Devin H Parry1, Jinling Xu, Gary Ruvkun. 1. Department of Genetics, Harvard Medical School, Richard B. Simches Research Building, 185 Cambridge Street, CPZN-7250, Boston, Massachusetts 02114-2790, USA.
Abstract
BACKGROUND: miRNAs are an abundant class of small, endogenous regulatory RNAs. Although it is now appreciated that miRNAs are involved in a broad range of biological processes, relatively little is known about the actual mechanism by which miRNAs downregulate target gene expression. An exploration of which protein cofactors are necessary for a miRNA to downregulate a target gene should reveal more fully the molecular mechanisms by which miRNAs are processed, trafficked, and regulate their target genes. RESULTS: A weak allele of the C. elegans miRNA gene let-7 was used as a sensitized genetic background for a whole-genome RNAi screen to detect miRNA pathway genes, and 213 candidate miRNA pathway genes were identified. About 2/3 of the 61 candidates with the strongest phenotype were validated through genetic tests examining the dependence of the let-7 phenotype on target genes known to function in the let-7 pathway. Biochemical tests for let-7 miRNA production place the function of nearly all of these new miRNA pathway genes downstream of let-7 expression and processing. By monitoring the downregulation of the protein product of the lin-14 mRNA, which is the target of the lin-4 miRNA, we have identified 19 general miRNA pathway genes. CONCLUSIONS: The 213 candidate miRNA pathway genes identified could act at steps that produce and traffic miRNAs or in downstream steps that detect miRNA::mRNA duplexes to regulate mRNA translation. The 19 validated general miRNA pathway genes are good candidates for genes that may define protein cofactors for sorting or targeting miRNA::mRNA duplexes, or for recognizing the miRNA base-paired to the target mRNA to downregulate translation.
BACKGROUND: miRNAs are an abundant class of small, endogenous regulatory RNAs. Although it is now appreciated that miRNAs are involved in a broad range of biological processes, relatively little is known about the actual mechanism by which miRNAs downregulate target gene expression. An exploration of which protein cofactors are necessary for a miRNA to downregulate a target gene should reveal more fully the molecular mechanisms by which miRNAs are processed, trafficked, and regulate their target genes. RESULTS: A weak allele of the C. elegans miRNA gene let-7 was used as a sensitized genetic background for a whole-genome RNAi screen to detect miRNA pathway genes, and 213 candidate miRNA pathway genes were identified. About 2/3 of the 61 candidates with the strongest phenotype were validated through genetic tests examining the dependence of the let-7 phenotype on target genes known to function in the let-7 pathway. Biochemical tests for let-7 miRNA production place the function of nearly all of these new miRNA pathway genes downstream of let-7 expression and processing. By monitoring the downregulation of the protein product of the lin-14 mRNA, which is the target of the lin-4 miRNA, we have identified 19 general miRNA pathway genes. CONCLUSIONS: The 213 candidate miRNA pathway genes identified could act at steps that produce and traffic miRNAs or in downstream steps that detect miRNA::mRNA duplexes to regulate mRNA translation. The 19 validated general miRNA pathway genes are good candidates for genes that may define protein cofactors for sorting or targeting miRNA::mRNA duplexes, or for recognizing the miRNA base-paired to the target mRNA to downregulate translation.
Authors: Blair R Szymczyna; John Bowman; Susan McCracken; Antonio Pineda-Lucena; Ying Lu; Brian Cox; Mark Lambermon; Brenton R Graveley; Cheryl H Arrowsmith; Benjamin J Blencowe Journal: Genes Dev Date: 2003-02-15 Impact factor: 11.361
Authors: B J Reinhart; F J Slack; M Basson; A E Pasquinelli; J C Bettinger; A E Rougvie; H R Horvitz; G Ruvkun Journal: Nature Date: 2000-02-24 Impact factor: 49.962
Authors: A Grishok; A E Pasquinelli; D Conte; N Li; S Parrish; I Ha; D L Baillie; A Fire; G Ruvkun; C C Mello Journal: Cell Date: 2001-07-13 Impact factor: 41.582
Authors: A E Pasquinelli; B J Reinhart; F Slack; M Q Martindale; M I Kuroda; B Maller; D C Hayward; E E Ball; B Degnan; P Müller; J Spring; A Srinivasan; M Fishman; J Finnerty; J Corbo; M Levine; P Leahy; E Davidson; G Ruvkun Journal: Nature Date: 2000-11-02 Impact factor: 49.962
Authors: Ravi S Kamath; Andrew G Fraser; Yan Dong; Gino Poulin; Richard Durbin; Monica Gotta; Alexander Kanapin; Nathalie Le Bot; Sergio Moreno; Marc Sohrmann; David P Welchman; Peder Zipperlen; Julie Ahringer Journal: Nature Date: 2003-01-16 Impact factor: 49.962