Functional cis-regulatory modules encoded by mouse-specific endogenous retrovirus

Cis-regulatory modules contain multiple transcription factor (TF)-binding sites and integrate the effects of each TF to control gene expression in specific cellular contexts. Transposable elements (TEs) are uniquely equipped to deposit their regulatory sequences across a genome, which could also contain cis-regulatory modules that coordinate the control of multiple genes with the same regulatory logic. We provide the first evidence of mouse-specific TEs that encode a module of TF-binding sites in mouse embryonic stem cells (ESCs). The majority (77%) of the individual TEs tested exhibited enhancer activity in mouse ESCs. By mutating individual TF-binding sites within the TE, we identified a module of TF-binding motifs that cooperatively enhanced gene expression. Interestingly, we also observed the same motif module in the in silico constructed ancestral TE that also acted cooperatively to enhance gene expression. Our results suggest that ancestral TE insertions might have brought in cis-regulatory modules into the mouse genome

An improved predictive recognition model for Cys2-His2 zinc finger proteins

Cys2-His2 zinc finger proteins (ZFPs) are the largest family of transcription factors in higher metazoans. They also represent the most diverse family with regards to the composition of their recognition sequences. Although there are a number of ZFPs with characterized DNA-binding preferences, the specificity of the vast majority of ZFPs is unknown and cannot be directly inferred by homology due to the diversity of recognition residues present within individual fingers. Given the large number of unique zinc fingers and assemblies present across eukaryotes, a comprehensive predictive recognition model that could accurately estimate the DNA-binding specificity of any ZFP based on its amino acid sequence would have great utility. Toward this goal, we have used the DNA-binding specificities of 678 two-finger modules from both natural and artificial sources to construct a random forest-based predictive model for ZFP recognition. We find that our recognition model outperforms previously described determinant-based recognition models for ZFPs, and can successfully estimate the specificity of naturally occurring ZFPs with previously defined specificities

Systematic Investigation of Alternative Splicing and Conserved Spliceosome Factors in Chlamydomonas reinhardtii

Splicing is a crucial step of processing pre-mRNA molecules for precise flow of genetic information from DNA to proteins, where introns are removed and exons of pre-mRNA are joined together to form mature mRNA. The variability in splicing pattern generate a wide array of mature mRNAs from a limited set of genes enabling greater protein diversity with different functions. This process is carried out by a megadalton complex called the spliceosome that consists of more than 200 proteins and snRNA molecules. Previous studies have shown that alterations to a DNA sequence of spliceosome proteins introduce errors in the splicing process that leads to incorrect splicing. Many spliceosome proteins have been implicated in diseases like neurodegenerative disorders, retinitis pigmentosa, cancer and spinal muscular atrophy. Thus, it is important to understand the role of individual spliceosome proteins in the splicing process and their effect on splicing fidelity. The spliceosome undergoes a series of transitions from a pre-catalytic state (complex A) to catalytically active state (complex C) during the splicing process. A subset of spliceosome proteins forms the core component of the spliceosome that are present throughout the splicing process, while other proteins are transient that bind and leave the complex at different points during the splicing process. Our lab previously characterized loss-of-function mutations in four different spliceosome proteins that act as suppressors of splice site mutations in Chlamydomonas reinhardtii. Interestingly, three out of four proteins are part of the small group of proteins that joins the catalytically active spliceosome complex C late in the splicing process. This dissertation focuses on understanding the role of two of these spliceosome proteins, DGCR14 and FRA10AC1. I analyzed the splicing patterns in dgr14 and fra10 mutants in a wild-type background and in double mutants with a mutation that affects nonsense mediated decay (NMD) to capture the breadth of global splicing changes incurred by the spliceosome mutants. The study demonstrates that NMD is an active pathway in C. reinhardtii and degrades non-functional transcripts as shown by analyzing a nonsense mutation in the SMG1 gene, which disrupts the NMD pathway. Next, I show that the two splicing factors affect the 3’ and 5’ splice site choice and their absence specifically weakens the selectivity of weak 3’ splice site. Also, the newly formed alternate 3’ splice site demonstrate significant decrease in the splicing fidelity at 3’ end. This suggests that the two splicing factors affect the splicing fidelity even though they join the spliceosome complex at late stage, pointing towards a potential proof-reading mechanism in splicing. Further work to investigate the interaction of these factors with other spliceosome proteins and 5’ and 3’ splice site is warranted. To capture the extent to which alternative splicing is active and functional in C. reinhardtii, I analyzed the existing RNA-seq data from Zones et al., 2015 study, obtained during the diurnal cell cycle of C. reinhardtii. The analysis shows that alternative splicing events are temporally regulated during the cell cycle and identified a subset of events that show periodic changes during the diurnal cell cycle. Many of these events introduce premature termination codon (PTC) in the transcript that potentially makes them NMD targets. This study also demonstrates a potential AS mediated regulation of ODC1 gene which encodes for ornithine decarboxylase enzyme, during light to dark transition during the diurnal cell cycle. Our findings are concordant with previous studies that show light-mediated activation of the ODC1 activity in C. reinhardtii and tobacco plants. Together my research work in this dissertation reveals new insights into the post-transcriptional regulation of genes by alternative splicing in C. reinhardtii and highlights the role of non-core spliceosome proteins in maintaining the splicing fidelity and selection of weak splice sites

Systematic Investigation of Alternative Splicing and Conserved Spliceosome Factors in Chlamydomonas reinhardtii

Splicing is a crucial step of processing pre-mRNA molecules for precise flow of genetic information from DNA to proteins, where introns are removed and exons of pre-mRNA are joined together to form mature mRNA. The variability in splicing pattern generate a wide array of mature mRNAs from a limited set of genes enabling greater protein diversity with different functions. This process is carried out by a megadalton complex called the spliceosome that consists of more than 200 proteins and snRNA molecules. Previous studies have shown that alterations to a DNA sequence of spliceosome proteins introduce errors in the splicing process that leads to incorrect splicing. Many spliceosome proteins have been implicated in diseases like neurodegenerative disorders, retinitis pigmentosa, cancer and spinal muscular atrophy. Thus, it is important to understand the role of individual spliceosome proteins in the splicing process and their effect on splicing fidelity. The spliceosome undergoes a series of transitions from a pre-catalytic state (complex A) to catalytically active state (complex C) during the splicing process. A subset of spliceosome proteins forms the core component of the spliceosome that are present throughout the splicing process, while other proteins are transient that bind and leave the complex at different points during the splicing process. Our lab previously characterized loss-of-function mutations in four different spliceosome proteins that act as suppressors of splice site mutations in Chlamydomonas reinhardtii. Interestingly, three out of four proteins are part of the small group of proteins that joins the catalytically active spliceosome complex C late in the splicing process. This dissertation focuses on understanding the role of two of these spliceosome proteins, DGCR14 and FRA10AC1. I analyzed the splicing patterns in dgr14 and fra10 mutants in a wild-type background and in double mutants with a mutation that affects nonsense mediated decay (NMD) to capture the breadth of global splicing changes incurred by the spliceosome mutants. The study demonstrates that NMD is an active pathway in C. reinhardtii and degrades non-functional transcripts as shown by analyzing a nonsense mutation in the SMG1 gene, which disrupts the NMD pathway. Next, I show that the two splicing factors affect the 3’ and 5’ splice site choice and their absence specifically weakens the selectivity of weak 3’ splice site. Also, the newly formed alternate 3’ splice site demonstrate significant decrease in the splicing fidelity at 3’ end. This suggests that the two splicing factors affect the splicing fidelity even though they join the spliceosome complex at late stage, pointing towards a potential proof-reading mechanism in splicing. Further work to investigate the interaction of these factors with other spliceosome proteins and 5’ and 3’ splice site is warranted. To capture the extent to which alternative splicing is active and functional in C. reinhardtii, I analyzed the existing RNA-seq data from Zones et al., 2015 study, obtained during the diurnal cell cycle of C. reinhardtii. The analysis shows that alternative splicing events are temporally regulated during the cell cycle and identified a subset of events that show periodic changes during the diurnal cell cycle. Many of these events introduce premature termination codon (PTC) in the transcript that potentially makes them NMD targets. This study also demonstrates a potential AS mediated regulation of ODC1 gene which encodes for ornithine decarboxylase enzyme, during light to dark transition during the diurnal cell cycle. Our findings are concordant with previous studies that show light-mediated activation of the ODC1 activity in C. reinhardtii and tobacco plants. Together my research work in this dissertation reveals new insights into the post-transcriptional regulation of genes by alternative splicing in C. reinhardtii and highlights the role of non-core spliceosome proteins in maintaining the splicing fidelity and selection of weak splice sites

Alternative Splicing During the Chlamydomonas reinhardtii Cell Cycle

Genome-wide analysis of transcriptome data in Chlamydomonas reinhardtii shows periodic patterns in gene expression levels when cultures are grown under alternating light and dark cycles so that G1 of the cell cycle occurs in the light phase and S/M/G0 occurs during the dark phase. However, alternative splicing, a process that enables a greater protein diversity from a limited set of genes, remains largely unexplored by previous transcriptome based studies in C. reinhardtii. In this study, we used existing longitudinal RNA-seq data obtained during the light-dark cycle to investigate the changes in the alternative splicing pattern and found that 3277 genes (19.75% of 17,746 genes) undergo alternative splicing. These splicing events include Alternative 5′ (Alt 5′), Alternative 3′ (Alt 3′) and Exon skipping (ES) events that are referred as alternative site selection (ASS) events and Intron retention (IR) events. By clustering analysis, we identified a subset of events (26 ASS events and 10 IR events) that show periodic changes in the splicing pattern during the cell cycle. About two-thirds of these 36 genes either introduce a pre-termination codon (PTC) or introduce insertions or deletions into functional domains of the proteins, which implicate splicing in altering gene function. These findings suggest that alternative splicing is also regulated during the Chlamydomonas cell cycle, although not as extensively as changes in gene expression. The longitudinal changes in the alternative splicing pattern during the cell cycle captured by this study provides an important resource to investigate alternative splicing in genes of interest during the cell cycle in Chlamydomonas reinhardtii and other eukaryotes