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Title: Characterization and functional analysis of a novel double-guide C/D box snoRNA in the fission yeast

Abstract

Ribose methylation of eukaryotic rRNA is directed by box C/D small nucleolar RNAs (snoRNAs), which pinpoint the nucleotide to be methylated in specific position within the rRNA sequence. Here, we report the identification of a novel double-guide C/D box snoRNA termed snR88 that directs methylation of two previously undetermined sites in 25S rRNA from the fission yeast. Knockout of the predicted TATA box of the snR88 gene resulted in the complete blocking of its expression, showing that snR88 is an independently transcribed gene and dispensable for yeast viability. The depletion of snR88 abolished 25S rRNA methylation at U2304 and U2497 simultaneously. Interestingly, an unusual pause of reverse transcription at U2495 was observed, which implies an unknown structure of 25S rRNA related to ribose methylation at U2497 in the fission yeast.

Authors:
 [1];  [1];  [2]
  1. Key Laboratory of Gene Engineering of the Ministry of Education, Biotechnology Research Center, Zhongshan University, Guangzhou 510275 (China)
  2. Key Laboratory of Gene Engineering of the Ministry of Education, Biotechnology Research Center, Zhongshan University, Guangzhou 510275 (China). E-mail: lsszh@zsu.edu.cn
Publication Date:
OSTI Identifier:
20979821
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 354; Journal Issue: 1; Other Information: DOI: 10.1016/j.bbrc.2006.12.207; PII: S0006-291X(07)00009-5; Copyright (c) 2007 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; FUNCTIONAL ANALYSIS; GENES; KNOCK-OUT REACTIONS; METHYLATION; NUCLEOTIDES; RIBOSE; RNA; TRANSCRIPTION; YEASTS

Citation Formats

Bi Yanzhen, Qu Lianghu, and Zhou Hui. Characterization and functional analysis of a novel double-guide C/D box snoRNA in the fission yeast. United States: N. p., 2007. Web.
Bi Yanzhen, Qu Lianghu, & Zhou Hui. Characterization and functional analysis of a novel double-guide C/D box snoRNA in the fission yeast. United States.
Bi Yanzhen, Qu Lianghu, and Zhou Hui. Fri . "Characterization and functional analysis of a novel double-guide C/D box snoRNA in the fission yeast". United States. doi:.
@article{osti_20979821,
title = {Characterization and functional analysis of a novel double-guide C/D box snoRNA in the fission yeast},
author = {Bi Yanzhen and Qu Lianghu and Zhou Hui},
abstractNote = {Ribose methylation of eukaryotic rRNA is directed by box C/D small nucleolar RNAs (snoRNAs), which pinpoint the nucleotide to be methylated in specific position within the rRNA sequence. Here, we report the identification of a novel double-guide C/D box snoRNA termed snR88 that directs methylation of two previously undetermined sites in 25S rRNA from the fission yeast. Knockout of the predicted TATA box of the snR88 gene resulted in the complete blocking of its expression, showing that snR88 is an independently transcribed gene and dispensable for yeast viability. The depletion of snR88 abolished 25S rRNA methylation at U2304 and U2497 simultaneously. Interestingly, an unusual pause of reverse transcription at U2495 was observed, which implies an unknown structure of 25S rRNA related to ribose methylation at U2497 in the fission yeast.},
doi = {},
journal = {Biochemical and Biophysical Research Communications},
number = 1,
volume = 354,
place = {United States},
year = {Fri Mar 02 00:00:00 EST 2007},
month = {Fri Mar 02 00:00:00 EST 2007}
}
  • Box C/D ribonucleoproteins (RNP) guide the 2'-O-methylation of targeted nucleotides in archaeal and eukaryotic rRNAs. The archaeal L7Ae and eukaryotic 15.5kD box C/D RNP core protein homologues initiate RNP assembly by recognizing kink-turn (K-turn) motifs. The crystal structure of the 15.5kD core protein from the primitive eukaryote Giardia lamblia is described here to a resolution of 1.8 {angstrom}. The Giardia 15.5kD protein exhibits the typical {alpha}-{beta}-{alpha} sandwich fold exhibited by both archaeal L7Ae and eukaryotic 15.5kD proteins. Characteristic of eukaryotic homologues, the Giardia 15.5kD protein binds the K-turn motif but not the variant K-loop motif. The highly conserved residues ofmore » loop 9, critical for RNA binding, also exhibit conformations similar to those of the human 15.5kD protein when bound to the K-turn motif. However, comparative sequence analysis indicated a distinct evolutionary position between Archaea and Eukarya. Indeed, assessment of the Giardia 15.5kD protein in denaturing experiments demonstrated an intermediate stability in protein structure when compared with that of the eukaryotic mouse 15.5kD and archaeal Methanocaldococcus jannaschii L7Ae proteins. Most notable was the ability of the Giardia 15.5kD protein to assemble in vitro a catalytically active chimeric box C/D RNP utilizing the archaeal M. jannaschii Nop56/58 and fibrillarin core proteins. In contrast, a catalytically competent chimeric RNP could not be assembled using the mouse 15.5kD protein. Collectively, these analyses suggest that the G. lamblia 15.5kD protein occupies a unique position in the evolution of this box C/D RNP core protein retaining structural and functional features characteristic of both archaeal L7Ae and higher eukaryotic 15.5kD homologues.« less
  • A predominantly pyrimidine-rich sequence (purine in the template strand, 32 of 37 bases) is located between a functional TATA element and the corresponding transcription start site region of the Saccharomyces cerevisiae iso-1-cytochrome c (CYC1) gene. By using linker deletions and gene fusion techniques, the functional characteristics of this pyrimidine sequence were examined. Results indicate that the function of this element is to limit the accumulation of full-length mRNAs with 5' ends which map upstream of the pyrimidine-rich sequence. Data suggest that the 5'-noncoding region of the CTC1 gene possesses signals for mRNA 3'-end processing.
  • The largest subunit of RNA polymerase II contains a repetitive C-terminal domain (CTD) consisting of tandem repeats of the consensus sequence Tyr{sub 1}Ser{sub 2}Pro{sub 3}Thr{sub 4}Ser{sub 5}Pro{sub 6}Ser{sub 7}. Substitution of nonphosphorylatable amino acids at positions two or five of the Saccharomyces cerevisiae CTD is lethal. We developed a selection system for isolating suppressors of this lethal phenotype and cloned a gene, SCA1 (suppressor of CTD alanine), which complements recessive suppressors of lethal multiple-substitution mutations. A partial deletion of SCA1 (sca1{Delta}::hisG) suppresses alanine or glutamate substitutions at position two of the consensus CTD sequence, and a lethal CTD truncation mutation,more » but SCA1 deletion does not suppress alanine or glutamate substitutions at position five. SCA1 is identical to SRB9, a suppressor of a cold-sensitive CTD truncation mutation. Strains carrying dominant SRB mutations have the same suppression properties as a sca1{Delta}::hisG strain. These results reveal a functional difference between positions two and five of the consensus CTD heptapeptide repeat. The ability of SCA1 and SRB mutant alleles to suppress CTD truncation mutations suggest that substitutions at position two, but not at position five, cause a defect in RNA polymerase II function similar to that introduced by CTD truncation. 46 refs., 6 figs., 3 tabs.« less
  • The bis(imido) complex of tungsten, W([double bond]NAr)[sub 2]Cl[sub 2](THF)[sub 2] (Ar = 2,6-C[sub 6]H[sub 3][sup i]Pr[sub 2]) reacts with Li[C[sub 5]Me[sub 5]] to provide burgundy ([eta][sup 5]-C[sub 5]Me[sub 5])W([double bond]NAr)[sub 2]Cl (1) in high yield. Compound 1 may be functionalized using MeLi, PhLi, or LiBEt[sub 3]H[sub *] (H[sup *] = H or D) to provide ([eta][sup 5]-C[sub 5]Me[sub 5])W([double bond]NAr)[sub 2]Me (2), ([eta][sup 5]-C[sub 5]Me[sub 5])W([double bond]NAr)[sub 2]Ph (3), ([eta][sup 5]-C[sub 5]Me[sub 5])W([double bond]NAr)[sub 2]H (4), and ([eta][sup 5]-C[sub 5]Me[sub 5])W([double bond]NAr)[sub 2]D (4-d) in moderate to high yields. ([eta][sup 5]-C[sub 5]Me[sub 5])W([double bond]NAr)[sub 2]Cl (1) crystallizes in the monoclinicmore » P2[sub 1]/n (no. 14) [Angstrom][sup 3] with Z = 4 and [rho][sub calcd] = 1.46 g cm[sup [minus]3]. The molecular structure of 1 is characterized by nearly identical imido ligands with W-N-C[sub ipso] angles averaging 169.3 (4)[degrees] and W-N bonds averaging 1.783 (4) [Angstrom]. Upon reaction of ([eta][sup 5]-C[sub 5]Me[sub 5])W([double bond]NAr)[sub 2]Cl (1) with LiNHAr, the tucked-in complex ([eta][sup 5],[eta][sup 1]-C[sub 5]Me[sub 4]CH[sub 2])W([double bond]NAr)[sub 2] (5) is formed in nearly quantitative yield. Experiments are presented which indicate the most likely mechanism of formation of 5 involves the intermediacy of the substituted complex, ([eta][sup 5]-C[sub 5]Me[sub 5])W([double bond]NAr)[sub 2](NHAr). 48 refs., 1 fig., 3 tabs.« less
  • No abstract prepared.