Project description:N6-methylation of 2’-O-methyladenosine (Am) in RNA occurs in eukaryotic cells to generate N6,2’-O-dimethyladenosine (m6Am). Identification of the methyltransferase responsible for m6Am catalysis has accelerated studies on the function of m6Am in RNA processing. While m6Am is generally found in the first transcribed nucleotide of mRNAs, the modification is also found internally within U2 snRNA. However, the writer required for catalyzing internal m6Am formation had remained elusive. By sequencing transcriptome-wide RNA methylation at single-base-resolution, we identified human METTL4 as the writer that directly methylates Am at U2 snRNA position 30 into m6Am. We found that METTL4 localizes to the nucleus and its conserved methyltransferase catalytic site is required for U2 snRNA methylation. By sequencing human cells with overexpressed Mettl4, we determined METTL4’s in vivo target RNA motif specificity. In the absence of Mettl4 in human cells, U2 snRNA lacks m6Am thereby affecting a subset of splicing events that exhibit specific features such as overall 3’ splice-site weakness with certain motif positions more affected than others. This study establishes that METTL4 methylation of U2 snRNA regulates splicing of specific pre-mRNA transcripts.
Project description:In this work we studied the role of U2 snRNA m6A(m)30 modification introduced by METTL4 methyltransferase on a model of METTL4 knockout human cell line. METTL4 gene inactivation resulted in dysregulation of gene expression and alternative splicing, as well as general decrease in splicing speed and accuracy.
Project description:In this work we studied the role of U2 snRNA m6A(m)30 modification introduced by METTL4 methyltransferase on a model of METTL4 knockout human cell line. METTL4 gene inactivation resulted in dysregulation of gene expression and alternative splicing, as well as general decrease in splicing speed and accuracy.
Project description:To investigate the global effects of perturbing U2 snRNA sequence on splicing and gene expression, we carried out transcriptomic analysis of HEK293T cell lines stably expressing U2 snRNA mutants C28U in the branchpoint interacting stem loop or T34A/ A35C/G36T to create an orthogonal branch point recognition sequence. With both mutants, the expression of many genes is generally repressed, potentially by nonsense mediated decay (NMD) arising from a global decrease in splicing efficiency. However, we also see upregulation of genes important for pre-mRNA processing, translation and protein folding. In several cases, the upregulation of certain genes can be linked to a shift in alternative splicing that favors the productive isoform relative to an NMD-targeted isoform.
Project description:In order to identify factors involved in transcription of human snRNA genes and 3’ end processing of the transcripts, we have carried out CRISPR affinity purification in situ of regulatory elements (CAPTURE), which is deadCas9-mediated pull-down, of the tandemly-repeated U2 snRNA genes in human cells. Pull-down proteins were identified by mass spectrometry-based proteomics analysis.
