U1, U2, and U4/U6 small nuclear ribonucleoproteins are required for in vitro splicing but not polyadenylation

Cell ◽  
1986 ◽  
Vol 46 (5) ◽  
pp. 691-696 ◽  
Author(s):  
Susan M. Berget ◽  
Barbara L. Robberson
Keyword(s):  
In Vitro Splicing ◽  
Small Nuclear Ribonucleoproteins

The splicing factor Prp17 interacts with the U2, U5 and U6 snRNPs and associates with the spliceosome pre- and post-catalysis

2008 ◽  
Vol 416 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Aparna K. Sapra ◽  
Piyush Khandelia ◽  
Usha Vijayraghavan
Keyword(s):  
Second Step ◽  
Splicing Factors ◽  
Temperature Sensitive ◽  
Biochemical Basis ◽  
Association Analyses ◽  
Small Nuclear Rnas ◽  
In Vitro Splicing ◽  
Mechanistic Basis ◽  
Small Nuclear Ribonucleoproteins

Saccharomyces cerevisiae PRP17-null mutants are temperature-sensitive for growth. In vitro splicing with extracts lacking Prp17 are kinetically slow for the first step of splicing and are arrested for the second step at temperatures greater than 34 °C. In the present study we show that these stalled spliceosomes are compromised for an essential conformational switch that is triggered by Prp16 helicase. These results suggest a plausible mechanistic basis for the second-step arrest in prp17Δ extracts and support a role for Prp17 in conjunction with Prp16. To understand the association of Prp17 with spliceosomes we used a functional epitope-tagged protein in co-immunoprecipitation experiments. Examination of co-precipitated snRNAs (small nuclear RNAs) show that Prp17 interacts with U2, U5 and U6 snRNPs (small nuclear ribonucleoproteins) but it is not a core component of any one snRNP. Prp17 association with in-vitro-assembled spliceosome complexes on actin pre-mRNAs was also investigated. Although the U5 snRNP proteins Prp8 and Snu114 are found in early pre-spliceosomes that contain all five snRNPs, Prp17 is not detectable at this step; however, Prp17 is present in the subsequent pre-catalytic A1 complex, containing unspliced pre-mRNA, formed after the dissociation of U4 snRNP. Thus Prp17 joins the spliceosome prior to both catalytic reactions. Our results indicate continued interactions in catalytic spliceosomes that contain reaction intermediates and in post-splicing complexes containing the lariat intron. These Prp17–spliceosome association analyses provide a biochemical basis for the delayed first step in prp17Δ and explain the previously known multiple genetic interactions between Prp17, factors of the Prp19-complex [NTC (nineteen complex)], functional elements in U2 and U5 snRNAs and other second-step splicing factors.

U1 small nuclear ribonucleoproteins are required early during spliceosome assembly

1987 ◽  
Vol 7 (8) ◽  
pp. 2877-2883
Author(s):  
M Zillmann ◽  
S D Rose ◽  
S M Berget
Keyword(s):  
Gel Electrophoresis ◽  
Early Recognition ◽  
Splice Junction ◽  
Specific Antibodies ◽  
U1 Snrnp ◽  
In Vitro Splicing ◽  
Precursor Rna ◽  
Splice Junctions ◽  
Small Nuclear Ribonucleoproteins

U1 small nuclear ribonucleoproteins (snRNPs) are required for in vitro splicing of pre-mRNA. Sequences within U1 RNA hybridize to, and thus recognize, 5' splice junctions. We have investigated the mechanism of association of U1 snRNPs with the spliceosome. U1-specific antibodies detected U1 association with precursor RNA early during assembly. Removal of the 5' terminal sequences of U1 RNA by oligo-directed cleavage or removal of U1 snRNPs by immunoprecipitation prior to the addition of precursor RNA depressed the association of all snRNPs with precursor RNA as detected by immunoprecipitation of splicing complexes by either Sm or U1-specific antibodies. Assembly of the spliceosome as monitored by gel electrophoresis was also depressed after cleavage of U1 RNA. The dependency of Sm precipitability of precursor RNA upon the presence of U1 snRNPs suggests that U1 snRNPs participate in the early recognition of substrate RNAs by U2 to U6 snRNPs. Although removal of the 5'-terminal sequences of U1 depressed U1 snRNP association with precursor RNA, it did not eliminate it, suggesting semistable association of U1 snRNPs with the assembling spliceosome in the absence of U1 RNA hybridization. This association was not dependent upon 5' splice junction sequences but was dependent upon 3' intronic sequences, indicating that U1 snRNPs interact with factors recognizing 3' intronic sequences. Mutual dependence of 5' and 3' recognition factors suggests significant snRNP-snRNP communication during early assembly.

scholarly journals U1 small nuclear ribonucleoproteins are required early during spliceosome assembly.

1987 ◽  
Vol 7 (8) ◽  
pp. 2877-2883 ◽  
Author(s):  
M Zillmann ◽  
S D Rose ◽  
S M Berget
Keyword(s):  
Gel Electrophoresis ◽  
Early Recognition ◽  
Splice Junction ◽  
Specific Antibodies ◽  
U1 Snrnp ◽  
In Vitro Splicing ◽  
Precursor Rna ◽  
Splice Junctions ◽  
Small Nuclear Ribonucleoproteins

U1 small nuclear ribonucleoproteins (snRNPs) are required for in vitro splicing of pre-mRNA. Sequences within U1 RNA hybridize to, and thus recognize, 5' splice junctions. We have investigated the mechanism of association of U1 snRNPs with the spliceosome. U1-specific antibodies detected U1 association with precursor RNA early during assembly. Removal of the 5' terminal sequences of U1 RNA by oligo-directed cleavage or removal of U1 snRNPs by immunoprecipitation prior to the addition of precursor RNA depressed the association of all snRNPs with precursor RNA as detected by immunoprecipitation of splicing complexes by either Sm or U1-specific antibodies. Assembly of the spliceosome as monitored by gel electrophoresis was also depressed after cleavage of U1 RNA. The dependency of Sm precipitability of precursor RNA upon the presence of U1 snRNPs suggests that U1 snRNPs participate in the early recognition of substrate RNAs by U2 to U6 snRNPs. Although removal of the 5'-terminal sequences of U1 depressed U1 snRNP association with precursor RNA, it did not eliminate it, suggesting semistable association of U1 snRNPs with the assembling spliceosome in the absence of U1 RNA hybridization. This association was not dependent upon 5' splice junction sequences but was dependent upon 3' intronic sequences, indicating that U1 snRNPs interact with factors recognizing 3' intronic sequences. Mutual dependence of 5' and 3' recognition factors suggests significant snRNP-snRNP communication during early assembly.

Design and synthesis of herboxidiene derivatives that potently inhibit in vitro splicing

2021 ◽  
Vol 19 (6) ◽  
pp. 1365-1377
Author(s):  
Arun K. Ghosh ◽  
Srinivasa Rao Allu ◽  
Guddeti Chandrashekar Reddy ◽  
Adriana Gamboa Lopez ◽  
Patricia Mendez ◽  
...  
Keyword(s):  
Biological Evaluation ◽  
Design And Synthesis ◽  
In Vitro Splicing ◽  
Enantioselective Syntheses ◽  
Derivatives Of

Enantioselective syntheses of C-6 modified derivatives of herboxidiene and their biological evaluation in splicing inhibitory assay.

Analysis of small nuclear ribonucleoproteins (RNPs) in Trypanosoma brucei: structural organization and protein components of the spliced leader RNP

1991 ◽  
Vol 11 (11) ◽  
pp. 5516-5526 ◽  
Author(s):  
M Cross ◽  
A Günzl ◽  
Z Palfi ◽  
A Bindereif
Keyword(s):  
Trypanosoma Brucei ◽  
Core Protein ◽  
Rnase H ◽  
Spliced Leader ◽  
The Core ◽  
In Vitro Reconstitution ◽  
U2 Snrnp ◽  
Protein Components ◽  
Small Nuclear Ribonucleoproteins

trans splicing in Trypanosoma brucei involves the ligation of the 40-nucleotide spliced leader (SL) to each of the exons of large, polycistronic pre-mRNAs and requires the function of small nuclear ribonucleoproteins (snRNPs). We have identified and characterized snRNP complexes of SL, U2, U4, and U6 RNAs in T. brucei extracts by a combination of glycerol gradient sedimentation, CsCl density centrifugation, and anti-m3G immunoprecipitation. Both the SL RNP and the U4/U6 snRNP contain salt-stable cores; the U2 snRNP, in contrast to other eucaryotic snRNPs, is not stable under stringent ionic conditions. Two distinct complexes of U6 RNA were found, a U6 snRNP and a U4/U6 snRNP. The structure of the SL RNP was analyzed in detail by oligonucleotide-directed RNase H protection and by in vitro reconstitution. Our results indicate that the 3' half of SL RNA constitutes the core protein-binding domain and that protein components of the SL RNP also bind to the U2 and U4 RNAs. Using antisense RNA affinity chromatography, we identified a set of low-molecular-mass proteins (14.8, 14, 12.5, and 10 kDa) as components of the core SL RNP.

Sign in / Sign up

Export Citation Format

Share Document