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Yi-Tao Yu

Publications and source records attributed to Yi-Tao Yu.

9 recordsLinked to original sources
PubMed2006

Incorporation of 5-fluorouracil into U2 snRNA blocks pseudouridylation and pre-mRNA splicing in vivo.

5-fluorouracil (5FU) is an effective anti-cancer drug, yet its mechanism of action remains unclear. Here, we examine the effect of 5FU on pre-mRNA splicing in vivo. Using RT-PCR, we show that the splicing of a number of pre-mRNAs is inhibited in HeLa cells that have been exposed to a low dose of 5FU. It appears that this inhibitory effect is not due to its incorporation into pre-mRNA, because partially or fully 5FU-substituted pre-mRNA, when injected into Xenopus oocytes, is spliced just as well as is the unsubstituted pre-mRNA. Detailed analyses of 5FU-treated cells indicate that 5FU is incorporated into U2 snRNA at important naturally occurring pseudouridylation sites. Remarkably, 5FU incorporation effectively blocks the formation of important pseudouridines in U2 snRNA, as only a trace of pseudouridine is detected when cells are exposed to a low dose of 5FU for 5 days. Injection of the hypopseudouridylated HeLa U2 snRNA into U2-depleted Xenopus oocytes fails to reconstitute pre-mRNA splicing, whereas control U2 isolated from untreated or uracil-treated HeLa cells completely reconstitutes the splicing. Our results demonstrate for the first time that 5FU incorporates into a spliceosomal snRNA at natural pseudouridylation sites in vivo, thereby inhibiting snRNA pseudouridylation and splicing. This mechanism may contribute substantially to 5FU-mediated cell death.

Animals↗
PubMed2006

Pre-mRNA splicing in the nuclei of Xenopus oocytes.

Xenopus oocytes have been utilized in a number of laboratories as an experimental system to study a variety of biological processes. Here, we describe its application to functional studies of spliceosomal small nuclear RNAs (snRNAs) in pre-messenger RNA (pre-mRNA) splicing, a process that occurs extremely efficiently in Xenopus oocytes. A DNA oligonucleotide complementary to an snRNA of interest is injected into the oocyte cytoplasm. The oligonucleotide subsequently diffuses into the nucleus and hybridizes to the target snRNA, thereby triggering snRNA degradation via endogenous RNase H activity. By the time the endogenous snRNA is depleted, the DNA oligonucleotide itself is degraded by endogenous deoxyribonuclease (DNase) activity. In principle, this procedure enables one to quantitatively deplete any snRNA of choice. Subsequently, a rescuing snRNA that is constructed in vitro may be injected into the snRNA-depleted oocytes to restore the splicing function. After reconstitution, a radiolabeled splicing substrate is injected into the nuclei of the oocytes. These oocyte nuclei are then manually isolated and used to prepare both nuclear RNA for splicing assays and nuclear extract for spliceosome assembly assays. The ability of an injected rescuing snRNA to reconstitute splicing can therefore be tested. Because all types of rescuing snRNAs (e.g., mutant snRNAs, snRNAs with or without modified nucleotides) can be constructed readily, the results obtained from this procedure provide valuable information on the function of a particular snRNA of interest in pre-mRNA splicing.

Animals↗
PubMed2005

Pseudouridylation of yeast U2 snRNA is catalyzed by either an RNA-guided or RNA-independent mechanism.

Yeast U2 small nuclear RNA (snRNA) contains three pseudouridines (Psi35, Psi42, and Psi44). Pus7p and Pus1p catalyze the formation of Psi35 and Psi44, respectively, but the mechanism of Psi42 formation remains unclear. Using a U2 substrate containing a single (32)P radiolabel at position 42, we screened a GST-ORF library for pseudouridylase activity. Surprisingly, we found a Psi42-specific pseudouridylase activity that coincided with Nhp2p, a protein component of a Box H/ACA sno/scaRNP (small nucleolar/Cajal body-specific ribonucleoprotein). When isolated by tandem affinity purification (TAP), the other protein components of the H/ACA sno/scaRNP also copurified with the pseudouridylase activity. Micrococcal nuclease-treated TAP preparations were devoid of pseudouridylase activity; however, activity was restored upon addition of RNAs from TAP preparations. Pseudouridylation reconstitution using RNAs from a Box H/ACA RNA library identified snR81, a snoRNA known to guide rRNA pseudouridylation, as the Psi42-specific guide RNA. Using the snR81-deletion strain, Nhp2p- or Cbf5p-conditional depletion strain, and a cbf5 mutation strain, we further demonstrated that the pseudouridylase activity is dependent on snR81 snoRNP in vivo. Our data indicate that snRNA pseudouridylation can be catalyzed by both RNA-dependent and RNA-independent mechanisms.

Catalysis↗
PubMed2004

Psi35 in the branch site recognition region of U2 small nuclear RNA is important for pre-mRNA splicing in Saccharomyces cerevisiae.

Pseudouridine 35 (psi35) in the branch site recognition region of yeast U2 small nuclear RNA is absolutely conserved in all eukaryotes examined. Pus7p catalyzes pseudouridylation at position 35 in Saccharomyces cerevisiae U2. The pus7 deletion strain, although viable in rich medium, is growth-disadvantaged under certain conditions. To clarify the function of U2 psi35 in yeast, we used this pus7 deletion strain to screen a collection of mutant U2 small nuclear RNAs, each containing a point mutation near the branch site recognition sequence, for a synthetic growth defect phenotype. The screen identified two U2 mutants, one containing a U40 --> G40 substitution (U40G) and another having a U40 deletion (U40Delta). Yeast strains carrying either of these U2 mutations grew as well as the wild-type strain in the selection medium, but they exhibited a temperature-sensitive growth defect phenotype when coupled with the pus7 deletion (pus7Delta). A subsequent temperature shift assay and a conditional pus7 depletion (via GAL promoter shutoff) in the U2-U40 mutant genetic background caused pre-mRNA accumulation, suggesting that psi35 is required for pre-mRNA splicing under certain conditions.

Mutation↗
PubMed2004

Detection and quantitation of RNA base modifications.

Using a new combination of previously published techniques, we developed a method for quantitating modified nucleotides in RNAs. First, an RNA is cleaved with RNase H at the 5' side of a nucleotide of interest. Next, 32P is substituted for the phosphate at the 5' end of this nucleotide. Finally, after nuclease P1 digestion, the released radiolabeled nucleotide is analyzed by thin layer chromatography and quantitated by PhosphorImager. Using this method, we showed that the analysis of a pseudouridine at a specific site within an in vitro synthesized U2 RNA is indeed quantitative. We also applied this technique to cellular U2 RNA isolated from mouse liver, and showed that position U34 is approximately 90% pseudouridylated. This method, combined with previously described reverse transcription-based methods, constitutes a powerful tool for detecting and quantifying modified nucleotides in RNAs. With minor modifications, this method can serve as an effective assay to study RNA modifying enzymes.

Animals↗
PubMed2003

Pseudouridylation (Psi) of U2 snRNA in S. cerevisiae is catalyzed by an RNA-independent mechanism.

Pseudouridylation of snRNAs in vertebrates is guided by small nucleolar/Cajal body-specific RNAs (sno/scaRNAs). We developed an in vitro system using cell extracts and single site-radiolabeled U2 snRNAs to study pseudouridylation in Saccharomyces cerevisiae. Micrococcal nuclease-treated cell extracts are fully competent to catalyze U2 pseudouridylation, suggesting an RNA-independent process. A pseudouridylase activity for Psi(35) within yeast U2 is identified via a screen of an S.cerevisiae GST-ORF protein library. This activity is associated with YOR243c ORF, which has not previously been assigned function. When the GST-YOR243c protein is expressed in Escherichia coli, pseudouridylation activity is comparable to that expressed in S.cerevisiae, demonstrating that this protein (designated Pus7) alone can catalyze Psi(35) formation in U2. Both in vitro and in vivo analyses using wild-type and pus7-Delta strains show that Pus7 is indispensable for Psi(35) formation in U2. Using site-specific radiolabeled U2 and U2 fragments, we show that Pus7 activity is specific for Psi(35) and that the U2 stem- loop II region is essential for the pseudouridylation reaction. A BLAST search revealed Pus7 homologs in various organisms.

Amino Acid Sequence↗
PubMed2003

The Saccharomyces cerevisiae U2 snRNA:pseudouridine-synthase Pus7p is a novel multisite-multisubstrate RNA:Psi-synthase also acting on tRNAs.

The Saccharomyces cerevisiae Pus7 protein was recently characterized as a novel RNA:pseudouridine (Psi)-synthase acting at position 35 in U2 snRNA. However, U2 snRNA was the only potential substrate tested for this enzyme. In this work, we demonstrated that although Pus7p is responsible for the formation of only one of the six Psi residues present in yeast UsnRNAs, it catalyzes U to Psi conversion at position 13 in cytoplasmic tRNAs and at position 35 in pre-tRNA(Tyr). Sites of RNA modification by Pus7p were identified by analysis of the in vivo RNA modification defects resulting from the absence of active Pus7p production and by in vitro tests using extracts from WT and genetically modified yeast cells. For demonstration of the direct implication of Pus7p in RNA modification, the activity of the WT and mutated Pus7p recombinant proteins was tested on in vitro produced tRNA and pre-tRNA transcripts. Mutation of an aspartic acid residue (D256) that is conserved in all Pus7 homologs abolishes the enzymatic activity both in vivo and in vitro. This suggests the direct involvement of D256 in catalysis. Target sites of Pus7p in RNAs share a common sequence Pu(G/C)UNPsiAPu (Pu = purine, N = any nucleotide), which is expected to be important for substrate recognition. Modification of tRNAs by Pus7p explains the presence of Pus7p homologs in archaea and some bacteria species, which do not have U2 snRNA, and in vertebrates, where Psi34 (equivalent to Psi35 in yeast) formation in U2 snRNA is an H/ACA snoRNA guided process. Our results increase the number of known RNA modification enzymes acting on different types of cellular RNAs.

Base Sequence↗
PubMed2002

An H/ACA guide RNA directs U2 pseudouridylation at two different sites in the branchpoint recognition region in Xenopus oocytes.

U2 is the most extensively modified of all spliceosomal snRNAs. We previously showed that at least some of the internally modified nucleotides in U2 snRNA are required for snRNP biogenesis and pre-mRNA splicing. Recent work from several laboratories suggests that nuclear guide RNAs facilitate U2 snRNA internal modification, including pseudouridylation and 2'-O-methylation. Here, we present a novel approach to identifying guide RNAs for U2 pseudouridylation. Several Xenopus oocyte nuclear RNAs were affinity selected with U2 snRNA substituted with 5-fluorouridine, a pseudouridylation inhibitor that sequesters pseudouridylases. One of these RNAs was sequenced and found to be a novel RNA of 134 nt. This small RNA contains an H/ACA motif and folds into a typical H/ACA RNA structure, and its authenticity as an H/ACA RNA was confirmed by immunoprecipitation analysis. The RNA contains two guide sequences for pseudouridylation (psi) of U2 snRNA at positions 34 and 44 in the branch-site recognition region, and we demonstrate that this RNA indeed guides the formation of psi34 and psi44 in U2 using a Xenopus oocyte reconstitution system. Therefore, this novel RNA was designated pugU2-34/44, for pseudouridylation guide for U2 snRNA U34 and U44. Intranuclear localization analyses indicate that pugU2-34/44 resides within the nucleoplasm rather than nucleoli or Cajal bodies where other guide RNAs have been localized. Our results clarify the mechanism of U2 snRNA pseudouridylation in Xenopus oocytes, and have interesting implications with regard to the intranuclear localization of U2 snRNA pseudouridylation.

Animals↗