title: MicroRNA-mediated target mRNA cleavage and 3'-uridylation in human cells.
abstract: MicroRNAs (miRNAs) play an important role in targeted gene silencing by facilitating posttranscriptional and translational repression. However, the precise mechanism of mammalian miRNA-mediated gene silencing remains to be elucidated. Here, we used a stem-loop array reverse-transcription polymerase chain reaction assay to analyse miRNA-induced mRNA recognition, cleavage, posttranscriptional modification, and degradation. We detected endogenous let-7 miRNA-induced and Argonaute-catalysed endonucleolytic cleavage on target mRNAs at various sites within partially paired miRNA:mRNA sequences. Most of the cleaved mRNA 5'-fragments were 3'-oligouridylated by activities of terminal uridylyl transferases (TUTases) in miRNA-induced silencing complexes and temporarily accumulated in the cytosol for 5'-3' degradation or other molecular fates. Some 3'-5' decayed mRNA fragments could also be captured by the miRNA-induced silencing complex stationed at the specific miRNA:mRNA target site and oligouridylated by other TUTases at its proximity without involving Argonaute-mediated RNA cleavage. Our findings provide new insights into the molecular mechanics of mammalian miRNA-mediated gene silencing by coordinated target mRNA recognition, cleavage, uridylation and degradation.
authors.affiliation: Section of Thoracic Molecular Oncology, Department of Thoracic and Cardiovascular Surgery, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Xu
authors.affiliation: Section of Thoracic Molecular Oncology, Department of Thoracic and Cardiovascular Surgery, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Lin
authors.affiliation: Section of Thoracic Molecular Oncology, Department of Thoracic and Cardiovascular Surgery, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA.
authors.forename: Roza
authors.initials: R
authors.lastname: Zandi
authors.affiliation: Section of Thoracic Molecular Oncology, Department of Thoracic and Cardiovascular Surgery, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA.
authors.forename: Jack A
authors.initials: JA
authors.lastname: Roth
authors.affiliation: Section of Thoracic Molecular Oncology, Department of Thoracic and Cardiovascular Surgery, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA.
authors.forename: Lin
authors.initials: L
authors.lastname: Ji
mesh_terms: D060565:Argonaute Proteins / Q000235:genetics* / Q000378:metabolism; D020029:Base Pairing; D001483:Base Sequence; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D020033:Protein Isoforms; D034622:RNA Interference; D012316:RNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D014542:Uridine Monophosphate / Q000378:metabolism*
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D035683:MicroRNAs; D020033:Protein Isoforms; D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; C487931:mirnlet7 microRNA, human; D049452:Green Fluorescent Proteins; D014542:Uridine Monophosphate; D012316:RNA Nucleotidyltransferases; C576505:TUT7 protein, human