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Improved annotation of C. elegans microRNAs by deep sequencing reveals structures associated with processing by Drosha and Dicer.

ABSTRACT: MicroRNAs (miRNAs) are small regulatory RNAs that are essential in all studied metazoans. Research has focused on the prediction and identification of novel miRNAs, while little has been done to validate, annotate, and characterize identified miRNAs. Using Illumina sequencing, ?20 million small RNA sequences were obtained from Caenorhabditis elegans. Of the 175 miRNAs listed on the miRBase database, 106 were validated as deriving from a stem-loop precursor with hallmark characteristics of miRNAs. This result suggests that not all sequences identified as miRNAs belong in this category of small RNAs. Our large data set of validated miRNAs facilitated the determination of general sequence and structural characteristics of miRNAs and miRNA precursors. In contrast to previous observations, we did not observe a preference for the 5' nucleotide of the miRNA to be unpaired compared to the 5' nucleotide of the miRNA*, nor a preference for the miRNA to be on either the 5' or 3' arm of the miRNA precursor stem-loop. We observed that steady-state pools of miRNAs have fairly homogeneous termini, especially at their 5' end. Nearly all mature miRNA-miRNA* duplexes had two nucleotide 3' overhangs, and there was a preference for a uracil in the first and ninth position of the mature miRNA. Finally, we observed that specific nucleotides and structural distortions were overrepresented at certain positions adjacent to Drosha and Dicer cleavage sites. Our study offers a comprehensive data set of C. elegans miRNAs and their precursors that significantly decreases the uncertainty associated with the identity of these molecules in existing databases.

SUBMITTER: Warf MB

PROVIDER: S-EPMC3062169 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Improved annotation of C. elegans microRNAs by deep sequencing reveals structures associated with processing by Drosha and Dicer.

Warf M Bryan MB Johnson W Evan WE Bass Brenda L BL

RNA (New York, N.Y.) 20110209 4

MicroRNAs (miRNAs) are small regulatory RNAs that are essential in all studied metazoans. Research has focused on the prediction and identification of novel miRNAs, while little has been done to validate, annotate, and characterize identified miRNAs. Using Illumina sequencing, ∼20 million small RNA sequences were obtained from Caenorhabditis elegans. Of the 175 miRNAs listed on the miRBase database, 106 were validated as deriving from a stem-loop precursor with hallmark characteristics of miRNAs ...[more]

PMID: 21307183

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Project description:BackgroundmicroRNAs (miRNAs) in fish have not been as extensively studied as those in mammals. The fish species Takifugu rubripes is an intensively studied model organism whose genome has been sequenced. The T. rubripes genome is approximately eight times smaller than the human genome, but has a similar repertoire of protein-coding genes. Therefore, it is useful for identifying non-coding genes, including miRNA genes. To identify miRNA expression patterns in different organs of T. rubripes and give fundamental information to aid understanding of miRNA populations in this species, we extracted small RNAs from tissues and performed deep sequencing analysis to profile T. rubripes miRNAs. These data will be of assistance in functional studies of miRNAs in T. rubripes.ResultsAfter analyzing a total of 139 million reads, we found miRNA species in nine tissues (fast and slow muscles, heart, eye, brain, intestine, liver, ovaries, and testes). We identified 1420 known miRNAs, many of which were strongly expressed in certain tissues with expression patterns similar to those described for other animals in previous reports. Most miRNAs were expressed in tissues other than the ovaries or testes. However, some miRNA families were highly abundant in the gonads, but expressed only at low levels in somatic tissue, suggesting specific function in germ cells. The most abundant isomiRs (miRNA variants) of many miRNAs had identical sequences in the 5' region. However, isomiRs of some miRNAs, including fru-miR-462-5p, varied in the 5' region in some tissues, suggesting that they may target different mRNA transcripts. Longer small RNAs (26-31 nt), which were abundant in the gonads, may be putative piRNAs because of their length and their origin from repetitive elements. Additionally, our data include possible novel classes of small RNAs.ConclusionsWe elucidated miRNA expression patterns in various organs of T. rubripes. Most miRNA sequences are conserved in vertebrates, indicating that the basic functions of vertebrate miRNAs share a common evolution. Some miRNA species exhibit different distributions of isomiRs between tissues, suggesting that they have a broad range of functions.

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Improved annotation of C. elegans microRNAs by deep sequencing reveals structures associated with processing by Drosha and Dicer.

Publications

Improved annotation of C. elegans microRNAs by deep sequencing reveals structures associated with processing by Drosha and Dicer.

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