Promiscuous splicing-derived hairpins are dominant substrates of tailing-mediated defense of miRNA biogenesis in mammals
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ABSTRACT:
PROVIDER: PRJNA849575 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR19662942_1.fastq.gz | Fastqsanger.gz | |||
| SRR19662942_2.fastq.gz | Fastqsanger.gz | |||
| SRR19662943_1.fastq.gz | Fastqsanger.gz | |||
| SRR19662943_2.fastq.gz | Fastqsanger.gz | |||
| SRR19662944_1.fastq.gz | Fastqsanger.gz |
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Project description:A canonical pathway generates microRNAs (miRNAs) via stepwise cleavage of hairpin precursors by RNase III enzymes Drosha and Dicer, followed by loading of resultant ~22 nucleotide (nt) RNAs into an Argonaute (Ago) effector complex. In addition, non-canonical substrates can bypass either RNase III enzyme to yield functional small RNAs, including hundreds of intron-derived hairpins that comprise pre-miRNA mimics that bypass Drosha (i.e., mirtrons). Here, we show that mirtron breadth is much larger than currently appreciated, since hundreds of novel splicing-derived hairpins are detected in intermediate (~50-100 nt) but not small (20-30nt) RNA data. Since mirtrons were originally defined on the basis of small RNA duplexes, we term the larger set of loci as structured, splicing-derived RNAs (ssdRNAs). Although individual species generally accumulate modestly, these are broadly detected in Dicer and/or Ago complexes and can mediate repression in directed assays. Nevertheless, their extremely poor conservation suggests they do not generally benefit regulatory networks, and may instead contaminate the canonical miRNA pathway. In aggregate, ssdRNAs/mirtrons comprise the dominant hairpin substrates for 3' tailing by multiple terminal nucleotidyltransferases, notably TUT4/7 and TENT2. We provide evidence that hairpin tailing is inhibitory for ssdRNA/mirtron biogenesis and/or function, and that this pathway has restricted the evolution of canonical pre-miRNAs that resemble splicing products. Overall, the rampant proliferation of evolutionary young mammalian mirtrons, coupled with an attendant molecular defense, comprises a Red Queen's race of intragenomic conflict.
2022-12-25 | GSE206186 | GEO
Project description:Canonical microRNA (miRNA) hairpins are processed by the RNase III enzymes Drosha and Dicer into ∼22 nt RNAs loaded into an Argonaute (Ago) effector. In addition, splicing generates numerous intronic hairpins that bypass Drosha (mirtrons) to yield mature miRNAs. Here, we identify hundreds of previously unannotated, splicing-derived hairpins in intermediate-length (∼50-100 nt) but not small (20-30 nt) RNA data. Since we originally defined mirtrons from small RNA duplexes, we term this larger set as structured splicing-derived RNAs (ssdRNAs). These associate with Dicer and/or Ago complexes, but generally accumulate modestly and are poorly conserved. We propose they contaminate the canonical miRNA pathway, which consequently requires defense against the siege of splicing-derived substrates. Accordingly, ssdRNAs/mirtrons comprise dominant hairpin substrates for 3' tailing by multiple terminal nucleotidyltransferases, notably TUT4/7 and TENT2. Overall, the rampant proliferation of young mammalian mirtrons/ssdRNAs, coupled with an inhibitory molecular defense, comprises a Red Queen's race of intragenomic conflict.
| S-EPMC10508058 | biostudies-literature
Project description:MicroRNA (miRNA) maturation is initiated by DROSHA, a double-stranded RNA (dsRNA)-specific RNase III enzyme. By cleaving primary miRNAs (pri-miRNAs) at specific positions, DROSHA serves as a main determinant of miRNA sequences and a highly selective gate-keeper for the canonical miRNA pathway. However, the sites of DROSHA-mediated processing have not been annotated on a genomic scale, and it remains unclear to what extent DROSHA functions outside the miRNA pathway. Here we establish a protocol termed ‘formaldehyde crosslinking and immunoprecipitation followed by sequencing (fCLIP-seq)’ that allows identification of DROSHA cleavage sites at single nucleotide resolution. fCLIP identifies numerous cleavage sites that do not match the ends of annotated mature miRNAs, particularly at the 3′ termini, suggesting widespread end modifications during miRNA maturation such as trimming and tailing. fCLIP also finds many pri-miRNAs undergoing alternative processing, yielding multiple miRNA isoforms. Moreover, we discover dozens of novel substrates that are bound and cleaved by DROSHA. These substrates are processed less efficiently than canonical pri-miRNAs, and produce only minute amounts of small RNAs. Depletion of DROSHA results in an increase of the hairpin-containing transcripts. Thus, the hairpins may serve mainly as cis-elements for DROSHA-mediated gene regulation rather than as miRNA genes. fCLIP-seq not only accurately maps the cleavage sites of DROSHA and suggests noncanonical functions of DROSHA, but also could be a general tool for investigating interactions between dsRNA binding proteins and structured RNAs.
2017-04-23 | GSE93650 | GEO
Project description:MicroRNA (miRNA) maturation is initiated by DROSHA, a double-stranded RNA (dsRNA)-specific RNase III enzyme. By cleaving primary miRNAs (pri-miRNAs) at specific positions, DROSHA serves as a main determinant of miRNA sequences and a highly selective gate-keeper for the canonical miRNA pathway. However, the sites of DROSHA-mediated processing have not been annotated on a genomic scale, and it remains unclear to what extent DROSHA functions outside the miRNA pathway. Here we establish a protocol termed ‘formaldehyde crosslinking and immunoprecipitation followed by sequencing (fCLIP-seq)’ that allows identification of DROSHA cleavage sites at single nucleotide resolution. fCLIP identifies numerous cleavage sites that do not match the ends of annotated mature miRNAs, particularly at the 3′ termini, suggesting widespread end modifications during miRNA maturation such as trimming and tailing. fCLIP also finds many pri-miRNAs undergoing alternative processing, yielding multiple miRNA isoforms. Moreover, we discover dozens of novel substrates that are bound and cleaved by DROSHA. These substrates are processed less efficiently than canonical pri-miRNAs, and produce only minute amounts of small RNAs. Depletion of DROSHA results in an increase of the hairpin-containing transcripts. Thus, the hairpins may serve mainly as cis-elements for DROSHA-mediated gene regulation rather than as miRNA genes. fCLIP-seq not only accurately maps the cleavage sites of DROSHA and suggests noncanonical functions of DROSHA, but also could be a general tool for investigating interactions between dsRNA binding proteins and structured RNAs.
2017-04-23 | GSE93651 | GEO
Project description:To analyse the impact of URT1-mediated uridylation on miRNA and siRNA tailing, we deep-sequenced small RNA libraries for WT and urt1 duplicate samples at the same developmental stage that was analyzed by TAIL-seq, i.e., two-week-old seedlings. Examination of miRNA and siRNA tailing in WT and urt1 samples.
2016-03-11 | E-GEOD-72456 | biostudies-arrayexpress
Project description:Uridylation of diverse RNA species represents an emerging theme in post-transcriptional gene regulation. In the microRNA pathway, such modifications regulate small RNA biogenesis and stability in plants, worms and mammals. Here, we report the first uridylyltransferase that acts on small RNAs in Drosophila, which we refer to as Tailor. Tailor is the source for the majority of 3´ terminal uridine-modifications in small RNAs and predominantly targets precursor-miRNAs. Uridylation modulates the characteristic two-nucleotide 3´ overhangs of miRNA hairpins, which regulates pre-miRNA processing by Dicer-1. Furthermore, Tailor preferentially uridylates mirtron hairpins, thereby impeding the production of non-canonical microRNAs. Mirtron-selectivity is explained by unique primary sequence specificity of Tailor, selecting RNA substrates ending with a 3´ guanosine, a feature not previously observed for terminal uridylyltransferases. In contrast to mirtrons, conserved Drosophila pre-miRNAs are significantly depleted in 3´ guanosine, thereby escaping regulatory uridylation.Our data support the hypothesis that evolutionary adaptation to pre-miRNA uridylation shapes the nucleotide composition of pre-miRNA 3´ ends and may serve as a barrier for the de novo creation of miRNAs in Drosophila.
2015-10-19 | GSE66213 | GEO
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