Slow transcriptional elongation causes embryonic lethality and perturbs kinetic coupling of long neural genes

Abstract

Running title: Transcriptional elongation rate controls gene expression and AS during development Maslon et al. 2 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 and impairs the differentiation of ESCs into the neural lineage. This is accompanied by changes in splicing and in gene expression in ESCs and along the pathway of neuronal differentiation. 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 more predominant 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. Supplemental material is available for this article. Maslon et al. 3Alternative splicing (AS) is a highly regulated process that generates RNA diversity and is a major contributor to protein isoform diversity. Its regulation not only depends on the interaction of trans-acting factors with regulatory RNA cis-acting sequences but also on multiple layers of regulation, which include DNA methylation, chromatin structure and modification, and transcription 1,2 . The co-transcriptional nature of pre-mRNA splicing led to the suggestion that the rate of transcription elongation acts to control AS in mammalian cells 3,4 . Notably, there is a functional relationship between the transcriptional and the splicing machineries, as evidenced by the role of splicing factors, such as TCERG1/CA150 5 and SRSF2 6 , in stimulating transcriptional elongation. A role for transcription elongation rate influencing splicing fidelity and cotranscriptionality was also observed in yeast 7,8 .Recent studies revisited the contribution of the kinetics of RNAPII elongation to the regulation of AS, giving rise to two complementary models 2,9 . The "window of opportunity" or kinetic model of AS regulation proposes that the rate of RNAPII elongation influences the outcome of alternative splicing selection. Use of a mutant form of RNAPII (C4/R749H) with a slower elongation rate leads to an increased 10 or decreased 11 inclusion of alternative cassette exons into mature mRNA. A complementary model, termed 'Goldilocks', concluded based on the study of RNAPII mutants with both slow and fast elongation rates, that an optimal rate of transcriptional elongation is required for normal co-transcriptional pre-mRNA splicing 12 . In both models, recruitment of splicing regulators to cis-acting RNA sequences as well as nascent RNA folding are influenced by the elongation rate of RNAPII [13][14][15] . The global impact of RNAP...