METTL4 catalyzes m6Am methylation in U2 snRNA to regulate pre-mRNA splicing

AbstractN6-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.

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The phylogenetically invariant ACAGAGA and AGC sequences of U6 small nuclear RNA are more tolerant of mutation in human cells than in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5377-5382.1993 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5377-5382

Author(s):

B Datta ◽

A M Weiner

Keyword(s):

Saccharomyces Cerevisiae ◽

Transient Expression ◽

Human Cells ◽

Mrna Splicing ◽

Small Nuclear Rna ◽

Transient Expression Assay ◽

U6 Snrna ◽

Nuclear Rna

U6 small nuclear RNA (snRNA) is the most highly conserved of the five spliceosomal snRNAs that participate in nuclear mRNA splicing. The proposal that U6 snRNA plays a key catalytic role in splicing [D. Brow and C. Guthrie, Nature (London) 337:14-15, 1989] is supported by the phylogenetic conservation of U6, the sensitivity of U6 to mutation, cross-linking of U6 to the vicinity of the 5' splice site, and genetic evidence for extensive base pairing between U2 and U6 snRNAs. We chose to mutate the phylogenetically invariant 41-ACAGAGA-47 and 53-AGC-55 sequences of human U6 because certain point mutations within the homologous regions of Saccharomyces cerevisiae U6 selectively block the first or second step of mRNA splicing. We found that both sequences are more tolerant to mutation in human cells (assayed by transient expression in vivo) than in S. cerevisiae (assayed by effects on growth or in vitro splicing). These differences may reflect different rate-limiting steps in the particular assays used or differential reliance on redundant RNA-RNA or RNA-protein interactions. The ability of mutations in U6 nucleotides A-45 and A-53 to selectively block step 2 of splicing in S. cerevisiae had previously been construed as evidence that these residues might participate directly in the second chemical step of splicing; an indirect, structural role seems more likely because the equivalent mutations have no obvious phenotype in the human transient expression assay.

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Incorporation of 5-fluorouracil into U2 snRNA blocks pseudouridylation and pre-mRNA splicing in vivo

Nucleic Acids Research ◽

10.1093/nar/gkl1084 ◽

2006 ◽

Vol 35 (2) ◽

pp. 550-558 ◽

Cited By ~ 39

Author(s):

X. Zhao ◽

Y.-T. Yu

Keyword(s):

Mrna Splicing ◽

U2 Snrna

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Kinetic competition during the transcription cycle results in stochastic RNA processing

eLife ◽

10.7554/elife.03939 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 109

Author(s):

Antoine Coulon ◽

Matthew L Ferguson ◽

Valeria de Turris ◽

Murali Palangat ◽

Carson C Chow ◽

...

Keyword(s):

Quality Control ◽

Point Mutation ◽

Single Molecule ◽

Rna Processing ◽

Globin Gene ◽

Mrna Splicing ◽

Splicing Factor ◽

Before And After ◽

Competing Pathways

Synthesis of mRNA in eukaryotes involves the coordinated action of many enzymatic processes, including initiation, elongation, splicing, and cleavage. Kinetic competition between these processes has been proposed to determine RNA fate, yet such coupling has never been observed in vivo on single transcripts. In this study, we use dual-color single-molecule RNA imaging in living human cells to construct a complete kinetic profile of transcription and splicing of the β-globin gene. We find that kinetic competition results in multiple competing pathways for pre-mRNA splicing. Splicing of the terminal intron occurs stochastically both before and after transcript release, indicating there is not a strict quality control checkpoint. The majority of pre-mRNAs are spliced after release, while diffusing away from the site of transcription. A single missense point mutation (S34F) in the essential splicing factor U2AF1 which occurs in human cancers perturbs this kinetic balance and defers splicing to occur entirely post-release.

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The branchpoint residue is recognized during commitment complex formation before being bulged out of the U2 snRNA-pre-mRNA duplex.

Molecular and Cellular Biology ◽

10.1128/mcb.17.7.3469 ◽

1997 ◽

Vol 17 (7) ◽

pp. 3469-3476 ◽

Cited By ~ 12

Author(s):

E Pascolo ◽

B Séraphin

Keyword(s):

Complex Formation ◽

Mrna Splicing ◽

Base Pairing ◽

Reporter Construct ◽

Spliceosome Assembly ◽

U2 Snrna ◽

Alternative Structures ◽

Splicing Defect ◽

Rule Out

We have analyzed the mechanism of branchpoint nucleotide selection during the first step of pre-mRNA splicing. It has previously been proposed that the branchpoint is selected as an adenosine residue bulged out of an RNA helix formed by the U2 snRNA-pre-mRNA base pairing. Although compatible with this bulge hypothesis, available data from both yeast and mammalian systems did not rule out alternative structures for the branch nucleotide. Mutating the residue preceding the branchpoint nucleotide in our reporter construct conferred a splicing defect that was suppressed in vivo by the complementary U2 snRNA mutants. In contrast, substitutions on the 3' side of the branchpoint could be suppressed by complementary U2 snRNA mutants only in a weakened intron context. To test why the identity of the branch nucleotide was important for its selection, we analyzed the effect of substitutions at this position on spliceosome assembly. We observed that these mutations block the formation of one of the two commitment complexes. Our results demonstrate that yeast branchpoint selection occurs in multiple steps. The nature of the branch residue is recognized, in the absence of U2 snRNA, during commitment complex formation. Then, base pairing with U2 snRNA constrains this residue into a bulge conformation.

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The phylogenetically invariant ACAGAGA and AGC sequences of U6 small nuclear RNA are more tolerant of mutation in human cells than in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5377 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5377-5382 ◽

Cited By ~ 14

Author(s):

B Datta ◽

A M Weiner

Keyword(s):

Saccharomyces Cerevisiae ◽

Transient Expression ◽

Human Cells ◽

Mrna Splicing ◽

Small Nuclear Rna ◽

Transient Expression Assay ◽

U6 Snrna ◽

Nuclear Rna

U6 small nuclear RNA (snRNA) is the most highly conserved of the five spliceosomal snRNAs that participate in nuclear mRNA splicing. The proposal that U6 snRNA plays a key catalytic role in splicing [D. Brow and C. Guthrie, Nature (London) 337:14-15, 1989] is supported by the phylogenetic conservation of U6, the sensitivity of U6 to mutation, cross-linking of U6 to the vicinity of the 5' splice site, and genetic evidence for extensive base pairing between U2 and U6 snRNAs. We chose to mutate the phylogenetically invariant 41-ACAGAGA-47 and 53-AGC-55 sequences of human U6 because certain point mutations within the homologous regions of Saccharomyces cerevisiae U6 selectively block the first or second step of mRNA splicing. We found that both sequences are more tolerant to mutation in human cells (assayed by transient expression in vivo) than in S. cerevisiae (assayed by effects on growth or in vitro splicing). These differences may reflect different rate-limiting steps in the particular assays used or differential reliance on redundant RNA-RNA or RNA-protein interactions. The ability of mutations in U6 nucleotides A-45 and A-53 to selectively block step 2 of splicing in S. cerevisiae had previously been construed as evidence that these residues might participate directly in the second chemical step of splicing; an indirect, structural role seems more likely because the equivalent mutations have no obvious phenotype in the human transient expression assay.

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Human Splicing Factor SF3a, but Not SF1, Is Essential for Pre-mRNA Splicing In Vivo

Molecular Biology of the Cell ◽

10.1091/mbc.e04-11-1034 ◽

2005 ◽

Vol 16 (3) ◽

pp. 1366-1377 ◽

Cited By ~ 81

Author(s):

Goranka Tanackovic ◽

Angela Krämer

Keyword(s):

Cell Viability ◽

Mrna Splicing ◽

Splicing Factor ◽

Cajal Bodies ◽

Nuclear Speckles ◽

U2 Snrna ◽

U2 Snrnp ◽

Constitutive Splicing

The three subunits of human splicing factor SF3a are essential for the formation of the functional 17S U2 snRNP and prespliceosome assembly in vitro. RNAi-mediated depletion indicates that each subunit is essential for viability of human cells. Knockdown of single subunits results in a general block in splicing strongly suggesting that SF3a is a constitutive splicing factor in vivo. In contrast, splicing of several endogenous and reporter pre-mRNAs is not affected after knockdown of SF1, which functions at the onset of spliceosome assembly in vitro and is essential for cell viability. Thus, SF1 may only be required for the splicing of a subset of pre-mRNAs. We also observe a reorganization of U2 snRNP components in SF3a-depleted cells, where U2 snRNA and U2-B″ are significantly reduced in nuclear speckles and the nucleoplasm, but still present in Cajal bodies. Together with the observation that the 17S U2 snRNP cannot be detected in extracts from SF3a-depleted cells, our results provide further evidence for a function of Cajal bodies in U2 snRNP biogenesis.

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Faculty Opinions recommendation of Interconnections Between RNA-Processing Pathways Revealed by a Sequencing-Based Genetic Screen for Pre-mRNA Splicing Mutants in Fission Yeast.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726351802.793520502 ◽

2016 ◽

Author(s):

Karla Neugebauer

Keyword(s):

Fission Yeast ◽

Rna Processing ◽

Genetic Screen ◽

Mrna Splicing ◽

Processing Pathways

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The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain

Nature Communications ◽

10.1038/s41467-021-21663-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Saikat Bhattacharya ◽

Michaella J. Levy ◽

Ning Zhang ◽

Hua Li ◽

Laurence Florens ◽

...

Keyword(s):

Rna Splicing ◽

Rna Binding ◽

Mrna Processing ◽

Key Factors ◽

Pol Ii ◽

Mrna Transcripts ◽

Hnrnp Protein ◽

Processing Factors

AbstractHeterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2-hnRNP Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2, by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery.

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Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-021-00558-8 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Miao-Miao Zhao ◽

Wei-Li Yang ◽

Fang-Yuan Yang ◽

Li Zhang ◽

Wei-Jin Huang ◽

...

Keyword(s):

Drug Development ◽

Cathepsin L ◽

Human Cells ◽

New Drugs ◽

Disease Course ◽

Promising Target ◽

First Time

AbstractTo discover new drugs to combat COVID-19, an understanding of the molecular basis of SARS-CoV-2 infection is urgently needed. Here, for the first time, we report the crucial role of cathepsin L (CTSL) in patients with COVID-19. The circulating level of CTSL was elevated after SARS-CoV-2 infection and was positively correlated with disease course and severity. Correspondingly, SARS-CoV-2 pseudovirus infection increased CTSL expression in human cells in vitro and human ACE2 transgenic mice in vivo, while CTSL overexpression, in turn, enhanced pseudovirus infection in human cells. CTSL functionally cleaved the SARS-CoV-2 spike protein and enhanced virus entry, as evidenced by CTSL overexpression and knockdown in vitro and application of CTSL inhibitor drugs in vivo. Furthermore, amantadine, a licensed anti-influenza drug, significantly inhibited CTSL activity after SARS-CoV-2 pseudovirus infection and prevented infection both in vitro and in vivo. Therefore, CTSL is a promising target for new anti-COVID-19 drug development.

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