scholarly journals Molecular Characterization of a Novel, Widespread Nuclear Protein That Colocalizes with Spliceosome Components

1998 ◽  
Vol 9 (1) ◽  
pp. 143-160 ◽  
Author(s):  
Marion S. Schmidt-Zachmann ◽  
Sylvia Knecht ◽  
Angela Krämer
Keyword(s):  
Molecular Characterization ◽  
Nuclear Protein ◽  
Cultured Cells ◽  
Splicing Factors ◽  
Nuclear Speckles ◽  
Sm Proteins ◽  
Small Nuclear Ribonucleoprotein ◽  
Ribonucleoprotein Particles ◽  
Nuclear Ribonucleoprotein

We report the identification and molecular characterization of a novel type of constitutive nuclear protein that is present in diverse vertebrate species, from Xenopus laevis to human. The cDNA-deduced amino acid sequence of the Xenopus protein defines a polypeptide of a calculated mass of 146.2 kDa and a isoelectric point of 6.8, with a conspicuous domain enriched in the dipeptide TP (threonine-proline) near its amino terminus. Immunolocalization studies in cultured cells and tissues sections of different origin revealed an exclusive nuclear localization of the protein. The protein is diffusely distributed in the nucleoplasm but concentrated in nuclear speckles, which represent a subnuclear compartment enriched in small nuclear ribonucleoprotein particles and other splicing factors, as confirmed by colocalization with certain splicing factors and Sm proteins. During mitosis, when transcription and splicing are downregulated, the protein is released from the nuclear speckles and transiently dispersed throughout the cytoplasm. Biochemical experiments have shown that the protein is recovered in a ∼12S complex, and gel filtration studies confirm that the protein is part of a large particle. Immunoprecipitation and Western blot analysis of chromatographic fractions enriched in human U2 small nuclear ribonucleoprotein particles of distinct sizes (12S, 15S, and 17S), reflecting their variable association with splicing factors SF3a and SF3b, strongly suggests that the 146-kDa protein reported here is a constituent of the SF3b complex.

scholarly journals Fanconi anemia FANCD2 and FANCI proteins regulate the nuclear dynamics of splicing factors

2017 ◽  
Vol 216 (12) ◽  
pp. 4007-4026 ◽  
Author(s):  
María Moriel-Carretero ◽  
Sara Ovejero ◽  
Marie Gérus-Durand ◽  
Dimos Vryzas ◽  
Angelos Constantinou
Keyword(s):  
Dna Replication ◽  
Fanconi Anemia ◽  
Splicing Factors ◽  
Nuclear Speckles ◽  
Nuclear Dynamics ◽  
Small Nuclear Ribonucleoprotein ◽  
Close Proximity ◽  
Dna Replication Stress ◽  
Cross Links ◽  
Nuclear Ribonucleoprotein

Proteins disabled in the cancer-prone disorder Fanconi anemia (FA) ensure the maintenance of chromosomal stability during DNA replication. FA proteins regulate replication dynamics, coordinate replication-coupled repair of interstrand DNA cross-links, and mitigate conflicts between replication and transcription. Here we show that FANCI and FANCD2 associate with splicing factor 3B1 (SF3B1), a key spliceosomal protein of the U2 small nuclear ribonucleoprotein (U2 snRNP). FANCI is in close proximity to SF3B1 in the nucleoplasm of interphase and mitotic cells. Furthermore, we find that DNA replication stress induces the release of SF3B1 from nuclear speckles in a manner that depends on FANCI and on the activity of the checkpoint kinase ATR. In chromatin, both FANCD2 and FANCI associate with SF3B1, prevent accumulation of postcatalytic intron lariats, and contribute to the timely eviction of splicing factors. We propose that FANCD2 and FANCI contribute to the organization of functional domains in chromatin, ensuring the coordination of DNA replication and cotranscriptional processes.

scholarly journals Spliceosomal Small Nuclear Ribonucleoprotein Particles Repeatedly Cycle through Cajal Bodies

2008 ◽  
Vol 19 (6) ◽  
pp. 2534-2543 ◽  
Author(s):  
David Staněk ◽  
Jarmila Přidalová-Hnilicová ◽  
Ivan Novotný ◽  
Martina Huranová ◽  
Michaela Blažíková ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Living Cells ◽  
Cajal Body ◽  
Cajal Bodies ◽  
Sm Proteins ◽  
Small Nuclear Ribonucleoprotein ◽  
Ribonucleoprotein Particles ◽  
Nuclear Extracts ◽  
Nuclear Ribonucleoprotein ◽  
Interfering Rna

The Cajal body (CB) is a nuclear structure closely associated with import and biogenesis of small nuclear ribonucleoprotein particles (snRNPs). Here, we tested whether CBs also contain mature snRNPs and whether CB integrity depends on the ongoing snRNP splicing cycle. Sm proteins tagged with photoactivatable and color-maturing variants of fluorescent proteins were used to monitor snRNP behavior in living cells over time; mature snRNPs accumulated in CBs, traveled from one CB to another, and they were not preferentially replaced by newly imported snRNPs. To test whether CB integrity depends on the snRNP splicing cycle, two human orthologues of yeast proteins involved in distinct steps in spliceosome disassembly after splicing, hPrp22 and hNtr1, were depleted by small interfering RNA treatment. Surprisingly, depletion of either protein led to the accumulation of U4/U6 snRNPs in CBs, suggesting that reassembly of the U4/U6·U5 tri-snRNP was delayed. Accordingly, a relative decrease in U5 snRNPs compared with U4/U6 snRNPs was observed in CBs, as well as in nuclear extracts of treated cells. Together, the data show that particular phases of the spliceosome cycle are compartmentalized in living cells, with reassembly of the tri-snRNP occurring in CBs.

scholarly journals DIS3L2 and LSm proteins are involved in the surveillance of Sm ring-deficient snRNAs

2020 ◽  
Vol 48 (11) ◽  
pp. 6184-6197
Author(s):  
Adriana Roithová ◽  
Zuzana Feketová ◽  
Štěpánka Vaňáčová ◽  
David Staněk
Keyword(s):  
Quality Control ◽  
Binding Site ◽  
Dependent Manner ◽  
Sm Proteins ◽  
Alternative Pathways ◽  
P Bodies ◽  
Small Nuclear Ribonucleoprotein ◽  
Ribonucleoprotein Particles ◽  
Small Nuclear Rnas ◽  
Nuclear Ribonucleoprotein

Abstract Spliceosomal small nuclear ribonucleoprotein particles (snRNPs) undergo a complex maturation pathway containing multiple steps in the nucleus and in the cytoplasm. snRNP biogenesis is strictly proofread and several quality control checkpoints are placed along the pathway. Here, we analyzed the fate of small nuclear RNAs (snRNAs) that are unable to acquire a ring of Sm proteins. We showed that snRNAs lacking the Sm ring are unstable and accumulate in P-bodies in an LSm1-dependent manner. We further provide evidence that defective snRNAs without the Sm binding site are uridylated at the 3′ end and associate with DIS3L2 3′→5′ exoribonuclease and LSm proteins. Finally, inhibition of 5′→3′ exoribonuclease XRN1 increases association of ΔSm snRNAs with DIS3L2, which indicates competition and compensation between these two degradation enzymes. Together, we provide evidence that defective snRNAs without the Sm ring are uridylated and degraded by alternative pathways involving either DIS3L2 or LSm proteins and XRN1.

Identification and characterization of the small nuclear ribonucleoprotein particle D' core protein

1990 ◽  
Vol 10 (9) ◽  
pp. 4480-4485
Author(s):  
J Andersen ◽  
R J Feeney ◽  
G W Zieve
Keyword(s):  
Electrophoretic Mobility ◽  
Protein Complexes ◽  
Ribonucleoprotein Particle ◽  
Small Nuclear Ribonucleoprotein ◽  
Core Proteins ◽  
Nuclear Ribonucleoprotein ◽  
Identification And Characterization ◽  
Snrnp Core ◽  
Small Nuclear Ribonucleoprotein Particle

The addition of urea to sodium dodecyl sulfate (SDS)-polyacrylamide gels has allowed the identification and characterization of the small nuclear ribonucleoprotein particle (snRNP) D' protein and has also improved resolution of the E, F, and G snRNP core proteins. In standard SDS-polyacrylamide gels, the D' and D snRNP core proteins comigrate at approximately 16 kilodaltons. The addition of urea to the separating gel caused the D' protein to shift to a slower electrophoretic mobility that is distinct from that of the D protein. The shift to a slower electrophoretic mobility in the presence of urea suggests that the D' protein has extensive secondary structure that is not totally disrupted by SDS alone. Both N-terminal sequencing and partial peptide maps indicate that the D and D' proteins are distinct gene products, and the sequence data have identified the faster moving of the two proteins as the previously cloned D protein (L. A. Rokeach, J. A. Haselby, and S. O. Hoch, Proc. Natl. Acad. Sci. USA 85:4832-4836, 1988). In the cytoplasm, the D protein is found primarily in the small-nuclear-RNA-free 6S protein complexes, while the D' protein is found primarily in the 20S protein complexes. Like the D protein, the D' protein is an autoantigen in patients with systemic lupus erythematosus and is recognized by some of the Sm class of autoimmune antisera.

scholarly journals Structurally related but functionally distinct yeast Sm D core small nuclear ribonucleoprotein particle proteins.

1995 ◽  
Vol 15 (1) ◽  
pp. 445-455 ◽  
Author(s):  
J Roy ◽  
B Zheng ◽  
B C Rymond ◽  
J L Woolford
Keyword(s):  
Protein Complexes ◽  
Mrna Splicing ◽  
Yeast Cells ◽  
Ribonucleoprotein Particle ◽  
Sm Proteins ◽  
Small Nuclear Ribonucleoprotein ◽  
Snrnp Protein ◽  
Nuclear Ribonucleoprotein ◽  
Precursor Mrna

Spliceosome assembly during pre-mRNA splicing requires the correct positioning of the U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) on the precursor mRNA. The structure and integrity of these snRNPs are maintained in part by the association of the snRNAs with core snRNP (Sm) proteins. The Sm proteins also play a pivotal role in metazoan snRNP biogenesis. We have characterized a Saccharomyces cerevisiae gene, SMD3, that encodes the core snRNP protein Smd3. The Smd3 protein is required for pre-mRNA splicing in vivo. Depletion of this protein from yeast cells affects the levels of U snRNAs and their cap modification, indicating that Smd3 is required for snRNP biogenesis. Smd3 is structurally and functionally distinct from the previously described yeast core polypeptide Smd1. Although Smd3 and Smd1 are both associated with the spliceosomal snRNPs, overexpression of one cannot compensate for the loss of the other. Thus, these two proteins have distinct functions. A pool of Smd3 exists in the yeast cytoplasm. This is consistent with the possibility that snRNP assembly in S. cerevisiae, as in metazoans, is initiated in the cytoplasm from a pool of RNA-free core snRNP protein complexes.

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