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.

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.

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.

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.

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.

Nuclear role for human Argonaute-1 as an estrogen-dependent transcription coactivator.

In mammals, argonaute (AGO) proteins have been characterized for their roles in small RNA-mediated posttranscriptional and also in transcriptional gene silencing. Here, we report a different role for AGO1 in estradiol-triggered transcriptional activation in human cells. We show that in MCF-7 mammary gland cells, AGO1 associates with transcriptional enhancers of estrogen receptor α (ERα) and that this association is up-regulated by treating the cells with estrogen (E2), displaying a positive correlation with the activation of these enhancers. Moreover, we show that AGO1 interacts with ERα and that this interaction is also increased by E2 treatment, but occurs in the absence of RNA. We show that AGO1 acts positively as a coactivator in estradiol-triggered transcription regulation by promoting ERα binding to its enhancers. Consistently, AGO1 depletion decreases long-range contacts between ERα enhancers and their target promoters. Our results point to a role of AGO1 in transcriptional regulation in human cells that is independent from small RNA binding.

The Histone Methyltransferase G9a Controls Axon Growth by Targeting the RhoA Signaling Pathway.

The generation of axonal and dendritic domains is critical for brain circuitry assembly and physiology. Negative players, such as the RhoA-Rho coiled-coil-associated protein kinase (ROCK) signaling pathway, restrain axon development and polarization. Surprisingly, the genetic control of neuronal polarity has remained largely unexplored. Here, we report that, in primary cultured neurons, expression of the histone methyltransferase G9a and nuclear translocation of its major splicing isoform (G9a/E10+) peak at the time of axon formation. RNAi suppression of G9a/E10+ or pharmacological blockade of G9a constrains neuronal migration, axon initiation, and the establishment of neuronal polarity in situ and in vitro. Inhibition of G9a function upregulates RhoA-ROCK activity by increasing the expression of Lfc, a guanine nucleotide exchange factor (GEF) for RhoA. Together, these results identify G9a as a player in neuronal polarization.

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.

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.

[Alternative splicing and the cure of spinal muscular atrophy]. / El splicing alternativo y la cura de la atrofia muscular espinal.

Alternative splicing of the messenger RNA plays a fundamental role in the flow of genetic information from DNA to proteins by expanding the coding capacity of the genome. The regulation of alternative splicing is as important as the regulation of transcription to determine the specific characteristics of cells and tissues, the normal functioning of cells and the responses of eukaryotic cells to external signals. Basic knowledge of the pre-mRNA sequences and splicing factors that recognize them has allowed scientists to design a therapeutic synthetic oligonucleotide for spinal muscular atrophy. This is an autosomal recessive inherited disease in which the SMN1 gene is mutated and affects one in 10,000 births. By blocking the binding of a negative splicing factor to the mRNA of a paralogue of the SMN1 gene, called SMN2, the Spinraza oligonucleotide corrects an abnormal alternative splicing event of the SMN2 gene and allows the synthesis of high levels of the SMN protein, constituting the first successful case of cure of a neurodegenerative disease.

El splicing alternativo del ácido ribonucleico mensajero (mRNA) juega un papel fundamental en el flujo de información genética desde el ADN a las proteínas al expandir la capacidad de codificación de los genomas. La regulación del splicing alternativo es tan importante como la regulación de la transcripción para determinar las características específicas de las células y los tejidos, el funcionamiento normal de las células y las respuestas de las células eucarióticas a las señales externas. El conocimiento básico de las secuencias del pre-mRNA y de los factores de splicing que las reconocen ha permitido a científicos diseñar un oligonucleótido sintético terapéutico para la atrofia muscular espinal. ésta es una enfermedad hereditaria autosómica recesiva en que el gen SMN1 se encuentra mutado y que afecta a uno de cada 10 000 nacimientos. Al bloquear la unión de un factor de splicing negativo al mRNA del gen parálogo del gen SMN1, denominado SMN2, el oligonucleótido Spinraza corrige un evento de splicing alternativo anormal del gen SMN2 y permite que se sinteticen altos niveles de la proteína SMN, constituyéndose en el primer caso exitoso de cura de una enfermedad neurodegenerativa.

Alternative Splicing and Transcription Elongation in Plants.

Alternative splicing and transcription elongation by RNA polymerase II (RNAPII) are two processes which are tightly connected. Splicing is a co-transcriptional process, and different experimental approaches show that splicing is coupled to transcription in Drosophila, yeast and mammals. However, little is known about coupling of transcription and alternative splicing in plants. The kinetic coupling explains how changes in RNAPII elongation rate influence alternative splicing choices. Recent work in Arabidopsis shows that expression of a dominant negative transcription elongation factor, TFIIS, enhances exon inclusion. Furthermore, the Arabidopsis transcription elongation complex has been recently described, providing new information about elongation factors that interact with elongating RNAPII. Light regulates alternative splicing in plants through a chloroplast retrograde signaling. We have recently shown that light promotes RNAPII elongation in the affected genes, while in darkness elongation is lower. These changes in transcription are consistent with elongation causing the observed changes in alternative splicing. Altogether, these findings provide evidence that coupling between transcription and alternative splicing is an important layer of gene expression regulation in plants.

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.

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.

Epigenetics at the base of alternative splicing changes that promote colorectal cancer.

Chromatin modification influences gene expression by either repressing or activating genes, depending on the specific histone mark. Chromatin structure can also influence alternative splicing of transcripts; however, the mechanisms by which epigenetic marks influence splicing are poorly understood. A report in the current issue of the JCI highlights the biological importance of the coordinated control of alternative pre-mRNA splicing by chromatin structure and transcriptional elongation. Yuan et al. found that mutation of the histone methyl transferase SEDT2 affects alternative splicing fates of several key regulatory genes, including those involved in Wnt signaling. As a consequence, loss of SEDT2 in the intestine aggravated Wnt/ß-catenin signaling effects, thereby leading to colorectal cancer.

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.

Major Roles for Pyrimidine Dimers, Nucleotide Excision Repair, and ATR in the Alternative Splicing Response to UV Irradiation.

We have previously found that UV irradiation promotes RNA polymerase II (RNAPII) hyperphosphorylation and subsequent changes in alternative splicing (AS). We show now that UV-induced DNA damage is not only necessary but sufficient to trigger the AS response and that photolyase-mediated removal of the most abundant class of pyrimidine dimers (PDs) abrogates the global response to UV. We demonstrate that, in keratinocytes, RNAPII is the target, but not a sensor, of the signaling cascade initiated by PDs. The UV effect is enhanced by inhibition of gap-filling DNA synthesis, the last step in the nucleotide excision repair pathway (NER), and reduced by the absence of XPE, the main NER sensor of PDs. The mechanism involves activation of the protein kinase ATR that mediates the UV-induced RNAPII hyperphosphorylation. Our results define the sequence UV-PDs-NER-ATR-RNAPII-AS as a pathway linking DNA damage repair to the control of both RNAPII phosphorylation and AS regulation.

Histone methylation, alternative splicing and neuronal differentiation.

Alternative splicing, as well as chromatin structure, greatly contributes to specific transcriptional programs that promote neuronal differentiation. The activity of G9a, the enzyme responsible for mono- and di-methylation of lysine 9 on histone H3 (H3K9me1 and H3K9me2) in mammalian euchromatin, has been widely implicated in the differentiation of a variety of cell types and tissues. In a recent work from our group (Fiszbein et al., 2016) we have shown that alternative splicing of G9a regulates its nuclear localization and, therefore, the efficiency of H3K9 methylation, which promotes neuronal differentiation. We discuss here our results in the light of a report from other group (Laurent et al. 2015) demonstrating a key role for the alternative splicing of the histone demethylase LSD1 in controlling specific gene expression in neurons. All together, these results illustrate the importance of alternative splicing in the generation of a proper equilibrium between methylation and demethylation of histones for the regulation of neuron-specific transcriptional programs.

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.