scholarly journals Chironomus tentans-Repressor Splicing Factor Represses SR Protein Function Locally on Pre-mRNA Exons and Is Displaced at Correct Splice Sites

2006 ◽  
Vol 17 (1) ◽  
pp. 32-42 ◽  
Author(s):  
Petra Björk ◽  
Ingela Wetterberg-Strandh ◽  
Göran Baurén ◽  
Lars Wieslander
Keyword(s):  
Protein Function ◽  
Splicing Factor ◽  
Chironomus Tentans ◽  
Splice Sites ◽  
Sr Protein ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Extracts ◽  
Interchromatin Granule ◽  
Nuclear Ribonucleoprotein

Chironomus tentans-repressor splicing factor (Ct-RSF) represses the activation of splicing by SR proteins in vitro. Ct-RSF colocalizes with the Ser-Arg-rich (SR) protein hrp45 in interchromatin granule clusters and coimmunoprecipitates with hrp45 in nuclear extracts. Ct-RSF and hrp45 can also interact directly in vitro. Ct-RSF and hrp45 are recruited together to transcribing genes and associate with growing pre-mRNAs. Ct-RSF and hrp45 colocalize at a large number of gene loci. Injection of anti-Ct-RSF antibodies into nuclei of living cells blocks association of both Ct-RSF and hrp45 with the growing pre-mRNA, whereas binding of U2 small nuclear ribonucleoprotein particle (snRNP) to the pre-mRNA is unaffected. On the intron-rich Balbiani ring (BR) 3 pre-mRNA, hrp45 as well as U1 and U2 snRNPs bind extensively, whereas relatively little Ct-RSF is present. In contrast, the BR1 and BR2 pre-mRNAs, dominated by exon sequences, bind relatively much Ct-RSF compared with hrp45 and snRNPs. Our data suggest that Ct-RSF represses SR protein function at exons and that the assembly of spliceosomes at authentic splice sites displaces Ct-RSF locally.

SR proteins promote the first specific recognition of Pre-mRNA and are present together with the U1 small nuclear ribonucleoprotein particle in a general splicing enhancer complex

1994 ◽  
Vol 14 (11) ◽  
pp. 7670-7682
Author(s):  
D Staknis ◽  
R Reed
Keyword(s):  
Protein Interactions ◽  
Splice Site ◽  
Sr Proteins ◽  
Cross Linking ◽  
Splice Sites ◽  
Sr Protein ◽  
Specific Recognition ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

We show that addition of SR proteins to in vitro splicing extracts results in a significant increase in assembly of the earliest prespliceosomal complex E and a corresponding decrease in assembly of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex H. In addition, SR proteins promote formation of the E5' and E3' complexes that assemble on RNAs containing only 5' and 3' splice sites, respectively. We conclude that SR proteins promote the earliest specific recognition of both the 5' and 3' splice sites and are limiting for this function in HeLa nuclear extracts. Using UV cross-linking, we demonstrate specific, splice site-dependent RNA-protein interactions of SR proteins in the E, E5', and E3' complexes. SR proteins do not UV cross-link in the H complex, and conversely, hnRNP cross-linking is largely excluded from the E-type complexes. We also show that a discrete complex resembling the E5' complex assembles on both purine-rich and non-purine-rich exonic splicing enhancers. This complex, which we have designated the Enhancer complex, contains U1 small nuclear RNP (snRNP) and is associated with different SR protein family members, depending on the sequence of the enhancer. We propose that both downstream 5' splice site enhancers and exonic enhancers function by establishing a network of pre-mRNA-protein and protein-protein interactions involving U1 snRNP, SR proteins, and U2AF that is similar to the interactions that bring the 5' and 3' splice sites together in the E complex.

scholarly journals SR proteins promote the first specific recognition of Pre-mRNA and are present together with the U1 small nuclear ribonucleoprotein particle in a general splicing enhancer complex.

1994 ◽  
Vol 14 (11) ◽  
pp. 7670-7682 ◽  
Author(s):  
D Staknis ◽  
R Reed
Keyword(s):  
Protein Interactions ◽  
Splice Site ◽  
Sr Proteins ◽  
Cross Linking ◽  
Splice Sites ◽  
Sr Protein ◽  
Specific Recognition ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

We show that addition of SR proteins to in vitro splicing extracts results in a significant increase in assembly of the earliest prespliceosomal complex E and a corresponding decrease in assembly of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex H. In addition, SR proteins promote formation of the E5' and E3' complexes that assemble on RNAs containing only 5' and 3' splice sites, respectively. We conclude that SR proteins promote the earliest specific recognition of both the 5' and 3' splice sites and are limiting for this function in HeLa nuclear extracts. Using UV cross-linking, we demonstrate specific, splice site-dependent RNA-protein interactions of SR proteins in the E, E5', and E3' complexes. SR proteins do not UV cross-link in the H complex, and conversely, hnRNP cross-linking is largely excluded from the E-type complexes. We also show that a discrete complex resembling the E5' complex assembles on both purine-rich and non-purine-rich exonic splicing enhancers. This complex, which we have designated the Enhancer complex, contains U1 small nuclear RNP (snRNP) and is associated with different SR protein family members, depending on the sequence of the enhancer. We propose that both downstream 5' splice site enhancers and exonic enhancers function by establishing a network of pre-mRNA-protein and protein-protein interactions involving U1 snRNP, SR proteins, and U2AF that is similar to the interactions that bring the 5' and 3' splice sites together in the E complex.

Recognition of mutant and cryptic 5' splice sites by the U1 small nuclear ribonucleoprotein in vitro

1987 ◽  
Vol 7 (2) ◽  
pp. 698-707
Author(s):  
B Chabot ◽  
J A Steitz
Keyword(s):  
Splice Site ◽  
Beta Globin ◽  
Splice Sites ◽  
Protection Assay ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoproteins

We examined the ability of U1 small nuclear ribonucleoproteins (U1 snRNPs) to recognize mutant and cryptic 5' splice sites on beta-globin pre-mRNA substrates using an RNase T1 protection assay. When U1 snRNPs were prebound to anti-(U1)RNP antibodies, we detected binding to mutant but not to cryptic 5' splice sites on several substrates. By contrast, in a splicing extract at 0 degree C, neither the mutated nor cryptic 5' splice sites of a human beta-globin transcript were selected as protected fragments with the same antibodies. However, after incubation of the transcript in the extract to yield splicing intermediates, fragments that included a cryptic 5' splice site were detected. The results of our analyses suggest that U1 snRNPs are involved in recognizing cryptic 5' splice sites but that interactions with other splicing components are required to stabilize the association.

scholarly journals In vitro splicing of pre-messenger RNA with extracts from 5-fluorouridine-treated cells

1994 ◽  
Vol 297 (2) ◽  
pp. 297-301 ◽  
Author(s):  
J R Patton
Keyword(s):  
Cell Growth ◽  
Messenger Rna ◽  
Rna Synthesis ◽  
Significant Inhibition ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Extracts ◽  
Specific Step ◽  
In Vitro Splicing ◽  
Nuclear Ribonucleoprotein

The effect of 5-fluorouridine (5-FU) treatment of cells on the splicing of pre-mRNA was determined using cellular extracts and splicing in vitro. Nuclear extracts from control cells and cells treated with 5-FU were prepared and used to splice pre-mRNAs in vitro. The drug treatment resulted in inhibition of cell growth but had little effect on RNA synthesis. The extracts from 5-FU-treated cells showed significant inhibition of splicing. This inhibition was the result of reduced efficiency and was not caused by a block at a specific step in the splicing pathway. There were no observable changes in the levels or physical properties of the small nuclear ribonucleoprotein particles that are essential cofactors in the splicing process. The deficiency in splicing in the extracts from 5-FU-treated cells could be supplemented by the addition of complementary fractions from a control extract.

Structure–function analysis of the U2 snRNP-associated splicing factor SF3a

2005 ◽  
Vol 33 (3) ◽  
pp. 439-442 ◽  
Author(s):  
A. Krämer ◽  
F. Ferfoglia ◽  
C.-J. Huang ◽  
F. Mulhaupt ◽  
D. Nesic ◽  
...  
Keyword(s):  
Structure Function ◽  
Function Analysis ◽  
Intracellular Localization ◽  
Splicing Factor ◽  
Branch Site ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
Constitutive Splicing ◽  
Nuclear Ribonucleoprotein

Human splicing factor SF3a is a part of the 17 S U2 snRNP (small nuclear ribonucleoprotein), which interacts with the pre-mRNA branch site early during spliceosome formation. The SF3a subunits of 60, 66 and 120 kDa are all required for SF3a function in vitro. Depletion of individual subunits from HeLa cells by RNA interference results in a global inhibition of splicing, indicating that SF3a is a constitutive splicing factor. Structure–function analyses have defined domains necessary for interactions within the SF3a heterotrimer, association with the U2 snRNP and spliceosome assembly. Studies aimed at the identification of regions in SF3a60 and SF3a66, required for proper intracellular localization, have led to a model for the final steps in U2 snRNP biogenesis and the proposal that SF3a is incorporated into the U2 snRNP in Cajal bodies.

scholarly journals Recognition of mutant and cryptic 5' splice sites by the U1 small nuclear ribonucleoprotein in vitro.

1987 ◽  
Vol 7 (2) ◽  
pp. 698-707 ◽  
Author(s):  
B Chabot ◽  
J A Steitz
Keyword(s):  
Splice Site ◽  
Beta Globin ◽  
Splice Sites ◽  
Protection Assay ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoproteins

We examined the ability of U1 small nuclear ribonucleoproteins (U1 snRNPs) to recognize mutant and cryptic 5' splice sites on beta-globin pre-mRNA substrates using an RNase T1 protection assay. When U1 snRNPs were prebound to anti-(U1)RNP antibodies, we detected binding to mutant but not to cryptic 5' splice sites on several substrates. By contrast, in a splicing extract at 0 degree C, neither the mutated nor cryptic 5' splice sites of a human beta-globin transcript were selected as protected fragments with the same antibodies. However, after incubation of the transcript in the extract to yield splicing intermediates, fragments that included a cryptic 5' splice site were detected. The results of our analyses suggest that U1 snRNPs are involved in recognizing cryptic 5' splice sites but that interactions with other splicing components are required to stabilize the association.

scholarly journals Conserved Loop I of U5 Small Nuclear RNA Is Dispensable for Both Catalytic Steps of Pre-mRNA Splicing in HeLa Nuclear Extracts

1999 ◽  
Vol 19 (4) ◽  
pp. 2782-2790 ◽  
Author(s):  
Véronique Ségault ◽  
Cindy L. Will ◽  
Maria Polycarpou-Schwarz ◽  
Iain W. Mattaj ◽  
Christiane Branlant ◽  
...  
Keyword(s):  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
Internal Loop ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Extracts ◽  
Nuclear Rna ◽  
Nuclear Ribonucleoprotein ◽  
U5 Snrna ◽  
Loop 2

ABSTRACT The function of conserved regions of the metazoan U5 snRNA was investigated by reconstituting U5 small nuclear ribonucleoprotein particles (snRNPs) from purified snRNP proteins and HeLa orXenopus U5 snRNA mutants and testing their ability to restore splicing to U5-depleted nuclear extracts. Substitution of conserved nucleotides comprising internal loop 2 or deletion of internal loop 1 had no significant effect on the ability of reconstituted U5 snRNPs to complement splicing. However, deletion of internal loop 2 abolished U5 activity in splicing and spliceosome formation. Surprisingly, substitution of the invariant loop 1 nucleotides with a GAGA tetraloop had no effect on U5 activity. Furthermore, U5 snRNPs reconstituted from an RNA formed by annealing the 5′ and 3′ halves of the U5 snRNA, which lacked all loop 1 nucleotides, complemented both steps of splicing. Thus, in contrast to yeast, loop 1 of the human U5 snRNA is dispensable for both steps of splicing in HeLa nuclear extracts. This suggests that its function can be compensated for in vitro by other spliceosomal components: for example, by proteins associated with the U5 snRNP. Consistent with this idea, immunoprecipitation studies indicated that several functionally important U5 proteins associate stably with U5 snRNPs containing a GAGA loop 1 substitution.

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.

Ribozyme, antisense RNA, and antisense DNA inhibition of U7 small nuclear ribonucleoprotein-mediated histone pre-mRNA processing in vitro

1989 ◽  
Vol 9 (10) ◽  
pp. 4479-4487
Author(s):  
M Cotten ◽  
G Schaffner ◽  
M L Birnstiel
Keyword(s):  
Antisense Rna ◽  
Nuclear Extract ◽  
Mrna Processing ◽  
Rna Molecules ◽  
Small Nuclear Ribonucleoprotein ◽  
In Vitro Processing ◽  
Antisense Dna ◽  
Nuclear Ribonucleoprotein ◽  
Fold Excess

A comparative analysis of ribozyme, antisense RNA, and antisense DNA inhibitors of the in vitro small nuclear ribonucleoprotein U7-dependent histone pre-mRNA processing reaction was performed. RNA molecules complementary to the U7 sequence inhibited in vitro processing of histone pre-mRNA at a sixfold excess over U7. Single-stranded DNA complementary to the entire U7 sequence inhibited the reaction at a 60-fold excess over U7, while a short, 18-nucleotide DNA molecule complementary to the 5' end of U7 inhibited the processing reaction at a 600-fold excess. A targeted ribozyme was capable of specifically cleaving the U7 small nuclear ribonucleoprotein in a nuclear extract and inhibited the U7-dependent processing reaction, but in our in vitro system it required a 1,000-fold excess over U7 for complete inhibition of processing.

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