RNA sequence analysis defines Dicer's role in mouse embryonic stem cells

Calabrese, J. Mauro; Seila, Amy C.; Yeo, G; Sharp, Phillip A.

doi:10.1073/pnas.0709193104

Cited by 301 publications

(295 citation statements)

References 47 publications

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“…Although planarian stem cells are collectively totipotent because they can give rise to both the somatic and germ lineages in the adult, our analyses indicate that planarians harbor only 1 miRNA (miR-92) known to be highly expressed in mammalian embryonic stem cells (30). However, we found no evidence that its 2 family members are up-regulated in neoblasts.…”

Section: Discussioncontrasting

confidence: 68%

High-resolution profiling and discovery of planarian small RNAs

Friedländer

Adamidi

Han

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

129

119

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Freshwater planarian flatworms possess uncanny regenerative capacities mediated by abundant and collectively totipotent adult stem cells. Key functions of these cells during regeneration and tissue homeostasis have been shown to depend on PIWI, a molecule required for Piwi-interacting RNA (piRNA) expression in planarians. Nevertheless, the full complement of piRNAs and microRNAs (miRNAs) in this organism has yet to be defined. Here we report on the large-scale cloning and sequencing of small RNAs from the planarian Schmidtea mediterranea, yielding altogether millions of sequenced, unique small RNAs. We show that piRNAs are in part organized in genomic clusters and that they share characteristic features with mammalian and fly piRNAs. We further identify 61 novel miRNA genes and thus double the number of known planarian miRNAs. Sequencing, as well as quantitative PCR of small RNAs, uncovered 10 miRNAs enriched in planarian stem cells. These miRNAs are downregulated in animals in which stem cells have been abrogated by irradiation, and thus constitute miRNAs likely associated with specific stem-cell functions. Altogether, we present the first comprehensive small RNA analysis in animals belonging to the third animal superphylum, the Lophotrochozoa, and single out a number of miRNAs that may function in regeneration. Several of these miRNAs are deeply conserved in animals.microRNAs ͉ miRNAs ͉ piRNAs ͉ regeneration ͉ stem cells P lanarians have become a molecularly tractable model system in which to study regeneration, tissue homeostasis, and stem-cell biology (1). Planaria are free-living, triploblastic flatworms of the phylum Platyhelminthes, which is presently considered to belong to the superphylum Lophotrochozoa. Model systems for modern molecular and developmental biology have almost exclusively focused on the other 2 superphyla, i.e., the Deuterostomes (which includes vertebrates) and the Ecdysozoa (e.g., Caenorhabditis elegans and Drosophila melanogaster). Unlike these model systems, planarians possess remarkable regeneration abilities. Decapitation, for example, results in the complete regeneration of the head within 7 days after amputation. Such robust restoration of missing body parts is mediated by adult stem cells known as neoblasts (2). Of the thousands of known planarian species, Schmidtea mediterranea is arguably the species of choice for modern molecular biology and high-throughput, genome-wide approaches because it is diploid, it exists in sexual and asexual strains, and its genome has recently been sequenced and annotated (3). The size of its genome is roughly a third of the human genome, and Ϸ80% of the Ϸ20,000 annotated planarian genes have orthologs in humans. Moreover, by morphology alone, neoblasts and their immediate division progeny comprise Ϸ25% of all cells in the adult animal (4). In addition, RNAi screens have identified hundreds of genes specifically linked to planarian regeneration and stem-cell biology (5). Many of these genes are conserved in humans, and thus understanding planarian ...

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Section: Discussioncontrasting

confidence: 68%

High-resolution profiling and discovery of planarian small RNAs

Friedländer

Adamidi

Han

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

129

119

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“…Annotating mature products from small RNA data libraries Small RNA reads were collected from several previously published studies of human (Landgraf et al 2007;Azuma-Mukai et al 2008;Ender et al 2008;Friedlander et al 2008;Morin et al 2008), mouse (Calabrese et al 2007;Babiarz et al 2008;Baek et al 2008;Marson et al 2008;Stark et al 2008;Tam et al 2008;Watanabe et al 2008), dog , and chicken (Glazov et al 2008;Rathjen et al 2009). Small RNA reads were clipped if necessary and mapped to known miRNA precursors (miRBase) using custom python scripts.…”

Section: Methodsmentioning

confidence: 99%

Widespread regulatory activity of vertebrate microRNA* species

Yang

Phillips

Betel

et al. 2010

RNA

315

242

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An obligate intermediate during microRNA (miRNA) biogenesis is an~22-nucleotide RNA duplex, from which the mature miRNA is preferentially incorporated into a silencing complex. Its partner miRNA* species is generally regarded as a passenger RNA, whose regulatory capacity has not been systematically examined in vertebrates. Our bioinformatic analyses demonstrate that a substantial fraction of miRNA* species are stringently conserved over vertebrate evolution, collectively exhibit greatest conservation in their seed regions, and define complementary motifs whose conservation across vertebrate 39-UTR evolution is statistically significant. Functional tests of 22 miRNA expression constructs revealed that a majority could repress both miRNA and miRNA* perfect match reporters, and the ratio of miRNA:miRNA* sensor repression was correlated with the endogenous ratio of miRNA:miRNA* reads. Analysis of microarray data provided transcriptome-wide evidence for the regulation of seedmatched targets for both mature and star strand species of several miRNAs relevant to oncogenesis, including mir-17, mir-34a, and mir-19. Finally, 39-UTR sensor assays and mutagenesis tests confirmed direct repression of five miR-19* targets via star seed sites. Overall, our data demonstrate that miRNA* species have demonstrable impact on vertebrate regulatory networks and should be taken into account in studies of miRNA functions and their contribution to disease states.

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“…Most canonical miRNA genes (61%) were clustered in the genome, falling within 50 kb of another miRNA gene, on the same genomic strand. Even when excluding the four known megaclusters (Calabrese et al 2007), which are on chromosomes 2, 12 (two clusters), and X (with 69, 35, 16, and 18 genes, respectively), a sizable fraction of the remaining genes (153 of 337) were in clusters of two to seven genes. As observed in humans (Baskerville and Bartel 2005), miRNAs from these loci within 50 kb of each other tended to have correlated expression, consistent with their processing from polycistronic pri-miRNA transcripts (Supplemental Fig.…”

Section: General Features Of Mammalian Mirnasmentioning

confidence: 99%

“…In mammals, high-throughput sequencing methods that have contributed to miRNA discovery efforts have included massively parallel signature sequencing (MPSS) (Mineno et al 2006), miRNA serial analysis of gene expression (miRAGE) (Cummins et al 2006), 454 pyrosequencing (Berezikov et al 2006a(Berezikov et al , 2007Calabrese et al 2007), and Illumina sequencing (Babiarz et al 2008;Kuchenbauer et al 2008). …”

mentioning

confidence: 99%

Mammalian microRNAs: experimental evaluation of novel and previously annotated genes

Chiang

Schoenfeld²,

Ruby³

et al. 2010

Genes Dev.

746

756

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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 398 annotated miRNA genes and identified 108 novel miRNA genes. More than 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 murine 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 precursor miRNA (pre-miRNA), consequential 59 heterogeneity, newly identified instances of miRNA editing, and evidence for widespread pre-miRNA uridylation reminiscent of miRNA regulation by Lin28.[Keywords: MicroRNA; miRNA biogenesis; noncoding RNA genes; high-throughput sequencing] Supplemental material is available at http://www.genesdev.org.

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RNA sequence analysis defines Dicer's role in mouse embryonic stem cells

Cited by 301 publications

References 47 publications

High-resolution profiling and discovery of planarian small RNAs

High-resolution profiling and discovery of planarian small RNAs

Widespread regulatory activity of vertebrate microRNA* species

Mammalian microRNAs: experimental evaluation of novel and previously annotated genes

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