scholarly journals A minimal spliceosomal complex A recognizes the branch site and polypyrimidine tract.

1997 ◽  
Vol 17 (5) ◽  
pp. 2944-2953 ◽  
Author(s):  
C C Query ◽  
P S McCaw ◽  
P A Sharp
Keyword(s):  
Specific Interactions ◽  
Polypyrimidine Tract ◽  
Detailed Understanding ◽  
Branch Site ◽  
U2 Snrnp ◽  
Dependent Activity ◽  
U1 Snrnp ◽  
Polypyrimidine Tract Binding Proteins ◽  
Per Se ◽  
Rna Complex

The association of U2 snRNP with the pre-mRNA branch region is a critical step in the assembly of spliceosomal complexes. We describe an assembly process that reveals both minimal requirements for formation of a U2 snRNP-substrate RNA complex, here designated the Amin complex, and specific interactions with the branch site adenosine. The substrate is a minimal RNA oligonucleotide, containing only a branch sequence and polypyrimidine tract. Interactions at the branch site adenosine and requirements for polypyrimidine tract-binding proteins for the Amin complex are the same as those of authentic prespliceosome complex A. Surprisingly, Amin complex formation does not require U1 snRNP or ATP, suggesting that these factors are not necessary for stable binding of U2 snRNP per se, but rather are necessary for accessibility of components on longer RNA substrates. Furthermore, there is an ATP-dependent activity that releases or destabilizes U2 snRNP from branch sequences. The simplicity of the Amin complex will facilitate a detailed understanding of the assembly of prespliceosomes.

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

1995 ◽  
Vol 15 (10) ◽  
pp. 5750-5756 ◽  
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.

scholarly journals Large-scale remodeling of a repressed exon ribonucleoprotein to an exon definition complex active for splicing

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Somsakul Pop Wongpalee ◽  
Ajay Vashisht ◽  
Shalini Sharma ◽  
Darryl Chui ◽  
James A Wohlschlegel ◽  
...  
Keyword(s):  
Large Scale ◽  
Nuclear Extract ◽  
Polypyrimidine Tract ◽  
Exon Definition ◽  
Hela Nuclear Extract ◽  
Polypyrimidine Tract Binding Protein ◽  
Hnrnp Proteins ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
Definition Phase

Polypyrimidine-tract binding protein PTBP1 can repress splicing during the exon definition phase of spliceosome assembly, but the assembly steps leading to an exon definition complex (EDC) and how PTBP1 might modulate them are not clear. We found that PTBP1 binding in the flanking introns allowed normal U2AF and U1 snRNP binding to the target exon splice sites but blocked U2 snRNP assembly in HeLa nuclear extract. Characterizing a purified PTBP1-repressed complex, as well as an active early complex and the final EDC by SILAC-MS, we identified extensive PTBP1-modulated changes in exon RNP composition. The active early complex formed in the absence of PTBP1 proceeded to assemble an EDC with the eviction of hnRNP proteins, the late recruitment of SR proteins, and binding of the U2 snRNP. These results demonstrate that during early stages of splicing, exon RNP complexes are highly dynamic with many proteins failing to bind during PTBP1 arrest.

Multiple interactions between the splicing substrate and small nuclear ribonucleoproteins in spliceosomes

1987 ◽  
Vol 7 (1) ◽  
pp. 281-293
Author(s):  
B Chabot ◽  
J A Steitz
Keyword(s):  
Splice Site ◽  
Beta Globin ◽  
Branch Site ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
Branch Formation ◽  
Splice Site Sequence ◽  
Multiple Interactions ◽  
Small Nuclear Ribonucleoproteins

Protection experiments with antibodies against small nuclear ribonucleoproteins (snRNPs) have elucidated the location of and requirements for interactions between snRNPs and human beta-globin transcripts during splicing in vitro. U2 snRNP association with the intron branch site continues after branch formation, requires intact U2 RNA, and is affected by some alterations of the 3' splice site sequence. U2 snRNP binding to the branched intermediate and U1 snRNP protection of an extended 5' splice region are detected exclusively in spliceosome fractions, indicating that both snRNPs are spliceosome components. While each snRNP associates specifically with the pre-mRNA, they also appear to interact with each other. The recovery of fragments mapping upstream of the 5' splice site suggests how the excised exon is held in the spliceosome.

scholarly journals Multiple interactions between the splicing substrate and small nuclear ribonucleoproteins in spliceosomes.

1987 ◽  
Vol 7 (1) ◽  
pp. 281-293 ◽  
Author(s):  
B Chabot ◽  
J A Steitz
Keyword(s):  
Splice Site ◽  
Beta Globin ◽  
Branch Site ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
Branch Formation ◽  
Splice Site Sequence ◽  
Multiple Interactions ◽  
Small Nuclear Ribonucleoproteins

Protection experiments with antibodies against small nuclear ribonucleoproteins (snRNPs) have elucidated the location of and requirements for interactions between snRNPs and human beta-globin transcripts during splicing in vitro. U2 snRNP association with the intron branch site continues after branch formation, requires intact U2 RNA, and is affected by some alterations of the 3' splice site sequence. U2 snRNP binding to the branched intermediate and U1 snRNP protection of an extended 5' splice region are detected exclusively in spliceosome fractions, indicating that both snRNPs are spliceosome components. While each snRNP associates specifically with the pre-mRNA, they also appear to interact with each other. The recovery of fragments mapping upstream of the 5' splice site suggests how the excised exon is held in the spliceosome.

scholarly journals 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.

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.

The spliceosome assembly pathway in mammalian extracts

1992 ◽  
Vol 12 (10) ◽  
pp. 4279-4287 ◽  
Author(s):  
S F Jamison ◽  
A Crow ◽  
M A Garcia-Blanco
Keyword(s):  
Spliceosome Assembly ◽  
Protein Factor ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
U2 Auxiliary Factor ◽  
Nuclear Ribonucleoprotein ◽  
Auxiliary Factor ◽  
First Time ◽  
Kinetics Of

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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