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The Role of Hud in Alternative Splicing and Alternative Polyadenylation in the Brain

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The Role of Hud in Alternative Splicing and Alternative Polyadenylation in the Brain University of New Mexico UNM Digital Repository Biomedical Sciences ETDs Electronic Theses and Dissertations Fall 11-3-2020 The Role of HuD in Alternative Splicing and Alternative Polyadenylation in the Brain Rebecca M. Sena University of New Mexico Follow this and additional works at: https://digitalrepository.unm.edu/biom_etds Part of the Medicine and Health Sciences Commons Recommended Citation Sena, Rebecca M.. "The Role of HuD in Alternative Splicing and Alternative Polyadenylation in the Brain." (2020). https://digitalrepository.unm.edu/biom_etds/216 This Thesis is brought to you for free and open access by the Electronic Theses and Dissertations at UNM Digital Repository. It has been accepted for inclusion in Biomedical Sciences ETDs by an authorized administrator of UNM Digital Repository. For more information, please contact [email protected], [email protected], [email protected]. Rebecca Sena Candidate Department of Neurosciences Department This thesis is approved, and it is acceptable in quality and form for publication: Approved by the Thesis Committee: Nora Perrone-Bizzozero, Ph.D., Chairperson David N. Linsenbardt, Ph.D. Fernando Valenzuela, M.D., Ph.D. Amy Gardiner, Ph.D. i THE ROLE OF HUD IN ALTERNATIVE SPLICING AND ALTERNATIVE POLYADENYLATION IN THE BRAIN by REBECCA SENA B.S. BIOLOGY NEW MEXICO HIGHLANDS UNIVERSITY, 2018 THESIS Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science Biomedical Sciences The University of New Mexico Albuquerque, New Mexico December 2020 ii THE ROLE OF HUD IN ALTERNATIVE SPLICING AND ALTERNATIVE POLYADENYLATION IN THE BRAIN By Rebecca Sena B.S. Biology, New Mexico Highlands University, 2018 M.S., Biomedical Sciences, The University of New Mexico, 2020 ABSTRACT RNA binding proteins (RBPs) regulate several processes in the cell, including alternative splicing and alternative polyadenylation. Hu proteins, a class of ELAV- like RBPs, are crucial for proper development and maintenance of the nervous system. Several Hu proteins, including HuD, have been shown to regulate alternative splicing and alternative polyadenylation of neuronal transcripts. However, previous studies have relied on molecular techniques to analyze individual transcripts, which do not provide a global overview of the transcriptome. The purpose of this study was to develop a bioinformatics pipeline to analyze splicing and polyadenylation using RNA-sequencing data. Once the pipeline was developed, it was utilized to analyze the effect of HuD KO on cortical transcripts. HuD KO was found to impact both alternative splicing and polyadenylation of genes that have been implicated in several nervous system and neuropsychiatric disorders. iii TABLE OF CONTENTS CHAPTER 1 INTRODUCTION ................................................................................ 1 1.1. RNA Binding Proteins ...................................................................................... 1 1.2. Alternative Splicing .......................................................................................... 1 1.3. Alternative Polyadenylation ............................................................................ 4 1.4. HU Proteins Regulate Alternative Splicing and Polyadenylation ................ 8 1.5. Goals of this Study .......................................................................................... 10 CHAPTER 2 METHODOLOGY ............................................................................ 13 2.1. Selection and Explanation of Alternative Splicing Tools ............................ 13 A. Estimating Alternative Splicing with PSI and PIR Metrics Using ASpli ..... 13 B. Estimating Alternative Splicing Using Replicate Multivariate Analysis of RNA-seq Data (rMATS) ...................................................................................... 16 2.2. Selection and Explanation of Alternative Polyadenylation Tools .............. 19 A. Estimation of Alternative Polyadenylation with InPAS ................................. 19 B. Estimation of Alternative Polyadenylation Using DaPars .............................. 19 2.3. RNA-sequencing .............................................................................................. 21 2.4. Developing the Alternative Splicing and Polyadenylation Pipeline ........... 21 A. Initial Quality Check ..................................................................................... 21 iv B. Removal of Adapter Sequences .................................................................... 22 C. Alignment of Raw RNA-seq Data with STAR ............................................. 23 D. Final Quality Check ...................................................................................... 24 E. Alternative Splicing Analysis ....................................................................... 24 F. Alternative Polyadenylation Analysis .......................................................... 26 G. Pathway Analysis .......................................................................................... 27 CHAPTER 3 RESULTS ........................................................................................... 28 3.1. Alternative Splicing of Transcripts in HUD KO Cortex ............................. 28 3.2. Alternative Polyadenylation of Transcripts in HUD KO Cortex ................ 44 CHAPTER 4 DISCUSSION ..................................................................................... 52 APPENDICES ............................................................................................................ 64 APPENDIX A: Alternative Splicing and Polyadenylation Datasets ................. 65 APPENDIX B: Utilizing the Pipeline on a RobUst Alcohol Dataset .................. 76 REFERENCES .......................................................................................................... 97 v Chapter 1 IntrodUction 1.1. RNA Binding Proteins RNA binding proteins (RBPs) serve important functions in the co- transcriptional and post-transcriptional control of several types of RNA, including messenger RNAs (mRNAs) 1,2. RBPs regulate several processes in the cell including alternative splicing and alternative polyadenylation, which occur in the nucleus, and mRNA stabilization, transport, and localization, which occur in the cytoplasm. These processes affect translation of mRNAs to proteins, ultimately regulating protein production and function. Many studies have focused on the role of RBPs in the cytoplasm, but much less is known about the role of RBPs in the nucleus. 1.2. Alternative Splicing Alternative splicing is perhaps the most studied RBP mechanism in the nucleus. Splicing is a highly regulated process that produces mRNA from pre-mRNA molecules. Pre-mRNA molecules consist of both introns, which are non-coding sequences, and exons, which encode the amino acid sequence of the protein. The canonical splicing mechanism consists of the removal of an intron between two exons through the formation of a lariat structure, which is first formed by a nucleophilic attack at the 5’ splice site of an exon to the adenosine residue in the branch point 3. 1 Then, two exons are joined together through a second nucleophilic attack at the 3’ splice site of the second exon, and the lariat is released 3. This mechanism is summarized in Figure 1. After additional pre-mRNA processing, such as the addition of a 5’ cap and poly(A) tail, mRNA is exported to the cytoplasm where it can be translated into protein. Figure 1. Canonical splicing mechanism in the cell. The splicing mechanism consists of the removal of an intron through the formation of a lariat structure, which is formed at the intron branch point. Two exons are then joined, and the lariat structure is released. Adapted from Baralle et. al., 2005. 2 In addition to the canonical splicing mechanism, there are several different forms of alternative splicing that may occur. These include exon skipping, mutually exclusive exons, alternative 5’ or 3’ splice sites, and intron retention. Exon skipping occurs when an exon is essentially left out of the final mRNA transcript. Mutually exclusive exons refer to a set of two exons where only one is retained and the other is not. Alternative 5’ or 3’ splice site usage can result in the boundaries of exons being either increased or decreased in the final transcript. Finally, intron retention occurs when introns are kept in the final mRNA transcript. Each of these events can result in mRNA isoforms with different exons from the same gene, or isoforms that include introns of the same gene. Changes in isoform expression can have a dramatic impact on protein function or lead to loss of function. For example, alternative splicing of PSD-95, a scaffolding protein that is essential for maturation and plasticity of excitatory synapses, determines the functional properties of the protein. Exon skipping of exon 18 causes the transcript to contain a premature stop codon, which targets the transcript for degradation through nonsense mediated decay (NMD) 4. Inhibition of PSD-95 expression in differentiated neurons was shown to impair development of glutamatergic synapses 4. This mechanism is important at different time periods in brain development, with lower levels of PSD-95 occurring during embryonic development and higher levels occurring later 4–6. This is one of many examples 3 illustrating that alternative splicing can have a considerable impact on nervous system function. It has been estimated that 92-95% of human genes undergo alternative splicing to produce
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