Search

Although splicing occurs largely co-transcriptionally, the order by which introns are removed does not necessarily follow the order in which they are transcribed. Whereas several genomic features are known to influence whether or not an intron is spliced before its downstream neighbor, multiple questions related to adjacent introns' splicing order (AISO) remain unanswered. Here, we present Insplico, the first standalone software for quantifying AISO that works with both short and long read sequencing technologies. We first demonstrate its applicability and effectiveness using simulated reads and by recapitulating previously reported AISO patterns, which unveiled overlooked biases associated with long read sequencing. We next show that AISO around individual exons is remarkably constant across cell and tissue types and even upon major spliceosomal disruption, and it is evolutionarily conserved between human and mouse brains. We also establish a set of universal features associated with AISO patterns across various animal and plant species. Finally, we used Insplico to investigate AISO in the context of tissue-specific exons, particularly focusing on SRRM4-dependent microexons. We found that the majority of such microexons have non-canonical AISO, in which the downstream intron is spliced first, and we suggest two potential modes of SRRM4 regulation of microexons related to their AISO and various splicing-related features. Insplico is available on gitlab.com/aghr/insplico. European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program [ERCCoG-LS2-101002275 to M.I.]; Spanish Ministry of Economy and Competitiveness [PID2020-115040GB-I00 to M.I.]; ‘Centro de Excelencia Severo Ochoa 2013–2017’ [SEV-2012-0208]. Funding for open access charge: European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program [ERCCoG-LS2-101002275 to M.I.].

Although splicing occurs largely co-transcriptionally, the order by which introns are removed does not necessarily follow the order in which they are transcribed. Whereas several genomic features are known to influence whether or not an intron is spliced before its downstream neighbour, multiple questions related to intron splicing order (ISO) remain unanswered. Here, we present Insplico, the first standalone tool to quantify ISO that works with both short and long read sequencing technologies. We first demonstrate its applicability and effectiveness recapitulating previously reported patterns, while unveiling overlooked biases associated with long read sequencing. We next show that ISO around individual exons is remarkably constant across cell and tissue types and even upon major spliceosomal disruption, and it is evolutionarily conserved between human and mouse brains. We also establish a set of universal features associated with ISO patterns across various animal and plant species. Finally, we used Insplico to investigate ISO in the context of tissue-specific exons, particularly focusing on SRRM4-dependent microexons. We found that the majority of such microexons have non-canonical ISO, in which the downstream intron is spliced first, and we revealed two potential modes of SRRM4 regulation of microexons related to their ISO and various splicing-related features. Insplico is available on gitlab.com/aghr/insplico.

RNA splicing is widely dysregulated in cancer, frequently due to altered expression or activity of splicing factors (SFs). Microexons are extremely small exons (3–27 nucleotides long) that are highly evolutionarily conserved and play critical roles in promoting neuronal differentiation and development. Inclusion of microexons in mRNA transcripts is mediated by the SF Serine/Arginine Repetitive Matrix 4 (SRRM4), whose expression is largely restricted to neural tissues. However, microexons have been largely overlooked in prior analyses of splicing in cancer, as their small size necessitates specialized computational approaches for their detection. Here, we demonstrate that despite having low expression in normal nonneural tissues, SRRM4 is further silenced in tumors, resulting in the suppression of normal microexon inclusion. Remarkably, SRRM4 is the most consistently silenced SF across all tumor types analyzed, implying a general advantage of microexon down-regulation in cancer independent of its tissue of origin. We show that this silencing is favorable for tumor growth, as decreased SRRM4 expression in tumors is correlated with an increase in mitotic gene expression, and up-regulation of SRRM4 in cancer cell lines dose-dependently inhibits proliferation in vitro and in a mouse xenograft model. Further, this proliferation inhibition is accompanied by induction of neural-like expression and splicing patterns in cancer cells, suggesting that SRRM4 expression shifts the cell state away from proliferation and toward differentiation. We therefore conclude that SRRM4 acts as a proliferation brake, and tumors gain a selective advantage by cutting off this brake., This project was funded in part by a grant from the Plan Estatal de Investigación Científica y Técnica y de Innovación to L.S. (PGC2018-101271-B-I00, http://www.ciencia.gob.es). S.A.H. is supported by a Marie Skłodowska-Curie Individual Fellowship from the European Union’s Horizon 2020 research and innovation programme (MSCA-IF-2017-794629, http://ec.europa.eu/). X.H. is supported by a PhD fellowship from the Fundación Ramón Areces (http://www.fundacionareces.es). We acknowledge the support of the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa’, the CERCA Programme / Generalitat de Catalunya, and the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership.

Cyanobacteria are important for fundamental studies of photosynthesis and have great biotechnological potential. In order to better study and fully exploit these organisms, the limited repertoire of genetic tools and parts must be expanded. A small number of inducible promoters have been used in cyanobacteria, allowing dynamic external control of gene expression through the addition of specific inducer molecules. However, the inducible promoters used to date suffer from various drawbacks including toxicity of inducers, leaky expression in the absence of inducer and inducer photolability, the latter being particularly relevant to cyanobacteria, which, as photoautotrophs, are grown under light. Here we introduce the rhamnose-inducible rhaBAD promoter of Escherichia coli into the model freshwater cyanobacterium Synechocystis sp. PCC 6803 and demonstrate it has superior properties to previously reported cyanobacterial inducible promoter systems, such as a non-toxic, photostable, non-metabolizable inducer, a linear response to inducer concentration and crucially no basal transcription in the absence of inducer.

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. Fil: Muñoz, Manuel Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Fondazione Ifom Istituto Firc Di Oncologia Molecolare; Italia Fil: Nieto Moreno, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Giono, Luciana Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Cambindo Botto, Adrian Edgardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Dujardin, Gwendal. Barcelona Institute Of Science And Technology; España. Universitat Pompeu Fabra; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Bastianello, Giulia. Fondazione Ifom Istituto Firc Di Oncologia Molecolare; Italia Fil: Lavore, Stefania. Fondazione Ifom Istituto Firc Di Oncologia Molecolare; Italia Fil: Torres-Méndez, Antonio. Barcelona Institute Of Science And Technology; España. Universitat Pompeu Fabra; España Fil: Menck, Carlos FM. Universidade de Sao Paulo; Brasil Fil: Blencowe, Benjamin. University of Toronto; Canadá Fil: Irimia, Manuel. Barcelona Institute Of Science And Technology; España. Universitat Pompeu Fabra; España Fil: Foiani, Marco. Fondazione Ifom Istituto Firc Di Oncologia Molecolare; Italia Fil: Kornblihtt, Alberto Rodolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina

The mechanisms by which entire programmes of gene regulation emerged during evolution are poorly understood. Neuronal microexons represent the most conserved class of alternative splicing in vertebrates, and are critical for proper brain development and function. Here, we discover neural microexon programmes in non-vertebrate species and trace their origin to bilaterian ancestors through the emergence of a previously uncharacterized ‘enhancer of microexons’ (eMIC) protein domain. The eMIC domain originated as an alternative, neural-enriched splice isoform of the pan-eukaryotic Srrm2/SRm300 splicing factor gene, and subsequently became fixed in the vertebrate and neuronal-specific splicing regulator Srrm4/nSR100 and its paralogue Srrm3. Remarkably, the eMIC domain is necessary and sufficient for microexon splicing, and functions by interacting with the earliest components required for exon recognition. The emergence of a novel domain with restricted expression in the nervous system thus resulted in the evolution of splicing programmes that qualitatively expanded the neuronal molecular complexity in bilaterians., This work has been funded by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (ERC-StG-LS2-637591 to M.Ir. and ERC-AdvG-670146 to J.V.), the Spanish Ministry of Economy and Competitiveness (BFU2014-55076-P and BFU2017-89201-P to M.Ir., BFU2014-005153 to J.V., and the ‘Centro de Excelencia Severo Ochoa 2013–2017’ (SEV-2012-0208)), AGAUR, Fundación Botín (to J.V.) and the Canadian Institutes of Health Research (to B.J.B. and A.-C.G.). RNP mass spectrometric analyses were performed at the CRG/UPF Proteomics Unit (part of ProteoRed-PRB3, supported by PE I+D+i 2013–2016 (PT17/0019) of the ISCIII and ERDF) by ‘Programa CERCA Generalitat de Catalunya’ and ‘Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement’ (2017SGR595). A.T.-M. held an FPI-SO fellowship, and Y.M. a Marie Skłodowska-Curie individual fellowship.

Cyanobacteria are important for fundamental studies of photosynthesis and have great biotechnological potential. In order to better study and fully exploit these organisms, the limited repertoire of genetic tools and parts must be expanded. A small number of inducible promoters have been used in cyanobacteria, allowing dynamic external control of gene expression through the addition of specific inducer molecules. However, the inducible promoters used to date suffer from various drawbacks including toxicity of inducers, leaky expression in the absence of inducer and inducer photolability, the latter being particularly relevant to cyanobacteria which, as photoautotrophs, are grown under light. Here we introduce the rhamnose-induciblerhaBADpromoter ofEscherichia coliinto the model freshwater cyanobacteriumSynechocystissp. PCC 6803 and demonstrate it has superior properties to previously reported cyanobacterial inducible promoter systems, such as a non-toxic, photostable, non-metabolizable inducer, a linear response to inducer concentration and crucially no basal transcription in the absence of inducer.

Alternative splicing of pre-mRNA is a major mechanism to diversify protein functionality in metazoans from a limited number of genes. The Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam) gene, which is important for neuronal wiring and phagocytosis of bacteria, can generate up to 38,016 isoforms by mutually exclusive alternative splicing in four clusters of variable exons. However, it is not understood how a specific exon is chosen from the many variables and how variable exons are prevented from being spliced together. A main role in the regulation of Dscam alternative splicing has been attributed to RNA binding proteins (RBPs), but how they impact on exon selection is not well understood. Serine-arginine rich (SR) proteins and hnRNP proteins are the two main types of RBPs with major roles in exon definition and splice site selection. Here, we analyzed the role of SR and hnRNP proteins in Dscam exon 9 alternative splicing in mutant Drosophila melanogaster embryos because of their essential function for development. Strikingly, loss or overexpression of canonical SR and hnRNP proteins even when multiple proteins are depleted together, does not affect Dscam alternative exon selection very dramatically. Conversely, noncanonical SR protein Serine–arginine repetitive matrix 2/3/4 (Srrm234) is a main determinant of exon inclusion in the Dscam exon 9 cluster. Since long-range base-pairings are absent in the exon 9 cluster, our data argue for a small complement of regulatory factors as main determinants of exon inclusion in the Dscam exon 9 cluster.

Alternative splicing increases neuronal transcriptomic complexity throughout animal phylogeny. To delve into the mechanisms controlling the assembly and evolution of this regulatory layer, we characterized the neuronal microexon program in Drosophila and compared it with that of mammals. In nonvertebrate bilaterians, this splicing program is restricted to neurons by the posttranscriptional processing of the enhancer of microexons (eMIC) domain in Srrm234. In Drosophila, this processing is dependent on regulation by Elav/Fne. eMIC deficiency or misexpression leads to widespread neurological alterations largely emerging from impaired neuronal activity, as revealed by a combination of neuronal imaging experiments and cell type–specific rescues. These defects are associated with the genome-wide skipping of short neural exons, which are strongly enriched in ion channels. We found no overlap of eMIC-regulated exons between flies and mice, illustrating how ancient posttranscriptional programs can evolve independently in different phyla to affect distinct cellular modules while maintaining cell-type specificity., The research has been funded primarily by the following: European Research Council ERC-StG-LS2-637591 (M.I.), European Research Council ERC-CoG-LS2-101002275 (M.I.), European Research Council ERC StG “EvoNeuroCircuit” 802531 (L.P.-G.), Spanish Ministerio de Ciencia BFU2017-89201-P (M.I.), NIH R01 grant R01GM122406 (S.K.), BBSRC (M.S.), Centro de Excelencia Severo Ochoa 2013-2017 SEV-2012-0208 (CRG), Cancer Research UK FC001594 (Francis Crick Institute), U.K. Medical Research Council FC001594 (Francis Crick Institute), Wellcome Trust FC001594 (Francis Crick Institute), FPI-Severo Ochoa PhD fellowship (A.T.-M.), Boehringer Ingelheim Fonds travel grant (A.T.-M.), and Boehringer Ingelheim Fonds PhD fellowship (R.J.V.R.).

RNA splicing is widely dysregulated in cancer, frequently due to altered expression or activity of splicing factors. Microexons are extremely small exons (3-27 nucleotides long) that are highly evolutionarily conserved and play critical roles in promoting neuronal differentiation and development. Inclusion of microexons in mRNA transcripts is mediated by the splicing factor SRRM4, whose expression is largely restricted to neural tissues. However, microexons have been largely overlooked in prior analyses of splicing in cancer, as their small size necessitates specialized computational approaches for their detection. Here we demonstrate that despite having low expression in normal non-neural tissues, SRRM4 is hypersilenced in tumors, resulting in the suppression of basal microexon inclusion. Remarkably, SRRM4 is the most consistently silenced splicing factor across all tumor types analyzed, implying a general advantage of microexon downregulation in cancer independent of its tissue of origin. We show that this silencing is favorable for tumor growth, as decreased SRRM4 expression in tumors is correlated with an increase in mitotic gene expression, and upregulation of SRRM4 in cancer cell lines dose-dependently inhibits proliferation in vitro and in a mouse xenograft model. Further, this proliferation inhibition is accompanied by induction of neural-like expression and splicing patterns in cancer cells, suggesting that SRRM4 expression shifts the cell state away from proliferation and towards differentiation. We therefore conclude that SRRM4 acts as a proliferation brake, and tumors gain a selective advantage by cutting off this brake.SignificanceMicroexons are extremely small exons enriched in the brain that play important roles in neural development. Their inclusion is mediated by the splicing factor SRRM4, also predominantly expressed in the brain. Surprisingly, we find that low expression of SRRM4 outside of the brain is further decreased in tumors, and in fact SRRM4 is the most consistently silenced splicing factor in tumors across tissue types. We demonstrate that SRRM4 inhibits cancer cell proliferation in vitro and in vivo by inducing a neuronal differentiation program. Our findings add a new element to the overall picture of splicing dysregulation in cancer, reveal an antiproliferative function for SRRM4 and microexons outside of the brain, and may present a common therapeutic intervention point across cancer types.

Epithelial-mesenchymal interactions are crucial for the development of numerous animal structures. Thus, unraveling how molecular tools are recruited in different lineages to control interplays between these tissues is key to understanding morphogenetic evolution. Here, we study Esrp genes, which regulate extensive splicing programs and are essential for mammalian organogenesis. We find that Esrp homologs have been independently recruited for the development of multiple structures across deuterostomes. Although Esrp is involved in a wide variety of ontogenetic processes, our results suggest ancient roles in non-neural ectoderm and regulating specific mesenchymal-to-epithelial transitions in deuterostome ancestors. However, consistent with the extensive rewiring of Esrp-dependent splicing programs between phyla, most developmental defects observed in vertebrate mutants are related to other types of morphogenetic processes. This is likely connected to the origin of an event in Fgfr, which was recruited as an Esrp target in stem chordates and subsequently co-opted into the development of many novel traits in vertebrates., This work has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No ERC-StG-LS2-637591 to M.I.), the Spanish Ministry of Economy and Competitiveness (grant BFU2014-58908P to J.G.-F, BFU2014-55076-P to M.I., and the ‘Centro de Excelencia Severo Ochoa 2013–2017’, SEV-2012-0208), and ICREA - Generalitat de Catalunya (Academia Prize to J.G.-F). We acknowledge the support of the CERCA Programme/Generalitat de Catalunya. D.B. held an APIF fellowship from University of Barcelona, Y.M. an EMBO Long Term postdoctoral fellowship (ALTF 1505-2015), C.R. an EMBO long-term fellowship (ALTF 1608-2014), ATM an FPI-SO fellowship.