined with the 5’UTR of RPL32 with increasing concentrations of protein from 10 nM to 20 µM. (b) The ratio of bound to unbound RNA was compared in each fraction toward the calculation of binding affinity. Full length LARP1 bound to RPL32 5’ UTR with an affinity of 110 nM, as calculated using ImageJ and Graphpad Prism software.

2.3 LARP1 species and their RNA shift patterns

Rationale: Polymerization by LARP1 may affect mRNA binding, shift patterns demonstrated by this protein in EMSA may indicate the species present.

A complication in the study of RNA binding arose with the detection of different homomeric complexes of LARP1. Purification of full length LARP1 consistently produced two peaks during size exclusion chromatography. The first peak to elute (Peak 1), representing the component of greatest size, corresponded to a molecular weight not consistent with the size of LARP1 or a cleanly isolated dimer, based on the elution of protein standards under the same conditions (Ferritin 400kDa and Aldolase 150kDa). This peak was in very high abundance and there were no contaminants observed at this mass. A second peak (Peak 2) appeared around the expected size for LARP1 and was thought to represent a monomer. The two peaks were dynamic and proportions changed to favor the putative monomer upon addition of salt or a reducing agent, though it was not possible to obtain LARP1 as only one species. Interestingly, different species of LARP1 (monomer, multimer) demonstrate different binding patterns when combined with radioactive RNA probes. The proposed monomer appears as a thin band at the top of the gel (as seen in Fonseca, et al., Figure 7, [7]), while the multimer species descends into the gel (Figure 10). Using protein preparations that have not been purified by size exclusion chromatography, both shifts can be observed. The physiological relevance of each species is not currently known, however, it has been shown that LARP1 contains a C-terminal dimerization domain and occurs naturally as a dimer [33].

In agreement with this prediction, the C-terminal fragment construct also demonstrates dual shifts, and sometimes produces a thin band at the top of the gel (Figure 11). As shown in

Lahr, et al., abolishing the dimerization domain in a C-terminal LARP1 construct, causes a change in the shift from a thick band within the gel to a thin band at the top of the gel [44].

0.3µM 20µM

10nM 20µM

Figure 11: Different species of LARP1 demonstrate different RNA shift patterns. LARP1 was consistently shown to form two peaks by size exclusion chromatography, likely to indicate the formation of a multimeric species in addition to the monomeric protein. Multimeric and monomeric LARP1 produces different shift patterns by EMSA. Counterintuitively, LARP1 monomers appear at the top of the gel near the wells while multimers descend into the gel and produce a thicker band. (a) Published work by Fonseca et al. used commercially

32 prepared protein purchased from Abnova (“50ng”), LARP1 shifts appear at the top of the gel

[7]. (b) Monomeric protein purified from bacteria also shifts to the top of the gel. (d)

Multimeric protein prepared in lab produces shifts within the gel (0.3 µM- 20 µM). (c)

Unpublished work also by Fonseca et al. used bacterial protein not purified by size exclusion.

The mixed protein appears to be predominantly in the monomeric form but also features a faint band corresponding to the multimer circled (protein 2 µM). Note that (a), (c), and (d) were produced using large 30cm gels while (b) is a 10cm mini gel, therefore distances in (c) and (d) can be compared but (a) and (b) cannot.

2.4 LARP1 TOP binding and sequence specificity

Rationale: It is insufficient to demonstrate binding to a single TOP 5’UTR, LARP1 may bind all TOP-containing transcripts or a subset. LARP1 may bind mRNA indiscriminately, non- specific interactions with mRNA homomers must be ruled out.

Having demonstrated that full length monomeric LARP1 binds to a known TOP sequence, we sought to refine the specificity of this interaction. In our hands a selection of ribosomal proteins with 5’UTR of different lengths were shown to bind directly to full length recombinant LARP1 (Figure 12, Figure 13). Protein concentrations sufficient to show binding to numerous TOP mRNA by either LARP1 species did not bind probes composed of stretches of individual nucleotides. PolyA/C/U/G substrates were radioactively labelled at the 5’ end using 32P-γATP and PNK and then combined with LARP1 up to 20uM (Figure 14).

Negligible binding was observed in every case. LARP1 has a higher affinity for TOP sequences than for ribonucleotide homopolymers under these conditions. However, a disparity in

33 length between the TOP sequences (27-53nt shown) and the polyA/C/U/G substrates (10nt) should be noted. Ribonucleotide polymers of this length were sufficient to bind LARP3

(polyU) and LARP4 (polyA) [31, 45], though the binding domain in LARP1 and importance of sequence length were later questioned. A lack of binding by full length LARP1 to 10nt polyA is in contrast to results presented by Aoki, et al. [20] using FLAG-conjugated RNA pulldowns in HEK293 cells.

Figure 12: Interaction is observed between full length LARP1 and a variety of TOP probes.

The 5’UTR of 4 different TOP transcripts were radioactively labelled and combined with full length LARP1. The 5’UTR of RPL27, RPL29, RPS16, and RPL32 are 27, 31, 53, and 42 nucleotides respectively [10].

Figure 13: LARP1 binds different TOP 5’ untranslated regions. An increasing concentration of full length SUMO-LARP1 was combined with TOP mRNA 5’ UTRs representing different ribosomal proteins. Binding affinity of different TOP 5’ UTR by LARP1 is thought to be highly variable. An unknown secondary feature of the UTRs, such as threshold length or a precise consensus sequence, may dictate LARP1 binding affinity.

Figure 14: LARP1 does not bind control oligonucleotides at TOP affinity. No binding was observed for LARP1 and a string of 10 adenine (a), uridine (b), guanosine (c), or cytosine (d) residues over a standard range of LARP1 protein concentrations that demonstrate strong binding to TOP 5’ UTRs.

2.5 TOP transcript binding by LARP1 domain fragments

Rationale: TOP mRNA binding by individual domains of LARP1 must be compared to elucidate which domain is primarily responsible for this function.

LARP1 is composed of the LA motif and an adjacent helix-turn-helix (LA-HTH), RRM-L5 toward the centre of the protein, and 3 DM15 repeats at the protein C-terminus. In this study the LA-HTH was expressed without the downstream RRM-L5, which occurs as a short segment within a poorly structured region. Additionally, LARP1 possesses a second RNA- binding domain, such activity has been documented for the DM15 repeats of C. elegans

LARP1. The LA-HTH and DM15 regions of LARP1 were expressed and purified as individual recombinant proteins and tested for TOP mRNA binding activity.

Binding of the LA-HTH segment or the DM15 to the 5’UTR of RPL32 was assessed and compared by EMSA (Figure 15). Shifts bands indicating RNA-protein interactions were observed for only fragments containing the DM15 repeats. Shifts were initially observed for a construct consisting of residues 799-959. However, the binding affinity of this construct was weak and the shift was difficult to interpret. The DM15-containing construct was extended toward the N- and C-termini to include residues 750-1019 and increases in shift clarity and binding affinity were observed. The extension of the DM15 construct seems to have included elements necessary for dimer formation as the modified recombinant fragment appears as 2 peaks by size exclusion chromatography (data not shown). Indeed, a

37 recent study established dimer formation and mutated the extreme C-terminus to abolish dimerization [44].

Figure 15: LARP1 C-terminal DM15 domain binds TOP mRNA. The complete, radioactively labelled 5’ UTR of RPL32 was combined with purified protein fragments representing different domains of LARP1. Fragments were prepared representing the LA-HTH and DM15 domains (a), referred to as “LA-HTH” and “DM15” in (b). A second DM15 construct containing additional amino acids on the N-terminal and C-terminal sides of the domain, referred to as “DM15-Extend.”, was also compared. Shift activity was observed for full length

LARP1, the DM15, and the DM15 constructs. It was therefore concluded that the DM15 domain binds TOP mRNA 5’UTR. No shift was observed for the LA-HTH. A higher degree of binding was observed for the DM15-Extend. construct, implying that adjacent sequences contribute to binding in some way.

2.6 The La Domain of LARP1

Rationale: LARP1 has also been shown to bind polyA RNA, this function may be attributed to an RNA binding domain of LARP1 not shown to bind TOP 5’UTR.

The LA-HTH fragment did not exhibit binding to TOP-containing sequences. In pursuit of an alternate role for this domain, it was recognized that the interaction of LARP1 and polyA has been previously described [20], though binding to full length LARP1 was not observed over the range of concentrations tested by EMSA (Figure 14). Cold polyA was used as a competitor to assess whether or not polyA and TOP mRNA bind at a shared site. Addition of cold polyA at a 5:1 ratio to LARP1 and labelled TOP 5’UTR did not drastically affect binding, indicating that these mRNA sequences do not bind to the same region of LARP1 (Figure 16).

Figure 16: Binding of additional targets by LARP1 does not interfere with TOP binding. Full length SUMO-LARP1 was combined with the radioactively labelled 5’ UTR of RPL32 with and without cold polyA RNA at a ratio of 1:5. Little change in the observed shifts occurs in the presence of polyA for both Peak 1/multimeric (a) and Peak 2/monomeric (b) protein.

Preliminary, qualitative interaction between LA-HTH and a 10 nucleotide stretch of adenine residues was demonstrated by isothermal titration calorimetry (ITC) and corroborated by nuclear magnetic resonance (NMR) of 15N-labelled protein (Figure 16). These techniques are complementary and allow affinity measurements over many orders of magnitude. ITC best demonstrates higher affinity interactions, allowing the decomposition of Gibbs free energy into enthalpic and entropic contributions. NMR is suitable for lower affinity interactions.

Unfortunately, due to an error in protein or RNA quantification that came to light at a late date, the LA-HTH-polyA Kd value associated with this data is inaccurate and therefore not provided.

Figure 17: Indications of an interaction between LA-HTH and polyA RNA. Thermal (left) and conformational (right) changes are observed when the La motif of LARP1 is combined with

PolyA mRNA. The thermogram of the buffer-polyA control was subtracted from the protein- polyA experiment. The HSQC spectrum of the 15N-LA-HTH protein alone (blue) was collected at 298K in a Bruker 600MHz, then RNA oligo (polyA 10-mer) was added at a ratio 1:1 to the protein and the spectrum collected again (green). The image shows a merge of the two spectra where chemical shift perturbations are observed upon addition of unlabeled RNA oligo (polyA-10mer) indicative of interaction. It was called to our attention that the addition of polyA to 15N-LA-HTH protein at the same pH caused many chemical shifts in the HSQC, however this effect was specific to the polyA oligo as the HSQC of 15N-LA-HTH was re-recorded at different pH with no major chemical shifts observed. We speculate that

42 since the LA-HTH domain is small, the conformational changes upon polyA binding could affect to some extent the whole domain. Binding to the LA-HTH fragment but not full length

LARP1 (Figure 14) might be explained by changes in conformation affecting the entire protein due to regulation by phosphorylation or other factors. These experiments were performed with Dr. Edna Matta-Camacho.

2.7 C-Terminal aromatic residues contribute to TOP binding by LARP1

Rationale: Computational modelling may reveal residues involved in TOP mRNA binding that may then be tested via site-directed mutagenesis of existing constructs.

Having demonstrated the DM15 domain of LARP1 is sufficient to bind TOP sequences in vitro, we aimed to further define which amino acids are important for this interaction.

Computational modelling of the DM15 domain of LARP1 by iTASSER (http://zhanglab. ccmb.med.umich.edu/I-TASSER/) and RaptoX (http://raptorx.uchicago.edu/) produced a 3- dimensional model revealing the most likely configuration of the 3 structural repeats of the

DM15 motif. The domain features a negative surface and a positive surface (red and blue, respectively), indicating where binding of molecules with negatively charged phosphate backbones would be expected. While the predicted positions of the two C-terminal DM15 repeats were toward the protein interior, the N-terminal most repeat was predicted in a surface-exposed position and is therefore a candidate for RNA interaction. In one known protein-RNA binding strategy, aromatic amino acids intercalate with nucleic acid residues

[46]. Two aromatic surface residues that demonstrate high species conservation, positioned within the repeat of interest, were mutated to alanine to assess their role in the interaction

43 between LARP1 and TOP transcripts. Conversion of tyrosine 816 to alanine slightly and non- significantly reduced LARP1-TOP interactions. However, conversion of phenylalanine 841 to alanine reduced binding drastically, identifying this residue as a component of the interaction between LARP1 and TOP (Figure 18).

WT Y816A F841A

Figure 18: LARP1 C-terminal aromatic residues are involved in TOP mRNA binding. (a) iTASSER and RaptoX modelling of residues 799-959. (b) Alignments of the DM15 domain across species and (c) of the 3 DM15 repeats of human LARP1

(http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) (d) Binding of RPL32 5’ UTR by the DM15 domain of LARP1 is compared in wild type and altered constructs is compared. (a)

Alanine replaces tyrosine (816) or phenylalanine (841) at key aromatic residues. Binding is greatly reduced when phenylalanine 841 is substituted.

2.8 Truncation of a TOP sequence from the 3’ end abolishes binding by LARP1

Rationale: Altering the sequence of a TOP 5’ UTR and re-performing assays may identify features of the RNA sequence important for this interaction.

Each sequence described as TOP contains three features: an invariable 5’ C residue, the 4-14 residue pyrimidine tract, and a downstream GC-rich region. The assemblage of these

45 features in different transcripts is highly variable, as are 5’UTR length and predicted secondary structure; we aimed to investigate which of these features impact binding by

LARP1. The 5’ UTR of RPL32, used for previous experiments, was truncated from the 3’ end to 30 residues, this truncation eliminated binding to LARP1. It follows that a further 3’ truncation, to 10 residues representing only the TOP sequence, abolished binding completely (Figure 19). This result may be explained by a direct interaction with the 3’ end of TOP mRNA 5’UTR by LARP1, or the requirement for this region in the formation of secondary structures appropriate for LARP1 binding. TOP mRNA less than 30nt are shown to bind LARP1 in Figure 13.

Figure 19: Truncation of TOP sequences from the 3’ end abolishes binding by LARP1. (a) 3’

Truncations of the 5’UTR of RPL32. (b) Full length SUMO-LARP1 was combined with the 5’

UTR of RPL32 at 3 lengths. Removing the most 3’ sections of 5’UTR abolishes binding. The

10nt length represents the TOP sequence alone, which does not bind LARP1. (c) Structure

47 predictions for the 53nt and 30nt demonstrate the differences in the most likely configurations for each molecule (colour scale represents 0-1 likelihood of match, red is 1, via http://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1.html).

2.9 LARP1 transcript levels in human breast tumors

Rationale: Comparing levels of LARP1 transcript expression in various breast cancer may provide clues to the role of this protein in cancer.

As a TOP mRNA regulator downstream of mTORC1, LARP1 functions upstream of protein translation machinery crucial to tumor progression. A role is suggested for LARP1 itself in cancer progression. Previous studies have resulted in conflicting conclusions as to the function of LARP1. LARP1 has been declared both an activator and a repressor of TOP mRNA translation [16, 17]. It is an important classification that would help to define LARP1 as a putative oncogene or tumor suppressor. Early studies in a limited number of specific human cancers (liver, cervical) [40, 42] have shown higher levels of LARP1 protein in cancerous tissues and have therefore appeared to agree with the theory of LARP1 as a translational activator, functioning to increase ribosomal protein levels and subsequently total translation. As for the effect of LARP1 on transcript stability, an increase in LARP1 has only been shown to decrease transcript levels [16]. LARP1 is phosphorylated by mTORC1 , thus far inhibition of phosphorylation by mTORC1 has been shown to increase LARP1’s association with RNA [16]. Overall, LARP1 in tumors merits further study.

During this study an opportunity to investigate LARP1 levels in human breast tumors arose.

No studies on LARP1 in breast cancer currently exist. RNA extracted from 36 human breast tumors at the Vall d’Hebron University Hospital in Barcelona, Spain was generously provided. Transcript levels of LARP1 itself was investigated in these samples by qRT-PCR.

Cycle threshold values were normalized to GAPDH and normal breast tissue and fold changes were calculated using the Livak method [47]. In this experiment 34 tumors had lower levels of LARP1 mRNA than the normal breast tissue. Many of the fold changes were modest (2 or below), though 7 tumors show fold decreases of 3 or greater. A finding of lower than normal levels of LARP1 transcript in cancerous tissue disagrees with some current findings [48], though it would support a proposed model in which LARP1 suppresses translation [16]. In this scenario reducing LARP1 enhances translation. The breast tumor samples were later reunited with data collected by the pathologist concerning the histological grade, type of tumor, and whether or not metastasis had occurred (Figure 20). However, the patient data set was incomplete at the time of writing and some conclusions are not yet considered. In this preliminary experiment, a link between metastasis and LARP1 appears unlikely.

49 a.

LARP1 Breast Tumor Expression 1

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

-1 CT))

-2

ΔΔ

( 2 -3

-4

-5

FoldChange (Log -6

-7

-8 Tumor # b.

Fold Change M Fold Change M Fold Change M Tumor 3 0.606146 + Tumor 35 -1.31239 + Tumor 38 -2.08491 + Tumor 5 -0.56218 - Tumor 17 -1.3846 - Tumor 10 -2.08937 + Tumor 25 -0.70939 - Tumor 29 -1.43232 - Tumor 36 -2.3729 - Tumor 1 -0.76717 + Tumor 32 -1.44175 - Tumor 31 -2.37727 + Tumor 9 -0.81473 - Tumor 30 -1.51833 - Tumor 20 -2.4748 - Tumor 18 -0.81952 - Tumor 11 -1.58384 - Tumor 22 -3.29549 - Tumor 26 -0.84014 - Tumor 15 -1.58399 - Tumor 13 -3.47015 + Tumor 7 -0.86145 - Tumor 24 -1.65383 - Tumor 8 -3.73876 + Tumor 34 -1.0823 + Tumor 19 -1.70437 + Tumor 2 -4.00908 + Tumor 33 -1.12529 + Tumor 16 -1.87714 + Tumor 6 -4.28224 - Tumor 27 -1.12897 - Tumor 14 -1.90219 - Tumor 4 -4.65925 + Tumor 28 -1.30854 - Tumor 37 -2.03034 + Tumor 21 -7.08017 -

Figure 20: LARP1 transcript levels and metastasis occurrence in human breast tumors. (a)

Fold changes calculated using the Livak method [47], #1 is a normal breast tissue control.

(b) Samples were arranged in order of increasing LARP1 levels and highlighted in red if the sample was obtained from a metastatic cancer.

Discussion

In July of 2015, a study that overlaps with many of these findings was published Lahr et al.

[44]. Their group similarly found that the DM15 domain of LARP1 bound TOP mRNA and they obtained crystals using a DM15 construct with slightly different boundaries (theirs:

796-946, ours: 799-959), into which TOP mRNA strands were modelled. Lahr et al. investigated an alternate strategy for mRNA binding, based on conserved residues within the positive surface formed by the DM15 domain. TOP mRNA binding was abolished by mutating arginine 840 to glutamine or tyrosine 883 to alanine. Our study pursued a different mutational strategy and observed reduced binding by substituting phenylalanine 841. The two studies agree that features outside the TOP sequence are important for binding by

LARP1, as certain 3’ truncated TOP 5’UTR also failed to interact with LARP1 in their hands, which they attributed to an optimal length of pyrimidine tract. Further is study is required to demonstrate the subset of TOP transcripts bound by LARP1 and their shared features in terms of RNA sequence or secondary structure.

Observations concerning dimerization were elaborated upon by Lahr et al., they identified residues involved in dimerization in the C-terminal region (908-909). Information regarding

LARP1 dimerization, or multimerization, remains shallow. Our study demonstrates a drastic differences in the migration patterns of RNA bound LARP1 monomers and multimers, indicating profound physical changes in LARP1-containing complexes upon multimerization. Understanding which factors (cellular concentration or regulatory modification of LARP1, for example) impact LARP1 multimerization and the physiological consequences of this change may merit further study. Within broad lines and with regards

51 to TOP mRNA binding and dimerization by LARP1, our study and the study by Lahr et al. corroborate each other.

The details of LARP1 function are gradually falling into place. We suspect the dual RNA binding activity of LARP1, TOP 5’ UTR and polyA, must coordinate with mTORC1 activation status to dictate LARP1 function. La-related proteins commonly bring together physically separate RNA binding domains to interact with their substrates [30, 37]. The LAM and DM15 domains of LARP1 may work together to bind certain substrates, which would help to explain any discrepancies in binding affinity demonstrated by fragment constructs (we show

Kd 110 nM for full length LARP1 and TOP mRNA while ~500 nM has been published for the

DM15 domain and TOP mRNA [44]). LARP1 may undergo context-dependent changes in overall conformation that cannot be executed by individual domains.

Our study fails to show polyA binding by full length LARP1, while an interaction is demonstrated between polyA and the LAM-HTH fragment. Unstructured regions within

LARP1 allow the protein flexibility and the conformation of full length LARP1 under experimental conditions may in some way block the LAM from RNA binding. Experiments demonstrating polyA binding by LARP1 under different conditions could support this model.

An interaction between LARP1 and PABP has been demonstrated in multiple studies, though whether or not this interaction is RNA-dependent is not agreed upon [16, 17]. In one study, binding to PABP is reduced by mutating a putative PAM2 site in the LAM of LARP1 [16]. The presence of PABP at this site may impact polyA binding by LARP1 at the same site, though further study is required to understand these interactions.

LARP1 features several putative mTORC1 phosphorylation sites in its C-terminus

(unpublished data by Fonseca et al.), near the DM15 domain, in addition to the binding site for RAPTOR [16, 17]. The inhibition of mTORC1 precedes a reduction in TOP mRNA translation, it follows that if LARP1 is a translational repressor, it binds mRNA in its unphosphorylated state. Phosphorylation by mTORC1 at sites near the DM15 motif may reduce TOP mRNA binding relieving translational suppression of these transcripts.

Phosphorylation may cause a protein to change its conformation completely such that interactions with RNA are affected, or, the negative charge of the phosphate group may compete with the RNA substrate directly [49]. Phosphorylation of LARP1, by mTORC1 or other kinases, may affect dimerization, conformation, and/or RNA binding at either site.

These effects may come into play if LARP1, alternatively, acts as a translational activator. The characterization of the domains of LARP1 as they TOP mRNA binding an important aspect of understanding function.

Additional experiments are necessary to clarify the role of LARP1 in TOP mRNA translation.

The knockdown of LARP1 by shRNA has been shown to increase or decrease the abundance of TOP mRNAs in actively translating polysomes, evidence that was contrasted with indirect data to produce two contrasting models. One study attributed this discrepancy to effects of the shRNA knockdown. The addition of recombinant LARP1 to an in vitro TOP translation mRNA assay might simplify LARP1’s contribution. However, in the case of LARP1, cellular context may be key. LARP1 binds RNA at two sites, affecting either the stability or the translation of TOP mRNA and additional 3’ targets, functions that may be sensitive to

53 mTORC1-dependant regulation. It may be impossible to classify LARP1 as a translational activator or repressor, its role may be simply too complicated.

While the link between LARP1 and cancer only strengthens [41, 42, 48, 50], the role of LARP1 as a regulator of translation and stability, toward an overall function in tumorigenesis is still under debate. LARP1 is thought to associate with up to 3000 different transcripts, with a different “interactome” in cancerous cells vs. non-cancerous cells [40, 48]. These associations may be via the 5’ UTR or the 3’ UTR, they may change upon mTORC1 activation, and they may exert positive, negative, or neutral effects on the translation and stability of the transcripts involved [16, 17, 20, 51]. Ours is the first study to suggest LARP1 transcript levels are reduced in some cancers, in apparent corroboration of the theory of LARP1 as factor suppressing cancerous states. A recent study that considers LARP1 to be a promotor of tumorigenesis nonetheless acknowledges that the effect of this protein on translation and stability may be context- or transcript-dependent [51]. Future studies on LARP1 should aim to connect existing structural and biochemical data to proposed models of LARP1 function in the cell. Future models of LARP1 function must consider nuance and complex, context- dependent explanations for cellular phenonmena.

Materials and Methods

4.1 Cloning All LARP1 bacterial expression constructs were produced from the pET-SUMO (Thermo

Fisher) modified 6His/SUMO tagged vector using HindII and XhoI insertion sites. The full length sequence of LARP1 was amplified from CMV-LARP1 (Origene) using primers (IDT)

CAA-GCT-TCC-ATG-CTT-TGG-AGG-GTG (1) and ACT-CGA-GCG-CTT-TCC-CAA-AGT-CTG

(1019). Primers CAA-GCT-TCC-TCC-CAT-GAA-CTG-CTC-AAG-GAA (799) and ACT-CGA-GCG-

GTG-GTT-GCC-CTC-CTC (959) were used to generate the DM15 construct using the same template, encompassing residues 799 to 959. The Extended DM15 was comprised of residues 750-1019 and was generated using primers CAA-GCT-TCC-CCC-TTG-GAG-AGC-

CAT-GTG (750) and 1019. The LAM-RRM construct spanned from 321 to 441 and was generated using primers AAG-CTT-CAA-GCT-TCC and CTC-GAG-ACT-CGA-GCG. Inserts and host plasmid were digested with the relevant Fast Digest (Thermo Fisher) restriction enzyme for 1 hour at 37°C. Digested host plasmids were gel extracted (Qiagen) following 1% agarose electrophoresis. Qaigen PCR clean-up protocols were used to prepare inserts following digestion. Digested plasmid and insert were combined in 1:5 ratio and joined using

T4 ligase (NEB) for 1 hour at room temperature.

4.2 Site-Directed Mutagenesis

Site-directed mutagenesis was used to generate the Y816A and F841A mutants of the 750-

1019 DM15 Extended construct. Y816A Forward: ACA-CAA-CAC-GTC-TAC-CAT-AAG-GCC-

CGT-AGG-CGC, Y816A Reverse: CAT-TAA-GGC-AGC-GCC-TAC-GGG-CCT-TAT-GGT-AGA. F841

Forward: AGA-TGA-ACA-CAC-TCT-TCC-GCG-CTT-GGT-CCT-TCT, F841A Reverse: TCT-CGG-

AGG-AAG-AAG-GAC-CAA-GCG-CGG-AAG-AGT. Primers (Biocorp) were combined with template plasmid at final concentrations of 200ng/uL and 1ng/uL respectively.

Amplification was performed using PfuUltra II (Agilent): 2 minutes at 94°C prior to 15 cycles of 30s at 94°C, 1 minute at 58°C, and 2 min/kB at 68°C, followed by 8 minutes extension at

68°C. Reactions were digested with Dpn1 (NEB) for 1 hour at 37°C, separated on 1% agarose gels in TAE, and mutant plasmids were gel extracted and transformed into DH5α bacteria and plated on ampicillin-containing LB agar (BioShop). Colonies were collected and grown overnight in 5mL liquid media prior to DNA extraction and sequencing.

4.3 Protein Expression and Purification

The full length sequence of LARP1 full length and fragment sequences were fused to SUMO and expressed in pET28. Recombinant protein was expressed in BL21 E. coli induced with 1 mM IPTG (BioShop) for 18 hours at 18⁰C. Pellets were lysed by sonication in a buffer of Tris-

HCl, 25 mM HEPES, 20% glycerol, 5 mM β-mercaptoethanol, 15 mM imidazole, 9ug/mL

RNase, 7ug/mL DNase, and protease inhibitors. Lysates were passed over 1mL nickel resin per litre culture and washed with 15 mM and 30 mM imidazole prior to elution with 1mL

500 mM imidazole per 1 litre culture. Eluted protein was dialyzed overnight into 100 mM

KCl and 25 mM HEPEs at pH 7.4 Full length SUMO-LARP1 was further purified by S200 size exclusion HPLC (GE Healthcare) in 100 mM KCl, 25 mM HEPES, and 0.5 mM TCEP at pH 7.4.

LARP1 elutes in 2 peaks representing, dimer and monomer forms respectively, under these conditions. The first eluted peak was concentrated using 100K MWCO Amicon Ultra

(Millipore) for use in further experiments. DM15 and DM15 Extended constructs were similarly purified though with S75 size exclusion chromatography.

4.4 Electrophoretic Gel Shift Mobility Assay

RNA probes were synthesized by IDT, Dharmacon, or Sigma and 5’ labelled using PNK

(Thermo Fisher) and P32 γATP with incubation at 37 for 30 minutes followed by purification with RNA columns (TE-10 Clontech or Roche) used to according to manufacturer specifications. Probes and protein were combined for 30 minutes at 30⁰C in 100 mM KCl, 5 mM MgCl2. Complexes were separated on 5% acrylamide gels in 0.5X TBE. Large gels, employed for high resolution separations, ran at 150V overnight and small gels ran at 70V for 2 hours. Gels were dried and exposed to film overnight at -80⁰C. For quantitative determinations band density was determined using ImageJ and curves were calculated using

GraphPad Prism.

4.5 Coomassie Protein Gels

Protein samples were combined with 4X loading buffer containing: 50 mM Tris-HCl pH 6.8,

2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA, 0.02 % bromophenol blue.

Samples were boiled for 5 minutes before loading. Acrylamide gels, 10-15%, were used to separate proteins at 120V for 1.5 hours. Gels were rinsed and stained overnight with gentle mixing in Coomassie G-250 stain containing 0.08% dye, 8% ammonium sulfate, 1.6% phosphoric acid and 20% methanol. Gels were destained with multiple water rinses.

4.6 Western Blot

Protein gels were transferred for 1.5 hours at 100V. Membranes were blocked with 5% skim milk for 40 minutes and rinsed before incubation with antibody overnight at 4°C. The antibody for human LARP1 was purchased from Abcam (ab86359).

4.7 RNA Sequences

The following TOP mRNA 5’ UTR were used in this study RPL32: CCU UU UCC GGU CCG CGG

CGC UGC GGU GGU GG (Dharmacon), RPS16: CUU UUC CGG UUG CGG CGC CGC GCG GUG AGG

UUG UCU AGU CCA CGC UCG GAG CC (Dharmacon), RPL27: CUU UCU GGU CUC GGC CGC AGA

AGC GAG (Sigma), RPL29: CCU UUU ACC UCG UUG CAC UGC UGA GAG CAA G (Sigma)

Ribonucleotide polymers were obtained from Integrated DNA Technologies (IDT).

4.8 Isothermal Titration Calorimetry

La-HTH was added to the sample cell of a MicroCal iTC200 titration calorimeter (MicroCal,

Northampton, MA) in 50 mM Tris-HCl buffer (pH 7.6), 50 mM NaCl at 20 °C. The reaction cell contained 200ul of 0.1 mM protein and was titrated with 19 injections of 2ul of 1 mM RNA oligo. A binding model that employs a single set of independent sites was used to fit the binding isotherm.

4.9 Nuclear Magnetic Resonance

N15 LA-HTH cultures were grown in M9 minimal media containing 33.7 mM Na2HPO4, 22 mM

KH2PO4, 8.55 mM NaCl, 9.35 mM N15H4Cl, 0.4% glycerol, 1 mM MgSO4, 0.3 mM CaCl2, 1ug/L biotin, 1ug/L thiamin, and trace elements. Overnight cultures of LA-HTH expressing BL21 were grown in LB and rinsed prior to introduction to minimal media to and OD of 0.1.

Cultures were grown at 37°C until OD 1.0 and induced with 1 mM IPTG. Protein was expressed overnight at 25°C prior to purification using protocols described above. The HSQC spectrum of the 15N-LaHTH protein alone (blue) was collected at 298K in a Bruker 600MHz, with and without 10nt polyA RNA at a 1:1 ratio.

4.10 Quantitative Real Time Polymerase Chain Reaction

Extracted RNA samples were obtained following conversion to cDNA by the pathology facility at Vall d’Hebron Research Institute. Each cDNA sample was normalized to 1mg/mL and equal volumes were used per qRT-PCR reaction. Primers were designed using Primer

Blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) and they were tested through the generation of a standard curve. SYBR Green (BioRad) PCR MasterMix was combined with

100 nM primers, 50ng template, and reactions were performed as per manufacturer’s instruction. As the relative amplification efficiencies of tumor and normal breast tissues were found to be approximately equal, the ΔΔCt method was applied to calculate transcript levels.

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