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

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.

scholarly journals USP42 drives nuclear speckle mRNA splicing via directing dynamic phase separation to promote tumorigenesis

2021 ◽  
Author(s):  
Shuyan Liu ◽  
Taishu Wang ◽  
Yulin Shi ◽  
Lu Bai ◽  
Shanshan Wang ◽  
...  
Keyword(s):  
Phase Separation ◽  
Posttranslational Modifications ◽  
Bioinformatic Analysis ◽  
Mrna Splicing ◽  
Dependent Manner ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
Dynamic Regulation ◽  
Intrinsically Disordered ◽  
The Impact

AbstractLiquid–liquid phase separation is considered a generic approach to organize membrane-less compartments, enabling the dynamic regulation of phase-separated assemblies to be investigated and pivotal roles of protein posttranslational modifications to be demonstrated. By surveying the subcellular localizations of human deubiquitylases, USP42 was identified to form nuclear punctate structures that are associated with phase separation properties. Bioinformatic analysis demonstrated that the USP42 C-terminal sequence was intrinsically disordered, which was further experimentally confirmed to confer phase separation features. USP42 is distributed to SC35-positive nuclear speckles in a positively charged C-terminal residue- and enzymatic activity-dependent manner. Notably, USP42 directs the integration of the spliceosome component PLRG1 into nuclear speckles, and its depletion interferes with the conformation of SC35 foci. Functionally, USP42 downregulation deregulates multiple mRNA splicing events and leads to deterred cancer cell growth, which is consistent with the impact of PLRG1 repression. Finally, USP42 expression is strongly correlated with that of PLRG1 in non-small-cell lung cancer samples and predicts adverse prognosis in overall survival. As a deubiquitylase capable of dynamically guiding nuclear speckle phase separation and mRNA splicing, USP42 inhibition presents a novel anticancer strategy by targeting phase separation.

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

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.

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

2005 ◽  
Vol 16 (3) ◽  
pp. 1366-1377 ◽  
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.

Modifications in small nuclear RNAs and their roles in spliceosome assembly and function

2018 ◽  
Vol 399 (11) ◽  
pp. 1265-1276 ◽  
Author(s):  
Markus T. Bohnsack ◽  
Katherine E. Sloan
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Current Knowledge ◽  
Mrna Splicing ◽  
Cajal Body ◽  
Modified Nucleotides ◽  
Spliceosome Assembly ◽  
Small Nuclear Rnas ◽  
And Function ◽  
Fine Tune

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.

Monochloramine induces reorganization of nuclear speckles and phosphorylation of SRp30 in human colonic epithelial cells: role of protein kinase C

2003 ◽  
Vol 285 (5) ◽  
pp. C1294-C1303 ◽  
Author(s):  
Ya-Qin Zhu ◽  
Yu Lu ◽  
Xiao-Di Tan
Keyword(s):  
Gastrointestinal Tract ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Morphological Changes ◽  
Sr Proteins ◽  
Mrna Splicing ◽  
Intestinal Epithelial ◽  
Nuclear Speckles ◽  
Colonic Epithelial Cells ◽  
Stimulation Of

Intestinal epithelial cells are constantly stimulated by reactive oxidant metabolites (ROMs) in inflamed mucosa. Monochloramine (NH2Cl), a cell-permeant ROM, is particularly relevant to the pathogenesis of inflammation in the gastrointestinal tract. Nuclear speckles, a unique nuclear subcompartment, accumulate a family of proteins, namely, serine- and arginine-rich (SR) proteins. They play important roles in regulation of pre-mRNA splicing. Currently, little is known about the link between inflammatory stimulation and the pre-mRNA splicing process, although gene expression is changed in inflamed tissues. The present study was designed to investigate whether stimulation of human colonic epithelial cells (HT-29 and Caco-2 cell lines) with NH2Cl affects nuclear speckles and their components. By indirect immunofluorescence, nuclear speckles have been shown to undergo rapid aggregation after NH2Cl stimulation. By utilizing Western blotting, SRp30 (a subset of SR proteins) in intestinal epithelial cells was found to be phosphorylated after NH2Cl treatment, whereas other SR proteins were not responsive to NH2Cl stimulation. The cytotoxic effect of NH2Cl was excluded by both negative lactate dehydrogenase assay and propidium iodide staining. Therefore, NH2Cl-induced morphological changes on nuclear speckles and phosphorylated SRp30 do not result from intestinal epithelial injury. Furthermore, the effect of NH2Cl on nuclear speckles and SRp30 was blocked by bisindolylmaleimide I, a selective PKC inhibitor. Together, the available data suggest that stimulation of intestinal epithelial cells with NH2Cl results in a consequent change on pre-mRNA splicing machinery via a distinctive signal pathway involving activation of PKC. This effect may contribute to oxidant-induced pathophysiological changes in the gastrointestinal tract.

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

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.

scholarly journals Whole-genome screening identifies proteins localized to distinct nuclear bodies

2013 ◽  
Vol 203 (1) ◽  
pp. 149-164 ◽  
Author(s):  
Ka-wing Fong ◽  
Yujing Li ◽  
Wenqi Wang ◽  
Wenbin Ma ◽  
Kunpeng Li ◽  
...  
Keyword(s):  
Posttranscriptional Regulation ◽  
Nuclear Bodies ◽  
Cajal Bodies ◽  
Nuclear Speckles ◽  
Genome Wide ◽  
A Genome ◽  
Human Proteins ◽  
Genome Screening ◽  
Interchromatin Space ◽  
Polycomb Bodies

The nucleus is a unique organelle that contains essential genetic materials in chromosome territories. The interchromatin space is composed of nuclear subcompartments, which are defined by several distinctive nuclear bodies believed to be factories of DNA or RNA processing and sites of transcriptional and/or posttranscriptional regulation. In this paper, we performed a genome-wide microscopy-based screening for proteins that form nuclear foci and characterized their localizations using markers of known nuclear bodies. In total, we identified 325 proteins localized to distinct nuclear bodies, including nucleoli (148), promyelocytic leukemia nuclear bodies (38), nuclear speckles (27), paraspeckles (24), Cajal bodies (17), Sam68 nuclear bodies (5), Polycomb bodies (2), and uncharacterized nuclear bodies (64). Functional validation revealed several proteins potentially involved in the assembly of Cajal bodies and paraspeckles. Together, these data establish the first atlas of human proteins in different nuclear bodies and provide key information for research on nuclear bodies.

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

2002 ◽  
Vol 22 (15) ◽  
pp. 5443-5450 ◽  
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.

scholarly journals Tau aggregates are RNA-protein assemblies that mis-localize multiple nuclear speckle components

2021 ◽  
Author(s):  
Evan Lester ◽  
Felicia K. Ooi ◽  
Nadine Bakkar ◽  
Jacob Ayers ◽  
Amanda L. Woerman ◽  
...  
Keyword(s):  
Cell Culture ◽  
Frontotemporal Dementia ◽  
Corticobasal Degeneration ◽  
Mrna Splicing ◽  
Protein Assemblies ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
Tau Aggregation ◽  
In Cells

AbstractTau aggregates contribute to neurodegenerative diseases including frontotemporal dementia and Alzheimer’s disease (AD). Although RNA promotes tau aggregation in vitro, whether tau aggregates in cells contain RNA is unknown. We demonstrate in cell culture and mouse brains that both cytosolic and nuclear tau aggregates contain RNA, with enrichment for snRNAs and snoRNAs. Nuclear tau aggregates colocalize with and alter the composition, dynamics, and organization of nuclear speckles, which are membraneless organelles involved in pre-mRNA splicing. Moreover, several nuclear speckle components, including SRRM2, mislocalize to cytosolic tau aggregates in cells, mouse brains, and patient brains with AD, frontotemporal dementia (FTD), and corticobasal degeneration (CBD). Consistent with these alterations we observe the presence of tau aggregates is sufficient to alter pre-mRNA splicing. This work identifies tau alteration of nuclear speckles as a feature of tau aggregation that may contribute to the pathology of tau aggregates.

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