The spliceosome assembly pathway in mammalian extracts

A mammalian splicing commitment complex was functionally defined by using a template commitment assay. This complex was partially purified and shown to be a required intermediate for complex A formation. The productive formation of this commitment complex required both splice sites and the polypyrimidine tract. U1 small nuclear ribonucleoprotein (snRNP) was the only spliceosomal U snRNP required for this formation. A protein factor, very likely U2AF, is probably involved in the formation of the splicing commitment complex. From the kinetics of appearance of complex A and complex B, it was previously postulated that complex A represents a functional intermediate in spliceosome assembly. Complex A was partially purified and shown to be a required intermediate for complex B (spliceosome) formation. Thus, a spliceosome pathway is for the first time supported by direct biochemical evidence: RNA+U1 snRNP+?U2 auxiliary factor+?Y----CC+U2 snRNP+Z----A+U4/6,5 snRNPs+ beta----B.

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Characterization of a U2AF-Independent Commitment Complex (E′) in the Mammalian Spliceosome Assembly Pathway

Molecular and Cellular Biology ◽

10.1128/mcb.25.1.233-240.2005 ◽

2005 ◽

Vol 25 (1) ◽

pp. 233-240 ◽

Cited By ~ 29

Author(s):

Oliver A. Kent ◽

Dustin B. Ritchie ◽

Andrew M. MacMillan

Keyword(s):

Splice Site ◽

Early Recognition ◽

Close Association ◽

Nuclear Extract ◽

Sr Protein ◽

Spliceosome Assembly ◽

Small Nuclear Ribonucleoprotein ◽

U2 Snrnp ◽

Nuclear Ribonucleoprotein ◽

Auxiliary Factor

ABSTRACT Early recognition of pre-mRNA during spliceosome assembly in mammals proceeds through the association of U1 small nuclear ribonucleoprotein particle (snRNP) with the 5′ splice site as well as the interactions of the branch binding protein SF1 with the branch region and the U2 snRNP auxiliary factor U2AF with the polypyrimidine tract and 3′ splice site. These factors, along with members of the SR protein family, direct the ATP-independent formation of the early (E) complex that commits the pre-mRNA to splicing. We report here the observation in U2AF-depleted HeLa nuclear extract of a distinct, ATP-independent complex designated E′ which can be chased into E complex and itself commits a pre-mRNA to the splicing pathway. The E′ complex is characterized by a U1 snRNA-5′ splice site base pairing, which follows the actual commitment step, an interaction of SF1 with the branch region, and a close association of the 5′ splice site with the branch region. These results demonstrate that both commitment to splicing and the early proximity of conserved sequences within pre-mRNA substrates can occur in a minimal complex lacking U2AF, which may function as a precursor to E complex in spliceosome assembly.

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U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor of 65 kDa, U2AF65, can promote U1 snRNP recruitment to 5′ splice sites

Biochemical Journal ◽

10.1042/bj20021202 ◽

2003 ◽

Vol 372 (1) ◽

pp. 235-240 ◽

Cited By ~ 13

Author(s):

Patrik FÖRCH ◽

Livia MERENDINO ◽

Concepción MARTÍNEZ ◽

Juan VALCÁRCEL

Keyword(s):

Splice Sites ◽

Ribonucleoprotein Particle ◽

Rich Domain ◽

Small Nuclear Ribonucleoprotein ◽

U2 Snrnp ◽

U1 Snrnp ◽

Nuclear Ribonucleoprotein ◽

Auxiliary Factor ◽

Small Nuclear Ribonucleoprotein Particle

The splicing factor U2AF65, U2 small nuclear ribonucleoprotein particle (snRNP) auxillary factor of 65 kDa, binds to pyrimidine-rich sequences at 3′ splice sites to recruit U2 snRNP to pre-mRNAs. We report that U2AF65 can also promote the recruitment of U1 snRNP to weak 5′ splice sites that are followed by uridine-rich sequences. The arginine- and serine-rich domain of U2AF65 is critical for U1 recruitment, and we discuss the role of its RNA–RNA annealing activity in this novel function of U2AF65.

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

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1258-1269.1991 ◽

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.

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Accumulation of a novel spliceosomal complex on pre-mRNAs containing branch site mutations.

Molecular and Cellular Biology ◽

10.1128/mcb.15.10.5750 ◽

1995 ◽

Vol 15 (10) ◽

pp. 5750-5756 ◽

Cited By ~ 19

Author(s):

P Champion-Arnaud ◽

O Gozani ◽

L Palandjian ◽

R Reed

Keyword(s):

Second Step ◽

Apparent Effect ◽

Ribonucleoprotein Particle ◽

Branch Site ◽

Small Nuclear Ribonucleoprotein ◽

U2 Snrnp ◽

U1 Snrnp ◽

Nuclear Ribonucleoprotein ◽

Branchpoint Sequence ◽

Independent Reaction

Pre-mRNA assembles into spliceosomal complexes in the stepwise pathway E-->A-->B-->C. We show that mutations in the metazoan branchpoint sequence (BPS) have no apparent effect on E complex formation but block the assembly of the A complex and the UV cross-linking of U2 small nuclear ribonucleoprotein particle (snRNP) proteins. Unexpectedly, a novel complex, designated E*, assembles on pre-mRNAs containing BPS mutations. Unlike the E complex, the E* complex accumulates in the presence of ATP. U1 snRNP and U2AF, which are tightly bound to pre-mRNA in the E complex, are not tightly bound in the E* complex. Significantly, previous work showed that U1 snRNP and U2AF become destabilized from pre-mRNA after E complex assembly on normal pre-mRNAs. Thus, our data are consistent with a model in which there are two steps in the transition from the E complex to the A complex (E-->E*-->A). In the first step, U1 snRNP and U2AF are destabilized in an ATP-dependent, BPS-independent reaction. In the second step, the stable binding of U2 snRNP occurs in a BPS-dependent reaction.

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

Molecular and Cellular Biology ◽

10.1128/mcb.21.9.3037-3046.2001 ◽

2001 ◽

Vol 21 (9) ◽

pp. 3037-3046 ◽

Cited By ~ 26

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.

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Recognition of U1 and U2 small nuclear RNAs can be altered by a 5-amino-acid segment in the U2 small nuclear ribonucleoprotein particle (snRNP) B" protein and through interactions with U2 snRNP-A' protein

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.1829-1839.1991 ◽

1991 ◽

Vol 11 (4) ◽

pp. 1829-1839

Author(s):

R C Bentley ◽

J D Keene

Keyword(s):

Amino Acid ◽

Rna Binding ◽

Rna Recognition ◽

Ribonucleoprotein Particle ◽

Small Nuclear Ribonucleoprotein ◽

U2 Snrnp ◽

U1 Snrnp ◽

Sequence Elements ◽

Nuclear Ribonucleoprotein ◽

Amino Acid Segment

We have investigated the sequence elements influencing RNA recognition in two closely related small nuclear ribonucleoprotein particle (snRNP) proteins, U1 snRNP-A and U2 snRNP-B". A 5-amino-acid segment in the RNA-binding domain of the U2 snRNP-B" protein was found to confer U2 RNA recognition when substituted into the corresponding position in the U1 snRNP-A protein. In addition, B", but not A, was found to require the U2 snRNP-A' protein as an accessory factor for high-affinity binding to U2 RNA. The pentamer segment in B" that conferred U2 RNA recognition was not sufficient to allow the A' enhancement of U2 RNA binding by B", thus implicating other sequences in this protein-protein interaction. Sequence elements involved in these interactions have been localized to variable loops of the RNA-binding domain as determined by nuclear magnetic resonance spectroscopy (D. Hoffman, C.C. Query, B. Golden, S.W. White, and J.D. Keene, Proc. Natl. Acad. Sci. USA, in press). These findings suggest a role for accessory proteins in the formation of RNP complexes and pinpoint amino acid sequences that affect the specificity of RNA recognition in two members of a large family of proteins involved in RNA processing.

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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

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1883-1891.1993 ◽

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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The 5′ end domain of U2 snRNA is required to establish the interaction of U2 snRNP with U2 auxiliary factor(s) during mammalian spliceosome assembly

Nucleic Acids Research ◽

10.1093/nar/19.4.877 ◽

1991 ◽

Vol 19 (4) ◽

pp. 877-884 ◽

Cited By ~ 6

Author(s):

Samy Khellil ◽

Marie-Claire Daugeron ◽

Christine Alibert ◽

Philippe Jeanteur ◽

Guy Cathala ◽

...

Keyword(s):

Spliceosome Assembly ◽

U2 Snrna ◽

U2 Snrnp ◽

U2 Auxiliary Factor ◽

Auxiliary Factor

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Recognition of U1 and U2 small nuclear RNAs can be altered by a 5-amino-acid segment in the U2 small nuclear ribonucleoprotein particle (snRNP) B" protein and through interactions with U2 snRNP-A' protein.

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.1829 ◽

1991 ◽

Vol 11 (4) ◽

pp. 1829-1839 ◽

Cited By ~ 44

Author(s):

R C Bentley ◽

J D Keene

Keyword(s):

Amino Acid ◽

Rna Binding ◽

Rna Recognition ◽

Ribonucleoprotein Particle ◽

Small Nuclear Ribonucleoprotein ◽

U2 Snrnp ◽

U1 Snrnp ◽

Sequence Elements ◽

Nuclear Ribonucleoprotein ◽

Amino Acid Segment

We have investigated the sequence elements influencing RNA recognition in two closely related small nuclear ribonucleoprotein particle (snRNP) proteins, U1 snRNP-A and U2 snRNP-B". A 5-amino-acid segment in the RNA-binding domain of the U2 snRNP-B" protein was found to confer U2 RNA recognition when substituted into the corresponding position in the U1 snRNP-A protein. In addition, B", but not A, was found to require the U2 snRNP-A' protein as an accessory factor for high-affinity binding to U2 RNA. The pentamer segment in B" that conferred U2 RNA recognition was not sufficient to allow the A' enhancement of U2 RNA binding by B", thus implicating other sequences in this protein-protein interaction. Sequence elements involved in these interactions have been localized to variable loops of the RNA-binding domain as determined by nuclear magnetic resonance spectroscopy (D. Hoffman, C.C. Query, B. Golden, S.W. White, and J.D. Keene, Proc. Natl. Acad. Sci. USA, in press). These findings suggest a role for accessory proteins in the formation of RNP complexes and pinpoint amino acid sequences that affect the specificity of RNA recognition in two members of a large family of proteins involved in RNA processing.

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