Counteracting chromatin effects of a splicing-correcting antisense oligonucleotide improves its therapeutic efficacy in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a motor-neuron disease caused by mutations of the SMN1 gene. The human paralog SMN2, whose exon 7 (E7) is predominantly skipped, cannot compensate for the lack of SMN1. Nusinersen is an antisense oligonucleotide (ASO) that upregulates E7 inclusion and SMN protein levels by displacing the splicing repressors hnRNPA1/A2 from their target site in intron 7. We show that by promoting transcriptional elongation, the histone deacetylase inhibitor VPA cooperates with a nusinersen-like ASO to promote E7 inclusion. Surprisingly, the ASO promotes the deployment of the silencing histone mark H3K9me2 on the SMN2 gene, creating a roadblock to RNA polymerase II elongation that inhibits E7 inclusion. By removing the roadblock, VPA counteracts the chromatin effects of the ASO, resulting in higher E7 inclusion without large pleiotropic effects. Combined administration of the nusinersen-like ASO and VPA in SMA mice strongly synergizes SMN expression, growth, survival, and neuromuscular function.

The physiology of alternative splicing.

Alternative splicing is a substantial contributor to the high complexity of transcriptomes of multicellular eukaryotes. In this Review, we discuss the accumulated evidence that most of this complexity is reflected at the protein level and fundamentally shapes the physiology and pathology of organisms. This notion is supported not only by genome-wide analyses but, mainly, by detailed studies showing that global and gene-specific modulations of alternative splicing regulate highly diverse processes such as tissue-specific and species-specific cell differentiation, thermal regulation, neuron self-avoidance, infrared sensing, the Warburg effect, maintenance of telomere length, cancer and autism spectrum disorders (ASD). We also discuss how mastering the control of alternative splicing paved the way to clinically approved therapies for hereditary diseases.

Regulation of alternative splicing through coupling with transcription and chromatin structure.

Alternative precursor messenger RNA (pre-mRNA) splicing plays a pivotal role in the flow of genetic information from DNA to proteins by expanding the coding capacity of genomes. Regulation of alternative splicing is as important as regulation of transcription to determine cell- and tissue-specific features, normal cell functioning, and responses of eukaryotic cells to external cues. Its importance is confirmed by the evolutionary conservation and diversification of alternative splicing and the fact that its deregulation causes hereditary disease and cancer. This review discusses the multiple layers of cotranscriptional regulation of alternative splicing in which chromatin structure, DNA methylation, histone marks, and nucleosome positioning play a fundamental role in providing a dynamic scaffold for interactions between the splicing and transcription machineries. We focus on evidence for how the kinetics of RNA polymerase II (RNAPII) elongation and the recruitment of splicing factors and adaptor proteins to chromatin components act in coordination to regulate alternative splicing.

Seeking the truth behind the myth: Argonaute tales from "nuclearland".

Argonaute proteins have been traditionally characterized as a highly evolutionary conserved family engaged in post-transcriptional gene silencing pathways. The Argonaute family is mainly grouped into the AGO and PIWI clades. The canonical role of Argonaute proteins relies on their ability to bind small-RNAs that recognize complementary sequences on target mRNAs to induce either mRNA degradation or translational repression. However, there is an increasing amount of evidence supporting that Argonaute proteins also exert multiple nuclear functions that subsequently regulate gene expression. In this line, genome-wide studies showed that members from the AGO clade regulate transcription, 3D chromatin organization, and splicing of active loci located within euchromatin. Here, we discuss recent work based on high-throughput technologies that have significantly contributed to shed light on the multivariate nuclear functions of AGO proteins in different model organisms. We also analyze data supporting that AGO proteins are able to execute these nuclear functions independently from small RNA pathways. Finally, we integrate these mechanistic insights with recent reports highlighting the clinical importance of AGO in breast and prostate cancer development.

Light Regulates Plant Alternative Splicing through the Control of Transcriptional Elongation.

Light makes carbon fixation possible, allowing plant and animal life on Earth. We have previously shown that light regulates alternative splicing in plants. Light initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing of a subset of Arabidopsis thaliana transcripts. Here, we show that light promotes RNA polymerase II (Pol II) elongation in the affected genes, whereas in darkness, elongation is lower. These changes in transcription are consistent with elongation causing the observed changes in alternative splicing, as revealed by different drug treatments and genetic evidence. The light control of splicing and elongation is abolished in an Arabidopsis mutant defective in the transcription factor IIS (TFIIS). We report that the chloroplast control of nuclear alternative splicing in plants responds to the kinetic coupling mechanism found in mammalian cells, providing unique evidence that coupling is important for a whole organism to respond to environmental cues.

Light regulates widespread plant alternative polyadenylation through the chloroplast.

Transcription of eukaryotic protein-coding genes generates immature mRNAs that are subjected to a series of processing events, including capping, splicing, cleavage, and polyadenylation (CPA), and chemical modifications of bases. Alternative polyadenylation (APA) greatly contributes to mRNA diversity in the cell. By determining the length of the 3' untranslated region, APA generates transcripts with different regulatory elements, such as miRNA and RBP binding sites, which can influence mRNA stability, turnover, and translation. In the model plant Arabidopsis thaliana, APA is involved in the control of seed dormancy and flowering. In view of the physiological importance of APA in plants, we decided to investigate the effects of light/dark conditions and compare the underlying mechanisms to those elucidated for alternative splicing (AS). We found that light controls APA in approximately 30% of Arabidopsis genes. Similar to AS, the effect of light on APA requires functional chloroplasts, is not affected in mutants of the phytochrome and cryptochrome photoreceptor pathways, and is observed in roots only when the communication with the photosynthetic tissues is not interrupted. Furthermore, mitochondrial and TOR kinase activities are necessary for the effect of light. However, unlike AS, coupling with transcriptional elongation does not seem to be involved since light-dependent APA regulation is neither abolished in mutants of the TFIIS transcript elongation factor nor universally affected by chromatin relaxation caused by histone deacetylase inhibition. Instead, regulation seems to correlate with changes in the abundance of constitutive CPA factors, also mediated by the chloroplast.

Beyond transcription: compelling open questions in plant RNA biology.

The study of RNAs has become one of the most influential research fields in contemporary biology and biomedicine. In the last few years, new sequencing technologies have produced an explosion of new and exciting discoveries in the field but have also given rise to many open questions. Defining these questions, together with old, long-standing gaps in our knowledge, is the spirit of this article. The breadth of topics within RNA biology research is vast, and every aspect of the biology of these molecules contains countless exciting open questions. Here, we asked 12 groups to discuss their most compelling question among some plant RNA biology topics. The following vignettes cover RNA alternative splicing; RNA dynamics; RNA translation; RNA structures; R-loops; epitranscriptomics; long non-coding RNAs; small RNA production and their functions in crops; small RNAs during gametogenesis and in cross-kingdom RNA interference; and RNA-directed DNA methylation. In each section, we will present the current state-of-the-art in plant RNA biology research before asking the questions that will surely motivate future discoveries in the field. We hope this article will spark a debate about the future perspective on RNA biology and provoke novel reflections in the reader.

Epigenetics in alternative pre-mRNA splicing.

Alternative splicing plays critical roles in differentiation, development, and disease and is a major source for protein diversity in higher eukaryotes. Analysis of alternative splicing regulation has traditionally focused on RNA sequence elements and their associated splicing factors, but recent provocative studies point to a key function of chromatin structure and histone modifications in alternative splicing regulation. These insights suggest that epigenetic regulation determines not only what parts of the genome are expressed but also how they are spliced.

Alternative splicing: a pivotal step between eukaryotic transcription and translation.

Alternative splicing was discovered simultaneously with splicing over three decades ago. Since then, an enormous body of evidence has demonstrated the prevalence of alternative splicing in multicellular eukaryotes, its key roles in determining tissue- and species-specific differentiation patterns, the multiple post- and co-transcriptional regulatory mechanisms that control it, and its causal role in hereditary disease and cancer. The emerging evidence places alternative splicing in a central position in the flow of eukaryotic genetic information, between transcription and translation, in that it can respond not only to various signalling pathways that target the splicing machinery but also to transcription factors and chromatin structure.

DNA damage regulates alternative splicing through inhibition of RNA polymerase II elongation.

DNA damage induces apoptosis and many apoptotic genes are regulated via alternative splicing (AS), but little is known about the control mechanisms. Here we show that ultraviolet irradiation (UV) affects cotranscriptional AS in a p53-independent way, through the hyperphosphorylation of RNA polymerase II carboxy-terminal domain (CTD) and a subsequent inhibition of transcriptional elongation, estimated in vivo and in real time. Phosphomimetic CTD mutants not only display lower elongation but also duplicate the UV effect on AS. Consistently, nonphosphorylatable mutants prevent the UV effect. Apoptosis promoted by UV in cells lacking p53 is prevented when the change in AS of the apoptotic gene bcl-x is reverted, confirming the relevance of this mechanism. Splicing-sensitive microarrays revealed a significant overlap of the subsets of genes that have changed AS with UV and those that have reduced expression, suggesting that transcriptional coupling to AS is a key feature of the DNA-damage response.

A slow transcription rate causes embryonic lethality and perturbs kinetic coupling of neuronal genes.

The rate of RNA polymerase II (RNAPII) elongation has an important role in the control of alternative splicing (AS); however, the in vivo consequences of an altered elongation rate are unknown. Here, we generated mouse embryonic stem cells (ESCs) knocked in for a slow elongating form of RNAPII We show that a reduced transcriptional elongation rate results in early embryonic lethality in mice. Focusing on neuronal differentiation as a model, we observed that slow elongation impairs development of the neural lineage from ESCs, which is accompanied by changes in AS and in gene expression along this pathway. In particular, we found a crucial role for RNAPII elongation rate in transcription and splicing of long neuronal genes involved in synapse signaling. The impact of the kinetic coupling of RNAPII elongation rate with AS is greater in ESC-differentiated neurons than in pluripotent cells. Our results demonstrate the requirement for an appropriate transcriptional elongation rate to ensure proper gene expression and to regulate AS during development.

How slow RNA polymerase II elongation favors alternative exon skipping.

Splicing is functionally coupled to transcription, linking the rate of RNA polymerase II (Pol II) elongation and the ability of splicing factors to recognize splice sites (ss) of various strengths. In most cases, slow Pol II elongation allows weak splice sites to be recognized, leading to higher inclusion of alternative exons. Using CFTR alternative exon 9 (E9) as a model, we show here that slowing down elongation can also cause exon skipping by promoting the recruitment of the negative factor ETR-3 onto the UG-repeat at E9 3' splice site, which displaces the constitutive splicing factor U2AF65 from the overlapping polypyrimidine tract. Weakening of E9 5' ss increases ETR-3 binding at the 3' ss and subsequent E9 skipping, whereas strengthening of the 5' ss usage has the opposite effect. This indicates that a delay in the cotranscriptional emergence of the 5' ss promotes ETR-3 recruitment and subsequent inhibition of E9 inclusion.

GSK-3 is an RNA polymerase II phospho-CTD kinase.

We have previously found that UV-induced DNA damage causes hyperphosphorylation of the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), inhibition of transcriptional elongation and changes in alternative splicing (AS) due to kinetic coupling between transcription and splicing. In an unbiased search for protein kinases involved in the AS response to DNA damage, we have identified glycogen synthase kinase 3 (GSK-3) as an unforeseen participant. Unlike Cdk9 inhibition, GSK-3 inhibition only prevents CTD hyperphosphorylation triggered by UV but not basal phosphorylation. This effect is not due to differential degradation of the phospho-CTD isoforms and can be reproduced, at the AS level, by overexpression of a kinase-dead GSK-3 dominant negative mutant. GSK-3 inhibition abrogates both the reduction in RNAPII elongation and changes in AS elicited by UV. We show that GSK-3 phosphorylates the CTD in vitro, but preferentially when the substrate is previously phosphorylated, consistently with the requirement of a priming phosphorylation reported for GSK-3 efficacy. In line with a role for GSK-3 in the response to DNA damage, GSK-3 inhibition prevents UV-induced apoptosis. In summary, we uncover a novel role for a widely studied kinase in key steps of eukaryotic transcription and pre-mRNA processing.

A DNA intercalating dye-based RT-qPCR alternative to diagnose SARS-CoV-2.

Early detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been proven crucial during the efforts to mitigate the effects of the COVID-19 pandemic. Several diagnostic methods have emerged in the past few months, each with different shortcomings and limitations. The current gold standard, RT-qPCR using fluorescent probes, relies on demanding equipment requirements plus the high costs of the probes and specific reaction mixes. To broaden the possibilities of reagents and thermocyclers that could be allocated towards this task, we have optimized an alternative strategy for RT-qPCR diagnosis. This is based on a widely used DNA-intercalating dye and can be implemented with several different qPCR reagents and instruments. Remarkably, the proposed qPCR method performs similarly to the broadly used TaqMan-based detection, in terms of specificity and sensitivity, thus representing a reliable tool. We think that, through enabling the use of vast range of thermocycler models and laboratory facilities for SARS-CoV-2 diagnosis, the alternative proposed here can increase dramatically the testing capability, especially in countries with limited access to costly technology and reagents.

Linking transcription, RNA polymerase II elongation and alternative splicing.

Gene expression is an intricately regulated process that is at the basis of cell differentiation, the maintenance of cell identity and the cellular responses to environmental changes. Alternative splicing, the process by which multiple functionally distinct transcripts are generated from a single gene, is one of the main mechanisms that contribute to expand the coding capacity of genomes and help explain the level of complexity achieved by higher organisms. Eukaryotic transcription is subject to multiple layers of regulation both intrinsic - such as promoter structure - and dynamic, allowing the cell to respond to internal and external signals. Similarly, alternative splicing choices are affected by all of these aspects, mainly through the regulation of transcription elongation, making it a regulatory knob on a par with the regulation of gene expression levels. This review aims to recapitulate some of the history and stepping-stones that led to the paradigms held today about transcription and splicing regulation, with major focus on transcription elongation and its effect on alternative splicing.

Reciprocal regulation between alternative splicing and the DNA damage response.

Splicing, the process that catalyzes intron removal and flanking exon ligation, can occur in different ways (alternative splicing) in immature RNAs transcribed from a single gene. In order to adapt to a particular context, cells modulate not only the quantity but also the quality (alternative isoforms) of their transcriptome. Since 95% of the human coding genome is subjected to alternative splicing regulation, it is expected that many cellular pathways are modulated by alternative splicing, as is the case for the DNA damage response. Moreover, recent evidence demonstrates that upon a genotoxic insult, classical DNA damage response kinases such as ATM, ATR and DNA-PK orchestrate the gene expression response therefore modulating alternative splicing which, in a reciprocal way, shapes the response to a damaging agent.

How Are Short Exons Flanked by Long Introns Defined and Committed to Splicing?

The splice sites (SSs) delimiting an intron are brought together in the earliest step of spliceosome assembly yet it remains obscure how SS pairing occurs, especially when introns are thousands of nucleotides long. Splicing occurs in vivo in mammals within minutes regardless of intron length, implying that SS pairing can instantly follow transcription. Also, factors required for SS pairing, such as the U1 small nuclear ribonucleoprotein (snRNP) and U2AF65, associate with RNA polymerase II (RNAPII), while nucleosomes preferentially bind exonic sequences and associate with U2 snRNP. Based on recent publications, we assume that the 5' SS-bound U1 snRNP can remain tethered to RNAPII until complete synthesis of the downstream intron and exon. An additional U1 snRNP then binds the downstream 5' SS, whereas the RNAPII-associated U2AF65 binds the upstream 3' SS to facilitate SS pairing along with exon definition. Next, the nucleosome-associated U2 snRNP binds the branch site to advance splicing complex assembly. This may explain how RNAPII and chromatin are involved in spliceosome assembly and how introns lengthened during evolution with a relatively minimal compromise in splicing.

Alternative splicing switches: Important players in cell differentiation.

Alternative splicing (AS) greatly expands the coding capacities of genomes by allowing the generation of multiple mature mRNAs from a limited number of genes. Although the massive switch in AS profiles that often accompanies variations in gene expression patterns occurring during cell differentiation has been characterized for a variety of models, their causes and mechanisms remain largely unknown. Here, we integrate foundational and recent studies indicating the AS switches that govern the processes of cell fate determination. We include some distinct AS events in pluripotent cells and somatic reprogramming and discuss new progresses on alternative isoform expression in adipogenesis, myogenic differentiation and stimulation of immune cells. Finally, we cover novel insights on AS mechanisms during neuronal differentiation, paying special attention to the role of chromatin structure.

Intragenic epigenetic changes modulate NCAM alternative splicing in neuronal differentiation.

Alternative splicing contributes to cell type-specific transcriptomes. Here, we show that changes in intragenic chromatin marks affect NCAM (neural cell adhesion molecule) exon 18 (E18) alternative splicing during neuronal differentiation. An increase in the repressive marks H3K9me2 and H3K27me3 along the gene body correlated with inhibition of polymerase II elongation in the E18 region, but without significantly affecting total mRNA levels. Treatment with the general DNA methylation inhibitor 5-azacytidine and BIX 01294, a specific inhibitor of H3K9 dimethylation, inhibited the differentiation-induced E18 inclusion, pointing to a role for repressive marks in sustaining NCAM splicing patterns typical of mature neurons. We demonstrate that intragenic deployment of repressive chromatin marks, induced by intronic small interfering RNAs targeting NCAM intron 18, promotes E18 inclusion in undifferentiated N2a cells, confirming the chromatin changes observed upon differentiation to be sufficient to induce alternative splicing. Combined with previous evidence that neuronal depolarization causes H3K9 acetylation and subsequent E18 skipping, our results show how two alternative epigenetic marks regulate NCAM alternative splicing and E18 levels in different cellular contexts.