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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 59 nucleotide of the miRNA to be unpaired compared to the 59 nucleotide of the miRNA*, nor a preference for the miRNA to be on either the 59 or 39 arm of the miRNA precursor stem–loop. We observed that steady-state pools of miRNAs have fairly homogeneous termini, especially at their 59 end. Nearly all mature miRNA–miRNA* duplexes had two nucleotide 39 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.

ORGANISM(S): Caenorhabditis elegans

PROVIDER: GSE24704 | GEO | 2011/02/01

SECONDARY ACCESSION(S): PRJNA132317

REPOSITORIES: GEO

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

Project description: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 59 nucleotide of the miRNA to be unpaired compared to the 59 nucleotide of the miRNA*, nor a preference for the miRNA to be on either the 59 or 39 arm of the miRNA precursor stem–loop. We observed that steady-state pools of miRNAs have fairly homogeneous termini, especially at their 59 end. Nearly all mature miRNA–miRNA* duplexes had two nucleotide 39 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. Deep sequencing of small RNAs in wild-type (N2) mixed stage C. elegans, roughly 20 million sequencing reads were obtained

2011-02-01 | E-GEOD-24704 | biostudies-arrayexpress

Conservation-independent identification of novel miRNAs

Project description:Motivation: MicroRNAs (miRNAs) are short regulatory RNAs derived from a longer precursor RNA. miRNA biogenesis has been studied in animals and plants, recently elucidating more diverse and complex aspects, such as non-conserved, speciesspecific, and heterogeneous miRNA precursor populations. Small RNA sequencing data can be used to computationally determine genomic loci of miRNA precursors. The challenge is to predict a valid miRNA precursor from inhomogeneous read coverage: while the mature miRNA typically produces hundreds of sequence reads, the remaining part of the precursor is covered very sparsely. Results: We introduce a new conservation-independent method for the identification of miRNA precursors, that allows for speciesspecific heterogeneous precursor populations. The algorithm requires small RNA sequencing data and evaluates precursor secondary structures, with key parameters that can be adjusted based on the specific organism under investigation (within animals, plants, algae). We illustrate the validity of results from our algorithm using sequencing data for the two Volvocine algae Chlamydomonas reinhardtii (Chlamydomonas) and Volvox carteri (Volvox). Both organisms show little cross-species miRNA sequence conservation, and a heterogeneous miRNA precursor population. We validate our list of Chlamydomonas miRNAs with annotated miRNAs, and demonstrate excellent agreement. Furthermore, we are able to identify additional novel miRNA precursors, with structures ranging from simple mammalian-like hairpins to precursor structures indicating the creation of multiple mature/star miRNA duplexes. Novel miRNAs identified in Volvox show no similarity to mature miRNAs in Chlamydomonas. These results confirm the need for conservationindependent miRNA identification methods. Examination of small RNAs of Volvox carteri during different stages of its life cycle

2016-03-02 | E-GEOD-58703 | biostudies-arrayexpress

Conservation-independent identification of novel miRNAs

2016-03-02 | GSE58703 | GEO

An Integrated miRNA Expression Atlas of Arabidopsis thaliana

Project description:MicroRNAs (miRNAs) are 21-24 nucleotide (nt) small non-coding RNAs that regulate a wide variety of biological processes at the posttranscriptional level. MiRNA expression often exhibits spatial and temporal specificity. However, genome-wide miRNA expression patterns in different Arabidopsis organs during plant development have not yet been fully investigated. In this study, we sequenced 59 small RNA libraries generated from different tissue types at different developmental stages of Arabidopsis. We then re-annotated Arabidopsis miRNAs based on the most recent criteria. Global analysis of miRNA expression patterns showed that most miRNAs are ubiquitously expressed in different organs or tissues. But a small set of miRNAs, either previously annotated or newly identified, show highly specific expression patterns. In addition, the expression of some miRNA members belonging to the same family is strictly regulated spatially and temporally. Unexpectedly, we found that quite a few miRNAs are produced from different arms of their hairpin precursors at different developmental stages, suggesting that arm switching could be a general and important mechanism in developmental regulation.

2017-12-05 | GSE79414 | GEO

Conserved miRNA discovery in switchgrass

Project description:Small RNA libraries were constructed, sequenced, and analyzed with our Sequence Homology Pipeline for miRNA discovery (Jeong et al. 2013) to identify 59 unique conserved miRNA sequences from 199 precursors in switchgrass.

2019-01-01 | GSE112228 | GEO

Multiple RNA recognition patterns during microRNA biogenesis in plants

Project description:MicroRNAs (miRNAs) are processed from longer precursors with fold-back structures. While animal MIRNA precursors have homogenous structures, plant precursors comprise a collection of fold-backs with variable size and shape. Here, we design an approach (SPARE) to systematically analyze miRNA processing intermediates and characterize the biogenesis of most of the evolutionary conserved miRNAs present in Arabidopsis thaliana. We found that plant MIRNAs are processed by four mechanisms, depending on the sequential direction of the processing machinery and the number of cuts required to release the miRNA. Classification of the precursors according to their processing mechanism revealed specific structural determinants for each group. We found that the complexity of the miRNA processing pathways occurs in both ancient and evolutionary young sequences, and that members of the same family can be processed in different ways. We observed that different structural determinants compete for the processing machinery and that alternative miRNAs can be generated from a single precursor. The results provide a mechanistic explanation for the structural diversity of MIRNA precursors in plants and new insights towards the understanding of the biogenesis of small RNAs.

2013-06-12 | GSE46429 | GEO

Multiple RNA recognition patterns during microRNA biogenesis in plants

2013-06-12 | E-GEOD-46429 | biostudies-arrayexpress

Construction and characterization of synthetic microRNA cluster for multiplex RNA interference in mammalian cells

Project description:RNA interference (RNAi) technology is widely used in basic and translational research. By mimicking natural primary microRNA (pri-miRNA) cluster, multiple engineered hairpins can be transcribed as a single transcript from the same Pol-II promoter, enabling multiplex RNAi in mammalian cells 1-5. However, constructing synthetic miRNA cluster is still time-consuming, and the processing and function of miRNA cluster are incompletely understood. Here, we identified a miRNA precursor architecture that allowed precise miRNA maturation. We established a hierarchical cloning method for efficient construction of synthetic miRNA cluster harboring up to 18 miRNA precursors. We demonstrated that maturation and function of individual miRNA precursors were independent on their positions in the cluster. We then analyzed the integration efficiency of miRNA clusters with varying number of miRNA precursors by using CRISPR/Cas9 mediated integration, piggyBac transposon system, and lentiviral system. This synthetic miRNA cluster system provides an important tool for multiplex RNAi in mammalian cells. Different kinds of synthetic miRNAs or miRNA clusters, including the same synthetic miRNA precursor embedded in a series of derivative backbones, 18 synthetic miRNA precursors, miRNA precursors with various stem lengths and miRNA clusters carrying different numbers of different pri-miRNA hairpins, were expressed in HEK293 cells. The microRNA maturation pattern of individual miRNA precursors was detected by small RNA sequencing.

2016-09-01 | E-GEOD-71088 | biostudies-arrayexpress

A Genome-wide RNAi screen identifies factors required for distinct stages of C. elegans piRNA biogenesis

Project description:In animals, piRNAs, and their associated Piwi proteins, guard germ cell genomes against mobile genetic elements via an RNAi-like mechanism. In C. elegans, 21U-RNAs comprise the piRNA class and these collaborate with 22G RNAs, via unclear mechanisms, to discriminate self from non-self and selectively and heritably silence the latter. Recent work indicates that 21U-RNAs are post-transcriptional processing products of individual transcription units that produce ~26 nucleotide capped precursors. Yet, nothing is known of how the expression of precursors is controlled or of how primary transcripts give rise to mature small RNAs. We conducted a genome-wide RNAi screen to identify components of the 21U biogenesis machinery. Screening by direct, qPCR-based measurements of mature 21U-RNA levels, we identified 22 genes important for 21U-RNA production, termed TOFUs (Twenty-One-u Fouled Up). We also identified 7 genes that normally repress 21U production. By measuring mature 21U-RNA and precursor levels for the 7 strongest hits from the screen, we have assigned factors to discrete stages of 21U-RNA production. Our work has identified factors separately required for the transcription of 21U precursors, and the processing of these precursors into mature 21U-RNAs, thereby providing an essential resource for studying the biogenesis of this important small RNA class. Small RNA and capped small RNA sequencing from total RNA of C. elegans subjected to different RNAi and different C. elegans mutants

2014-04-01 | E-GEOD-53970 | biostudies-arrayexpress

Understanding the structure/sequence preference of plant miRNA biogenesis machinery

Project description:MicroRNAs (miRNAs) are ~21nt long endogenous non-coding RNAs that are involved in post-transcriptional silencing of target mRNAs. miRNAs are processed from precursors having a stem-loop structure that are recognized and cleaved by Dicer-like 1 (DCL1) with the help of dsRNA binding protein Hyponastic leaves 1 (HYL1) and a zinc finger protein named Serrate (SE). Plant miRNA stem-loops are very diverse in length and structure than animal miRNAs. Hence, mechanism of miRNA biogenesis from their precursors is poorly understood. Our bioinformatic analysis predicted that plant miRNAs have unique sequence signature. An artificial precursor with different predicted sequence signatures was constructed. This construct was expressed transiently in tobacco leaves and small RNAs were sequenced. Our analysis revealed that indeed the predicted sequence signature is prefered by plant miRNA biogenesis machinery.

2017-07-15 | GSE99094 | GEO

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