Characterization of Sm-like proteins in yeast and their association with U6 snRNA.
(25/1744)
Seven Sm proteins associate with U1, U2, U4 and U5 spliceosomal snRNAs and influence snRNP biogenesis. Here we describe a novel set of Sm-like (Lsm) proteins in Saccharomyces cerevisiae that interact with each other and with U6 snRNA. Seven Lsm proteins co-immunoprecipitate with the previously characterized Lsm4p (Uss1p) and interact with each other in two-hybrid analyses. Free U6 and U4/U6 duplexed RNAs co-immunoprecipitate with seven of the Lsm proteins that are essential for the stable accumulation of U6 snRNA. Analyses of U4/U6 di-snRNPs and U4/U6.U5 tri-snRNPs in Lsm-depleted strains suggest that Lsm proteins may play a role in facilitating conformational rearrangements of the U6 snRNP in the association-dissociation cycle of spliceosome complexes. Thus, Lsm proteins form a complex that differs from the canonical Sm complex in its RNA association(s) and function. We discuss the possible existence and functions of alternative Lsm complexes, including the likelihood that they are involved in processes other than pre-mRNA splicing. (+info)
Congenital end-plate acetylcholinesterase deficiency caused by a nonsense mutation and an A-->G splice-donor-site mutation at position +3 of the collagenlike-tail-subunit gene (COLQ): how does G at position +3 result in aberrant splicing?
(26/1744)
Congenital end-plate acetylcholinesterase (AChE) deficiency (CEAD), the cause of a disabling myasthenic syndrome, arises from defects in the COLQ gene, which encodes the AChE triple-helical collagenlike-tail subunit that anchors catalytic subunits of AChE to the synaptic basal lamina. Here we describe a patient with CEAD with a nonsense mutation (R315X) and a splice-donor-site mutation at position +3 of intron 16 (IVS16+3A-->G) of COLQ. Because both A and G are consensus nucleotides at the +3 position of splice-donor sites, we constructed a minigene that spans exons 15-17 and harbors IVS16+3A-->G for expression in COS cells. We found that the mutation causes skipping of exon 16. The mutant splice-donor site of intron 16 harbors five discordant nucleotides (at -3, -2, +3, +4, and +6) that do not base-pair with U1 small-nuclear RNA (snRNA), the molecule responsible for splice-donor-site recognition. Versions of the minigene harboring, at either +4 or +6, nucleotides complementary to U1 snRNA restore normal splicing. Analysis of 1,801 native splice-donor sites reveals that presence of a G nucleotide at +3 is associated with preferential usage, at positions +4 to +6, of nucleotides concordant to U1 snRNA. Analysis of 11 disease-associated IVS+3A-->G mutations indicates that, on average, two of three nucleotides at positions +4 to +6 fail to base-pair, and that the nucleotide at +4 never base-pairs, with U1 snRNA. We conclude that, with G at +3, normal splicing generally depends on the concordance that residues at +4 to +6 have with U1 snRNA, but other cis-acting elements may also be important in assuring the fidelity of splicing. (+info)
Allele-specific genetic interactions between Prp8 and RNA active site residues suggest a function for Prp8 at the catalytic core of the spliceosome.
(27/1744)
The highly conserved spliceosomal protein Prp8 is known to cross-link the critical sequences at both the 5' (GU) and 3' (YAG) ends of the intron. We have identified prp8 mutants with the remarkable property of suppressing exon ligation defects due to mutations in position 2 of the 5' GU, and all positions of the 3' YAG. The prp8 mutants also suppress mutations in position A51 of the critical ACAGAG motif in U6 snRNA, which has been observed previously to cross-link position 2 of the 5' GU. Other mutations in the 5' splice site, branchpoint, and neighboring residues of the U6 ACAGAG motif are not suppressed. Notably, the suppressed residues are specifically conserved from yeast to man, and from U2- to U12-dependent spliceosomes. We propose that Prp8 participates in a previously unrecognized tertiary interaction between U6 snRNA and both the 5' and 3' ends of the intron. This model suggests a mechanism for positioning the 3' splice site for catalysis, and assigns a fundamental role for Prp8 in pre-mRNA splicing. (+info)
Functional interactions of Prp8 with both splice sites at the spliceosomal catalytic center.
(28/1744)
A U5 snRNP protein, hPrp8, interacts closely with the GU dinucleotide at the 5' splice site (5'SS), forming a specific UV-inducible cross-link. To test if this physical contact between the 5'SS and the carboxy-terminal region of Prp8 reflects a functional recognition of the 5'SS during spliceosome assembly, we mutagenized the corresponding region of yeast Prp8 and screened the resulting mutants for suppression of 5'SS mutations in vivo. All of the isolated prp8 alleles not only suppress 5'SS but also 3'SS mutations, affecting the second catalytic step. Suppression of the 5'SS mutations by prp8 alleles was also tested in the presence of U1-7U snRNA, a predicted suppressor of the U+2A mutation. As expected, U1-7U efficiently suppresses prespliceosome formation, and the first, but not the second, step of U+2A pre-mRNA splicing. Independently, Prp8 functionally interacts with both splice sites at the later stage of splicing, affecting the efficiency of the second catalytic step. The striking proximity of two of the prp8 suppressor mutations to the site of the 5'SS:hPrp8 cross-link suggests that some protein:5'SS contacts made before the first step may be subsequently extended to accommodate the 3'SS for the second catalytic step. Together, these results strongly implicate Prp8 in specific interactions at the catalytic center of the spliceosome. (+info)
The Rev protein is able to transport to the cytoplasm small nucleolar RNAs containing a Rev binding element.
(29/1744)
Small nucleolar RNAs (snoRNAs) were utilized to express Rev-binding sequences inside the nucleolus and to test whether they are substrates for Rev binding and transport. We show that U16 snoRNA containing the minimal binding site for Rev stably accumulates inside the nucleolus maintaining the interaction with the basic C/D snoRNA-specific factors. Upon Rev expression, the chimeric RNA is exported to the cytoplasm, where it remains bound to Rev in a particle devoid of snoRNP-specific factors. These data indicate that Rev can elicit the functions of RNA binding and transport inside the nucleolus. (+info)
Pac1p, an RNase III homolog, is required for formation of the 3' end of U2 snRNA in Schizosaccharomyces pombe.
(30/1744)
Like its homologs in higher eukaryotes, the U2 snRNA in Schizosaccharomyces pombe is transcribed by RNA polymerase II and is not polyadenylated. Instead, an RNA stem-loop structure located downstream of the U2 snRNA coding sequence and transcribed as part of a 3' extended precursor serves as a signal for 3'-end formation. We have identified three mutants that have temperature-sensitive defects in U2 snRNA 3'-end formation. In these mutants, the synthesis of the major snRNAs is also affected and unprocessed rRNA precursors accumulate at the restrictive temperature. Two of these mutants contain the same G-to-A transition within the pac1 gene, whereas the third contains a lesion outside the pac1 locus, indicating that at least two genes are involved. The pac1+ gene is codominant with the mutant allele and can rescue the temperature-sensitive phenotype and the defects in snRNA and rRNA synthesis, if overexpressed. In vitro, Pac1p, an RNase III homolog, can cleave a synthetic U2 precursor within the signal for 3'-end formation, generating a product that is a few nucleotides longer than mature U2 snRNA. In addition, U2 precursors are cleaved and trimmed to the mature size in extracts made from wild-type S. pombe cells. However, extracts made from pac1 mutant cells are unable to do so unless they are supplemented with purified recombinant Pac1p. Thus, the 3' end of S. pombe U2 snRNA is generated by a processing reaction that requires Pac1p and an additional component, and can be dissociated from transcription in vitro. (+info)
A candidate U1 small nuclear RNA for trypanosomatid protozoa.
(31/1744)
In trypanosomatid protozoa, all mRNAs obtain identical 5'-ends by trans-splicing of the 5'-terminal 39 nucleotides of a small spliced leader RNA to appropriate acceptor sites in pre-mRNA. Although this process involves spliceosomal small nuclear (sn) RNAs, it is thought that trypanosomatids do not contain a homolog of the cis-spliceosomal U1 snRNA. We show here that a trypanosomatid protozoon, Crithidia fasciculata, contains a novel small RNA that displays several features characteristic of a U1 snRNA, including (i) a methylguanosine cap and additional 5'-terminal modifications, (ii) a potential binding site for common core proteins that are present in other trans-spliceosomal ribonucleoproteins, (iii) a U1-like 5'-terminal sequence, and (iv) a U1-like stem/loop I structure. Because trypanosomatid pre-mRNAs do not appear to contain cis-spliced introns, we argue that this previously unrecognized RNA species is a good candidate to be a trans-spliceosomal U1 snRNA. (+info)
Flexible zinc finger requirement for binding of the transcriptional activator staf to U6 small nuclear RNA and tRNA(Sec) promoters.
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The transactivator Staf, which contains seven zinc finger motifs, exerts its effect on gene expression by binding to specific targets in small nuclear RNA (snRNA) and snRNA-type gene promoters. In this work, binding site selection allowed us to identify the 21-base pair ATTACCCATAATGCATYGCGG sequence as the high affinity consensus binding site for Staf. It shows a high sequence divergence with Staf-responsive elements in the Xenopus selenocysteine tRNA (tRNA(Sec)) and human U6 snRNA promoters. By using a combination of approaches, we analyzed the interaction of wild-type and truncated Staf zinc finger domains with the consensus, Xenopus tRNA(Sec), and human U6 sites. Two main conclusions emerged from our data. First, the data clearly indicate that zinc finger 7 does not establish base-specific contacts in Staf-DNA complexes. The second conclusion concerns zinc finger 1, which is required for the binding to the Xenopus tRNA(Sec) site but is dispensable in the case of the human U6 site. Taking into account the sequence differences in the two sites, these findings demonstrate that Staf utilizes zinc finger 1 in a rather flexible manner, illustrating how a protein can interact with DNAs containing targets of different sequences. (+info)