scholarly journals Essential Role of a Trypanosome U4-Specific Sm Core Protein in Small Nuclear Ribonucleoprotein Assembly and Splicing

2010 ◽  
Vol 9 (3) ◽  
pp. 379-386 ◽  
Author(s):  
Nicolas Jaé ◽  
Pingping Wang ◽  
Tianpeng Gu ◽  
Martin Hühn ◽  
Zsofia Palfi ◽  
...  
Keyword(s):  
Essential Role ◽  
Core Protein ◽  
Affinity Purification ◽  
Rnase H ◽  
Essential Step ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoproteins

ABSTRACT Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring or variant Sm cores with snRNA-specific Sm subunits. Here we show biochemically by a combination of RNase H cleavage and tandem affinity purification that the U4 snRNP contains a variant Sm heteroheptamer core in which only SmD3 is replaced by SSm4. This U4-specific, nuclear-localized Sm core protein is essential for growth and splicing. As shown by RNA interference (RNAi) knockdown, SSm4 is specifically required for the integrity of the U4 snRNA and the U4/U6 di-snRNP in trypanosomes. In addition, we demonstrate by in vitro reconstitution of Sm cores that under stringent conditions, the SSm4 protein suffices to specify the assembly of U4 Sm cores. Together, these data indicate that the assembly of the U4-specific Sm core provides an essential step in U4/U6 di-snRNP biogenesis and splicing in trypanosomes.

scholarly journals PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle

1989 ◽  
Vol 9 (9) ◽  
pp. 3710-3719
Author(s):  
J Banroques ◽  
J N Abelson
Keyword(s):  
Essential Role ◽  
Mrna Splicing ◽  
Ribonucleoprotein Particle ◽  
Specific Antibodies ◽  
Small Nuclear Ribonucleoprotein ◽  
Assembly Pathway ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoprotein Particle

The Saccharomyces cerevisiae prp mutants (prp2 through prp11) are known to be defective in pre-mRNA splicing at nonpermissive temperatures. We have sequenced the PRP4 gene and shown that it encodes a 52-kilodalton protein. We obtained PRP4 protein-specific antibodies and found that they inhibited in vitro pre-mRNA splicing, which confirms the essential role of PRP4 in splicing. Moreover, we found that PRP4 is required early in the spliceosome assembly pathway. Immunoprecipitation experiments with anti-PRP4 antibodies were used to demonstrate that PRP4 is a protein of the U4/U6 small nuclear ribonucleoprotein particle (snRNP). Furthermore, the U5 snRNP could be immunoprecipitated through snRNP-snRNP interactions in the large U4/U5/U6 complex.

scholarly journals PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle.

1989 ◽  
Vol 9 (9) ◽  
pp. 3710-3719 ◽  
Author(s):  
J Banroques ◽  
J N Abelson
Keyword(s):  
Essential Role ◽  
Mrna Splicing ◽  
Ribonucleoprotein Particle ◽  
Specific Antibodies ◽  
Small Nuclear Ribonucleoprotein ◽  
Assembly Pathway ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoprotein Particle

The Saccharomyces cerevisiae prp mutants (prp2 through prp11) are known to be defective in pre-mRNA splicing at nonpermissive temperatures. We have sequenced the PRP4 gene and shown that it encodes a 52-kilodalton protein. We obtained PRP4 protein-specific antibodies and found that they inhibited in vitro pre-mRNA splicing, which confirms the essential role of PRP4 in splicing. Moreover, we found that PRP4 is required early in the spliceosome assembly pathway. Immunoprecipitation experiments with anti-PRP4 antibodies were used to demonstrate that PRP4 is a protein of the U4/U6 small nuclear ribonucleoprotein particle (snRNP). Furthermore, the U5 snRNP could be immunoprecipitated through snRNP-snRNP interactions in the large U4/U5/U6 complex.

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.

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.

Uncoupling two functions of the U1 small nuclear ribonucleoprotein particle during in vitro splicing

1993 ◽  
Vol 13 (6) ◽  
pp. 3135-3145
Author(s):  
S D Seiwert ◽  
J A Steitz
Keyword(s):  
Splice Site ◽  
Rnase H ◽  
Ribonucleoprotein Particle ◽  
Additional Function ◽  
Small Nuclear Ribonucleoprotein ◽  
U1 Snrna ◽  
In Vitro Splicing ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoprotein Particle

To probe functions of the U1 small nuclear ribonucleoprotein particle (snRNP) during in vitro splicing, we have used unusual splicing substrates which replace the 5' splice site region of an adenovirus substrate with spliced leader (SL) RNA sequences from Leptomonas collosoma or Caenorhabditis elegans. In agreement with previous results (J.P. Bruzik and J.A. Steitz, Cell 62:889-899, 1990), we find that oligonucleotide-targeted RNase H destruction of the 5' end of U1 snRNA inhibits the splicing of a standard adenovirus splicing substrate but not of the SL RNA-containing substrates. However, use of an antisense 2'-O-methyl oligoribonucleotide that disrupts the first stem of U1 snRNA as well as stably sequestering positions of U1 snRNA involved in 5' and 3' splice site recognition inhibits the splicing of both the SL constructs and the standard adenovirus substrate. The 2'-O-methyl oligoribonucleotide is no more effective than RNase H pretreatment in preventing pairing of U1 with the 5' splice site, as assessed by inhibition of psoralen cross-link formation between the SL RNA-containing substrate and U1. The 2'-O-methyl oligoribonucleotide does not alter the protein composition of the U1 monoparticle or deplete the system of essential splicing factors. Native gel analysis indicates that the 2'-O-methyl oligoribonucleotide inhibits splicing by diminishing the formation of splicing complexes. One interpretation of these results is that removal of the 5' end of U1 inhibits base pairing in a different way than sequestering the same sequence with a complementary oligoribonucleotide. Alternatively, our data may indicate that two elements near the 5' end of U1 RNA normally act during spliceosome assembly; the extreme 5' end base pairs with the 5' splice site, while the sequence or structural integrity of stem I is essential for some additional function. It follows that different introns may differ in their use of the repertoire of U1 snRNP functions.

scholarly journals Uncoupling two functions of the U1 small nuclear ribonucleoprotein particle during in vitro splicing.

1993 ◽  
Vol 13 (6) ◽  
pp. 3135-3145 ◽  
Author(s):  
S D Seiwert ◽  
J A Steitz
Keyword(s):  
Splice Site ◽  
Rnase H ◽  
Ribonucleoprotein Particle ◽  
Additional Function ◽  
Small Nuclear Ribonucleoprotein ◽  
U1 Snrna ◽  
In Vitro Splicing ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoprotein Particle

To probe functions of the U1 small nuclear ribonucleoprotein particle (snRNP) during in vitro splicing, we have used unusual splicing substrates which replace the 5' splice site region of an adenovirus substrate with spliced leader (SL) RNA sequences from Leptomonas collosoma or Caenorhabditis elegans. In agreement with previous results (J.P. Bruzik and J.A. Steitz, Cell 62:889-899, 1990), we find that oligonucleotide-targeted RNase H destruction of the 5' end of U1 snRNA inhibits the splicing of a standard adenovirus splicing substrate but not of the SL RNA-containing substrates. However, use of an antisense 2'-O-methyl oligoribonucleotide that disrupts the first stem of U1 snRNA as well as stably sequestering positions of U1 snRNA involved in 5' and 3' splice site recognition inhibits the splicing of both the SL constructs and the standard adenovirus substrate. The 2'-O-methyl oligoribonucleotide is no more effective than RNase H pretreatment in preventing pairing of U1 with the 5' splice site, as assessed by inhibition of psoralen cross-link formation between the SL RNA-containing substrate and U1. The 2'-O-methyl oligoribonucleotide does not alter the protein composition of the U1 monoparticle or deplete the system of essential splicing factors. Native gel analysis indicates that the 2'-O-methyl oligoribonucleotide inhibits splicing by diminishing the formation of splicing complexes. One interpretation of these results is that removal of the 5' end of U1 inhibits base pairing in a different way than sequestering the same sequence with a complementary oligoribonucleotide. Alternatively, our data may indicate that two elements near the 5' end of U1 RNA normally act during spliceosome assembly; the extreme 5' end base pairs with the 5' splice site, while the sequence or structural integrity of stem I is essential for some additional function. It follows that different introns may differ in their use of the repertoire of U1 snRNP functions.

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.

The low-abundance U11 and U12 small nuclear ribonucleoproteins (snRNPs) interact to form a two-snRNP complex

1992 ◽  
Vol 12 (3) ◽  
pp. 1276-1285
Author(s):  
K M Wassarman ◽  
J A Steitz
Keyword(s):  
Secondary Structure ◽  
Direct Interaction ◽  
Nuclear Extract ◽  
Rnase H ◽  
Small Nuclear Ribonucleoprotein ◽  
Cell Extracts ◽  
Detectable Difference ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoproteins ◽  
Selection Of

A novel small nuclear ribonucleoprotein (snRNP) complex containing both U11 and U12 RNAs has been identified in HeLa cell extracts. This U11/U12 snRNP complex can be visualized on glycerol gradients, on native polyacrylamide gels, and by selection with antisense 2'-O-methyl oligoribonucleotides. RNase H-mediated degradation of the U12 snRNA confirmed a direct interaction between the U11 and U12 snRNPs. This snRNP complex is the first to be identified involving low-abundance snRNPs. Selection of the U11/U12 snRNP complex is sensitive to high salt, suggestive of a protein-mediated interaction. Secondary structure analyses revealed several regions of the U11 snRNP accessible for interaction with other RNAs or proteins but no detectable difference between the accessibility of these regions in the U11 monoparticle compared with the U11/U12 snRNP complex. There are also several accessible single-stranded regions in the U12 snRNP, and oligonucleotide-directed RNase H digestion identified nucleotides 28 to 36 of U12 as containing sequences required for the U11/U12 interaction. Both the U12 snRNP and the U11/U12 snRNP complex can be disrupted without altering the cleavage/polyadenylation activity of a nuclear extract.

scholarly journals 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.

scholarly journals The low-abundance U11 and U12 small nuclear ribonucleoproteins (snRNPs) interact to form a two-snRNP complex.

1992 ◽  
Vol 12 (3) ◽  
pp. 1276-1285 ◽  
Author(s):  
K M Wassarman ◽  
J A Steitz
Keyword(s):  
Secondary Structure ◽  
Direct Interaction ◽  
Nuclear Extract ◽  
Rnase H ◽  
Small Nuclear Ribonucleoprotein ◽  
Cell Extracts ◽  
Detectable Difference ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoproteins ◽  
Selection Of

A novel small nuclear ribonucleoprotein (snRNP) complex containing both U11 and U12 RNAs has been identified in HeLa cell extracts. This U11/U12 snRNP complex can be visualized on glycerol gradients, on native polyacrylamide gels, and by selection with antisense 2'-O-methyl oligoribonucleotides. RNase H-mediated degradation of the U12 snRNA confirmed a direct interaction between the U11 and U12 snRNPs. This snRNP complex is the first to be identified involving low-abundance snRNPs. Selection of the U11/U12 snRNP complex is sensitive to high salt, suggestive of a protein-mediated interaction. Secondary structure analyses revealed several regions of the U11 snRNP accessible for interaction with other RNAs or proteins but no detectable difference between the accessibility of these regions in the U11 monoparticle compared with the U11/U12 snRNP complex. There are also several accessible single-stranded regions in the U12 snRNP, and oligonucleotide-directed RNase H digestion identified nucleotides 28 to 36 of U12 as containing sequences required for the U11/U12 interaction. Both the U12 snRNP and the U11/U12 snRNP complex can be disrupted without altering the cleavage/polyadenylation activity of a nuclear extract.

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