Conditional depletion of transcriptional kinases Ctk1 and Bur1 and effects on co-transcriptional spliceosome assembly and pre-mRNA splicing

Abstract Modifications in cellular RNAs have emerged as key regulators of all aspects of gene expression, including pre-mRNA splicing. During spliceosome assembly and function, the small nuclear RNAs (snRNAs) form numerous dynamic RNA-RNA and RNA-protein interactions, which are required for spliceosome assembly, correct positioning of the spliceosome on substrate pre-mRNAs and catalysis. The human snRNAs contain several base methylations as well as a myriad of pseudouridines and 2′-O-methylated nucleotides, which are largely introduced by small Cajal body-specific ribonucleoproteins (scaRNPs). Modified nucleotides typically cluster in functionally important regions of the snRNAs, suggesting that their presence could optimise the interactions of snRNAs with each other or with pre-mRNAs, or may affect the binding of spliceosomal proteins. snRNA modifications appear to play important roles in snRNP biogenesis and spliceosome assembly, and have also been proposed to influence the efficiency and fidelity of pre-mRNA splicing. Interestingly, alterations in the modification status of snRNAs have recently been observed in different cellular conditions, implying that some snRNA modifications are dynamic and raising the possibility that these modifications may fine-tune the spliceosome for particular functions. Here, we review the current knowledge on the snRNA modification machinery and discuss the timing, functions and dynamics of modifications in snRNAs.

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Arabidopsis exoribonuclease USB1 interacts with the PPR-domain protein SOAR1 to negatively regulate abscisic acid signaling

Journal of Experimental Botany ◽

10.1093/jxb/eraa315 ◽

2020 ◽

Vol 71 (19) ◽

pp. 5837-5851

Author(s):

Yu Ma ◽

Shang Zhang ◽

Chao Bi ◽

Chao Mei ◽

Shang-Chuan Jiang ◽

...

Keyword(s):

Abscisic Acid ◽

Regulation Of Gene Expression ◽

Mrna Splicing ◽

Plant Responses ◽

Aba Signaling ◽

Pentatricopeptide Repeat ◽

Spliceosome Assembly ◽

Early Seedling ◽

Post Transcriptional Regulation ◽

Early Seedling Development

Abstract Signaling by the phytohormone abscisic acid (ABA) involves pre-mRNA splicing, a key process of post-transcriptional regulation of gene expression. However, the regulatory mechanism of alternative pre-mRNA splicing in ABA signaling remains largely unknown. We previously identified a pentatricopeptide repeat protein SOAR1 (suppressor of the ABAR-overexpressor 1) as a crucial player downstream of ABAR (putative ABA receptor) in ABA signaling. In this study, we identified a SOAR1 interaction partner USB1, which is an exoribonuclease catalyzing U6 production for spliceosome assembly. We reveal that together USB1 and SOAR1 negatively regulate ABA signaling in early seedling development. USB1 and SOAR1 are both required for the splicing of transcripts of numerous genes, including those involved in ABA signaling pathways, suggesting that USB1 and SOAR1 collaborate to regulate ABA signaling by affecting spliceosome assembly. These findings provide important new insights into the mechanistic control of alternative pre-mRNA splicing in the regulation of ABA-mediated plant responses to environmental cues.

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Partial purification of the yeast U2 snRNP reveals a novel yeast pre-mRNA splicing factor required for pre-spliceosome assembly

The EMBO Journal ◽

10.1093/emboj/18.12.3463 ◽

1999 ◽

Vol 18 (12) ◽

pp. 3463-3474 ◽

Cited By ~ 46

Author(s):

F. Caspary

Keyword(s):

Mrna Splicing ◽

Splicing Factor ◽

Partial Purification ◽

Spliceosome Assembly ◽

U2 Snrnp

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p68 RNA Helicase Is an Essential Human Splicing Factor That Acts at the U1 snRNA-5′ Splice Site Duplex

Molecular and Cellular Biology ◽

10.1128/mcb.22.15.5443-5450.2002 ◽

2002 ◽

Vol 22 (15) ◽

pp. 5443-5450 ◽

Cited By ~ 87

Author(s):

Zhi-Ren Liu

Keyword(s):

Splice Site ◽

Early Stage ◽

Rna Helicase ◽

Mrna Splicing ◽

Cross Linking ◽

Spliceosome Assembly ◽

U1 Snrna ◽

U1 Snrnp ◽

P68 Rna Helicase

ABSTRACT Modulation of the interaction between U1 snRNP and the 5′ splice site (5′ss) is a key event that governs 5′ss recognition and spliceosome assembly. Using the methylene blue-mediated cross-linking method (Z. R. Liu, A. M. Wilkie, M. J. Clemens, and C. W. Smith, RNA 2:611-621, 1996), a 65-kDa protein (p65) was shown to interact with the U1-5′ss duplex during spliceosome assembly (Z. R. Liu, B. Sargueil, and C. W. Smith, Mol. Cell. Biol. 18:6910-6920, 1998). In this report, p65 was identified as p68 RNA helicase and shown to be essential for in vitro pre-mRNA splicing. Depletion of endogenous p68 RNA helicase does not affect the loading of the U1 snRNP to the 5′ss during early stage of splicing. However, dissociation of the U1 from the 5′ss is largely inhibited. The data suggest that p68 RNA helicase functions in destabilizing the U1-5′ss interactions. Furthermore, depletion of p68 RNA helicase arrested spliceosome assembly at the prespliceosome stage, suggesting that p68 may play a role in the transition from prespliceosome to spliceosome.

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SANS (USH1G) regulates pre-mRNA splicing by mediating the intra-nuclear transfer of tri-snRNPs from Cajal bodies to nuclear speckles for spliceosome assembly

10.1101/2020.11.11.378448 ◽

2020 ◽

Author(s):

Adem Yildirim ◽

Sina Mozaffari-Jovin ◽

Ann-Kathrin Wallisch ◽

Jessica Ries ◽

Sebastian Ludwig ◽

...

Keyword(s):

Nuclear Transfer ◽

Usher Syndrome ◽

Mrna Splicing ◽

Cajal Bodies ◽

Nuclear Speckles ◽

Spliceosome Assembly ◽

Common Cause ◽

Intrinsically Disordered ◽

Assembly Kinetics ◽

Cytoplasmic Processes

AbstractSplicing is catalyzed by the spliceosome, a compositionally dynamic complex assembled stepwise on pre-mRNA. We reveal the link between splicing machinery components with the intrinsically disordered ciliopathy protein SANS. Pathogenic mutations in SANS/USH1G lead to Usher syndrome – the most common cause of deaf-blindness. SANS functions has been associated with cytoplasmic processes so far. Here, we show SANS localization in Cajal bodies and nuclear speckles. There SANS interacts with components of spliceosomal complexes and the large splicing cofactor SON and PRPFs of the tri-snRNP complex. SANS is required for the release of tri-snRNPs from Cajal bodies and their recruitment to nuclear speckles. SANS depletion alters spliceosome assembly kinetics, leading to stalled complex A formation, which can be chased to spliced products by the addition of tri-snRNPs. SANS deficiency and USH1G mutations affects splicing of genes related to cell proliferation and USH. We provide the first evidence that splicing deregulation may participate at the pathophysiology of Usher syndrome.

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A synthetic small molecule stalls pre-mRNA splicing by promoting an early-stage U2AF2–RNA complex

10.1101/2020.09.28.317727 ◽

2020 ◽

Author(s):

Rakesh Chatrikhi ◽

Callen F. Feeney ◽

Mary J. Pulvino ◽

Georgios Alachouzos ◽

Andrew J. MacRae ◽

...

Keyword(s):

Small Molecule ◽

Rna Binding ◽

Early Stage ◽

Mrna Splicing ◽

Spliceosome Assembly ◽

Rna Recognition Motifs ◽

Computational Docking ◽

Macromolecular Assemblies ◽

Multi Stage ◽

Recognition Motifs

AbstractDysregulated pre-mRNA splicing is an emerging Achilles heel of cancers and myelodysplasias. To expand the currently limited portfolio of small molecule drug leads, we screened for chemical modulators of the U2AF complex, which nucleates spliceosome assembly and is mutated in myelodysplasias. A hit compound specifically enhances RNA binding by a U2AF2 subunit. Remarkably, the compound inhibits splicing of representative substrates in cells and stalls spliceosome assembly at the stage of U2AF function. Computational docking, together with structure-guided mutagenesis, indicates that the compound bridges an active conformation of the U2AF2 RNA recognition motifs via hydrophobic and electrostatic moieties. Altogether, our results highlight the potential of trapping early spliceosome assembly as an effective pharmacological means to manipulate pre-mRNA splicing. By extension, we suggest that stabilizing inactive checkpoints may offer a breakthrough approach for small molecule inhibition of multi-stage macromolecular assemblies.

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PKR activation and eIF2α phosphorylation mediate human globin mRNA splicing at spliceosome assembly

Cell Research ◽

10.1038/cr.2017.39 ◽

2017 ◽

Vol 27 (5) ◽

pp. 688-704 ◽

Cited By ~ 17

Author(s):

Lena Ilan ◽

Farhat Osman ◽

Lise Sarah Namer ◽

Einav Eliahu ◽

Smadar Cohen-Chalamish ◽

...

Keyword(s):

Mrna Splicing ◽

Globin Mrna ◽

Spliceosome Assembly ◽

Eif2α Phosphorylation

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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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NS1-Binding Protein (NS1-BP): a Novel Human Protein That Interacts with the Influenza A Virus Nonstructural NS1 Protein Is Relocalized in the Nuclei of Infected Cells

Journal of Virology ◽

10.1128/jvi.72.9.7170-7180.1998 ◽

1998 ◽

Vol 72 (9) ◽

pp. 7170-7180 ◽

Cited By ~ 88

Author(s):

Thorsten Wolff ◽

Robert E. O’Neill ◽

Peter Palese

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Genetic Interaction ◽

Binding Protein ◽

Mrna Splicing ◽

Mutant Protein ◽

Dominant Negative Effect ◽

Ns1 Protein ◽

Spliceosome Assembly ◽

Infected Cells

ABSTRACT We used the yeast interaction trap system to identify a novel human 70-kDa protein, termed NS1-binding protein (NS1-BP), which interacts with the nonstructural NS1 protein of the influenza A virus. The genetic interaction was confirmed by the specific coprecipitation of the NS1 protein from solution by a glutathioneS-transferase–NS1-BP fusion protein and glutathione-Sepharose. NS1-BP contains an N-terminal BTB/POZ domain and five kelch-like tandem repeat elements of ∼50 amino acids. In noninfected cells, affinity-purified antibodies localized NS1-BP in nuclear regions enriched with the spliceosome assembly factor SC35, suggesting an association of NS1-BP with the cellular splicing apparatus. In influenza A virus-infected cells, NS1-BP relocalized throughout the nucleoplasm and appeared distinct from the SC35 domains, which suggests that NS1-BP function may be disturbed or altered. The addition of a truncated NS1-BP mutant protein to a HeLa cell nuclear extract efficiently inhibited pre-mRNA splicing but not spliceosome assembly. This result could be explained by a possible dominant-negative effect of the NS1-BP mutant protein and suggests a role of the wild-type NS1-BP in promoting pre-mRNA splicing. These data suggest that the inhibition of splicing by the NS1 protein may be mediated by binding to NS1-BP.

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DHX15-independent roles for TFIP11 in U6 snRNA modification, U4/U6.U5 tri-snRNP assembly and pre-mRNA splicing fidelity

Nature Communications ◽

10.1038/s41467-021-26932-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Amandine Duchemin ◽

Tina O’Grady ◽

Sarah Hanache ◽

Agnès Mereau ◽

Marc Thiry ◽

...

Keyword(s):

Rna Helicase ◽

Mrna Splicing ◽

Cajal Bodies ◽

Human Homolog ◽

Spliceosome Assembly ◽

The Core ◽

U6 Snrna

AbstractThe U6 snRNA, the core catalytic component of the spliceosome, is extensively modified post-transcriptionally, with 2’-O-methylation being most common. However, how U6 2’-O-methylation is regulated remains largely unknown. Here we report that TFIP11, the human homolog of the yeast spliceosome disassembly factor Ntr1, localizes to nucleoli and Cajal Bodies and is essential for the 2’-O-methylation of U6. Mechanistically, we demonstrate that TFIP11 knockdown reduces the association of U6 snRNA with fibrillarin and associated snoRNAs, therefore altering U6 2′-O-methylation. We show U6 snRNA hypomethylation is associated with changes in assembly of the U4/U6.U5 tri-snRNP leading to defects in spliceosome assembly and alterations in splicing fidelity. Strikingly, this function of TFIP11 is independent of the RNA helicase DHX15, its known partner in yeast. In sum, our study demonstrates an unrecognized function for TFIP11 in U6 snRNP modification and U4/U6.U5 tri-snRNP assembly, identifying TFIP11 as a critical spliceosome assembly regulator.

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