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Phosphate-independent small RNA sequencing of WT and mir-249 mutant C. elegans

ABSTRACT: MicroRNAs (miRNA), discovered in C. elegans, are short non-coding RNAs that bind and regulate the expression of target mRNAs in animals and plants. C. elegans miRNAs bind to partially complementary sequences in the 3' UTR of the target mRNA, which results in translational repression through mRNA destabilization. The high-throughput sequencing of RNA cleavage fragments was performed to directly detect cleaved miRNA targets in C. elegans. Based on this analysis, miR-249 was identified as a potential miRNA that regulates a ZK637.6 pseudogene, paralogous to asna-1 (ZK637.5), by a cleavage mechanism with extensive, evolutionary conserved complementarity. Additionally, we validated miR-249-directed cleavage of the ZK637.6 by a gene-specific 5’ rapid amplification of cDNA ends and observed notable difference in expression of ZK637.6 in wild-type versus mir-249 mutant C. elegans. Furthermore, phosphate-independent small-RNA sequencing analysis revealed that miR-249 target genes, including ZK637.6 pseudogene, showed 22G-RNA productions dependent on miR-249 targeting. These findings may lead to a better understanding of the biological roles of miRNAs for pseudogenes in C. elegans.

ORGANISM(S): Caenorhabditis elegans

PROVIDER: GSE41900 | GEO | 2013/08/08

SECONDARY ACCESSION(S): PRJNA178470

REPOSITORIES: GEO

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Phosphate-independent small RNA sequencing of WT and mir-249 mutant C. elegans

Project description:MicroRNAs (miRNA), discovered in C. elegans, are short non-coding RNAs that bind and regulate the expression of target mRNAs in animals and plants. C. elegans miRNAs bind to partially complementary sequences in the 3' UTR of the target mRNA, which results in translational repression through mRNA destabilization. The high-throughput sequencing of RNA cleavage fragments was performed to directly detect cleaved miRNA targets in C. elegans. Based on this analysis, miR-249 was identified as a potential miRNA that regulates a ZK637.6 pseudogene, paralogous to asna-1 (ZK637.5), by a cleavage mechanism with extensive, evolutionary conserved complementarity. Additionally, we validated miR-249-directed cleavage of the ZK637.6 by a gene-specific 5M-bM-^@M-^Y rapid amplification of cDNA ends and observed notable difference in expression of ZK637.6 in wild-type versus mir-249 mutant C. elegans. Furthermore, phosphate-independent small-RNA sequencing analysis revealed that miR-249 target genes, including ZK637.6 pseudogene, showed 22G-RNA productions dependent on miR-249 targeting. These findings may lead to a better understanding of the biological roles of miRNAs for pseudogenes in C. elegans. Total small RNAs from wild-type and mir-249 mutant in adult stage worms were subjected to small RNA sequencing using an Illumina platform with Tobacco Acid Pyrophosphatase (TAP) treatment, allowing detection of secondary siRNAs carrying 5M-bM-^@M-^Y-tri-phosphate.

2013-08-08 | E-GEOD-41900 | biostudies-arrayexpress

Analysis of 22G siRNA triggered siRNA amplification and 3' 2'-O-methylated small RNAs in Caenorhabditis elegans

Project description:Small RNAs, including piRNAs, miRNAs and endogenous siRNAs, bind Argonaute proteins to form RNA-silencing complexes that target coding genes, transposons and aberrant RNAs. To assess the requirements for endogenous siRNA formation and activity in C. elegans, we developed a GFP-based sensor for the endogenous siRNA 22G siR-1, one of a set of abundant siRNAs processed from a precursor RNA mapping to the X chromosome, the X-cluster. Silencing of the sensor is also dependent on the partially complementary, unlinked 26G siR-O7 siRNA. We show that 26G siR-O7 acts in trans to initiate 22G siRNA formation from the X-cluster. The presence of several mispairs between 26G siR-O7 and the X-cluster mRNA, as well as mutagenesis of the siRNA sensor, indicates that siRNA target recognition is permissive to a degree of mispairing. From a candidate reverse genetic screen, we identified several factors required for 22G siR-1 activity, including the Argonaute ergo-1 and the 3' methyltransferase henn-1. Quantitative RT-PCR of small RNAs in a henn-1 mutant and deep sequencing of methylated small RNAs indicate that siRNAs and piRNAs that associate with PIWI clade Argonautes are methylated by HENN-1, while siRNAs and miRNAs that associate with non-PIWI clade Argonautes are not. Thus, PIWI-class Argonaute proteins are specifically adapted to associate with methylated small RNAs in C. elegans. This SuperSeries is composed of the following subset Series: GSE34320: Analysis of 22G siRNA triggered siRNA amplification in Caenorhabditis elegans GSE34321: Analysis of 3' 2'-O-methylated small RNAs in Caenorhabditis elegans Refer to individual Series

2012-02-03 | E-GEOD-35550 | biostudies-arrayexpress

MicroRNA-seq of cell lines expressing pri-miR-9 paralog constructs

Project description:MicroRNA (miRNA) biogenesis begins with Drosha cleavage, the fidelity of which is critical for downstream processing and mature miRNA target specificity. To understand how pri-miRNA sequence and structure influence Drosha cleavage, we studied the processing of three pri-miR-9 paralogs (pri-miR-9-1, pri-miR-9-2 and pri-miR-9-3), which encode the same miRNA but differ in the surrounding scaffold. We show that pri-miR-9-1 has a unique Drosha cleavage profile due to its kinked tertiary structure. Cleavage of pri-miR-9-1, but not pri-miR-9-2 or pri-miR-9-3, generates an alternative-miR-9 with a shifted seed sequence that expands the scope of its target RNAs. Here, we report the the miRNA-seq data of HEK293T and HeLa cell lines transfected with different pri-miR-9 constructs.

2018-12-10 | GSE108893 | GEO

Mir-146a wild-type 3ʹ sequence identity is dispensable for proper innate immune function in vivo

Project description:A long-prevailing model has held that the “seed” region (nucleotides 2-8) of a microRNA is typically sufficient to mediate target recognition and repression. However, numerous recent studies, both within the context of defining miRNA/target pairs by direct physical association and by directly assessing this model in vivo in C. elegans have brought this model into question. To test the importance of miRNA 3' pairing in vivo, in a mammalian system, we engineered a mutant murine mir-146a allele in which the 5' half of the mature microRNA retains the sequence of the wild-type mir-146a but the 3ʹ half has been altered to be anti-complementary to the wild-type miR-146a sequence. Mice homozygous or hemizygous for this mutant allele are phenotypically indistinguishable from wild-type controls and do not recapitulate any of the immunopathology previously described for mir-146a-null mice. Our results strongly support the conclusion that 3ʹ pairing is dispensable in the context of the function of a key mammalian microRNA.

2019-02-09 | PXD011413 | Pride

Cooperative target mRNA destabilization and translation inhibition by miR-58 microRNA family in C. elegans [RNA-Seq]

Project description:In animals, microRNAs frequently form families with related sequences. The functional relevance of miRNA families and the relative contribution of family members to target repression have remained, however, largely unexplored. Here, we used the C. elegans miR-58 miRNA family, comprised primarily of four highly abundant members: miR-58.1, miR-80, miR-81 and miR-82, as a model to investigate the redundancy of miRNA family members and their impact on target expression in an in vivo setting. RNA was extracted from different miR-58 family mutants (mir-58.1, mir-80; mir-58.1 and mir-80; mir-58.1; mir-81-82) and wild-type Bristol C. elegans strain at late L4 stage and submitted to transcriptome sequencing with Illumina HiSeq2000. The goal was to compare miR-58 target RNA expression and system-wide perturbations across various samples.

2015-06-05 | E-GEOD-60420 | biostudies-arrayexpress

MicroRNA-seq of cell lines expressing pri-miR-9 paralog constructs II

Project description:MicroRNA (miRNA) biogenesis begins with Drosha cleavage, the fidelity of which is critical for downstream processing and mature miRNA target specificity. To understand how pri-miRNA sequence and structure influence Drosha cleavage, we studied the processing of pri-miR-9-1 sequence variants, which encode the same mature miRNA but differ in the surrounding scaffold. We show that, in addition to previously known features, the overall structural flexibility of pri-miRNA impacts Drosha cleavage fidelity. Internal loops and nearby G·U wobble pairs on the pri-miRNA stem induce the use of non-canonical cleavage sites by Drosha, resulting in 5’ isomiR production. Here, we report the the miRNA-seq data of HEK293T cell lines transfected with different pri-miR-9 constructs.

2022-01-27 | GSE172446 | GEO

Cooperative target mRNA destabilization and translation inhibition by miR-58 microRNA family in C. elegans [RNA-Seq]

2015-06-05 | GSE60420 | GEO

Cooperative target mRNA destabilization and translation inhibition by miR-58 microRNA family in C. elegans [miRNA-Seq]

2015-06-05 | GSE60419 | GEO

Cooperative target mRNA destabilization and translation inhibition by miR-58 microRNA family in C. elegans [miRNA-Seq]

Project description:In animals, microRNAs frequently form families with related sequences. The functional relevance of miRNA families and the relative contribution of family members to target repression have remained, however, largely unexplored. Here, we used the C. elegans miR-58 miRNA family, comprised primarily of four highly abundant members: miR-58.1, miR-80, miR-81 and miR-82, as a model to investigate the redundancy of miRNA family members and their impact on target expression in an in vivo setting. RNA was extracted from different miR-58 family mutants (mir-58.1, mir-80; mir-58.1 and mir-80; mir-58.1; mir-81-82) and wild-type Bristol C. elegans strain at late L4 stage and submitted to small RNA profiling with Illumina HiSeq2000. The goal was to see whether miR-58 family members can compensate each other's expression.

2015-06-05 | E-GEOD-60419 | biostudies-arrayexpress

Analysis of 22G siRNA triggered siRNA amplification and 3' 2'-O-methylated small RNAs in Caenorhabditis elegans

2012-02-03 | GSE35550 | GEO

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