title: Splicing remodels the let-7 primary microRNA to facilitate Drosha processing in Caenorhabditis elegans.
abstract: MicroRNAs (miRNAs) are a class of small noncoding RNAs that use partial base-pairing to recognize and regulate the expression of messenger RNAs (mRNAs). Mature miRNAs arise from longer primary transcripts (pri-miRNAs) that are processed to a shorter hairpin precursor miRNA (pre-miRNA) by the Microprocessor complex. In Caenorhabditis elegans the primary let-7 (pri-let-7) transcript undergoes trans-splicing, where pri-let-7 is cleaved at a 3' splice site and the splice-leader-1 (SL1) sequence is appended at the 5' end. Here we investigate the role of this splicing event in the biogenesis of let-7 miRNA. We hypothesized that splicing changes the secondary structure of the pri-let-7 transcript, creating a more favorable substrate for recognition by the Microprocessor. Supporting this idea, we detected conspicuous structural differences between unspliced and SL1-spliced pri-let-7 transcripts using in vitro ribonuclease (RNase) assays. Through the generation of transgenic worm strains, we found that the RNA secondary structure produced by splicing, as opposed to the act of splicing itself, optimizes processing of pri-let-7 by the Microprocessor in vivo. We also observed that the endogenous spliced, but not the unspliced, pri-let-7 transcripts bind to the Microprocessor and accumulate upon its depletion. We conclude that splicing is a key step in generating pri-let-7 transcripts with a structure that enables downstream processing events to produce appropriate levels of mature let-7.
authors.affiliation: Division of Biology, University of California, San Diego, La Jolla, California 92093-0349, USA.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Mondol
authors.affiliation: Division of Biology, University of California, San Diego, La Jolla, California 92093-0349, USA.
authors.forename: Byoung Chan
authors.initials: BC
authors.lastname: Ahn
authors.affiliation: Division of Biology, University of California, San Diego, La Jolla, California 92093-0349, USA.
authors.forename: Amy E
authors.initials: AE
authors.lastname: Pasquinelli
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000235:genetics; D035683:MicroRNAs / Q000737:chemistry* / Q000235:genetics*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D043244:Ribonuclease III / Q000378:metabolism; D020040:Trans-Splicing
chemical_list: D029742:Caenorhabditis elegans Proteins; D035683:MicroRNAs; C474488:let-7 microRNA, C elegans; D043244:Ribonuclease III; C493336:drsh-1 protein, C elegans
keywords: C. elegans; Drosha; let-7; miRNA; pri-miRNA; trans-splicing