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Functional importance of individual rRNA 2'-O-ribose methylations revealed by high-resolution phenotyping.

ABSTRACT: Ribosomal RNAs contain numerous modifications at specific nucleotides. Despite their evolutionary conservation, the functional role of individual 2'-O-ribose methylations in rRNA is not known. A distinct family of small nucleolar RNAs, box C/D snoRNAs, guides the methylating complex to specific rRNA sites. Using a high-resolution phenotyping approach, we characterized 20 box C/D snoRNA gene deletions for altered growth dynamics under a wide array of environmental perturbations, encompassing intraribosomal antibiotics, inhibitors of specific cellular features, as well as general stressors. Ribosome-specific antibiotics generated phenotypes indicating different and long-ranging structural effects of rRNA methylations on the ribosome. For all studied box C/D snoRNA mutants we uncovered phenotypes to extraribosomal growth inhibitors, most frequently reflected in alteration in growth lag (adaptation time). A number of strains were highly pleiotropic and displayed a great number of sensitive phenotypes, e.g., deletion mutants of snR70 and snR71, which both have clear human homologues, and deletion mutants of snR65 and snR68. Our data indicate that individual rRNA ribose methylations can play either distinct or general roles in the workings of the ribosome.

SUBMITTER: Esguerra J

PROVIDER: S-EPMC2271359 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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Functional importance of individual rRNA 2'-O-ribose methylations revealed by high-resolution phenotyping.

Esguerra Jonathan J Warringer Jonas J Blomberg Anders A

RNA (New York, N.Y.) 20080206 4

Ribosomal RNAs contain numerous modifications at specific nucleotides. Despite their evolutionary conservation, the functional role of individual 2'-O-ribose methylations in rRNA is not known. A distinct family of small nucleolar RNAs, box C/D snoRNAs, guides the methylating complex to specific rRNA sites. Using a high-resolution phenotyping approach, we characterized 20 box C/D snoRNA gene deletions for altered growth dynamics under a wide array of environmental perturbations, encompassing intr ...[more]

PMID: 18256246

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Project description:The major information-carrying macromolecules in the cell, DNA, RNA, and protein, carry an additional layer of information on top of their sequence in the form of modifications of residues. The modifications provide additional functional groups and impact the structure and function of the molecules. Cellular RNA molecules contain more than 100 different modifications and are found in all domains of life and in all major classes of RNA in eukaryotic organisms. Together these modifications constitute the epitranscriptome of which two-thirds are methylations with 2’-O methylation of the ribose moiety of the nucleotide as the most abundant. Many aspects of ribose methylation are underexplored because the existing methods for their detection are laborious and can only address a few modification sites at a time. Here, we introduce RiboMeth-Seq, a high-throughput sequencing based method and applies it to yeast ribosomal RNA. We detect all of the known as well as one new methylation site and provide evidence for hypomethylation at specific residues. Furthermore we demonstrate that many methylation events are interdependent and outline the timing of modifications during ribosome biogenesis. Our results demonstrate a novel and efficient approach to understanding of the role of modifications in ribosomal RNA folding and ribosome function. Recent evidence point to changes in ribose methylation patterns in cancer ribosomes and we anticipate that RiboMeth-Seq can be applied here as well as to other diseases in which ribosomes are affected, including the heritable ribosomopathies. Yeast RNA was degraded at denaturing conditions into small fragments, long enough to be mapped by sequence alignment to the reference genome (20-40 nucleotides). The fragments were ligated to RNA oligos using a tRNA ligase that was mutated to remove its kinase activity, reverse transcribed and the cDNA used as input for Ion semiconductor sequencing. The first and last nucleotides of the inserts were recorded using the full sequence for mapping and the read-ends plotted against the sequence. 2'-O-Methylated RNA positions are protected from cleavage, and read ends from such positions are therefore underrepresented compared to the surrounding positions.

Project description:Single-base resolution DNA methylomes have been accomplished for both Arabidopsis and human cells which have high genome methylation levels by Illumina ultra-high-throughput bisulfite sequencing technology (MethylC-Seq). Here by combining MethylC-Seq and biological replicate strategies we generated single-base resolution methylome for the silkworm which has low genome methylation levels like other insects. Our conservative estimation showed that methylcytosines (mCs) accout for about 0.11% of genomic cytosines, exclusively in CG context. The CG methylation is significantly enriched in gene bodies and positively correlated with gene expression levels, suggesting its positive role in gene transcription in silkworms. However, the well-documented functions of methylation on promoters and rDNAs in plants and mammals do not seem to have effects in insects. Methylated genes are enriched in functions involved in cellular metabolism and biosynthesis. Small RNA (smRNA) loci are also significantly enriched in gene bodies, and moreover, the smRNA loci and the predicted target sites of microRNA have high level of CG methylation, indicating functional involvement of smRNAs in the genic methylation This first methylome for silkworms provides a foundation for further studies on the epigenetic gene regulation of silkworms’ or even insects’ gene methylation. Each silk gland of 5th instar larvae of two individuals (called Biological Replicate 1 and 2, respectively) of the silkworm (Bombyx mori) strain Dazao was ground into powder in liquid nitrogen. Half of the powder from each silk gland was used to extract total DNAs using DNeasy Blood & Tissue Kit (Qiagen) and another half was used to extract total RNAs using RNeasy Mini Kit (Qiagen). We sequenced bisulfite-treated total DNA extracted from the silk glands of the two individuals, using Illumina Ultra-High-Throughput Sequencing, generating the Single-Base Resolution Methylomes. To reveal functional consequences of gene body methylation, we generated expression profiles for the two individuals’ silk glands using Digital Gene Expression tag profiling (DGE) technology, which combines classic SAGE (Serial Analysis of Gene Expression) and Illumina ultra-high-throughput sequencing technology.

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Functional importance of individual rRNA 2'-O-ribose methylations revealed by high-resolution phenotyping.

Publications

Functional importance of individual rRNA 2'-O-ribose methylations revealed by high-resolution phenotyping.

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