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

Abstract N 6-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 3′ splice-site weakness and an increase in exon inclusion. These findings suggest that METTL4 methylation of U2 snRNA regulates splicing of specific pre-mRNA transcripts.

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

10.1101/2020.01.24.917575 ◽

2020 ◽

Cited By ~ 1

Author(s):

Yeek Teck Goh ◽

Casslynn W. Q. Koh ◽

Donald Yuhui Sim ◽

Xavier Roca ◽

W. S. Sho Goh

Keyword(s):

Rna Processing ◽

Human Cells ◽

Mrna Splicing ◽

U2 Snrna ◽

Mrna Transcripts ◽

Single Base Resolution ◽

Accelerated Studies ◽

Rna Motif ◽

Target Rna

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 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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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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PLRG1 Is an Essential Regulator of Cell Proliferation and Apoptosis during Vertebrate Development and Tissue Homeostasis

Molecular and Cellular Biology ◽

10.1128/mcb.01807-08 ◽

2009 ◽

Vol 29 (11) ◽

pp. 3173-3185 ◽

Cited By ~ 32

Author(s):

André Kleinridders ◽

Hans-Martin Pogoda ◽

Sigrid Irlenbusch ◽

Neil Smyth ◽

Csaba Koncz ◽

...

Keyword(s):

Cell Cycle Progression ◽

Tissue Homeostasis ◽

Mrna Splicing ◽

Vertebrate Development ◽

Adult Tissue ◽

Serum Stimulation ◽

Evolutionarily Conserved ◽

Proliferation And Apoptosis ◽

Dependent Cell

ABSTRACT PLRG1, an evolutionarily conserved component of the spliceosome, forms a complex with Pso4/SNEV/Prp19 and the cell division and cycle 5 homolog (CDC5L) that is involved in both pre-mRNA splicing and DNA repair. Here, we show that the inactivation of PLRG1 in mice results in embryonic lethality at 1.5 days postfertilization. Studies of heart- and neuron-specific PLRG1 knockout mice further reveal an essential role of PLRG1 in adult tissue homeostasis and the suppression of apoptosis. PLRG1-deficient mouse embryonic fibroblasts (MEFs) fail to progress through S phase upon serum stimulation and exhibit increased rates of apoptosis. PLRG1 deficiency causes enhanced p53 phosphorylation and stabilization in the presence of increased γ-H2AX immunoreactivity as an indicator of an activated DNA damage response. p53 downregulation rescues lethality in both PLRG1-deficient MEFs and zebrafish in vivo, showing that apoptosis resulting from PLRG1 deficiency is p53 dependent. Moreover, the deletion of PLRG1 results in the relocation of its interaction partner CDC5L from the nucleus to the cytoplasm without general alterations in pre-mRNA splicing. Taken together, the results of this study identify PLRG1 as a critical nuclear regulator of p53-dependent cell cycle progression and apoptosis during both embryonic development and adult tissue homeostasis.

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A Functional Interaction between the Carboxy-Terminal Domain of RNA Polymerase II and Pre-mRNA Splicing

The Journal of Cell Biology ◽

10.1083/jcb.136.1.5 ◽

1997 ◽

Vol 136 (1) ◽

pp. 5-18 ◽

Cited By ~ 91

Author(s):

Lei Du ◽

Stephen L. Warren

Keyword(s):

Rna Polymerase Ii ◽

Mammalian Cells ◽

Mrna Splicing ◽

Splicing Factors ◽

Functional Interaction ◽

Carboxy Terminal Domain ◽

Localization Signal ◽

Nuclear Domains ◽

Carboxy Terminal

In the preceding study we found that Sm snRNPs and SerArg (SR) family proteins co-immunoprecipitate with Pol II molecules containing a hyperphosphorylated CTD (Kim et al., 1997). The association between Pol IIo and splicing factors is maintained in the absence of pre-mRNA, and the polymerase need not be transcriptionally engaged (Kim et al., 1997). The latter findings led us to hypothesize that a phosphorylated form of the CTD interacts with pre-mRNA splicing components in vivo. To test this idea, a nested set of CTD-derived proteins was assayed for the ability to alter the nuclear distribution of splicing factors, and to interfere with splicing in vivo. Proteins containing heptapeptides 1-52 (CTD52), 1-32 (CTD32), 1-26 (CTD26), 1-13 (CTD13), 1-6 (CTD6), 1-3 (CTD3), or 1 (CTD1) were expressed in mammalian cells. The CTD-derived proteins become phosphorylated in vivo, and accumulate in the nucleus even though they lack a conventional nuclear localization signal. CTD52 induces a selective reorganization of splicing factors from discrete nuclear domains to the diffuse nucleoplasm, and significantly, it blocks the accumulation of spliced, but not unspliced, human β-globin transcripts. The extent of splicing factor disruption, and the degree of inhibition of splicing, are proportional to the number of heptapeptides added to the protein. The above results indicate a functional interaction between Pol II's CTD and pre-mRNA splicing.

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Interaction of proteins with promoter elements of the human U2 snRNA genes in vivo

Gene ◽

10.1016/s0378-1119(03)00717-0 ◽

2003 ◽

Vol 315 ◽

pp. 103-112 ◽

Cited By ~ 9

Author(s):

Diana C. Boyd ◽

Ana Pombo ◽

Shona Murphy

Keyword(s):

Promoter Elements ◽

U2 Snrna

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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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An mRNA-type intron is present in the Rhodotorula hasegawae U2 small nuclear RNA gene

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5613-5619.1993 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5613-5619

Author(s):

Y Takahashi ◽

S Urushiyama ◽

T Tani ◽

Y Ohshima

Keyword(s):

Mrna Splicing ◽

Small Nuclear Rna ◽

U2 Snrna ◽

U6 Snrna ◽

Branch Site ◽

Snrna Gene ◽

Nuclear Rna ◽

Catalytic Role ◽

Mrna Precursor ◽

Site Recognition

Splicing an mRNA precursor requires multiple factors involving five small nuclear RNA (snRNA) species called U1, U2, U4, U5, and U6. The presence of mRNA-type introns in the U6 snRNA genes of some yeasts led to the hypothesis that U6 snRNA may play a catalytic role in pre-mRNA splicing and that the U6 introns occurred through reverse splicing of an intron from an mRNA precursor into a catalytic site of U6 snRNA. We characterized the U2 snRNA gene of the yeast Rhodotorula hasegawae, which has four mRNA-type introns in the U6 snRNA gene, and found an mRNA-type intron of 60 bp. The intron of the U2 snRNA gene is present in the highly conserved region immediately downstream of the branch site recognition domain. Interestingly, we found that this region can form a novel base pairing with U6 snRNA. We discuss the possible implications of these findings for the mechanisms of intron acquisition and for the role of U2 snRNA in pre-mRNA splicing.

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Elucidation of the BMI1 interactome identifies novel regulatory roles in glioblastoma

NAR Cancer ◽

10.1093/narcan/zcab009 ◽

2021 ◽

Vol 3 (1) ◽

Author(s):

Verónica Freire-Benéitez ◽

Nicola Pomella ◽

Thomas O Millner ◽

Anaëlle A Dumas ◽

Maria Victoria Niklison-Chirou ◽

...

Keyword(s):

Chromatin Immunoprecipitation ◽

Protein Composition ◽

Mrna Splicing ◽

Cholesterol Transport ◽

Normal Brain ◽

Regulatory Functions ◽

Polycomb Repressive Complex 1 ◽

Normal Brain Development

Abstract Glioblastoma (GBM) is the most common and aggressive intrinsic brain tumour in adults. Epigenetic mechanisms controlling normal brain development are often dysregulated in GBM. Among these, BMI1, a structural component of the Polycomb Repressive Complex 1 (PRC1), which promotes the H2AK119ub catalytic activity of Ring1B, is upregulated in GBM and its tumorigenic role has been shown in vitro and in vivo. Here, we have used protein and chromatin immunoprecipitation followed by mass spectrometry (MS) analysis to elucidate the protein composition of PRC1 in GBM and transcriptional silencing of defining interactors in primary patient-derived GIC lines to assess their functional impact on GBM biology. We identify novel regulatory functions in mRNA splicing and cholesterol transport which could represent novel targetable mechanisms in GBM.

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