Mammalian microRNAs: Experimental evaluation of novel and previously annotated genes
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus Homo sapiens
PROVIDER: GSE20384 | GEO | 2010/04/26
SECONDARY ACCESSION(S): PRJNA125509
REPOSITORIES: GEO
Similar Datasets
Project description:MicroRNAs (miRNAs) are small regulatory RNAs that derive from distinctive hairpin transcripts. To learn more about the miRNAs of mammals, we sequenced 60 million small RNAs from mouse brain, ovary, testes, embryonic stem cells, three embryonic stages, and whole newborns. Analysis of these sequences confirmed 387 annotated miRNA genes and identified 110 novel miRNA genes. Over 150 previously annotated miRNAs and hundreds of candidates failed to yield sequenced RNAs with miRNA-like features. Ectopically expressing these previously proposed miRNA hairpins also did not yield small RNAs, whereas ectopically expressing the confirmed and newly identified hairpins usually did yield small RNAs with the classical miRNA features, including dependence on the Drosha endonuclease for processing. These experiments, which suggest that previous estimates of conserved mammalian miRNAs were inflated, provide a substantially revised list of confidently identified mammalian miRNAs from which to infer the general features of mammalian miRNAs. Our analyses also revealed new aspects of miRNA biogenesis and modification, including tissue-specific strand preferences, sequential Dicer cleavage of a metazoan pre-miRNA, newly identified instances of miRNA editing, and evidence for widespread Lin28-like miRNA regulation. For miRNA discovery, small RNAs were sequenced from mouse brain, ovary, testes, three embryonic stages, and whole newborns; for ectopic over-expression assays, pre-miRNA hairpins and the surrounding regions were transfected into HEK293T, and the small RNA were sequenced from the transfected cells 39-48 hours after transfection.
2010-04-26 | E-GEOD-20384 | biostudies-arrayexpress
Project description:Drosha is a type III RNAse, which plays a critical role in miRNA biogenesis. Drosha and its double-stranded RNA-binding partner protein Pasha/DGCR8 likely recognize and cleave miRNA precursor RNAs or pri-miRNA hairpins co-transcriptionally. To identify RNAs processed by Drosha, we used tiling microarrays to examine transcripts after depletion of drosha mRNA with dsRNA in Drosophila Schneider S2 cells. This strategy identified 137 Drosha-regulated RNAs, including 11 putative pri-miRNAs comprising 15 annotated miRNAs. Most of the identified pri-miRNAs seem extremely large, >10 kilobases as revealed by both the Drosha knock down strategy and by RNA PolII chromatin IP followed by Drosophila tiling microarrays. Surprisingly, more than a hundred additional RNAs not annotated as miRNAs are under Drosha control and are likely to be direct targets of Drosha action. This is because many of them encode annotated genes, and unlike bona fide pri-miRNAs, they are not affected by depletion of the miRNA processing factor, dicer-1. Moreover, application of the evofold analysis software indicates that at least 25 of the Drosha-regulated RNAs contain evolutionarily conserved hairpins similar to those recognized by the Drosha-Pasha/DGCR8 complex in pri-miRNAs. One of these hairpins is located in the 5′ UTR of both pasha and mammalian DGCR8. These observations suggest that a negative feedback loop acting on pasha mRNA may regulate the miRNA-biogenesis pathway: i.e., excess Drosha cleaves pasha/DGCR8 primary transcripts and leads to a reduction in pasha/DGCR8 mRNA levels and Pasha/DGCR8 synthesis. Keywords: time course, ChIP-chip
2008-12-25 | GSE14215 | GEO
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
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: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.
2016-03-02 | GSE58703 | GEO
Project description:Canonical microRNAs (miRNAs) require two processing steps: the first by the Microprocessor, a complex of DGCR8 and Drosha, and the second by Dicer. dgcr8delta/delta mouse embryonic stem cells (mESCs) have less severe phenotypes than dicer1delta/delta mESCs, suggesting a physiological role for Microprocessor-independent, Dicer-dependent small RNAs. To identify these small RNAs with unusual biogenesis, we performed high-throughput sequencing from wild type, dgcr8delta/delta, and dicer1delta/delta mESCs. Several of the DGCR8-independent, Dicer-dependent RNAs were non-canonical miRNAs. These derived from mirtrons and a newly identified subclass of miRNA precursors, which appears to be the endogenous counterpart of short hairpin RNAs (shRNAs). Our analyses also revealed endogenous siRNAs resulting from Dicer cleavage of long hairpins, the vast majority of which originated from one genomic locus with tandem, inverted short interspersed nuclear elements (SINEs). Our results extend the known diversity of mammalian small-RNA generating pathways and show that mammalian siRNAs exist in tissues outside of oocytes.
2008-09-10 | GSE12521 | GEO
Project description:The Microprocessor plays an essential role in canonical miRNA biogenesis by facilitating cleavage of stem-loop structures in primary transcripts to yield pre-miRNAs. Although miRNA biogenesis has been extensively studied through biochemical and molecular genetic approaches, it has yet to be addressed to what extent the current miRNA biogenesis models hold true in intact cells. To address the issues of in vivo recognition and cleavage by the Microprocessor, we investigate RNAs that are associated with DGCR8 and Drosha by using immunoprecipitation coupled with next-generation sequencing. Here, we present global protein-RNA interactions with unprecedented sensitivity and specificity. Our data indicate that precursors of canonical miRNAs and miRNA-like hairpins are the major substrates of the Microprocessor. As a result of specific enrichment of nascent cleavage products, we are able to pinpoint the Microprocessor-mediated cleavage sites per se at single-nucleotide resolution. Unexpectedly, a 2-nt 3’ overhang invariably exists at the ends of cleaved bases instead of nascent pre-miRNAs. Besides canonical miRNA precursors, we find that two novel miRNA-like structures embedded in mRNAs are cleaved to yield pre-miRNA-like hairpins, uncoupled from miRNA maturation. Our data provide a framework for in vivo Microprocessor-mediated cleavage and a foundation for experimental and computational studies on miRNA biogenesis in living cells.
2014-10-02 | GSE61979 | GEO
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
Project description:Canonical microRNAs (miRNAs) require two processing steps: the first by the Microprocessor, a complex of DGCR8 and Drosha, and the second by Dicer. dgcr8delta/delta mouse embryonic stem cells (mESCs) have less severe phenotypes than dicer1delta/delta mESCs, suggesting a physiological role for Microprocessor-independent, Dicer-dependent small RNAs. To identify these small RNAs with unusual biogenesis, we performed high-throughput sequencing from wild type, dgcr8delta/delta, and dicer1delta/delta mESCs. Several of the DGCR8-independent, Dicer-dependent RNAs were non-canonical miRNAs. These derived from mirtrons and a newly identified subclass of miRNA precursors, which appears to be the endogenous counterpart of short hairpin RNAs (shRNAs). Our analyses also revealed endogenous siRNAs resulting from Dicer cleavage of long hairpins, the vast majority of which originated from one genomic locus with tandem, inverted short interspersed nuclear elements (SINEs). Our results extend the known diversity of mammalian small-RNA generating pathways and show that mammalian siRNAs exist in tissues outside of oocytes. Small RNAs were sequenced from wt, dgcr8(-), and dicer(-) mouse ES cells and the frequencies of small RNA types compared between the three. This record includes Illumina-platform-generated datasets from all three samples and 454-platform-generated datasets from wt and dgcr8(-) samples. [raw data files are unavailable]
2008-09-09 | E-GEOD-12521 | biostudies-arrayexpress
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