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Query: UNIPROT:P06889 (Mol)
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Maturation of mRNAs in trypanosomes involves trans splicing of the 5' end of the spliced leader RNA and the exons of polycistronic pre-mRNAs, requiring small nuclear ribonucleoproteins (snRNPs) as cofactors. We have mapped protein-binding sites in the U2 and U4/U6 snRNPs by a combination of RNase H protection analysis, native gel electrophoresis, and CsCl density gradient centrifugation. In the U2 snRNP, protein binding occurs primarily in the 3'-terminal domain; through U2 snRNP reconstitution and chemical modification-interference assays, we have identified discrete positions within stem-loop IV of Trypanosoma brucei U2 RNA that are essential for protein binding; significantly, some of these positions differ from the consensus sequence derived from cis-spliceosomal U2 RNAs. In the U4/U6 snRNP, the major protein-binding region is contained within the 3'-terminal half of U4 RNA. In sum, while the overall domain structure of the U2 and U4/U6 snRNPs is conserved between cis- and trans-splicing systems, our data suggest that there are also trans-spliceosomal specific determinants of RNA-protein binding.
Mol Cell Biol 1992 Feb
PMID:Domain structure of U2 and U4/U6 small nuclear ribonucleoprotein particles from Trypanosoma brucei: identification of trans-spliceosomal specific RNA-protein interactions. 131 Jan 47

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.
Mol Cell Biol 1992 Feb
PMID:Three novel functional variants of human U5 small nuclear RNA. 131 Jan 51

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.
Mol Cell Biol 1992 Mar
PMID:The low-abundance U11 and U12 small nuclear ribonucleoproteins (snRNPs) interact to form a two-snRNP complex. 137 90

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.
Mol Cell Biol 1992 Oct
PMID:The spliceosome assembly pathway in mammalian extracts. 138 87

The major small nuclear ribonucleoproteins (snRNPs) U1, U2, U5 and U4/U6 participate in the splicing of pre-mRNA. U1, U2, U4 and U5 RNAs share a highly conserved sequence motif PuA(U)nGPu, termed the Sm site, which is normally flanked by two hairpin loops. The Sm site provides the major binding site for the group of common proteins, B', B, D1, D2, D3, E, F and G, which are shared by the spliceosomal snRNPs. We have investigated the ability of common snRNP proteins to recognize the Sm site of snRNA by using ultraviolet light-induced RNA-protein cross-linking within U1 snRNP particles. The U1 snRNP particles, reconstituted in vitro, contained U1 snRNA labelled with 32P. Cross-linking of protein to this U1 snRNA occurred only in the presence of the single-stranded stretch of snRNA that makes up the conserved Sm site. Characterization of the cross-linked protein by one and two-dimensional gel electrophoresis indicated that snRNP protein G had become cross-linked to the U1 snRNA. This was confirmed by specific immunoprecipitation of the cross-linked RNA-protein complex with an anti-G antiserum. The cross-link was located on the U1 snRNA by fingerprint analysis with RNases T1 and A; this demonstrated that the protein G has been cross-linked to the AAU stretch within the 5'-terminal half of the Sm site (AAUUUGUGG). These results suggest that the snRNP protein G may be involved in the direct recognition of the Sm site.
J Mol Biol 1992 Sep 05
PMID:Direct binding of small nuclear ribonucleoprotein G to the Sm site of small nuclear RNA. Ultraviolet light cross-linking of protein G to the AAU stretch within the Sm site (AAUUUGUGG) of U1 small nuclear ribonucleoprotein reconstituted in vitro. 138 14

To investigate the function of the U5 small nuclear ribonucleoprotein (snRNP) in pre-mRNA splicing, we have screened for factors that genetically interact with Saccharomyces cerevisiae U5 snRNA. We isolated trans-acting mutations that exacerbate the phenotypes of conditional alleles of the U5 snRNA and named these genes SLU, for synergistically lethal with U5 snRNA. SLU1 and SLU2 are essential for the first catalytic step of splicing, while SLU7 and SLU4 (an allele of PRP17 [U. Vijayraghavan, M. Company, and J. Abelson, Genes Dev. 3:1206-1216, 1989]) are required only for the second step of splicing. Furthermore, slu4-1 and slu7-1 are lethal in combination with mutations in PRP16 and PRP18, which also function in the second step, but not with mutations in factors required for the first catalytic step, such as PRP8 and PRP4. We infer from these data that SLU4, SLU7, PRP18, PRP16, and the U5 snRNA interact functionally and that a major role of the U5 snRNP is to coordinate a set of factors that are required for the completion of the second catalytic step of splicing.
Mol Cell Biol 1992 Nov
PMID:Synthetic lethal mutations suggest interactions between U5 small nuclear RNA and four proteins required for the second step of splicing. 140 91

An essential pre-mRNA splicing factor, the product of the PRP38 gene, has been genetically identified in a screen of temperature-sensitive mutants of Saccharomyces cerevisiae. Shifting temperature-sensitive prp38 cultures from 23 to 37 degrees C prevents the first cleavage-ligation event in the excision of introns from mRNA precursors. In vitro splicing inactivation and complementation studies suggest that the PRP38-encoded factor functions, at least in part, after stable splicing complex formation. The PRP38 locus contains a 726-bp open reading frame coding for an acidic 28-kDa polypeptide (PRP38). While PRP38 lacks obvious structural similarity to previously defined splicing factors, heat inactivation of PRP38, PRP19, or any of the known U6 (or U4/U6) small nuclear ribonucleoprotein-associating proteins (i.e., PRP3, PRP4, PRP6, and PRP24) leads to a common, unexpected consequence: intracellular U6 small nuclear RNA (snRNA) levels decrease as splicing activity is lost. Curiously, U4 snRNA, normally extensively base paired with U6 snRNA, persists in the virtual absence of U6 snRNA.
Mol Cell Biol 1992 Sep
PMID:PRP38 encodes a yeast protein required for pre-mRNA splicing and maintenance of stable U6 small nuclear RNA levels. 150 95

U1 small nuclear RNAs (U1snRNAs) occur in the nucleus of plants and animals where, complexed with several proteins in the form of U1 small nuclear ribonucleoprotein particles (U1snRNPs), they play an important role in precursor messenger RNA (pre-mRNA) splicing. Ten potato U1snRNA genes have been isolated on two genomic clones illustrating the clustering of this multigene family on the potato genome. Based on both the sequence of their coding regions and upstream regulatory elements, seven of the genes are potentially functional. The other three genes were pseudogenes with defective promoter or coding region sequences. Analysis of expression of individual cloned U1snRNA genes in transfected tobacco protoplasts was impossible due to the similarity of U1snRNA sequences in tobacco. However, by marking the coding regions with oligonucleotides or constructing chimaeric genes consisting of a potato U1snRNA promoter region and maize U5snRNA coding region, three of the U1 promoter regions were shown to be transcriptionally active.
Plant Mol Biol 1992 Sep
PMID:Characterization and expression of U1snRNA genes from potato. 151 Nov 41

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.
Mol Cell Biol 1991 Nov
PMID:Analysis of small nuclear ribonucleoproteins (RNPs) in Trypanosoma brucei: structural organization and protein components of the spliced leader RNP. 165 32

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites.
Mol Cell Biol 1991 Dec
PMID:A novel protein factor is required for use of distal alternative 5' splice sites in vitro. 165 20


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