The translational landscape of the splicing factor SRSF1 and its role in mitosis

The shuttling serine/arginine rich (SR) protein SRSF1 (previously known as SF2/ASF) is a splicing regulator that also activates translation in the cytoplasm. In order to dissect the gene network that is translationally regulated by SRSF1, we performed a high-throughput deep sequencing analysis of polysomal fractions in cells overexpressing SRSF1. We identified approximately 1500 mRNAs that are translational targets of SRSF1. These include mRNAs encoding proteins involved in cell cycle regulation, such as spindle, kinetochore, and M phase proteins, which are essential for accurate chromosome segregation. Indeed, we show that translational activity of SRSF1 is required for normal mitotic progression. Furthermore, we found that mRNAs that display alternative splicing changes upon SRSF1 overexpression are also its translational targets, strongly suggesting that SRSF1 couples pre-mRNA splicing and translation. These data provide insights on the complex role of SRSF1 in the control of gene expression at multiple levels and its implications in cancer

Histone H1 regulates non-coding RNA turnover on chromatin in a m6A-dependent manner

Linker histones are highly abundant chromatin-associated proteins with well-established structural roles in chromatin and as general transcriptional repressors. In addition, it has been long proposed that histone H1 exerts context-specific effects on gene expression. Here, we identify a function of histone H1 in chromatin structure and transcription using a range of genomic approaches. In the absence of histone H1, there is an increase in the transcription of non-coding RNAs, together with reduced levels of m6A modification leading to their accumulation on chromatin and causing replication-transcription conflicts. This strongly suggests that histone H1 prevents non-coding RNA transcription and regulates non-coding transcript turnover on chromatin. Accordingly, altering the m6A RNA methylation pathway rescues the replicative phenotype of H1 loss. This work unveils unexpected regulatory roles of histone H1 on non-coding RNA turnover and m6A deposition, highlighting the intimate relationship between chromatin conformation, RNA metabolism, and DNA replication to maintain genome performance.Work at the M.G. lab was supported by the Spanish Ministry of Sciences and Innovation (BFU2016-78849-P and PID2019-105949GB-I00, co-financed by the European Union FEDER funds), a CSIC grant (2019AEP004), and a Salvador de Madariaga mobility grant (PRX19/00293). J.M.F.-J., C.S.-M., and J.I.-A. were supported by the Spanish Ministry of Sciences and Innovation fellowships (BES-2014-070050, BES-2017-079897, and PRE2020-095071, respectively); S.M.-V. was supported by a predoctoral fellowship from the Spanish Ministry of Education and Universities (FPU18/04794); and M.S.-P. was supported by an AGAUR-FI predoctoral fellowship co-financed by Generalitat de Catalunya and the European Social Fund. A.J. was supported by the Spanish Ministry of Sciences and Innovation (BFU2017-82805-C2-1-P and PID2020-112783GB-C21) and J.F.C. by core funding to the MRC Human Genetics Unit from the Medical Research Council (UK)

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.Fil: Maslon, M.. University of Edinburgh; Reino UnidoFil: Braunschweig, U.. University of Toronto; CanadáFil: Aitken, S.. University of Edinburgh; Reino UnidoFil: Mann, A.R.. University of Edinburgh; Reino UnidoFil: Kilanowski, F.. University of Edinburgh; Reino UnidoFil: Hunter, C.H.. University of Edinburgh; Reino UnidoFil: Blencowe, B.J.. University of Toronto; CanadáFil: 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; ArgentinaFil: Adams, I.. University of Edinburgh; Reino UnidoFil: Cáceres, J.F.. University of Edinburgh; Reino Unid

Slow RNAPII Transcription Elongation Rate, Low Levels of RNAPII Pausing, and Elevated Histone H1 Content at Promoters Associate with Higher m6A Deposition on Nascent mRNAs

N6-methyladenosine modification (m6A) fine-tunes RNA fate in a variety of ways, thus regulating multiple fundamental biological processes. m6A writers bind to chromatin and interact with RNA polymerase II (RNAPII) during transcription. To evaluate how the dynamics of the transcription process impact m6A deposition, we studied RNAPII elongation rates in mouse embryonic stem cells with altered chromatin configurations, due to reductions in linker histone H1 content. We found that genes transcribed at slow speed are preferentially methylated and display unique signatures at their promoter region, namely high levels of histone H1, together with marks of bivalent chromatin and low RNAPII pausing. They are also highly susceptible to m6A loss upon histone H1 reduction. These results indicate that RNAPII velocity links chromatin structure and the deposition of m6A, highlighting the intricate relationship between different regulatory layers on nascent mRNA molecules

Data from: The translational landscape of the splicing factor SRSF1 and its role in mitosis

The shuttling Serine/Arginine rich (SR) protein SRSF1 (previously known as SF2/ASF) is a splicing regulator that also activates translation in the cytoplasm. In order to dissect the gene network that is translationally regulated by SRSF1, we performed a high-throughput deep sequencing analysis of polysomal fractions in cells overexpressing SRSF1. We identified approximately 1,500 mRNAs that are translational targets of SRSF1. These include mRNAs encoding proteins involved in cell cycle regulation, such as spindle, kinetochore and M phase proteins, which are essential for accurate chromosome segregation. Indeed, we show that translational activity of SRSF1 is required for normal mitotic progression. Furthermore, we found that mRNAs that display alternative splicing changes upon SRSF1 overexpression are also its translational targets; strongly suggesting that SRSF1 couples pre-mRNA splicing and translation. These data provide insights on the complex role of SRSF1 in the control of gene expression at multiple levels and its implications in cancer

The translational landscape of the splicing factor SRSF1 and its role in mitosis

The shuttling serine/arginine rich (SR) protein SRSF1 (previously known as SF2/ASF) is a splicing regulator that also activates translation in the cytoplasm. In order to dissect the gene network that is translationally regulated by SRSF1, we performed a high-throughput deep sequencing analysis of polysomal fractions in cells overexpressing SRSF1. We identified approximately 1500 mRNAs that are translational targets of SRSF1. These include mRNAs encoding proteins involved in cell cycle regulation, such as spindle, kinetochore, and M phase proteins, which are essential for accurate chromosome segregation. Indeed, we show that translational activity of SRSF1 is required for normal mitotic progression. Furthermore, we found that mRNAs that display alternative splicing changes upon SRSF1 overexpression are also its translational targets, strongly suggesting that SRSF1 couples pre-mRNA splicing and translation. These data provide insights on the complex role of SRSF1 in the control of gene expression at multiple levels and its implications in cancer.MRC Core Funding Sara R Heras, Javier F Cáceres. Wellcome Trust Senior Investigator Award Grant 095518/Z/11/Z Magdalena M Maslon, Javier F Cáceres. Marie Curie IntraEuropean fellowship Sara R Heras. Consolider RNAReg CSD2009-00080 Nicolas Bellora, Eduardo Eyras. Ministerio de Economia y Competitividad BIO2011-23920 Nicolas Bellora, Eduardo Eyras. Sandra Ibarra Foundation FSI2011-35 Nicolas Bellora, Eduardo Eyras

Supplementary file 3-Alternative splicing changes following SRSF1 overexpression

List of 382 SRSF1-regulated cassette exons determined by exon arrays (cassette exons tab) and list of SRSF1-regulated cassette exons with SRSF1-clip-tags CLIP tag over casette exon (clip tag over cassette exon tab). Data are supplied as an excel spreadsheet

Supplementary file 3-Alternative splicing changes following SRSF1 overexpression

List of 382 SRSF1-regulated cassette exons determined by exon arrays (cassette exons tab) and list of SRSF1-regulated cassette exons with SRSF1-clip-tags CLIP tag over casette exon (clip tag over cassette exon tab). Data are supplied as an excel spreadsheet

Supplementary file 1-SRSF1 translational targets

Data containing list of 1576 mRNAs that showed a 1.85-fold increase in polysomal-association in response to SRSF1 overexpression (translational targets tab). Data are supplied as an excel spreadsheet. Descriptions of columns are included in additional tab (descriptions of columns). Negative controls tab contains information on cellular abundance of mRNAs used as negative controls in all validation experiments

Supplementary file 2-SILAC analysis following SRSF1 overexpression

List of proteins identified in SILAC experiment (SILAC tab). Data are supplied as an excel spreadsheet. Descriptions of columns are included in the additional tab (description of columns tab)