Mechanism of Translation Regulation of BTG1 by eIF3

Master’s Thesis

Presented to

The Faculty of the Graduate School of Arts and Sciences Brandeis University Department of Biology Amy S.Y. Lee, Advisor

In Partial Fulfillment of the Requirements for the Degree

Master of Science in Biology

by Shih-Ming (Annie) Huang

May 2019

Copyright by

Shih-Ming (Annie) Huang

© 2019

ACKNOWLEDGEMENT

I would like to express my deepest gratitude to Dr. Amy S.Y. Lee for her continuous patience, support, encouragement, and guidance throughout this journey.

I am very thankful for all the members of the Lee Lab for providing me with this caring and warm environment to complete my work. I would also like to thank Dr.

James Nuñez for collaborating with us on this project and helping us in any shape or form.

iii ABSTRACT

Mechanism of Translation Regulation of BTG1 by eIF3

A thesis presented to the Department of Biology

Graduate School of Arts and Sciences Brandeis University Waltham, Massachusetts

By Shih-Ming (Annie) Huang

REDACTED

iv TABLE OF CONTENTS REDACTED

v LIST OF FIGURES REDACTED

vi INTRODUCTION

I. Regulation

All cells in our bodies contain the same genome, but distinct cell types express very different sets of . The sets of gene expressed under specific conditions determine what the cell can do, by controlling the and functional RNAs the cell contains. The process of controlling which genes are expressed is known as gene regulation. Any step along the pathway can be controlled, from DNA transcription, translation of mRNAs into proteins, to post-translational modifications. Compared to transcription regulation, translation regulation can control existing mRNAs, allowing more rapid changes in the cellular concentration of proteins. Thus, translational regulation plays a crucial role in maintaining homeostasis and controlling cell growth and proliferation (Hershey et al. 2012).

II. Translation Regulation in Eukaryotes

Translation can be divided into initiation, elongation, termination, and ribosome recycling. The canonical mechanism of translation initiation includes the formation of 43S preinitiation complexes, the attachment of 43S complexes to mRNA, and the ribosome scanning of mRNA 5′ untranslated regions (UTRs) (Jackson et al., 2010). The 43S preinitiation complex is formed when the methionine-tRNA is delivered by eIF2 to the P site of the 40S ribosomal subunit (Sonenberg and Hinnebusch, 2009). The complex is then recruited to the 5′ end of the mRNA by eIF3 and eIF4

(Kumar et al., 2016). After attachment, the 43S preinitiation complex scans the 5′ UTR and recognizes the AUG start codon. Through scanning, the secondary structure of the 5′ UTR is unwound while the ribosome moves along it (López-Lastra et al., 2005). Once the initiation codon is recognized, the 60S subunit joins to form the 80S initiation complex for subsequent elongation.

1 Notably, the interactions between the eukaryotic initiation factors, ribosomes, and mRNA for the formation of the initiation complexes are potential steps for regulation.

Regulation by RNA-binding Proteins

RNA-binding proteins (RBPs) are proteins that can bind to single stranded or structured

RNAs. RBPs can direct mRNA-specific translation regulation through interactions with the 5′ or

3′ UTRs. For example, the gamma interferon-activated inhibitor of translation (GAIT) complex binds to specific 3′ UTR elements and suppresses translation by inhibiting the recruitment of the

43S translation initiation complex (Mukhopadhyay et al., 2009). Another RBP, CNBP/ZNF9 binds to G-rich elements in target mRNAs to prevent G-quadruplex formation. Thus, promoting translation by stabilizing mRNA structures (Benhalevy et al., 2017). With its versatile modes of action, RBPs can regulate many aspects of mRNA stability, localization, and export.

Regulation by Secondary Structure

Other than serving as a translation regulatory element by interacting with RBPs, secondary structure in the 5ʹ UTR of an mRNA can also directly play a functional role in translation regulation. For example, genes encoding protooncogenes, growth factors, and differentiation often have stable secondary structures in the 5ʹ UTR and are poorly translated under normal conditions

(Davuluri et al., 2000). It is thought that since efficient translation requires scanning of the 5ʹ UTR by the preinitiation complex, stable secondary structures inhibit translation by blocking ribosome movement (Rozen et al., 1990; Pickering and Willis, 2005).

Upstream Open Reading Frames Regulate Gene-Specific Translation

Upstream open reading frames (uORF) are one of the most common regulatory RNA elements. In fact, 49% of the human transcriptome contains uORFs (Suzuki et al., 2000). uORFs are short ORFs within the 5' UTR, upstream of the main coding sequence (CDS). Typically, uORFs

2 are thought to be inhibitory to translation of the downstream CDS. When an uORF is recognized by a scanning ribosome, the translation machinery can start translation at this inappropriate start codon, and then dissociates at the next stop codon. After this, the ribosome can either stall during elongation to block recruitment of additional ribosomes, trigger mRNA decay, or can continue to scan to reinitiate downstream translation at the appropriate start codon (Meijer and Thomas, 2002).

All these different mechanisms suggest that uORFs can be major regulators of translation.

III. Eukaryotic Initiation Factor 3 (eIF3)

eIF3 is a multiprotein complex with 13 subunits (eIF3a to eIF3m) (Figure 1). It is involved in almost every step of translation initiation. During canonical translation, eIF3 interacts with many of the eukaryotic initiation factors for ribosome recruitment, attachment of 43S complexes to mRNA, and the scanning of the 5′ UTR (Yin et al., 2018). Besides its prominent role in canonical translation, the subunits of eIF3 have been shown to have specialized functions.

For example, eIF3a plays a crucial role in regulating progression and cell proliferation (Dong et al., 2009). Its expression also has been linked to different types of cancer, although the exact mechanism by which eIF3 controls cell proliferation remains unknown

(Pincheira et al., 2001; Chen and Burger, 2004). Furthermore, downregulation of eIF3b is linked to cell arrest and apoptosis (Liang et al., 2012; Choi et al., 2017). Elevated expression of eIF3b has been detected in several tumors (Wang et al., 2013; Choi et al., 2017). eIF3d has also been shown to play an oncogenic role in many different types of cancer (Fan and Guo, 2015; Lin et al., 2015).

Recent research has shown that eIF3d can serve as a cap-binding for eIF4E-independent translation by binding to the 5′ cap structure of specific mRNAs (Lee et al., 2016). Additionally, eIF3g contains an RNA recognition motif that is responsible for mediating the interaction of the

3 43S preinitiation complex and the mRNA (Zhou et al., 2008). eIF3g has also been shown to interact with nuclear and cytoskeleton proteins (Zheng et al., 2016).

Figure 1. EM structure of mammalian eIF3 Ribbon representation of the eIF3 core subunits in the context of the 43S preinitiation complex (Des-Georges et al., 2015).

IV. Translation Regulation by eIF3

Previous research by Lee et al. showed that eIF3 is responsible for regulating cell proliferation and differentiation by interacting with select mRNAs, including c-JUN and

BTG1(Figure 2). As shown by photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP), eIF3a, b, d and g subunits bind to the 5′ UTR of specific mRNAs (Figure 3). By using selective 2′-hydroxyl acylation analysed by primer extension

(SHAPE), Lee et al. experimentally determined the secondary structure of eIF3-binding sites and showed that eIF3 binds to RNA stem–loop elements in the 5′ UTR of the target mRNAs. However, native agarose gel electrophoresis shows that eIF3 interacts with c-JUN and BTG1 with different modes of action. eIF3 interacts with c-JUN both in vivo and in vitro, indicating this is a binary-

4 interaction. In contrast, eIF3 only interacts with BTG1 in vivo but not in vitro, suggesting that another co-factor(s) is needed for the interaction to occur.

Figure 2. Validation of eIF3 PAR-CLIP targets eIF3 PAR-CLIP targets validated by eIF3 immunoprecipitation followed by RT-PCR (Data from Lee et al., 2015).

Figure 3. eIF3 subunits crosslinked to RNA SDS gel showing eIF3 subunits crosslinked to RNA from PAR-CLIP (Data from Lee et al., 2015).

5 To investigate the effect of the eIF3–RNA interaction on translation, a luciferase reporter assay was used to measure translation driven by the 5′ UTR of BTG1 or c-JUN with and without eIF3-binding sites. Data showed that eIF3 serves as an activator for c-JUN translation and a repressor for BTG1 translation (Figure 4). Together, Lee et al showed that besides serving as a scaffold protein during translation initiation, eIF3 also acts as transcript-specific translation regulator by binding to specific mRNAs with different modes of action.

Figure 4. Luciferase reporter assay showing eIF3 as a translation regulator Luciferase activities measuring translation efficiency of mRNA driven by the c-JUN 5′ UTR or BTG1 5′ UTR with or without eIF3-binding site (Data from Lee et al., 2015).

V. BTG anti-proliferation factor 1 (BTG1)

BTG anti-proliferation factor 1(BTG1) is an antiproliferative gene that is involved in regulating cell growth and differentiation. It is part of the BTG/Tob family, which share a conserved N‐terminal BTG domain that has been implicated in protein-protein interactions

(Matsuda et al., 2001). BTG Anti-Proliferation Factor 2 (BTG2), a homolog of BTG1, has been shown to control cell cycle progression by responding to DNA damage (Rouault et al., 1996).

Despite their differences in their role in cell cycle regulation, BTG1 and BTG2 share many similarities in terms of their structure and expression regulation.

6 The BTG1 gene in human is located on 12q22 while BTG2 is located on 1q32

(Rimokh et al., 1991; Rouault et al., 1996). The transcripts of both genes are subject to regulation by microRNAs through binding sites in the 3′ UTR (Li et al., 2014; Mao et al., 2015). The expression pattern of BTG1 and BTG2 differs, with BTG1 expressed highly in the heart and pancreases while BTG2 is abundant in the kidney, lung and other various organs (Rouault et al.,

1992, 1996).

BTG1 as Transcriptional Coactivator

BTG1 has been found to be associated with several cellular targets. By using yeast two- hybrid system, it has been shown that BTG1 interact with Hoxb9, a that is involved in regulating cell proliferation and differentiation (Prévôt et al., 2000, Shrestha et al.,

2012). The interaction between BTG1 and Hoxb9 enhances Hoxb9-dependent transcription, suggesting that BTG1 functions as a transcriptional coactivator. Furthermore, BTG1 contains an

LXXLL motif, which is found in most of the nuclear receptor coactivators that are essential for interacting with nuclear receptors (Hu et al., 2011; Heery et al., 1997). Other than serving as a transcriptional coactivator, BTG1 also contains a domain that bind to PRMT1, (Lin et al., 1996).

Even though it is still unclear what role BTG1 exerts on PRMT1, intriguingly, one of the biological roles of PRMT1‐mediated arginine methylation is to coactivate transcription (Liu et al., 2016).

BTG1 in Posttranscriptional Regulation

In addition to serving as a transcriptional coactivator, BTG1 can regulate gene expression by controlling mRNA levels. The BTG1 APRO domain interacts with PABPC1 to stimulate CAF1 deadenylase activity and promote mRNA degradation by the removal of the poly(A) tail (Stupfler et al., 2016; Matsuda et al., 2001). BTG1 also interacts with the CNOT7 and CNOT8 deadenylase

7 subunits of the CCR4–NOT complex. This interaction has been shown to play an important role in regulating proliferation (Aslam et al., 2009).

Biological Processes in Relation to BTG1

BTG1 is a key regulator in cellular processes including cell differentiation and apoptosis.

Overexpression of BTG1 in NIH3T3 or breast cancer cells increases apoptosis (Corjay, 1998;

Nahta et al., 2006). In mice, knockdown of BTG1 decreases proliferating stem and progenitor cells, eventually leading to apoptosis (Farioli-Vecchioli et al., 2012). BTG1 has also been implicated in the differentiation of myoblasts and endothelial cells (Micheli et al., 2017).

Furthermore, BTG1 is needed for the vertebral patterning of the axial skeleton in mice (Tijchon et al., 2015).

VI. Regulation of BTG1 Expression

The expression of BTG1 is tightly regulated through phases of the cell cycle, with the highest level observed in quiescence. BTG1 levels decrease as the cell progresses through G1 phase, with the minimum expression as cells enter the (Rouault et al., 1992). There has been much research into the mechanisms by which BTG1 expression is regulated. MiR-19a and miR-454-3p bind to the 3′ UTR of BTG1 and repress its expression (Lu et al., 2015). Interestingly, overexpression of miR-454-3p in cells shifts cell cycle arrest from G2/M phase to S, which correlates with the observation that BTG1 is downregulated in the S phase (Wu et al., 2014).

Despite its importance in many cellular processes, the regulation of BTG1 expression remains poorly understood.

BTG1 contains two uORFs: uORF1 is located upstream of eIF3-binding site while uORF2 is located within this region (Figure 5). Currently, while there is no data suggesting that either of

8 these two uORFs serve as regulator for BTG1 translation, in vivo ribosome profiling suggests that uORF2 is not used as a start codon under normal cellular conditions.

Figure 5. SHAPE structure of BTG1 5′-UTR Secondary structure of BTG1 5′-UTR determined experimentally showing eIF3-binding site as a stem-loop structure (Lee et al., 2015). Two uORFs are labeled.

VII. CRISPR interference (CRISPRi)

CRISPRi is a modified system of the genome editing technology, CRISPR-Cas9. In the

CRISPRi system, the endonuclease activity of CRISPR associated endonuclease enzyme is removed by mutations to yield a catalytically dead Cas9 (dCas9). Coupling of dCas9 to a KRAB effector domain can repress transcription when complexes with single guide RNA (sgRNA)

(Gilbert et al., 2013).

9 MATERIAL AND METHODS REDACTED

10 RESULTS REDACTED

11 DISCUSSION REDACTED

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