scholarly journals Identification and characterization ofDrosophilaSnurportin reveals a role for the import receptor Moleskin/importin-7 in snRNP biogenesis

2013 ◽  
Vol 24 (18) ◽  
pp. 2932-2942 ◽  
Author(s):  
Amanda Hicks Natalizio ◽  
A. Gregory Matera
Keyword(s):  
Fruit Fly ◽  
Survival Motor Neuron ◽  
Cajal Body ◽  
Small Nuclear Ribonucleoprotein ◽  
Small Nuclear Rnas ◽  
Assembly Factor ◽  
Nuclear Ribonucleoprotein ◽  
Identification And Characterization ◽  
Transport Receptor ◽  
Import Receptor

Nuclear import is an essential step in small nuclear ribonucleoprotein (snRNP) biogenesis. Snurportin1 (SPN1), the import adaptor, binds to trimethylguanosine (TMG) caps on spliceosomal small nuclear RNAs. Previous studies indicated that vertebrate snRNP import requires importin-β, the transport receptor that binds directly to SPN1. We identify CG42303/snup as the Drosophila orthologue of human snurportin1 (SNUPN). Of interest, the importin-β binding (IBB) domain of SPN1, which is essential for TMG cap–mediated snRNP import in humans, is not well conserved in flies. Consistent with its lack of an IBB domain, we find that Drosophila SNUP (dSNUP) does not interact with Ketel/importin-β. Fruit fly snRNPs also fail to bind Ketel; however, the importin-7 orthologue Moleskin (Msk) physically associates with both dSNUP and spliceosomal snRNPs and localizes to nuclear Cajal bodies. Strikingly, we find that msk-null mutants are depleted of the snRNP assembly factor, survival motor neuron, and the Cajal body marker, coilin. Consistent with a loss of snRNP import function, long-lived msk larvae show an accumulation of TMG cap signal in the cytoplasm. These data indicate that Ketel/importin-β does not play a significant role in Drosophila snRNP import and demonstrate a crucial function for Msk in snRNP biogenesis.

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 U1 small nuclear RNA-promoted exon selection requires a minimal distance between the position of U1 binding and the 3' splice site across the exon.

1997 ◽  
Vol 17 (12) ◽  
pp. 7099-7107 ◽  
Author(s):  
D Y Hwang ◽  
J B Cohen
Keyword(s):  
Splice Site ◽  
Minimum Size ◽  
Minimal Distance ◽  
Small Nuclear Rna ◽  
Small Nuclear Ribonucleoprotein ◽  
Optimal Distance ◽  
Small Nuclear Rnas ◽  
Constitutive Splicing ◽  
Nuclear Ribonucleoprotein ◽  
Lower Size

Both experimental work and surveys of the lengths of internal exons in nature have suggested that vertebrate internal exons require a minimum size of approximately 50 nucleotides for efficient inclusion in mature mRNA. This phenomenon has been ascribed to steric interference between complexes involved in recognition of the splicing signals at the two ends of short internal exons. To determine whether U1 small nuclear ribonucleoprotein, a multicomponent splicing factor that is involved in the first recognition of splice sites, contributes to the lower size limit of vertebrate internal exons, we have taken advantage of our previous observation that U1 small nuclear RNAs (snRNAs) which bind upstream or downstream of the 5' splice site (5'SS) stimulate splicing of the upstream intron. By varying the position of U1 binding relative to the 3'SS, we show that U1-dependent splicing of the upstream intron becomes inefficient when U1 is positioned 48 nucleotides or less downstream of the 3'SS, suggesting a minimal distance between U1 and the 3'SS of approximately 50 nucleotides. This distance corresponds well to the suggested minimum size of internal exons. The results of experiments in which the 3'SS region of the reporter was duplicated suggest an optimal distance of greater than 72 nucleotides. We have also found that inclusion of a 24-nucleotide miniexon is promoted by the binding of U1 to the downstream intron but not by binding to the 5'SS. Our results are discussed in the context of models to explain constitutive splicing of small exons in nature.

scholarly journals Splicing-independent recruitment of spliceosomal small nuclear RNPs to nascent RNA polymerase II transcripts

2007 ◽  
Vol 178 (6) ◽  
pp. 937-949 ◽  
Author(s):  
Snehal Bhikhu Patel ◽  
Natalya Novikova ◽  
Michel Bellini
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Messenger Rnas ◽  
Stem Loop ◽  
Small Nuclear Ribonucleoprotein ◽  
Small Nuclear Rnas ◽  
Transcriptional Units ◽  
Necessary And Sufficient ◽  
Nuclear Ribonucleoprotein ◽  
Nascent Transcripts

In amphibian oocytes, most lateral loops of the lampbrush chromosomes correspond to active transcriptional sites for RNA polymerase II. We show that newly assembled small nuclear ribonucleoprotein (RNP [snRNP]) particles, which are formed upon cytoplasmic injection of fluorescently labeled spliceosomal small nuclear RNAs (snRNAs), target the nascent transcripts of the chromosomal loops. With this new targeting assay, we demonstrate that nonfunctional forms of U1 and U2 snRNAs still associate with the active transcriptional units. In particular, we find that their association with nascent RNP fibrils is independent of their base pairing with pre–messenger RNAs. Additionally, stem loop I of the U1 snRNA is identified as a discrete domain that is both necessary and sufficient for association with nascent transcripts. Finally, in oocytes deficient in splicing, the recruitment of U1, U4, and U5 snRNPs to transcriptional units is not affected. Collectively, these data indicate that the recruitment of snRNPs to nascent transcripts and the assembly of the spliceosome are uncoupled events.

scholarly journals SMN-independent Subunits of the SMN Complex

2007 ◽  
Vol 282 (38) ◽  
pp. 27953-27959 ◽  
Author(s):  
Daniel J. Battle ◽  
Mumtaz Kasim ◽  
Jin Wang ◽  
Gideon Dreyfuss
Keyword(s):  
Motor Neurons ◽  
Atp Hydrolysis ◽  
Muscular Atrophy ◽  
Small Nuclear Rna ◽  
Small Nuclear Ribonucleoprotein ◽  
Cell Extracts ◽  
Smn Complex ◽  
Small Nuclear Rnas ◽  
Survival Of Motor Neurons ◽  
Nuclear Ribonucleoprotein

The survival of motor neurons (SMN) complex is essential for the biogenesis of small nuclear ribonucleoprotein (snRNP) complexes in eukaryotic cells. Reduced levels of SMN cause the motor neuron degenerative disease, spinal muscular atrophy. We identify here stable subunits of the SMN complex that do not contain SMN. Sedimentation and immunoprecipitation experiments using cell extracts reveal at least three complexes composed of Gemin3, -4, and -5; Gemin6, -7, and unrip; and SMN with Gemin2, as well as free Gemin5. Complexes containing Gemin3-Gemin4-Gemin5 and Gemin6-Gemin7-unrip persist at similar levels when SMN is reduced. In cells, immunofluorescence microscopy shows differential localization of Gemin5 after cell stress. We further show that the Gemin5-containing subunits bind small nuclear RNA independently of the SMN complex and without a requirement for exogenous ATP. ATP hydrolysis is, however, required for displacement of small nuclear RNAs from the Gemin5-containing subunits and their assembly into snRNPs. These findings demonstrate a modular nature of the SMN complex and identify a new intermediate in the snRNP assembly process.

scholarly journals Dephosphorylation of survival motor neurons (SMN) by PPM1G/PP2Cγ governs Cajal body localization and stability of the SMN complex

2007 ◽  
Vol 179 (3) ◽  
pp. 451-465 ◽  
Author(s):  
Sebastian Petri ◽  
Matthias Grimmler ◽  
Sabine Over ◽  
Utz Fischer ◽  
Oliver J. Gruss
Keyword(s):  
Motor Neurons ◽  
Survival Motor Neuron ◽  
Cajal Body ◽  
Sm Proteins ◽  
Interacting Protein ◽  
Survival Motor Neurons ◽  
Small Nuclear Ribonucleoprotein ◽  
Smn Complex ◽  
Catalytically Active ◽  
And Function

The survival motor neuron (SMN) complex functions in maturation of uridine-rich small nuclear ribonucleoprotein (RNP) particles. SMN mediates the cytoplasmic assembly of Sm proteins onto uridine-rich small RNAs, and then participates in targeting RNPs to nuclear Cajal bodies (CBs). Recent studies have suggested that phosphorylation might control localization and function of the SMN complex. Here, we show that the nuclear phosphatase PPM1G/PP2Cγ interacts with and dephosphorylates the SMN complex. Small interfering RNA knockdown of PPM1G leads to an altered phosphorylation pattern of SMN and Gemin3, loss of SMN from CBs, and reduced stability of SMN. Accumulation in CBs is restored upon overexpression of catalytically active, but not that of inactive, PPM1G. This demonstrates that PPM1G's phosphatase activity is necessary to maintain SMN subcellular distribution. Concomitant knockdown of unr interacting protein (unrip), a component implicated in cytoplasmic retention of the SMN complex, also rescues the localization defects. Our data suggest that an interplay between PPM1G and unrip determine compartment-specific phosphorylation patterns, localization, and function of the SMN complex.

scholarly journals Survival Motor Neuron Function in Motor Axons Is Independent of Functions Required for Small Nuclear Ribonucleoprotein Biogenesis

2006 ◽  
Vol 26 (43) ◽  
pp. 11014-11022 ◽  
Author(s):  
T. L. Carrel ◽  
M. L. McWhorter ◽  
E. Workman ◽  
H. Zhang ◽  
E. C. Wolstencroft ◽  
...  
Keyword(s):  
Motor Neuron ◽  
Survival Motor Neuron ◽  
Motor Axons ◽  
Small Nuclear Ribonucleoprotein ◽  
Neuron Function ◽  
Nuclear Ribonucleoprotein

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.

The yeast PRP19 protein is not tightly associated with small nuclear RNAs, but appears to associate with the spliceosome after binding of U2 to the pre-mRNA and prior to formation of the functional spliceosome

1993 ◽  
Vol 13 (3) ◽  
pp. 1883-1891
Author(s):  
W Y Tarn ◽  
K R Lee ◽  
S C Cheng
Keyword(s):  
Micrococcal Nuclease ◽  
Splicing Factors ◽  
Amino Acid Residues ◽  
Spliceosome Assembly ◽  
Protein Factor ◽  
Small Nuclear Ribonucleoprotein ◽  
Small Nuclear Rnas ◽  
Snrnp Protein ◽  
Precursor Rna ◽  
Nuclear Ribonucleoprotein

We have previously shown that the yeast PRP19 protein is associated with the spliceosome during the splicing reaction by immunoprecipitation studies with anti-PRP19 antibody. We have extended such studies by using extracts depleted of specific splicing factors to investigate the step of the spliceosome assembly process that PRP19 is involved in. PRP19 was not associated with the splicing complexes formed in U2- or U6-depleted extracts but was associated with the splicing complex formed in heat-inactivated prp2 extracts. This finding indicates that PRP19 becomes associated with the splicing complexes after or concomitant with binding of the U6 small nuclear ribonucleoprotein particle (snRNP) to the precursor RNA and before formation of the functional spliceosome. We further analyzed whether PRP19 is an integral component of snRNPs. We have constructed a strain in which an epitope of nine amino acid residues recognized by a well-characterized monoclonal antibody, 12CA5, is linked to the carboxyl terminus of the wild-type PRP19 protein. Immunoprecipitation of the splicing extracts with anti-PRP19 antibody or precipitation of the extracts prepared from the epitope-tagged strain with the 12CA5 antibody did not precipitate significant amounts of snRNAs. Addition of micrococcal nuclease-treated extracts to the PRP19-depleted extract restored its splicing activity. These results indicate that PRP19 is not tightly associated with any of the snRNAs required for the splicing reaction. No non-snRNP protein factor has been demonstrated to participate in either step of the spliceosome assembly pathway that PRP19 might be involved in. Thus, PRP19 represents a novel splicing factor.

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