The changing paradigm of intron retention: regulation, ramifications and recipes

Intron retention (IR) is a form of alternative splicing that has long been neglected in mammalian systems although it has been studied for decades in non-mammalian species such as plants, fungi, insects and viruses. It was generally assumed that mis-splicing, leading to the retention of introns, would have no physiological consequence other than reducing gene expression by nonsense-mediated decay. Relatively recent landmark discoveries have highlighted the pivotal role that IR serves in normal and disease-related human biology. Significant technical hurdles have been overcome, thereby enabling the robust detection and quantification of IR. Still, relatively little is known about the cis- and trans-acting modulators controlling this phenomenon. The fate of an intron to be, or not to be, retained in the mature transcript is the direct result of the influence exerted by numerous intrinsic and extrinsic factors at multiple levels of regulation. These factors have altered current biological paradigms and provided unexpected insights into the transcriptional landscape. In this review, we discuss the regulators of IR and methods to identify them. Our focus is primarily on mammals, however, we broaden the scope to non-mammalian organisms in which IR has been shown to be biologically relevant

Post-Transcriptional Regulation Of The Eulkaryotic Transcriptome By The Covalent Rna Modicication N6-Methyladenosine

Post-Transcriptional regulation of the eukaryotic transcriptome by the covalent RNA modification N6-methyladenosine Stephen James Anderson Brian Gregory Once a messenger RNA molecule is transcribed, a myriad of RNA fate decisions must be made. How these fate decisions are made is often unclear, and elucidating factors determining these fate outcomes is an essential task in order to fully understand gene regulation. One poorly- understood but undoubtedly important factor in post-transcriptional gene regulation is the covalent modification of ribonucleotides. Much like DNA can have chemical groups added to a nucleotide within its primary sequence, RNA can be modified in a similar manner. These covalent modifications of RNA are a ubiquitous feature found within the RNA of all organisms. Dozens of these modifications have been described to date, yet the function or importance of most of these modifications remains unclear. One crucial RNA modification is N6-methyladenosine (m6A), as it is the most abundant known non-cap modification within the eukaryotic transcriptome. In this work, we characterize the role of m6A in the Arabidopsis transcriptome using various sequencing methods that demonstrate that m6A is an abundant mark that is largely maintained across differing Arabidopsis tissues and developmental stages. This prevalent mark promotes transcript stability in mNRAs involved in many important and diverse biological processes, such as salt stress. The absence of this mark results in endonucleolytic cleavage and degradation of the transcript in a highly specific and local manner. We further demonstrate that this modification modulates secondary structure throughout the transcriptome, and that m6A is associated with changes in RNA-binding protein association. Lastly, we turn our view to how an association between m6A and the m6A-specific binding protein YTHDC1 influences the development and transcriptome-wide splicing and polyadenylation pattern in the mouse germline. We demonstrate that in the absence of YTHDC1, widespread developmental, splicing, and polyadenylation defects occur, resulting in non-functional gametes. In total, this work greatly expands our knowledge and understanding of the biological importance and mechanisms of m6A-mediated post-transcriptional regulation

Next-generation sequencing identifies mechanisms of tumourigenesis caused by loss of SMARCB1 in Malignant Rhabdoid Tumours

PhD ThesisIntroduction: Malignant Rhabdoid Tumours (MRT) are unique malignancies caused by biallelic inactivation of a single gene (SMARCB1). SMARCB1 encodes for a protein that is part of the SWI/SNF chromatin remodelling complex, responsible for the regulation of hundreds of downstream genes/pathways. Despite the simple biology of these tumours, no studies have identified the critical pathways involved in tumourigenesis. The understanding of downstream effects is essential to identifying therapeutic targets that can improve the outcome of MRT patients. Methods: RNA-seq and 450K-methylation analyses have been performed in MRT human primary malignancies (n > 39) and in 4 MRT cell lines in which lentivirus was used to re-express SMARCB1 (G401, A204, CHLA-266, and STA-WT1). The MRT cell lines were treated with 5-aza-2 -deoxycytidine followed by global gene transcription analysis (RNA-seq and 450K-methylation) to investigate how changes in methylation lead to tumourigenesis. Results: We show that primary Malignant Rhabdoid Tumours present a unique and distinct expression/methylation profile which confirms that MRT broadly constitute a single and different tumour type from other paediatric malignancies. However, despite their common cause MRT can be can sub-group by location (i.e. CNS or kidney). We observe that re-expression of SMARCB1 in MRT cell lines determines activation/inactivation of specific downstream pathways such as IL-6/TGF beta. We also observe a direct correlation between alterations in methylation and gene expression in CD44, GLI2, GLI3, CDKN1A, CDKN2A and JARID after SMARB1 re-expression. Loss of SMARCB1 also promotes expression of aberrant isoforms and novel transcripts and causes genome-wide changes in SWI/SNF binding. Conclusion: Next generation transcriptome and methylome analysis in primary MRT and in functional models give us detailed downstream effects of SMARCB1 loss in Malignant Rhabdoid Tumours. The integration of data from both primary and functional models has provided, for the first time, a genome-wide catalogue of SMARCB1 tumourigenic changes (validated using systems biology). Here we show how a single V deletion of SMARCB1 is responsible for deregulation of expression, methylation status and binding at the promoter regions of potent tumour-suppressor genes. The genes, pathways and biological mechanisms indicated as key in tumour development may ultimately be targetable therapeutically and will lead to better treatments for what is currently one of the most lethal paediatric cancers.NECCR, Children with cancer UK, Brain Trust, Love Oliver, CCLG, Karen and Iain Wark, The Smiley Ridley Fund, whose financial support made this project possible

Molecular and physiological roles of long 3′ UTR mRNA isoforms in neurons

The brain is an organ where the greatest proportion of genes are expressed compared to any other part of the body. To add even more complexity, gene expression in the brain is subject to various layers of regulation through RNA processing mechanisms including alternative splicing (AS) and alternative cleavage and polyadenylation (APA). These RNA processing mechanisms contribute to increased transcriptome diversity in the brain. APA often induces the synthesis of mRNA isoforms that harbor the same protein-coding sequence but different length 3′ untranslated regions (3′ UTRs) from a single gene. Alternative 3′ UTRs regulate gene expression post-transcriptionally by modulating transcript stability, translation efficiency, or subcellular localization. In Chapter 1, we reviewed all of the reported functions of 3′ UTRs in the nervous system. Despite the fact 3′ UTR is highly regarded in gene regulation, evidence of impacts of long 3′ UTR loss on in vivo animal is scarce. To study the physiological relevance of long 3′ UTR mRNA isoforms, we have driven our attention to the Calm1 gene. Calm1 is one of the three genes that encode Calmodulin which is required for proper neural development and function. In Chapter 2, we found that the expression of the long 3′ UTR mRNA isoform of Calm1 was necessary for mouse nervous system development and function. Disruption of the Calm1 long 3′ UTR isoform impaired dorsal root ganglion axon development in mouse embryos and neuronal activation upon novel environment exposure in young adult mice. Our results presented direct evidence for the physiological importance of the Calm1 long 3′ UTR mRNA isoform in vivo. To screen molecular and cellular functions of long 3′ UTRs in a fast and efficient manner, establishing an in vitro cell system is warranted. In Chapter 3, we presented mouse embryonic stem cell (mESC)-derived neurons as a suitable cell-culture system. The transcriptomic profile of the mESC-derived neurons closely resembled the profile in the mouse cerebral cortex, showing the suitability of using this system for studying long 3′ UTRs. The mESC system is amenable to genetic manipulation via CRISPR-Cas9, thus providing as good avenue for fast generation of long 3′ UTR isoform knockout lines. As a proof of principle, a workflow for the generation of Myosin phosphatase Rho interacting protein (Mprip) long 3′ UTR isoform knockout cell lines, differentiation into glutamatergic neurons, and confirmation of the long 3′ UTR expression abolishment is presented. Taking advantage of the convenient culture cell system we have established, we next aimed to explore more functions of long 3′ UTRs. A recent discovery in our lab suggested that APA and AS are closely linked RNA processing mechanisms in which long 3′ UTRs modulate upstream AS. In Chapter 4, we explored the coupling events between AS and APA in mouse neurons using Pull-a-Long-Seq (PL-Seq) pipeline, which presents a particular utility in quantifying the coordination of tandem 3′ UTR APA events with upstream cassette exon AS. PL-Seq performed on the Endonuclease V (Endov) gene reveals that expression of its long 3′ UTR in neurons is preferentially associated with an exon skipping event located far upstream of the terminal exon

The intricate interplay between epigenetic events, alternative splicing and noncoding RNA deregulation in colorectal cancer

Colorectal cancer (CRC) results from a transformation of colonic epithelial cells into adenocarcinoma cells due to genetic and epigenetic instabilities, alongside remodelling of the surrounding stromal tumour microenvironment. Epithelial-specific epigenetic variations escorting this process include chromatin remodelling, histone modifications and aberrant DNA methylation, which influence gene expression, alternative splicing and function of non-coding RNA. In this review, we first highlight epigenetic modulators, modifiers and mediators in CRC, then we elaborate on causes and consequences of epigenetic alterations in CRC pathogenesis alongside an appraisal of the complex feedback mechanisms realized through alternative splicing and non-coding RNA regulation. An emphasis in our review is put on how this intricate network of epigenetic and post-transcriptional gene regulation evolves during the initiation, progression and metastasis formation in CRC

Distinct expression and methylation patterns for genes with different fates following a single whole-genome duplication in flowering plants

For most sequenced flowering plants, multiple whole-genome duplications (WGDs) are found. Duplicated genes following WGD often have different fates that can quickly disappear again, be retained for long(er) periods, or subsequently undergo small-scale duplications. However, how different expression, epigenetic regulation, and functional constraints are associated with these different gene fates following a WGD still requires further investigation due to successive WGDs in angiosperms complicating the gene trajectories. In this study, we investigate lotus (Nelumbo nucifera), an angiosperm with a single WGD during the K–pg boundary. Based on improved intraspecific-synteny identification by a chromosome-level assembly, transcriptome, and bisulfite sequencing, we explore not only the fundamental distinctions in genomic features, expression, and methylation patterns of genes with different fates after a WGD but also the factors that shape post-WGD expression divergence and expression bias between duplicates. We found that after a WGD genes that returned to single copies show the highest levels and breadth of expression, gene body methylation, and intron numbers, whereas the long-retained duplicates exhibit the highest degrees of protein–protein interactions and protein lengths and the lowest methylation in gene flanking regions. For those long-retained duplicate pairs, the degree of expression divergence correlates with their sequence divergence, degree in protein–protein interactions, and expression level, whereas their biases in expression level reflecting subgenome dominance are associated with the bias of subgenome fractionation. Overall, our study on the paleopolyploid nature of lotus highlights the impact of different functional constraints on gene fate and duplicate divergence following a single WGD in plant

Pattern of DNA methylation in daphnia : evolutionary perspective

DNA methylation is an evolutionary ancient epigenetic modification that is phylogenetically widespread. Comparative studies of the methylome across a diverse range of non-conventional and conventional model organisms is expected to help reveal how the landscape of DNA methylation and its functions have evolved. Here, we explore the DNA methylation profile of two species of the crustacean Daphnia using whole genome bisulfite sequencing. We then compare our data with the methylomes of two insects and two mammals to achieve a better understanding of the function of DNA methylation in Daphnia. Using RNA-sequencing data for all six species, we investigate the correlation between DNA methylation and gene expression. DNA methylation in Daphnia is mainly enriched within the coding regions of genes, with the highest methylation levels observed at exons 2-4. In contrast, vertebrate genomes are globally methylated, and increase towards the highest methylation levels observed at exon 2, and maintained across the rest of the gene body. Although DNA methylation patterns differ among all species, their methylation profiles share a bimodal distribution across the genomes. Genes with low levels of CpG methylation and gene expression are mainly enriched for species specific genes. In contrast, genes associated with high methylated CpG sites are highly transcribed and evolutionary conserved across all species. Finally, the positive correlation between internal exons and gene expression potentially points to an evolutionary conserved mechanism, whereas the negative regulation of gene expression via methylation of promoters and exon 1 is potentially a secondary mechanism that has been evolved in vertebrates

U6 snRNA m6A modification is required for accurate and efficient cis- and trans-splicing of <i>C. elegans</i> mRNAs

pre-mRNA splicing is a critical feature of eukaryotic gene expression. Many eukaryotes use cis-splicing to remove intronic sequences from pre-mRNAs. In addition to cis-splicing, many organisms use trans-splicing to replace the 5′ ends of mRNAs with a non-coding spliced-leader RNA. Both cis- and trans-splicing rely on accurately recognising splice site sequences by spliceosomal U snRNAs and associated proteins. Spliceosomal snRNAs carry multiple RNA modifications with the potential to affect different stages of pre-mRNA splicing. Here, we show that m6A modification of U6 snRNA A43 by the RNA methyltransferase METT-10 is required for accurate and efficient cis- and trans-splicing of C. elegans pre-mRNAs. The absence of U6 snRNA m6A modification primarily leads to alternative splicing at 5′ splice sites. Furthermore, weaker 5′ splice site recognition by the unmodified U6 snRNA A43 affects splicing at 3′ splice sites. U6 snRNA m6A43 and the splicing factor SNRNP27K function to recognise an overlapping set of 5′ splice sites with an adenosine at +4 position. Finally, we show that U6 snRNA m6A43 is required for efficient SL trans-splicing at weak 3′ trans-splice sites. We conclude that the U6 snRNA m6A modification is important for accurate and efficient cis- and trans-splicing in C. elegans