scholarly journals 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

1991 ◽  
Vol 19 (4) ◽  
pp. 877-884 ◽  
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

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.

scholarly journals 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.

Early commitment of yeast pre-mRNA to the spliceosome pathway

1988 ◽  
Vol 8 (9) ◽  
pp. 3755-3760 ◽  
Author(s):  
P Legrain ◽  
B Seraphin ◽  
M Rosbash
Keyword(s):  
Consensus Sequence ◽  
Rnase H ◽  
Small Nuclear Rna ◽  
Spliceosome Assembly ◽  
U2 Snrna ◽  
Consensus Sequences ◽  
U2 Snrnp ◽  
Site Consensus ◽  
Sequence Requirements

Pre-mRNA splicing in vitro is preceded by complex formation (spliceosome assembly). U2 small nuclear RNA (snRNA) is found in the earliest form of the spliceosome detected by native gel electrophoresis, both in Saccharomyces cerevisiae and in metazoan extracts. To examine the requirements for the formation of this early complex (band III) in yeast extracts, we cleaved the U2 snRNA by oligonucleotide-directed RNase H digestion. U2 snRNA depletion by this means inhibits both splicing and band III formation. Using this depleted extract, we were able to design a chase experiment which shows that a pre-mRNA substrate is committed to the spliceosome assembly pathway in the absence of functional U2 snRNP. Interactions occurring during the commitment step are highly resistant to the addition of an excess of unlabeled substrate and require little or no ATP. Sequence requirements for this commitment step have been analyzed by competition experiments with deletion mutants: both the 5' splice site consensus sequence and the branch point TACTAAC box sequence are necessary. These experiments strongly suggest that the initial assembly process requires a trans-acting factor(s) (RNA and/or proteins) that recognizes and stably binds to the two consensus sequences of the pre-mRNA prior to U2 snRNP binding.

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

2005 ◽  
Vol 25 (1) ◽  
pp. 233-240 ◽  
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.

scholarly journals Early commitment of yeast pre-mRNA to the spliceosome pathway.

1988 ◽  
Vol 8 (9) ◽  
pp. 3755-3760 ◽  
Author(s):  
P Legrain ◽  
B Seraphin ◽  
M Rosbash
Keyword(s):  
Consensus Sequence ◽  
Rnase H ◽  
Small Nuclear Rna ◽  
Spliceosome Assembly ◽  
U2 Snrna ◽  
Consensus Sequences ◽  
U2 Snrnp ◽  
Site Consensus ◽  
Sequence Requirements

Pre-mRNA splicing in vitro is preceded by complex formation (spliceosome assembly). U2 small nuclear RNA (snRNA) is found in the earliest form of the spliceosome detected by native gel electrophoresis, both in Saccharomyces cerevisiae and in metazoan extracts. To examine the requirements for the formation of this early complex (band III) in yeast extracts, we cleaved the U2 snRNA by oligonucleotide-directed RNase H digestion. U2 snRNA depletion by this means inhibits both splicing and band III formation. Using this depleted extract, we were able to design a chase experiment which shows that a pre-mRNA substrate is committed to the spliceosome assembly pathway in the absence of functional U2 snRNP. Interactions occurring during the commitment step are highly resistant to the addition of an excess of unlabeled substrate and require little or no ATP. Sequence requirements for this commitment step have been analyzed by competition experiments with deletion mutants: both the 5' splice site consensus sequence and the branch point TACTAAC box sequence are necessary. These experiments strongly suggest that the initial assembly process requires a trans-acting factor(s) (RNA and/or proteins) that recognizes and stably binds to the two consensus sequences of the pre-mRNA prior to U2 snRNP binding.

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.

scholarly journals Functional properties of p54, a novel SR protein active in constitutive and alternative splicing.

1996 ◽  
Vol 16 (10) ◽  
pp. 5400-5408 ◽  
Author(s):  
W J Zhang ◽  
J Y Wu
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Sr Proteins ◽  
Dependent Manner ◽  
Sr Protein ◽  
Protein Protein Interaction ◽  
Small Nuclear Ribonucleoprotein ◽  
S100 Extract ◽  
U2 Auxiliary Factor ◽  
Auxiliary Factor

The p54 protein was previously identified by its reactivity with an autoantiserum. We report here that p54 is a new member of the SR family of splicing factors, as judged from its structural, antigenic, and functional characteristics. Consistent with its identification as an SR protein, p54 can function as a constitutive splicing factor in complementing splicing-deficient HeLa cell S100 extract. However, p54 also shows properties distinct from those of other SR family members, p54 can directly interact with the 65-kDa subunit of U2 auxiliary factor (U2AF65), a protein associated with the 3' splice site. In addition, p54 interacts with other SR proteins but does not interact with the U1 small nuclear ribonucleoprotein U1-70K or the 35-kDa subunit of U2 auxiliary factor (U2AF35). This protein-protein interaction profile is different from those of prototypical SR proteins SC35 and ASF/SF2, both of which interact with U1-70K and U2AF35 but not with U2AF65. p54 promotes the use of the distal 5' splice site in E1A pre-mRNA alternative splicing, while the same site is suppressed by ASF/SF2 and SC35. These findings and the differential tissue distribution of p54 suggest that this novel SR protein may participate in regulation of alternative splicing in a tissue- and substrate-dependent manner.

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.

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