Three novel functional variants of human U5 small nuclear RNA

1992 ◽  
Vol 12 (2) ◽  
pp. 734-746
Author(s):  
E J Sontheimer ◽  
J A Steitz
Keyword(s):  
Hela Cells ◽  
Short Form ◽  
Northern Blotting ◽  
Rnase H ◽  
Small Nuclear Rna ◽  
Small Nuclear Ribonucleoprotein ◽  
Functional Variants ◽  
Nuclear Rna ◽  
The Individual ◽  
Nuclear Ribonucleoprotein

We have identified and characterized three new variants of U5 small nuclear RNA (snRNA) from HeLa cells, called U5D, U5E, and U5F. Each variant has a 2,2,7-trimethylguanosine cap and is packaged into an Sm-precipitable small nuclear ribonucleoprotein (snRNP) particle. All retain the evolutionarily invariant 9-base loop at the top of stem 1; however, numerous base changes relative to the abundant forms of U5 snRNA are present in other regions of the RNAs, including a loop that is part of the yeast U5 minimal domain required for viability and has been shown to bind a protein in HeLa extracts. U5E and U5F each constitute 7% of the total U5 population in HeLa cells and are slightly longer than the previously characterized human U5 (A, B, and C) species. U5D, which composes 5% of HeLa cell U5 snRNAs, is present in two forms: a full-length species, U5DL, and a shorter species, U5DS, which is truncated by 15 nucleotides at its 3' end and therefore resembles the short form of U5 (snR7S) in Saccharomyces cerevisiae. We have established conditions that allow specific detection of the individual U5 variants by either Northern blotting (RNA blotting) or primer extension; likewise, U5E and U5F can be specifically and completely degraded in splicing extracts by oligonucleotide-directed RNase H cleavage. All variant U5 snRNAs are assembled into functional particles, as indicated by their immunoprecipitability with anti-(U5) RNP antibodies, their incorporation into the U4/U5/U6 tri-snRNP complex, and their presence in affinity-purified spliceosomes. The higher abundance of these U5 variants in 293 cells compared with that in HeLa cells suggests possible roles in alternative splicing.

scholarly journals Three novel functional variants of human U5 small nuclear RNA.

1992 ◽  
Vol 12 (2) ◽  
pp. 734-746 ◽  
Author(s):  
E J Sontheimer ◽  
J A Steitz
Keyword(s):  
Hela Cells ◽  
Short Form ◽  
Northern Blotting ◽  
Rnase H ◽  
Small Nuclear Rna ◽  
Small Nuclear Ribonucleoprotein ◽  
Functional Variants ◽  
Nuclear Rna ◽  
The Individual ◽  
Nuclear Ribonucleoprotein

We have identified and characterized three new variants of U5 small nuclear RNA (snRNA) from HeLa cells, called U5D, U5E, and U5F. Each variant has a 2,2,7-trimethylguanosine cap and is packaged into an Sm-precipitable small nuclear ribonucleoprotein (snRNP) particle. All retain the evolutionarily invariant 9-base loop at the top of stem 1; however, numerous base changes relative to the abundant forms of U5 snRNA are present in other regions of the RNAs, including a loop that is part of the yeast U5 minimal domain required for viability and has been shown to bind a protein in HeLa extracts. U5E and U5F each constitute 7% of the total U5 population in HeLa cells and are slightly longer than the previously characterized human U5 (A, B, and C) species. U5D, which composes 5% of HeLa cell U5 snRNAs, is present in two forms: a full-length species, U5DL, and a shorter species, U5DS, which is truncated by 15 nucleotides at its 3' end and therefore resembles the short form of U5 (snR7S) in Saccharomyces cerevisiae. We have established conditions that allow specific detection of the individual U5 variants by either Northern blotting (RNA blotting) or primer extension; likewise, U5E and U5F can be specifically and completely degraded in splicing extracts by oligonucleotide-directed RNase H cleavage. All variant U5 snRNAs are assembled into functional particles, as indicated by their immunoprecipitability with anti-(U5) RNP antibodies, their incorporation into the U4/U5/U6 tri-snRNP complex, and their presence in affinity-purified spliceosomes. The higher abundance of these U5 variants in 293 cells compared with that in HeLa cells suggests possible roles in alternative splicing.

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 U5 small nuclear ribonucleoprotein: RNA structure analysis and ATP-dependent interaction with U4/U6.

1989 ◽  
Vol 9 (8) ◽  
pp. 3350-3359 ◽  
Author(s):  
D L Black ◽  
A L Pinto
Keyword(s):  
Rna Structure ◽  
Rna Secondary Structure ◽  
Rnase H ◽  
Rna Sequences ◽  
Large Loop ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Extracts ◽  
The Individual ◽  
Nuclear Ribonucleoprotein ◽  
Dependent Interaction

To understand how the U5 small nuclear ribonucleoprotein (snRNP) interacts with other spliceosome components, its structure and binding to the U4/U6 snRNP were analyzed. The interaction of the U5 snRNP with the U4/U6 snRNP was studied by separating the snRNPs in HeLa cell nuclear extracts on glycerol gradients. A complex running at 25S and containing U4, U5, and U6 but not U1 or U2 snRNAs was identified. In contrast to results with native gel electrophoresis to separate snRNPs, this U4/U5/U6 snRNP complex requires ATP to assemble from the individual snRNPs. The structure of the U5 RNA within the U5 snRNP and the U4/5/6 snRNP complexes was then compared. Oligonucleotide-targeted RNase H digestion identified one RNA sequence in the U5 snRNP capable of base pairing to other nucleic acid sequences. Chemical modification experiments identified this sequence as well as two other U5 RNA sequences as accessible to modification within the U5 RNP. One of these regions is a large loop in the U5 RNA secondary structure whose sequence is conserved from Saccharomyces cerevisiae to humans. Interestingly, no differences in modification of free U5 snRNP as compared to U5 in the U4/U5/U6 snRNP complex were observed, suggesting that recognition of specific RNA sequences in the U5 snRNP is not required for U4/U5/U6 snRNP assembly.

scholarly journals Loop I of U1 small nuclear RNA is the only essential RNA sequence for binding of specific U1 small nuclear ribonucleoprotein particle proteins.

1988 ◽  
Vol 8 (11) ◽  
pp. 4787-4791 ◽  
Author(s):  
J Hamm ◽  
V L van Santen ◽  
R A Spritz ◽  
I W Mattaj
Keyword(s):  
Protein Interactions ◽  
Protein C ◽  
Small Nuclear Rna ◽  
Structural Element ◽  
Rna Sequence ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Rna ◽  
Egg Extracts ◽  
Specific Proteins ◽  
Nuclear Ribonucleoprotein

The binding of the U1 small nuclear ribonucleoprotein (snRNP)-specific proteins C, A, and 70K to U1 small nuclear RNA (snRNA) was analyzed. Assembly of U1 snRNAs from bean and soybean and a set of mutant Xenopus U1 snRNAs into U1 snRNPs in Xenopus egg extracts was studied. The ability to bind proteins was analyzed by immunoprecipitation with monospecific antibodies and by a protein-sequestering assay. The only sequence essential for binding of the U1-specific proteins was the conserved loop sequence in the 5' hairpin of U1. Further analysis suggested that protein C binds directly to the loop and that the assembly of proteins A and 70K into the RNP requires mainly protein-protein interactions. Protein C apparently recognizes a specific RNA sequence rather than a secondary structural element in the RNA.

Functional analysis of the sea urchin U7 small nuclear RNA

1988 ◽  
Vol 8 (3) ◽  
pp. 1076-1084
Author(s):  
G M Gilmartin ◽  
F Schaufele ◽  
G Schaffner ◽  
M L Birnstiel
Keyword(s):  
Sea Urchin ◽  
Micrococcal Nuclease ◽  
Small Nuclear Rna ◽  
Histone Mrna ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Rna ◽  
U7 Snrnp ◽  
Structure Relationship ◽  
Nuclear Ribonucleoprotein ◽  
Sm Protein

U7 small nuclear RNA (snRNA) is an essential component of the RNA-processing machinery which generates the 3' end of mature histone mRNA in the sea urchin. The U7 small nuclear ribonucleoprotein particle (snRNP) is classified as a member of the Sm-type U snRNP family by virtue of its recognition by both anti-trimethylguanosine and anti-Sm antibodies. We analyzed the function-structure relationship of the U7 snRNP by mutagenesis experiments. These suggested that the U7 snRNP of the sea urchin is composed of three important domains. The first domain encompasses the 5'-terminal sequences, up to about nucleotides 7, which are accessible to micrococcal nuclease, while the remainder of the RNA is highly protected and hence presumably bound by proteins. This region contains the sequence complementarities between the U7 snRNA and the histone pre-mRNA which have previously been shown to be required for 3' processing (F. Schaufele, G. M. Gilmartin, W. Bannwarth, and M. L. Birnstiel, Nature [London] 323:777-781, 1986). Nucleotides 9 to 20 constitute a second domain which includes sequences for Sm protein binding. The complementarities between the U7 snRNA sequences in this region and the terminal palindrome of the histone mRNA appear to be fortuitous and play only a secondary, if any, role in 3' processing. The third domain is composed of the terminal palindrome of U7 snRNA, the secondary structure of which must be maintained for the U7 snRNP to function, but its sequence can be drastically altered without any observable effect on snRNP assembly or 3' processing.

Loop I of U1 small nuclear RNA is the only essential RNA sequence for binding of specific U1 small nuclear ribonucleoprotein particle proteins

1988 ◽  
Vol 8 (11) ◽  
pp. 4787-4791
Author(s):  
J Hamm ◽  
V L van Santen ◽  
R A Spritz ◽  
I W Mattaj
Keyword(s):  
Protein Interactions ◽  
Protein C ◽  
Small Nuclear Rna ◽  
Structural Element ◽  
Rna Sequence ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Rna ◽  
Egg Extracts ◽  
Specific Proteins ◽  
Nuclear Ribonucleoprotein

The binding of the U1 small nuclear ribonucleoprotein (snRNP)-specific proteins C, A, and 70K to U1 small nuclear RNA (snRNA) was analyzed. Assembly of U1 snRNAs from bean and soybean and a set of mutant Xenopus U1 snRNAs into U1 snRNPs in Xenopus egg extracts was studied. The ability to bind proteins was analyzed by immunoprecipitation with monospecific antibodies and by a protein-sequestering assay. The only sequence essential for binding of the U1-specific proteins was the conserved loop sequence in the 5' hairpin of U1. Further analysis suggested that protein C binds directly to the loop and that the assembly of proteins A and 70K into the RNP requires mainly protein-protein interactions. Protein C apparently recognizes a specific RNA sequence rather than a secondary structural element in the RNA.

Model of the interaction between the UI A small nuclear ribonucleoprotein and UI small nuclear RNA stem/loop II

1993 ◽  
Vol 21 (3) ◽  
pp. 605-609 ◽  
Author(s):  
P. R. Evans ◽  
C. Oubridge ◽  
T.-H. Jessen ◽  
J. Li ◽  
C. H. Teo ◽  
...  
Keyword(s):  
Small Nuclear Rna ◽  
Stem Loop ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Rna ◽  
Nuclear Ribonucleoprotein

scholarly journals Functional analysis of the sea urchin U7 small nuclear RNA.

1988 ◽  
Vol 8 (3) ◽  
pp. 1076-1084 ◽  
Author(s):  
G M Gilmartin ◽  
F Schaufele ◽  
G Schaffner ◽  
M L Birnstiel
Keyword(s):  
Sea Urchin ◽  
Micrococcal Nuclease ◽  
Small Nuclear Rna ◽  
Histone Mrna ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Rna ◽  
U7 Snrnp ◽  
Structure Relationship ◽  
Nuclear Ribonucleoprotein ◽  
Sm Protein

U7 small nuclear RNA (snRNA) is an essential component of the RNA-processing machinery which generates the 3' end of mature histone mRNA in the sea urchin. The U7 small nuclear ribonucleoprotein particle (snRNP) is classified as a member of the Sm-type U snRNP family by virtue of its recognition by both anti-trimethylguanosine and anti-Sm antibodies. We analyzed the function-structure relationship of the U7 snRNP by mutagenesis experiments. These suggested that the U7 snRNP of the sea urchin is composed of three important domains. The first domain encompasses the 5'-terminal sequences, up to about nucleotides 7, which are accessible to micrococcal nuclease, while the remainder of the RNA is highly protected and hence presumably bound by proteins. This region contains the sequence complementarities between the U7 snRNA and the histone pre-mRNA which have previously been shown to be required for 3' processing (F. Schaufele, G. M. Gilmartin, W. Bannwarth, and M. L. Birnstiel, Nature [London] 323:777-781, 1986). Nucleotides 9 to 20 constitute a second domain which includes sequences for Sm protein binding. The complementarities between the U7 snRNA sequences in this region and the terminal palindrome of the histone mRNA appear to be fortuitous and play only a secondary, if any, role in 3' processing. The third domain is composed of the terminal palindrome of U7 snRNA, the secondary structure of which must be maintained for the U7 snRNP to function, but its sequence can be drastically altered without any observable effect on snRNP assembly or 3' processing.

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