Functional redundancy of worm spliceosomal proteins U1A and U2B (original) (raw)
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RNA-Protein Interactions in the U12-Dependent Spliceosome
2016
Nuclear precursor messenger RNA (Pre-mRNA) splicing is an important regulatory step in metazoan gene expression. More than 99% of nuclear pre-mRNA introns are U2-type that are spliced by U2-dependent spliceosome containing U1, U2, U4, U5 and U6 snRNAs. Only less than 1% of the introns are U12-type and spliced by U11, U12, U4atac, U5 and U6atac snRNAs. U12 and U6atac snRNAs play a central role in the splicing of U12-dependent introns. Our previous work demonstrated that the conserved 3′ stem-loop region of U6atac snRNA contains a U12-dependent spliceosome-specific targeting activity, however any potential molecular mechanism was unclear. We discovered that the distal 3′ stem-loop of U6atac has structural and sequence similarities with stemloop III of U12 snRNA. These observations convinced us to investigate the structure-function requirement of the substructure of the U6atac 3′ stem-loop in U12-dependent in vivo splicing. Our results show that the C-terminal RNA recognition motif of p65, a U12 snRNA binding protein, also binds to the distal 3′ stem-loop of U6atac. Using in vivo genetic suppressor assay, we demonstrate that stem-loop III of U12 snRNA which binds to p65 protein can be functionally replaced by U6atac distal stem-loop and vice-versa. Furthermore, we tested the compatibility of the U6atac 3' end from phylogenetically distant species in a human U6atac suppressor background to establish the evolutionary relatedness of these ix structures and in vivo functionality. In conclusion, we demonstrate that p65 Cterminal RNA recognition motif interacts with the U6atac distal 3′ stem-loop. Although the significance of p65 binding to U6atac snRNA is not clear, our study suggests that both the helix structure, as well as the sequence of U6atac distal 3′ stem-loop is important for snRNA-protein interactions and U12-dependent intron splicing. x CHAPTER I
The two similarly expressed genes encoding U3 snRNA in Schizosaccharomyces pombe lack introns
Molecular Biology and Evolution
Both genes encoding U3 small nuclear RNA (snRNA) from the budding yeast Saccharomyces cerevisiae were recently shown to be interrupted by introns of the type removed by the pre-mRNA splicing machinery. We previously described one of the two U3 genes from the fission yeast Schizosaccharomyces pombe. In the present work, the second S. pombe U3 coding sequence was identified, and direct RNA sequence analysis was used to show that neither the U3A nor the U3B gene from this organism contains an intervening sequence. Our data also demonstrate that, as expected, the two RNAs exhibit great primary-and secondary-structure conservation. These similarities are not likely to be the result of a recent gene duplication or conversion event, because the DNA sequences flanking the U3A and U3B genes have diverged substantially. A notable exception is a 19-bp block, centered 36 nucleotides upstream from the transcriptional start site, in which the two loci match in 15 positions; this motif may represent an RNA polymerase II upstream regulatory element, because related sequences are found preceding fission yeast Ul, U2, U4, and U5 snRNA genes. The significance of a short conserved sequence just downstream of the U3A and U3B genes is unknown, as it is not found 3' to other snRNA coding sequences in S. pombe. The 5' one-third of U3B RNA can be folded into a dual hairpin structure, as we previously proposed for Schizosaccharomyces pombe U3A and for other lower eukaryotic U3 homologues. Quantitation of fission yeast U3A and U3B indicates that, in contrast to snR17A and B in Saccharomyces cerevisiae, these RNAs accumulate to similar levels.
A comprehensive biochemical and genetic analysis of the yeast U1 snRNP reveals five novel proteins
RNA (New York, N.Y.), 1998
The U1 snRNP is essential for recognition of the pre-mRNA 5'-splice site and the subsequent assembly of the spliceosome. Yeast U1 snRNP is considerably more complex than its metazoan counterpart, which suggests possible differences between yeast and metazoa in early splicing events. We have comprehensively analyzed the composition of yeast U1 snRNPs using a combination of biochemical, mass spectrometric, and genetic methods. We demonstrate the specific association of four novel U1 snRNP proteins, Snu71p, Snu65p, Nam8p, and Snu56p, that have no known metazoan homologues. A fifth protein, Npl3p, is an abundant cellular component that reproducibly co-purifies with the U1 snRNP, but its association is salt-sensitive. Therefore, we are unable to establish conclusively whether it binds specifically to the U1 snRNP. Interestingly, Nam8p and Npl3p were previously assigned functions in (pre-m)RNA-metabolism; however, so far, no association with U1 snRNP has been demonstrated or proposed....
RNA, 2004
The general splicing factor U2AF65 recognizes the polypyrimidine tract (Py tract) that precedes 3′ splice sites and has three RNA recognition motifs (RRMs). The C-terminal RRM (RRM3), which is highly conserved, has been proposed to contribute to Py-tract binding and establish protein–protein contacts with splicing factors mBBP/SF1 and SAP155. Unexpectedly, we find that the human RRM3 domain is dispensable for U2AF65 activity in vitro. However, it has an essential function in Schizosaccharomyces pombe distinct from binding to the Py tract or to mBBP/SF1 and SAP155. First, deletion of RRM3 from the human protein has no effect on Py-tract binding. Second, RRM123 and RRM12 select similar sequences from a random pool of RNA. Third, deletion of RRM3 has no effect on the splicing activity of U2AF65 in vitro. However, deletion of the RRM3 domain of S. pombe U2AF59 abolishes U2AF function in vivo. In addition, certain amino acid substitutions on the four-stranded β-sheet surface of RRM3 comp...
Phylogenetic Comparison and Splicing Analysis of the U1 snRNP-specific Protein U1C in Eukaryotes
Frontiers in Molecular Biosciences
As a pivotal regulator of 5’ splice site recognition, U1 small nuclear ribonucleoprotein (U1 snRNP)-specific protein C (U1C) regulates pre-mRNA splicing by interacting with other components of the U1 snRNP complex. Previous studies have shown that U1 snRNP and its components are linked to a variety of diseases, including cancer. However, the phylogenetic relationships and expression profiles of U1C have not been studied systematically. To this end, we identified a total of 110 animal U1C genes and compared them to homologues from yeast and plants. Bioinformatics analysis shows that the structure and function of U1C proteins is relatively conserved and is found in multiple copies in a few members of the U1C gene family. Furthermore, the expression patterns reveal that U1Cs have potential roles in cancer progression and human development. In summary, our study presents a comprehensive overview of the animal U1C gene family, which can provide fundamental data and potential cues for fur...
Structure, 1996
Background: Human U1A protein binds to hairpin II of U1 small nuclear RNA (snRNA) and, together with other proteins, forms the U1 snRNP essential in pre-mRNA splicing. U1A protein also binds to the 3′ untranslated region (3′UTR) of its own pre-mRNA, inhibiting polyadenylation of the 3′ end and thereby downregulating its own expression. The 3′UTR folds into an evolutionarily conserved secondary structure with two internal loops; one loop contains the sequence AUUGCAC and the other its variant AUUGUAC. The sequence AUUGCAC is also found in hairpin II of U1 snRNA; hence, U1A protein recognizes the same heptanucleotide sequence in two different structural contexts. In order to better understand the control mechanism of the polyadenylation process, we have built a model of the U1A protein-3′UTR complex based on the crystal structure of the U1A protein-hairpin II RNA complex which we determined previously.
U small nuclear ribonucleoprotein requirements for nematode cis- and trans-splicing in vitro
Journal of Biological Chemistry, 1992
In nematodes, a fraction of mRNAs acquires a common 22-nucleotide 5"terminal spliced leader sequence via a trans-splicing reaction. The same premessenger RNAs which receive the spliced leader are also processed by conventional cis-splicing. Whole cell extracts prepared from synchronous embryos of the parasitic nematode Ascaris lumbricoides catalyze both cis-and trans-splicing. We have used this cell-free system and oligodeoxynucleotide directed RNase H digestion to assess the U small nuclear RNA requirements for nematode cis-and trans-splicing. These experiments indicated that both cis-and trans-splicing require intact U2 and U4/U6 small nuclear ribonucleoproteins (sn-RNPs). However, whereas cis-splicing displays the expected requirement for an intact U1 snRNP, transsplicing is unaffected when -90% of U1 snRNP is degraded. These results suggest that 5' splice site identification differs in nematode cis-and trans-splicing.
1990
List of publications General introduction Identification of RNA binding segment of human U1A protein and definition of its binding site on U1 snRNA. Major determinants of the specificity of interaction between small nuclear ribonucleoproteins U1A and U2B" and their cognate RNAs. Analysis of in vitro binding of U1A protein mutants toU1 snRNA. Conserved amino acid residues within and outside of the N-terminal ribonucleoprotein motif of U1A small nuclear ribonucleoprotein involved in U1 RNA binding. The U2B" RNP motif as a site of protein-protein interaction. A weak interaction between U2A' protein and U2 snRNA helps to stabilize their complex with the U2B" protein. The U1 snRNP-specific U1A protein inhibits polyadenylation of its own pre-mRNA.
Biochimie, 2008
U3 small nucleolar RNA (snoRNA) is one of the members of the box C/D class of snoRNA and is essential for ribosomal RNA (rRNA) processing to generate 18S rRNA in the nucleolus. Although U3 snoRNA is abundant, and is well conserved from yeast to mammals, the genes encoding U3 snoRNA in C. elegans have long remained unidentified. A recent RNomics study in C. elegans predicted five distinct U3 snoRNA genes. However, characterization of these candidates for U3 snoRNA has yet to be performed. In this study, we isolated and characterized four candidate RNAs for U3 snoRNA from the immunoprecipitated RNAs of C. elegans using an antibody against the 2,2,7-trimethylguanosine (TMG) cap. The sequences were identical to the predicted U3 sequences in the RNomics study. Here, we show the several lines of evidence that the isolated RNAs are the true U3 snoRNAs of C. elegans. Moreover, we report the novel expression pattern of U3 snoRNA and fibrillarin, which is an essential component of U3 small nucleolar ribonucleoprotein complex, during early embryo development of C. elegans. To our knowledge, this is the first observation of the inconsistent localization U3 snoRNA and fibrillarin during early embryogenesis, providing novel insight into the mechanisms of nucleologenesis and ribosome production during early embryogenesis.
Evolutionary conservation of the spliceosaomal protein, U2 B”
Nucleic Acids Research, 1991
Ul and U2snRNPs play key roles in pre-mRNA splicing. The interactions between the Ul and U2snRNP-specific proteins, UMA, U2A' and U2B" and their respective UsnRNAs are of interest both to elucidate their roles in splicing, and as models to study RNA-protein interactions. We have cloned a full-length cDNA, encoding U2B1, from potato. This is the first report of a sequence for a plant UsnRNP protein. The plant U2B" sequence exhibits extensive similarity with the human U2B1 protein at both the DNA and amino acid levels. The evolutionary conservation at the protein level, particularly in sequences implicated in determining specific binding to U2snRNA, suggests conservation of U2B" function from plants to man. The significance of amino acid substitutions in the RNP-80 motif with respect to U2snRNA binding in plants is discussed.