Single cell measurement of telomerase expression and splicing using microfluidic emulsion cultures.

Telomerase is a reverse transcriptase that maintains telomeres on the ends of chromosomes, allowing rapidly dividing cells to proliferate while avoiding senescence and apoptosis. Understanding telomerase gene expression and splicing at the single cell level could yield insights into the roles of telomerase during normal cell growth as well as cancer development. Here we use droplet-based single cell culture followed by single cell or colony transcript abundance analysis to investigate the relationship between cell growth and transcript abundance of the telomerase genes encoding the RNA component (hTR) and protein component (hTERT) as well as hTERT splicing. Jurkat and K562 cells were examined under normal cell culture conditions and during exposure to curcumin, a natural compound with anti-carcinogenic and telomerase activity-reducing properties. Individual cells predominantly express single hTERT splice variants, with the α+/β- variant exhibiting significant transcript abundance bimodality that is sustained through cell division. Sub-lethal curcumin exposure results in reduced bimodality of all hTERT splice variants and significant upregulation of alpha splicing, suggesting a possible role in cellular stress response. The single cell culture and transcript abundance analysis method presented here provides the tools necessary for multiparameter single cell analysis which will be critical for understanding phenotypes of heterogeneous cell populations, disease cell populations and their drug response

Mechanisms of MiRNA-based Gene Regulation in C. elegans and Human Cells

abstract: Multicellular organisms use precise gene regulation, executed throughout development, to build and sustain various cell and tissue types. Post-transcriptional gene regulation is essential for metazoan development and acts on mRNA to determine its localization, stability, and translation. MicroRNAs (miRNAs) and RNA binding proteins (RBPs) are the principal effectors of post-transcriptional gene regulation and act by targeting the 3'untranslated regions (3'UTRs) of mRNA. MiRNAs are small non-coding RNAs that have the potential to regulate hundreds to thousands of genes and are dysregulated in many prevalent human diseases such as diabetes, Alzheimer's disease, Duchenne muscular dystrophy, and cancer. However, the precise contribution of miRNAs to the pathology of these diseases is not known. MiRNA-based gene regulation occurs in a tissue-specific manner and is implemented by an interplay of poorly understood and complex mechanisms, which control both the presence of the miRNAs and their targets. As a consequence, the precise contributions of miRNAs to gene regulation are not well known. The research presented in this thesis systematically explores the targets and effects of miRNA-based gene regulation in cell lines and tissues. I hypothesize that miRNAs have distinct tissue-specific roles that contribute to the gene expression differences seen across tissues. To address this hypothesis and expand our understanding of miRNA-based gene regulation, 1) I developed the human 3'UTRome v1, a resource for studying post-transcriptional gene regulation. Using this resource, I explored the targets of two cancer-associated miRNAs miR-221 and let-7c. I identified novel targets of both these miRNAs, which present potential mechanisms by which they contribute to cancer. 2) Identified in vivo, tissue-specific targets in the intestine and body muscle of the model organism Caenorhabditis elegans. The results from this study revealed that miRNAs regulate tissue homeostasis, and that alternative polyadenylation and miRNA expression patterns modulate miRNA targeting at the tissue-specific level. 3) Explored the functional relevance of miRNA targeting to tissue-specific gene expression, where I found that miRNAs contribute to the biogenesis of mRNAs, through alternative splicing, by regulating tissue-specific expression of splicing factors. These results expand our understanding of the mechanisms that guide miRNA targeting and its effects on tissue-specific gene expression.Dissertation/ThesisDoctoral Dissertation Molecular and Cellular Biology 201

A Gene-Centered Method For Mapping 3’UTR-RBP Interactions: A Dissertation

Interactions between 3´ untranslated regions (UTRs) and RNA-binding proteins (RBPs) play critical roles in post-transcriptional gene regulation. Metazoan genomes encode hundreds of RBPs and thousands of 3’ UTRs have been experimentally identified, yet the spectrum of interactions between 3´UTRs and RBPs remains largely unknown. Several methods are available to map these interactions, including protein-centered methods such as RBP immunoprecipitation (RIP) and cross-link immunoprecipitation (CLIP), yeast three-hybrid assays and RNAcompete. However, there is a paucity of RNA-centered approaches for assaying an RNA element of interest against multiple RBPs in a parallel, scalable manner. Here, I present a strategy for delineating protein-RNA interaction networks using a gene centered approach. This approach includes annotating RBPs and identifying physical interactions between an RNA of interest and these RBPs using the Protein-RNA Interaction Mapping Assay (PRIMA). Few RBPs have been experimentally determined in most eukaryotic organisms. Therefore I show that existing RBP annotations can be supplemented using computational predictions of RNA binding domains (RBD) from protein sequences. A single RNA of interest can be tested using PRIMA against a library of RBPs constructed from these annotations. PRIMA utilizes the green fluorescent protein (GFP) in yeast as a reporter. PRIMA is based on reconstitution of the interaction between the 5´ and 3´ ends of an mRNA, which increases mRNA stability and enhances translation. PRIMA recapitulates known and uncovers new interactions involving RBPs from human, Caenorhabditis elegans and bacteriophage with short RNA fragments and full-length 3´UTRs. The development of RBP prey libraries will enable the testing of 3´UTRs against the hundreds of RBPs, which is essential to gain broad insights into post-transcriptional gene regulation at a systems level

Functional characterisation of mutations in the SRRM2 gene associated with risk of Non-Syndromic familial Non-Medullary thyroid cancer

Non-syndromic familial non-medullary thyroid cancer (FNMTC) is the most common malignancy of the endocrine system and is defined by the presence of non-medullary thyroid cancer (NMTC) in 2 or more first-degree relatives in the absence of predisposing environmental factors and a recognized thyroid cancer syndrome. There is a clear genetic basis to FNMTC, however, details of the genes involved is limited; a number of susceptibility genes have been proposed but these remain controversial. SRRM2 is a splicing factor that promotes exonic enhancer-dependent splicing by mediating critical interactions between the spliceosomal U2 snRNP and additional splicing factors bound directly to pre-mRNA. SRRM2 is also associated with cell cycle control, where has been shown to associate with the histone H4 subtype transcriptional regulator HiNF-P and is hypothesized that the SRRM2/ HiNF-P complex may regulate histone production via a p220NPAT- dependent pathway. SRRM2 was previously identified as a putative susceptibility loci for FNMTC, where a missense variant (S346F) was found to co-segregate with FNMTC in a well-documented FNMTC family. RNA-seq of leukocytes revealed altered splicing patterns for 1,642 exons in the FNMTC patients. The altered splicing pattern for 7 exons was experimentally verified using semi-quantitative PCR. In addition to the S346F mutation, 6 SRRM2 variants have been identified in the Western Australian FNMTC cohort: one variant co-segregated with FNMTC and was predicted by in silico methods to be pathogenic (R1805W), and the others of unknown significance. The first aim of this study was to generate model cell systems of SRRM2 mutations previously associated with FNMTC predisposition. The second aim was to investigate whether these mutant cell systems show differences in the splicing patterns of specific pre-mRNAs through quantitative real-time PCR. The final aim was to employ flow cytometry techniques to assess the proliferative characteristics of the mutant cell systems and determine whether these mutations prevent proper regulation of the cell cycle. The original project plan was to generate the model cell systems by performing a transient over-expression analysis in HEK293 and Nthy-ori 3-1 cell lines transfected vector constructs containing the wild type or mutant SRRM2 sequences. However, during the construction of the mutant plasmids, the transient over-expression analysis was abandoned due to challenges associated with the size of the SRRM2 ORF. The CRISPR/Cas9 system was used instead to generate model cell systems of SRRM2 mutations in the HAP1 cell line. CRISPR/Cas9 was successfully implemented to generate a mutant HAP1 cell line harboring the R1805W mutation, however, the cell line was heterozygous for the mutation and the wild type sequence. RT-qPCR was performed to characterise the splicing pattern of the wild type and mutant cell lines to determine whether there was a difference in splicing for 7 exons previously shown to be differentially spliced in FNMTC patients heterozygous for the S346F mutation. No significant differences were identified between the wild type and mutant cell lines, suggesting that the R1805W mutation does not alter the splicing pattern of the 7 exons investigated. Cell cycle analysis was performed to quantify the percentages of cells in the G0/G1 and G2/M phases of the cell cycle using propidium iodide staining and quantification of G0/G1 and G2/M peak fluorescence intensity. The experiments were completed successfully in this regard; however, no significant differences were observed between the wild type and mutant cell lines. The proliferation kinetics of the two cell lines were characterised with a proliferation assay using carboxyfluorescein succinimidyl ester staining and the dye dilution method, however, no significant differences were observed in the proliferation metrics quantified. Whilst preliminary, these findings suggest that the R1805W mutation does not predispose to FNMTC through the altered splicing of the 7 exons investigated, or by altering proper regulation of the cell cycle. Further investigations are to properly interrogate the association of this this mutation to FNMTC, as well as the other mutatio

Design, Construction, and Screening of an shRNA Library Targeting Human Circular RNAs

Recent advances in next-generation sequencing (NGS) methods and computational analyses have identified a large class of non-polyadenylated RNA molecules. Further, analysis of these RNAs revealed several unexpected junctions, which do not map to mRNAs, leading to the discovery of circular RNAs (circRNAs). Unlike linear RNAs, circRNAs have no ends and are not sensitive to exoribonucleases, endowing circRNAs with a longer half-life. This enhanced stability has prompted the study of circRNAs as cancer biomarkers. While expression studies are becoming widely used to profile circRNAs in multiple cancers, there are no genome-wide tools available to decrease their levels in cells. Methods that investigate the role of circRNAs through measuring their expression are prone to artifacts as bypassing transcription termination results in RNA concatamers. To better characterize the function of circRNAs, we developed a novel pooled library of ~ 15,000 shRNAs targeting ~ 5,000 circRNAs. We performed a loss-of-function screen with the circRNA shRNA library in a colorectal cancer cell line to systematically identify circRNAs that were required for cell proliferation and survival. During the construction and validation of this library, we also developed a method to improve NGS quality by reducing sequencing failure due to shRNA hairpin and/or heteroduplex formation. Using this approach, we identified and validated several circRNAs essential for the survival of colorectal cancer cell lines. We believe that these essential circRNAs will provide an opportunity to understand cancer biology in a more detailed way, and to design effective cancer therapeutics and diagnostics

Elongation Factor TFIIS Prevents Transcription Stress and R-Loop Accumulation to Maintain Genome Stability

Although correlations between RNA polymerase II (RNAPII) transcription stress, R-loops, and genome instability have been established, the mechanisms underlying these connections remain poorly understood. Here, we used a mutant version of the transcription elongation factor TFIIS (TFIISmut), aiming to specifically induce increased levels of RNAPII pausing, arrest, and/or backtracking in human cells. Indeed, TFIISmut expression results in slower elongation rates, relative depletion of polymerases from the end of genes, and increased levels of stopped RNAPII; it affects mRNA splicing and termination as well. Remarkably, TFIISmut expression also dramatically increases R-loops, which may form at the anterior end of backtracked RNAPII and trigger genome instability, including DNA strand breaks. These results shed light on the relationship between transcription stress and R-loops and suggest that different classes of R-loops may exist, potentially with distinct consequences for genome stability.Cancer Research UK FC001166UK Medical Research Council FC001166Wellcome Trust FC001166European Research Council 693327, ERC2014 AdG669898Ministerio de Economía y Competitividad BFU2013-42918-P, BFU2016-75058-

A Role for Pre-mRNA-PROCESSING PROTEIN 40C in the Control of Growth, Development, and Stress Tolerance in Arabidopsis thaliana

Because of their sessile nature, plants have adopted varied strategies for growing and reproducing in an ever-changing environment. Control of mRNA levels and pre-mRNA alternative splicing are key regulatory layers that contribute to adjust and synchronize plant growth and development with environmental changes. Transcription and alternative splicing are thought to be tightly linked and coordinated, at least in part, through a network of transcriptional and splicing regulatory factors that interact with the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II. One of the proteins that has been shown to play such a role in yeast and mammals is pre-mRNA-PROCESSING PROTEIN 40 (PRP40, also known as CA150, or TCERG1). In plants, members of the PRP40 family have been identified and shown to interact with the CTD of RNA Pol II, but their biological functions remain unknown. Here, we studied the role of AtPRP40C, in Arabidopsis thaliana growth, development and stress tolerance, as well as its impact on the global regulation of gene expression programs. We found that the prp40c knockout mutants display a late-flowering phenotype under long day conditions, associated with minor alterations in red light signaling. An RNA-seq based transcriptome analysis revealed differentially expressed genes related to biotic stress responses and also differentially expressed as well as differentially spliced genes associated with abiotic stress responses. Indeed, the characterization of stress responses in prp40c mutants revealed an increased sensitivity to salt stress and an enhanced tolerance to Pseudomonas syringae pv. maculicola (Psm) infections. This constitutes the most thorough analysis of the transcriptome of a prp40 mutant in any organism, as well as the first characterization of the molecular and physiological roles of a member of the PRP40 protein family in plants. Our results suggest that PRP40C is an important factor linking the regulation of gene expression programs to the modulation of plant growth, development, and stress responses.Fil: Hernando, Carlos Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: García Hourquet, Mariano. Fundación Instituto Leloir; ArgentinaFil: de Leone, María José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Careno, Daniel Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Iserte, Javier Alonso. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Mora Garcia, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Yanovsky, Marcelo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Understanding the Imprinting Mechanism of UBE3A for Therapeutic Intervention

Human chromosome 15q11-q13 contains a cluster of imprinted genes that are associated with a number of neurological disorders that exhibit non-Mendelian patterns of inheritance, such as Angelman syndrome (AS) and Prader-Willi syndrome. Angelman syndrome is caused by the loss-of-expression of maternally inherited ubiquitin E3A protein ligase gene (UBE3A). Prader-Willi syndrome is caused by loss-of-function of paternally inherited SNORD116 snoRNAs (small nucleolar RNAs), which are expressed as part of a long polycistronic transcriptional unit (PTU) comprised of SNURF-SNRPN, additional orphan C/D box snoRNA clusters, and the UBE3A antisense transcript (UBE3A-AS). The full-length transcript of PTU, including UBE3A-AS, is only expressed in neurons causing the imprinting of paternal UBE3A. Why this occurs in only neurons remains largely unknown. Furthermore, this neuron-specific imprinting adds additional difficulty for therapeutic intervention. In this dissertation, the imprinting mechanism of UBE3A is examined in detail, while an alternative high-throughput screening (HTS) method for drug discovery in neurons is developed. A combination of bioinformatic and molecular analysis of the human and mouse PTU revealed that UBE3A-AS/Ube3a-AS is extensively processed via 5’ capping 3’polyadenyation and alternative splicing, suggesting that the antisense may have regulatory functions apart from imprinting UBE3A in neurons. Following this discovery, the transcriptional profiles and processing of mouse paternal Ube3a was investigated as literature suggested that imprinted paternal Ube3a, unlike other imprinted genes, was transcribed up to intron 4. This analysis unveiled a fourth Ube3a isoform that terminates within intron 4. Moreover, expression of this isoform correlated with Ube3a-AS expression, suggesting alternative reasons for the imprinting of Ube3a. In addition to the analysis of the imprinting of Ube3a, an alternative solution for drug discovery for central nervous system disorders was developed and validated. Here, an embryonic stem cell-derived neuronal culture system was developed for HTS and tested using the paternal Ube3a^Y FP reporter cell-line. Using a known reactivator of paternal Ube3a, Topotecan - a topoisomerase inhibitor, as a positive control a proof-of-concept study demonstrated the utility of this method for HTS drug discovery. Collectively, these results advance the field and understanding of antisense lncRNAs and provide a versatile tool for drug discovery for neurological disorders

Prmt5 is a regulator of muscle stem cell expansion in adult mice.

Skeletal muscle stem cells (MuSC), also called satellite cells, are indispensable for maintenance and regeneration of adult skeletal muscles. Yet, a comprehensive picture of the regulatory events controlling the fate of MuSC is missing. Here, we determine the proteome of MuSC to design a loss-of-function screen, and identify 120 genes important for MuSC function including the arginine methyltransferase Prmt5. MuSC-specific inactivation of Prmt5 in adult mice prevents expansion of MuSC, abolishes long-term MuSC maintenance and abrogates skeletal muscle regeneration. Interestingly, Prmt5 is dispensable for proliferation and differentiation of Pax7(+) myogenic progenitor cells during mouse embryonic development, indicating significant differences between embryonic and adult myogenesis. Mechanistic studies reveal that Prmt5 controls proliferation of adult MuSC by direct epigenetic silencing of the cell cycle inhibitor p21. We reason that Prmt5 generates a poised state that keeps MuSC in a standby mode, thus allowing rapid MuSC amplification under disease conditions