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 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 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.

scholarly journals Structural/functional properties of a mammalian multi-component structure containing all major spliceosomal small nuclear ribonucleoprotein particles

1998 ◽  
Vol 332 (1) ◽  
pp. 135-144 ◽  
Author(s):  
Marina MORAITOU ◽  
Meropi PATRINOU-GEORGOULA ◽  
Apostolia GUIALIS
Keyword(s):  
Rat Liver ◽  
Rna Splicing ◽  
Micrococcal Nuclease ◽  
Component Structure ◽  
Small Nuclear Ribonucleoprotein ◽  
Ribonucleoprotein Particles ◽  
Nuclear Extracts ◽  
U2 Snrnp ◽  
Stable Association ◽  
Nuclear Ribonucleoprotein

An approx. 40 S multi-component structure, consisting of all major spliceosomal small nuclear ribonucleoprotein particles (snRNP) (U1, U2, U4/U6 and U5) in stable association with a large number of polypeptides, mainly in the range 50–210 kDa, has been reported to exist within rat liver nuclear extracts [Guialis, Moraitou, Patrinou-Georgoula and Dangli (1991) Nucleic Acids Res. 19, 287–296]. Using a new polyclonal antibody recognizing a 63 kDa protein component of the complex, this multi-snRNP assembly was detected within rat liver nuclear extracts as efficiently as with the antibody for the U2 snRNP-specific B´ polypeptide. The 63 kDa protein was found to correspond to the 66 kDa subunit of the splicing factor SF3a, a known integral component of the HeLa 17 S U2 snRNP. Anti-2,2,7-trimethylguanosine affinity chromatography was an easy and efficient way of purifying the multi-snRNP complex from rat liver 40 S heterogeneous nuclear ribonucleoprotein particle (hnRNP)-containing sucrose gradient fractions. By subsequent glycerol-gradient sedimentation, all known snRNP forms active in RNA splicing were identified among its constituents. A complex structurally similar to the rat multi-snRNP was also identified in HeLa nuclear extracts. Preservation of hnRNP–snRNP interactions was observed within HeLa 40 S fractions. Moreover, these fractions were capable of restoring splicing activity when applied in reconstitution studies to supplement a micrococcal nuclease-treated splicing extract.

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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