Splicing factor Prp8 governs U4/U6 RNA unwinding during activation of the spliceosome.
(9/3478)
The pre-mRNA 5' splice site is recognized by the ACAGA box of U6 spliceosomal RNA prior to catalysis of splicing. We previously identified a mutant U4 spliceosomal RNA, U4-cs1, that masks the ACAGA box in the U4/U6 complex, thus conferring a cold-sensitive splicing phenotype in vivo. Here, we show that U4-cs1 blocks in vitro splicing in a temperature-dependent, reversible manner. Analysis of splicing complexes that accumulate at low temperature shows that U4-cs1 prevents U4/U6 unwinding, an essential step in spliceosome activation. A novel mutation in the evolutionarily conserved U5 snRNP protein Prp8 suppresses the U4-cs1 growth defect. We propose that wild-type Prp8 triggers unwinding of U4 and U6 RNAs only after structurally correct recognition of the 5' splice site by the U6 ACAGA box and that the mutation (prp8-201) relaxes control of unwinding. (+info)
Hybridization of antisense oligonucleotides with the 3'part of tRNA(Phe).
(10/3478)
The interaction of antisense oligodeoxyribonucleotides with yeast tRNA(Phe) was investigated. 14-15-mers complementary to the 3'-terminal sequence including the ACCA end bind to the tRNA under physiological conditions. At low oligonucleotide concentrations the binding occurs at the unique complementary site. At higher oligonucleotide concentrations, the second oligonucleotide molecule binds to the complex due to non-perfect duplex formation in the T-loop stabilized by stacking between the two bound oligonucleotides. In these complexes the acceptor stem is open and the 5'-terminal sequence of the tRNA is accessible for binding of a complementary oligonucleotide. The results prove that the efficient binding of oligonucleotides to the 3'-terminal sequence of the tRNA occurs through initial binding to the single-stranded sequence ACCA followed by invasion in the acceptor stem and strand displacement. (+info)
Identification of eight proteins that cross-link to pre-mRNA in the yeast commitment complex.
(11/3478)
In the yeast commitment complex and the mammalian E complex, there is an important base-pairing interaction between the 5' end of U1 snRNA and the conserved 5' splice site region of pre-mRNA. But no protein contacts between splicing proteins and the pre-mRNA substrate have been defined in or near this region of early splicing complexes. To address this issue, we used 4-thiouridine-substituted 5' splice site-containing RNAs as substrates and identified eight cross-linked proteins, all of which were identified previously as commitment complex components. The proteins were localized to three domains: the exon, the six nucleotides of the 5' ss region, and the downstream intron. The results indicate that the 5' splice site region and environs are dense with protein contacts in the commitment complex and suggest that some of them make important contributions to formation or stability of the U1 snRNP-pre-mRNA complex. (+info)
Uracil-induced down-regulation of the yeast uracil permease.
(12/3478)
In Saccharomyces cerevisiae the FUR4-encoded uracil permease catalyzes the first step of the pyrimidine salvage pathway. The availability of uracil has a negative regulatory effect upon its own transport. Uracil causes a decrease in the level of uracil permease, partly by decreasing the FUR4 mRNA level in a promoter-independent fashion, probably by increasing its instability. Uracil entry also triggers more rapid degradation of the existing permease by promoting high efficiency of ubiquitination of the permease that signals its internalization. A direct binding of intracellular uracil to the permease is possibly involved in this feedback regulation, as the behavior of the permease is similar in mutant cells unable to convert intracellular uracil into UMP. We used cells impaired in the ubiquitination step to show that the addition of uracil produces rapid inhibition of uracil transport. This may be the first response prior to the removal of the permease from the plasma membrane. Similar down-regulation of uracil uptake, involving several processes, was observed under adverse conditions mainly corresponding to a decrease in the cellular content of ribosomes. These results suggest that uracil of exogenous or catabolic origin down-regulates the cognate permease to prevent buildup of excess intracellular uracil-derived nucleotides. (+info)
LB-AUT7, a novel symbiosis-regulated gene from an ectomycorrhizal fungus, Laccaria bicolor, is functionally related to vesicular transport and autophagocytosis.
(13/3478)
We have identified LB-AUT7, a gene differentially expressed 6 h after ectomycorrhizal interaction between Laccaria bicolor and Pinus resinosa. LB-Aut7p can functionally complement its Saccharomyces cerevisiae homolog, which is involved in the attachment of autophagosomes to microtubules. Our findings suggest the induction of an autophagocytosis-like vesicular transport process during ectomycorrhizal interaction. (+info)
The ski7 antiviral protein is an EF1-alpha homolog that blocks expression of non-Poly(A) mRNA in Saccharomyces cerevisiae.
(14/3478)
We mapped and cloned SKI7, a gene that negatively controls the copy number of L-A and M double-stranded RNA viruses in Saccharomyces cerevisiae. We found that it encodes a nonessential 747-residue protein with similarities to two translation factors, Hbs1p and EF1-alpha. The ski7 mutant was hypersensitive to hygromycin B, a result also suggesting a role in translation. The SKI7 product repressed the expression of nonpolyadenylated [non-poly(A)] mRNAs, whether capped or uncapped, thus explaining why Ski7p inhibits the propagation of the yeast viruses, whose mRNAs lack poly(A). The dependence of the Ski7p effect on 3' RNA structures motivated a study of the expression of capped non-poly(A) luciferase mRNAs containing 3' untranslated regions (3'UTRs) differing in length. In a wild-type strain, increasing the length of the 3'UTR increased luciferase expression due to both increased rates and duration of translation. Overexpression of Ski7p efficiently cured the satellite virus M2 due to a twofold-increased repression of non-poly(A) mRNA expression. Our experiments showed that Ski7p is part of the Ski2p-Ski3p-Ski8p antiviral system because a single ski7 mutation derepresses the expression of non-poly(A) mRNA as much as a quadruple ski2 ski3 ski7 ski8 mutation, and the effect of the overexpression of Ski7p is not obtained unless other SKI genes are functional. ski1/xrn1Delta ski2Delta and ski1/xrn1Delta ski7Delta mutants were viable but temperature sensitive for growth. (+info)
In vitro suicide inhibition of self-splicing of a group I intron from Pneumocystis carinii by an N3' --> P5' phosphoramidate hexanucleotide.
(15/3478)
Binding enhancement by tertiary interactions is a strategy that takes advantage of the higher order folding of functionally important RNAs to bind short nucleic acid-based compounds tightly and more specifically than possible by simple base pairing. For example, tertiary interactions enhance binding of specific hexamers to a group I intron ribozyme from the opportunistic pathogen Pneumocystis carinii by 1,000- to 100,000-fold relative to binding by only base pairing. One such hexamer, d(AnTnGnAnCn)rU, contains an N3' --> P5' phosphoramidate deoxysugar-phosphate backbone (n) that is resistant to chemical and enzymatic decay. Here, it is shown that this hexamer is also a suicide inhibitor of the intron's self-splicing reaction in vitro. The hexamer is ligated in trans to the 3' exon of the precursor, producing dead-end products. At 4 mM Mg2+, the fraction of trans-spliced product is greater than normally spliced product at hexamer concentrations as low as 200 nM. This provides an additional level of specificity for compounds that can exploit the catalytic potential of complexes with RNA targets. (+info)
The cis acting sequences responsible for the differential decay of the unstable MFA2 and stable PGK1 transcripts in yeast include the context of the translational start codon.
(16/3478)
A general pathway of mRNA turnover has been described for yeast in which the 3' poly(A) tail is first deadenylated to an oligo(A) length, leading to decapping and subsequent 5'-3' exonucleolytic decay. The unstable MFA2 mRNA and the stable PGK1 mRNAs both decay through this pathway, albeit at different rates of deadenylation and decapping. To determine the regions of the mRNAs that are responsible for these differences, we examined the decay of chimeric mRNAs derived from the 5' untranslated, coding, and 3' untranslated regions of these two mRNAs. These experiments have led to the identification of the features of these mRNAs that lead to their different stabilities. The MFA2 mRNA is unstable solely because its 3' UTR promotes the rates of deadenylation and decapping; all other features of this mRNA are neutral with respect to mRNA decay rates. The PGK1 mRNA is stable because the sequence context of the PGK1 translation start codon and the coding region function together to stabilize the transcript, whereas the PGK13' UTR is neutral with respect to decay. Importantly, changes in the PGK1 start codon context that destabilized the transcript also reduced its translational efficiency. This observation suggests that the nature of the translation initiation complex modulates the rates of mRNA decapping and decay. (+info)