Type IV collagen is an integral component of basement membranes. Mutations in COL4A1, one of the key genes encoding Type IV collagen, can result in a variety of diseases. It is clear that a significant proportion of mutations that affect splicing can cause disease directly or contribute to the susceptibility or severity of disease. Here, we analyzed exonic mutations and intronic mutations described in the COL4A1 gene using bioinformatics programs and identified candidate mutations that may alter the normal splicing pattern through a minigene system. We identified seven variants that induce splicing alterations by disrupting normal splice sites, creating new ones, or altering splice regulatory elements. These mutations are predicted to impact protein function. Our results help in the correct molecular characterization of variants in COL4A1 and may help develop more personalized treatment options.

Disruptions in spatiotemporal gene expression can result in atypical brain function. Specifically, autism spectrum disorder (ASD) is characterized by abnormalities in pre-mRNA splicing. Abnormal splicing patterns have been identified in the brains of individuals with ASD, and mutations in splicing factors have been found to contribute to neurodevelopmental delays associated with ASD. Here we review studies that shed light on the importance of splicing observed in ASD and that explored the intricate relationship between splicing factors and ASD, revealing how disruptions in pre-mRNA splicing may underlie ASD pathogenesis. We provide an overview of the research regarding all splicing factors associated with ASD and place a special emphasis on five specific splicing factors-HNRNPH2, NOVA2, WBP4, SRRM2, and RBFOX1-known to impact the splicing of ASD-related genes. In the discussion of the molecular mechanisms influenced by these splicing factors, we lay the groundwork for a deeper understanding of ASD\'s complex etiology. Finally, we discuss the potential benefit of unraveling the connection between splicing and ASD for the development of more precise diagnostic tools and targeted therapeutic interventions. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Evolution and Genomics > Computational Analyses of RNA RNA-Based Catalysis > RNA Catalysis in Splicing and Translation.

RNA splicing has emerged as a critical player in the DNA damage response (DDR). However, the underlying mechanism(s) by which pre-mRNA splicing is coordinately regulated by genotoxic stress has remained largely unclear. Here, we show that a DDR factor, DNA-dependent protein kinase (DNA-PK), participates in the modulation of pre-mRNA splicing in the presence of DNA double-strand break (DSB)-induced genotoxic stress. Through indirect immunostaining, we made the surprising discovery that DNA-PK catalytic subunits (DNA-PKcs) autophosphorylated at serine 2056 (S2056) accumulate at nuclear speckles (dynamic nuclear structures that are enriched with splicing factors), following their dissociation from DSB lesions. Inactivation of DNA-PKcs, either using a small molecule inhibitor or by RNA interference, alters alternative splicing of a set of pre-mRNAs in A549 cells treated with the topoisomerase II inhibitor mitoxantrone, indicative of an involvement of DNA-PKcs in modulating pre-mRNA splicing following genotoxic stress. These findings indicate a novel physical and functional connection between the DNA damage response and pre-mRNA splicing, and enhance our understanding of how mRNA splicing is involved in the cellular response to DSB lesions.

N6-methyladenosine (m6A) is a highly dynamic RNA modification that has recently emerged as a key regulator of gene expression. While many m6A modifications are installed by the METTL3-METTL14 complex, others appear to be introduced independently, implying that additional human m6A methyltransferases remain to be identified. Using crosslinking and analysis of cDNA (CRAC), we reveal that the putative human m6A \"writer\" protein METTL16 binds to the U6 snRNA and other ncRNAs as well as numerous lncRNAs and pre-mRNAs. We demonstrate that METTL16 is responsible for N6-methylation of A43 of the U6 snRNA and identify the early U6 biogenesis factors La, LARP7 and the methylphosphate capping enzyme MEPCE as METTL16 interaction partners. Interestingly, A43 lies within an essential ACAGAGA box of U6 that base pairs with 5\' splice sites of pre-mRNAs during splicing, suggesting that METTL16-mediated modification of this site plays an important role in splicing regulation. The identification of METTL16 as an active m6A methyltransferase in human cells expands our understanding of the mechanisms by which the m6A landscape is installed on cellular RNAs.

Initiation of zygotic transcription in mammals is poorly understood. In mice, zygotic transcription is first detected shortly after pronucleus formation in 1-cell embryos, but the identity of the transcribed loci and mechanisms regulating their expression are not known. Using total RNA-Seq, we have found that transcription in 1-cell embryos is highly promiscuous, such that intergenic regions are extensively expressed and thousands of genes are transcribed at comparably low levels. Striking is that transcription can occur in the absence of defined core-promoter elements. Furthermore, accumulation of translatable zygotic mRNAs is minimal in 1-cell embryos because of inefficient splicing and 3\' processing of nascent transcripts. These findings provide novel insights into regulation of gene expression in 1-cell mouse embryos that may confer a protective mechanism against precocious gene expression that is the product of a relaxed chromatin structure present in 1-cell embryos. The results also suggest that the first zygotic transcription itself is an active component of chromatin remodeling in 1-cell embryos.

Although splicing is essential for the expression of most eukaryotic genes, inactivation of splicing factors causes specific defects in mitosis. The molecular cause of this defect is unknown. Here, we show that the spliceosome subunits SNW1 and PRPF8 are essential for sister chromatid cohesion in human cells. A transcriptome-wide analysis revealed that SNW1 or PRPF8 depletion affects the splicing of specific introns in a subset of pre-mRNAs, including pre-mRNAs encoding the cohesion protein sororin and the APC/C subunit APC2. SNW1 depletion causes cohesion defects predominantly by reducing sororin levels, which causes destabilisation of cohesin on DNA. SNW1 depletion also reduces APC/C activity and contributes to cohesion defects indirectly by delaying mitosis and causing \"cohesion fatigue\". Simultaneous expression of sororin and APC2 from intron-less cDNAs restores cohesion in SNW1-depleted cells. These results indicate that the spliceosome is required for mitosis because it enables expression of genes essential for cohesion. Our transcriptome-wide identification of retained introns in SNW1- and PRPF8-depleted cells may help to understand the aetiology of diseases associated with splicing defects, such as retinosa pigmentosum and cancer.

UBL5 is an atypical ubiquitin-like protein, whose function in metazoans remains largely unexplored. We show that UBL5 is required for sister chromatid cohesion maintenance in human cells. UBL5 primarily associates with spliceosomal proteins, and UBL5 depletion decreases pre-mRNA splicing efficiency, leading to globally enhanced intron retention. Defective sister chromatid cohesion is a general consequence of dysfunctional pre-mRNA splicing, resulting from the selective downregulation of the cohesion protection factor Sororin. As the UBL5 yeast orthologue, Hub1, also promotes spliceosome functions, our results show that UBL5 plays an evolutionary conserved role in pre-mRNA splicing, the integrity of which is essential for the fidelity of chromosome segregation.

In Arabidopsis, pre-mRNAs of serine/arginine-rich (SR) proteins undergo extensive alternative splicing (AS). However, little is known about the cis-elements and trans-acting proteins involved in regulating AS. Using a splicing reporter (GFP-intron-GFP), consisting of the GFP coding sequence interrupted by an alternatively spliced intron of SCL33, we investigated whether cis-elements within this intron are sufficient for AS, and which SR proteins are necessary for regulated AS. Expression of the splicing reporter in protoplasts faithfully produced all splice variants from the intron, suggesting that cis-elements required for AS reside within the intron. To determine which SR proteins are responsible for AS, the splicing pattern of the GFP-intron-GFP reporter was investigated in protoplasts of three single and three double mutants of SR genes. These analyses revealed that SCL33 and a closely related paralog, SCL30a, are functionally redundant in generating specific splice variants from this intron. Furthermore, SCL33 protein bound to a conserved sequence in this intron, indicating auto-regulation of AS. Mutations in four GAAG repeats within the conserved region impaired generation of the same splice variants that are affected in the scl33 scl30a double mutant. In conclusion, we have identified the first intronic cis-element involved in AS of a plant SR gene, and elucidated a mechanism for auto-regulation of AS of this intron.