m3G cap hypermethylation of U1 small nuclear ribonucleoprotein (snRNP) in vitro: evidence that the U1 small nuclear RNA-(guanosine-N2)-methyltransferase is a non-snRNP cytoplasmic protein that requires a binding site on the Sm core domain

1994 ◽  
Vol 14 (6) ◽  
pp. 4160-4172
Author(s):  
G Plessel ◽  
U Fischer ◽  
R Lührmann
Keyword(s):  
Nuclear Transport ◽  
Iodoacetic Acid ◽  
Small Nuclear Rna ◽  
Vitro Assay ◽  
Core Domain ◽  
Small Nuclear Ribonucleoprotein ◽  
U1 Snrnp ◽  
Snrnp Protein ◽  
Localization Signal

The RNA components of small nuclear ribonucleoproteins (U snRNPs) possess a characteristic 5'-terminal trimethylguanosine cap structure (m3G cap). This cap is an important component of the nuclear localization signal of U snRNPs. It arises by hypermethylation of a cotranscriptionally added m7G cap. Here we describe an in vitro assay for the hypermethylation, which employs U snRNP particles reconstituted in vitro from purified components and subsequent analysis by m3G cap-specific immunoprecipitation. Complementation studies in vitro revealed that both cytosol and S-adenosylmethionine are required for the hypermethylation of an m7G-capped U1 snRNP reconstituted in vitro, indicating that the U1 snRNA-(guanosine-N2)-methyltransferase is a trans-active non-snRNP protein. Chemical modification revealed one cytoplasmic component required for hypermethylation and one located on the snRNP: these components have different patterns of sensitivity to modification by N-ethylmaleimide and iodoacetic acid (IAA). In the presence of cytosol and S-adenosylmethionine, an intact Sm core domain is a necessary and sufficient substrate for cap hypermethylation. These data, together with our observation that isolated native U1 snRNPs but not naked U1 RNA inhibit the trimethylation of in vitro-reconstituted U1 snRNP, indicate that the Sm core binds the methyltransferase specifically. Moreover, isolated native U2 snRNP also inhibits trimethylation of U1 snRNP, suggesting that other Sm-class U snRNPs might share the same methyltransferase. IAA modification of m7G-capped U1 snRNPs inhibited hypermethylation when they were microinjected into Xenopus oocytes and consequently also inhibited nuclear import. In contrast, modification with IAA of m3G-capped U1 snRNPs reconstituted in vitro did not interfere with their nuclear transport in oocytes. These data suggest that m3G cap formation and nuclear transport of U1 snRNPs are mediated by distinct factors, which require distinct binding sites on the Sm core of U1 snRNP.

scholarly journals m3G cap hypermethylation of U1 small nuclear ribonucleoprotein (snRNP) in vitro: evidence that the U1 small nuclear RNA-(guanosine-N2)-methyltransferase is a non-snRNP cytoplasmic protein that requires a binding site on the Sm core domain.

1994 ◽  
Vol 14 (6) ◽  
pp. 4160-4172 ◽  
Author(s):  
G Plessel ◽  
U Fischer ◽  
R Lührmann
Keyword(s):  
Nuclear Transport ◽  
Iodoacetic Acid ◽  
Small Nuclear Rna ◽  
Vitro Assay ◽  
Core Domain ◽  
Small Nuclear Ribonucleoprotein ◽  
U1 Snrnp ◽  
Snrnp Protein ◽  
Localization Signal

The RNA components of small nuclear ribonucleoproteins (U snRNPs) possess a characteristic 5'-terminal trimethylguanosine cap structure (m3G cap). This cap is an important component of the nuclear localization signal of U snRNPs. It arises by hypermethylation of a cotranscriptionally added m7G cap. Here we describe an in vitro assay for the hypermethylation, which employs U snRNP particles reconstituted in vitro from purified components and subsequent analysis by m3G cap-specific immunoprecipitation. Complementation studies in vitro revealed that both cytosol and S-adenosylmethionine are required for the hypermethylation of an m7G-capped U1 snRNP reconstituted in vitro, indicating that the U1 snRNA-(guanosine-N2)-methyltransferase is a trans-active non-snRNP protein. Chemical modification revealed one cytoplasmic component required for hypermethylation and one located on the snRNP: these components have different patterns of sensitivity to modification by N-ethylmaleimide and iodoacetic acid (IAA). In the presence of cytosol and S-adenosylmethionine, an intact Sm core domain is a necessary and sufficient substrate for cap hypermethylation. These data, together with our observation that isolated native U1 snRNPs but not naked U1 RNA inhibit the trimethylation of in vitro-reconstituted U1 snRNP, indicate that the Sm core binds the methyltransferase specifically. Moreover, isolated native U2 snRNP also inhibits trimethylation of U1 snRNP, suggesting that other Sm-class U snRNPs might share the same methyltransferase. IAA modification of m7G-capped U1 snRNPs inhibited hypermethylation when they were microinjected into Xenopus oocytes and consequently also inhibited nuclear import. In contrast, modification with IAA of m3G-capped U1 snRNPs reconstituted in vitro did not interfere with their nuclear transport in oocytes. These data suggest that m3G cap formation and nuclear transport of U1 snRNPs are mediated by distinct factors, which require distinct binding sites on the Sm core of U1 snRNP.

scholarly journals U1 small nuclear ribonucleoprotein studied by in vitro assembly.

1983 ◽  
Vol 96 (6) ◽  
pp. 1751-1755 ◽  
Author(s):  
E D Wieben ◽  
S J Madore ◽  
T Pederson
Keyword(s):  
Direct Analysis ◽  
Small Nuclear Rna ◽  
In Vitro System ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Extracts ◽  
U1 Snrnp ◽  
Nuclear Rna ◽  
Specific Complex ◽  
Reticulocyte Lysate

The small nuclear RNAs are known to be complexed with proteins in the cell (snRNP). To learn more about these proteins, we developed an in vitro system for studying their interactions with individual small nuclear RNA species. Translation of HeLa cell poly(A)+ mRNA in an exogenous message-dependent reticulocyte lysate results in the synthesis of snRNP proteins. Addition of human small nuclear RNA U1 to the translation products leads to the formation of a U1 RNA-protein complex that is recognized by a human autoimmune antibody specific for U1 snRNP. This antibody does not react with free U1 RNA. Moreover, addition of a 10- to 20-fold molar excess of transfer RNA instead of U1 RNA does not lead to the formation of an antibody-recognized RNP. The proteins forming the specific complex with U1 RNA correspond to the A, B1, and B2 species (32,000, 27,000, and 26,000 mol wt, respectively) observed in previous studies with U1 snRNP obtained by antibody-precipitation of nuclear extracts. The availability of this in vitro system now permits, for the first time, direct analysis of snRNA-protein binding interactions and, in addition, provides useful information on the mRNAs for snRNP proteins.

scholarly journals The importin-β binding domain of snurportin1 is responsible for the Ran- and energy-independent nuclear import of spliceosomal U snRNPs in vitro

2002 ◽  
Vol 156 (3) ◽  
pp. 467-479 ◽  
Author(s):  
Jochen Huber ◽  
Achim Dickmanns ◽  
Reinhard Lührmann
Keyword(s):  
Nuclear Import ◽  
Structural Similarity ◽  
Nuclear Pore ◽  
Independent Manner ◽  
Core Domain ◽  
U1 Snrnp ◽  
Importin Α ◽  
Localization Signal ◽  
Import Receptor

The nuclear localization signal (NLS) of spliceosomal U snRNPs is composed of the U snRNA's 2,2,7-trimethyl-guanosine (m3G)-cap and the Sm core domain. The m3G-cap is specifically bound by snurportin1, which contains an NH2-terminal importin-β binding (IBB) domain and a COOH-terminal m3G-cap–binding region that bears no structural similarity to known import adaptors like importin-α (impα). Here, we show that recombinant snurportin1 and importin-β (impβ) are not only necessary, but also sufficient for U1 snRNP transport to the nuclei of digitonin-permeabilized HeLa cells. In contrast to impα–dependent import, single rounds of U1 snRNP import, mediated by the nuclear import receptor complex snurportin1–impβ, did not require Ran and energy. The same Ran- and energy-independent import was even observed for U5 snRNP, which has a molecular weight of more than one million. Interestingly, in the presence of impβ and a snurportin1 mutant containing an impα IBB domain (IBBimpα), nuclear U1 snRNP import was Ran dependent. Furthermore, β-galactosidase (βGal) containing a snurportin1 IBB domain, but not IBBimpα-βGal, was imported into the nucleus in a Ran-independent manner. Our results suggest that the nature of the IBB domain modulates the strength and/or site of interaction of impβ with nucleoporins of the nuclear pore complex, and thus whether or not Ran is required to dissociate these interactions.

scholarly journals Interactions between highly conserved U2 small nuclear RNA structures and Prp5p, Prp9p, Prp11p, and Prp21p proteins are required to ensure integrity of the U2 small nuclear ribonucleoprotein in Saccharomyces cerevisiae.

1994 ◽  
Vol 14 (9) ◽  
pp. 6337-6349 ◽  
Author(s):  
S E Wells ◽  
M Ares
Keyword(s):  
Synthetic Lethality ◽  
Small Nuclear Rna ◽  
Rna Structures ◽  
Stem Loop ◽  
U2 Snrna ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
Nuclear Rna ◽  
Nuclear Ribonucleoprotein

Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA is an early and important step in spliceosome assembly. We searched for evidence of cooperative function between yeast U2 small nuclear RNA (snRNA) and several genetically identified splicing (Prp) proteins required for the first chemical step of splicing, using the phenotype of synthetic lethality. We constructed yeast strains with pairwise combinations of 28 different U2 alleles with 10 prp mutations and found lethal double-mutant combinations with prp5, -9, -11, and -21 but not with prp3, -4, -8, or -19. Many U2 mutations in highly conserved or invariant RNA structures show no phenotype in a wild-type PRP background but render mutant prp strains inviable, suggesting that the conserved but dispensable U2 elements are essential for efficient cooperative function with specific Prp proteins. Mutant U2 snRNA fails to accumulate in synthetic lethal strains, demonstrating that interaction between U2 RNA and these four Prp proteins contributes to U2 snRNP assembly or stability. Three of the proteins (Prp9p, Prp11p, and Prp21p) are associated with each other and pre-mRNA in U2-dependent splicing complexes in vitro and bind specifically to synthetic U2 snRNA added to crude splicing extracts depleted of endogenous U2 snRNPs. Taken together, the results suggest that Prp9p, -11p, and -21p are U2 snRNP proteins that interact with a structured region including U2 stem loop IIa and mediate the association of the U2 snRNP with pre-mRNA.

Nuclear transport of the U2 snRNP-specific U2B'' protein is mediated by both direct and indirect signalling mechanisms

1994 ◽  
Vol 107 (7) ◽  
pp. 1807-1816 ◽  
Author(s):  
C. Kambach ◽  
I.W. Mattaj
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Nuclear Transport ◽  
U2 Snrna ◽  
Central Segment ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
Localization Signal ◽  
Protein Amino Acids

Experiments investigating the nuclear import of the U2 snRNP-specific B'' protein (U2B'') are presented. U2B'' nuclear transport is shown to be able to occur independently of binding to U2 snRNA. The central segment of the protein (amino acids 90–146) encodes an unusual nuclear localization signal (NLS) that is related to that of the U1 snRNP-specific A protein. However, nuclear import of U2B'' does not depend on this NLS. Sequences in the N-terminal RNP motif of the protein are sufficient to direct nuclear transport, and evidence is presented that the interaction of U2B'' with the U2A' protein mediates this effect. This suggests that U2B'' can ‘piggy-back’ to the nucleus in association with U2A’, and thus be imported to the nucleus by two different mechanisms. U2A' nuclear transport, on the other hand, can occur independently of both U2B'' binding and of U2 snRNA.

Differential block of U small nuclear ribonucleoprotein particle interactions during in vitro splicing of adenovirus E1A transcripts containing abnormally short introns

1991 ◽  
Vol 11 (3) ◽  
pp. 1258-1269
Author(s):  
M Himmelspach ◽  
R Gattoni ◽  
C Gerst ◽  
K Chebli ◽  
J Stévenin
Keyword(s):  
Donor Site ◽  
Ribonucleoprotein Particle ◽  
Adenovirus E1a ◽  
Branch Site ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
The Common ◽  
Nuclear Ribonucleoprotein

We have studied the consequences of decreasing the donor site-branch site distance on splicing factor-splice site interactions by analyzing alternative splicing of adenovirus E1A pre-mRNAs in vitro. We show that the proximal 13S donor site has a cis-inhibiting effect on the 9S and 12S mRNA reactions when it is brought too close to the common branch site, suggesting that the factor interactions in the common 3' part of the intron are impaired by the U1 small nuclear ribonucleoprotein particle (snRNP) binding to the displaced 13S donor site. Further analysis of the interactions was carried out by studying complex assembly and the accessibility to micrococcal nuclease digestion of 5'-truncated E1A substrates containing only splice sites for the 13S mRNA reaction. A deletion which brings the donor site- branch site distance to 49 nucleotides, which is just below the minimal functional distance, results in a complete block of the U4-U5-U6 snRNP binding, whereas a deletion 15 nucleotides larger results in a severe inhibition of the formation of the U2 snRNP-containing complexes. Sequence accessibility analyses performed by using the last mini-intron-containing transcript demonstrate that the interactions of U2 snRNP with the branch site are strongly impaired whereas the initial bindings of U1 snRNP to the donor site and of specific factors to the 3' splice site are not significantly modified. Our results strongly suggest that the interaction of U1 snRNP with the donor site of a mini-intron is stable enough in vitro to affect the succession of events leading to U2 snRNP binding with the branch site.

scholarly journals The Balance of RanBP1 and RCC1 Is Critical for Nuclear Assembly and Nuclear Transport

1997 ◽  
Vol 8 (10) ◽  
pp. 1955-1970 ◽  
Author(s):  
Robert T. Pu ◽  
Mary Dasso
Keyword(s):  
Dna Replication ◽  
Protein Import ◽  
Nuclear Transport ◽  
Small Gtpase ◽  
Stable Complex ◽  
Guanine Nucleotide ◽  
Vitro Assay ◽  
Nuclear Assembly ◽  
Egg Extracts

Ran is a small GTPase that is essential for nuclear transport, mRNA processing, maintenance of structural integrity of nuclei, and cell cycle control. RanBP1 is a highly conserved Ran guanine nucleotide dissociation inhibitor. We sought to use Xenopus egg extracts for the development of an in vitro assay for RanBP1 activity in nuclear assembly, protein import, and DNA replication. Surprisingly, when we used anti-RanBP1 antibodies to immunodeplete RanBP1 fromXenopus egg extracts, we found that the extracts were also depleted of RCC1, Ran’s guanine nucleotide exchange factor, suggesting that these proteins form a stable complex. In contrast to previous observations using extracts that had been depleted of RCC1 only, extracts lacking both RanBP1 and RCC1 (codepleted extracts) did not exhibit defects in assays of nuclear assembly, nuclear transport, or DNA replication. Addition of either recombinant RanBP1 or RCC1 to codepleted extracts to restore only one of the depleted proteins caused abnormal nuclear assembly and inhibited nuclear transport and DNA replication in a manner that could be rescued by further addition of RCC1 or RanBP1, respectively. Exogenous mutant Ran proteins could partially rescue nuclear function in extracts without RanBP1 or without RCC1, in a manner that was correlated with their nucleotide binding state. These results suggest that little RanBP1 or RCC1 is required for nuclear assembly, nuclear import, or DNA replication in the absence of the other protein. The results further suggest that the balance of GTP- and GDP-Ran is critical for proper nuclear assembly and function in vitro.

scholarly journals Nuclear transport of U1 snRNP in somatic cells: differences in signal requirement compared with Xenopus laevis oocytes.

1994 ◽  
Vol 125 (5) ◽  
pp. 971-980 ◽  
Author(s):  
U Fischer ◽  
J Heinrich ◽  
K van Zee ◽  
E Fanning ◽  
R Lührmann
Keyword(s):  
Xenopus Laevis ◽  
Nuclear Import ◽  
Nuclear Transport ◽  
Somatic Cells ◽  
Xenopus Laevis Oocytes ◽  
Xenopus Laevis Oocyte ◽  
Nuclear Targeting ◽  
Transport Pathway ◽  
U1 Snrnp

The signal requirement for the nuclear import of U1 RNA in somatic cells from different species was investigated by microinjection of both digoxygenin-labeled wild type and mutant U1 RNA molecules and in vitro reconstituted U1 snRNPs. U1 RNA was shown to be targeted to the nucleus by a temperature-dependent process that requires the prior assembly of RNPs from the common proteins and the microinjected RNA. Competition in the cell between immunoaffinity-purified U1 snRNPs and digoxygenin-labeled U1 snRNPs reconstituted in vitro showed that the transport is saturable and should therefore be a mediated process. The transport of a karyophilic protein under the same conditions was not affected, indicating the existence of a U snRNP-specific transport pathway in somatic cells, as already seen in the Xenopus laevis oocyte system. Surprisingly, the signal requirement for nuclear transport of U1 snRNP was found to differ between oocytes and somatic cells from mouse, monkey and Xenopus, in that the m3GGpppG-cap is no longer an essential signaling component in somatic cells. However, as shown by investigation of the transport kinetics of m3GpppG- and ApppG-capped U1 snRNPs, the m3GpppG-cap accelerates the rate of U1 snRNP import significantly indicating that it has retained a signaling role for nuclear targeting of U1 snRNP in somatic cells. Moreover, our data strongly suggest that cell specific rather than species specific differences account for the differential m3G-cap requirement in nuclear import of U1 snRNPs.

scholarly journals A Novel Yeast U2 snRNP Protein, Snu17p, Is Required for the First Catalytic Step of Splicing and for Progression of Spliceosome Assembly

2001 ◽  
Vol 21 (9) ◽  
pp. 3037-3046 ◽  
Author(s):  
Alexander Gottschalk ◽  
Cornelia Bartels ◽  
Gitte Neubauer ◽  
Reinhard Lührmann ◽  
Patrizia Fabrizio
Keyword(s):  
Affinity Purification ◽  
Rna Recognition Motif ◽  
Spliceosome Assembly ◽  
Exon 2 ◽  
Small Nuclear Ribonucleoprotein ◽  
U1 Snrnp ◽  
Catalytically Active ◽  
Snrnp Protein ◽  
Nuclear Ribonucleoprotein

ABSTRACT We have isolated and microsequenced Snu17p, a novel yeast protein with a predicted molecular mass of 17 kDa that contains an RNA recognition motif. We demonstrate that Snu17p binds specifically to the U2 small nuclear ribonucleoprotein (snRNP) and that it is part of the spliceosome, since the pre-mRNA and the lariat-exon 2 are specifically coprecipitated with Snu17p. Although the SNU17gene is not essential, its knockout leads to a slow-growth phenotype and to a pre-mRNA splicing defect in vivo. In addition, the first step of splicing is dramatically decreased in extracts prepared from thesnu17 deletion (snu17Δ) mutant. This defect is efficiently reversed by the addition of recombinant Snu17p. To investigate the step of spliceosome assembly at which Snu17p acts, we have used nondenaturing gel electrophoresis. In Snu17p-deficient extracts, the spliceosome runs as a single slowly migrating complex. In wild-type extracts, usually at least two distinct complexes are observed: the prespliceosome, or B complex, containing the U2 but not the U1 snRNP, and the catalytically active spliceosome, or A complex, containing the U2, U6, and U5 snRNPs. Northern blot analysis and affinity purification of the snu17Δ spliceosome showed that it contains the U1, U2, U6, U5, and U4 snRNPs. The unexpected stabilization of the U1 snRNP and the lack of dissociation of the U4 snRNP suggest that loss of Snu17p inhibits the progression of spliceosome assembly prior to U1 snRNP release and after [U4/U6.U5] tri-snRNP addition.

scholarly journals Reconstitution of functional mammalian U4 small nuclear ribonucleoprotein: Sm protein binding is not essential for splicing in vitro.

1992 ◽  
Vol 12 (4) ◽  
pp. 1460-1468 ◽  
Author(s):  
C Wersig ◽  
A Bindereif
Keyword(s):  
Mutational Analysis ◽  
Nuclear Extract ◽  
Small Nuclear Rna ◽  
Interaction Domain ◽  
Sequence Element ◽  
Spliceosome Assembly ◽  
Small Nuclear Ribonucleoprotein ◽  
U4 Rna ◽  
Nuclear Ribonucleoprotein

We have developed an in vitro splicing complementation assay to investigate the domain structure of the mammalian U4 small nuclear RNA (snRNA) through mutational analysis. The addition of affinity-purified U4 snRNP or U4 RNA to U4-depleted nuclear extract efficiently restores splicing activity. In the U4-U6 interaction domain of U4 RNA, only stem II was found to be essential for splicing activity; the 5' loop is important for spliceosome stability. In the central domain, we have identified a U4 RNA sequence element that is important for splicing and spliceosome assembly. Surprisingly, an intact Sm domain is not essential for splicing in vitro. Our data provide evidence that several distinct regions of U4 RNA contribute to snRNP assembly, spliceosome assembly and stability, and splicing activity.

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