title: Identification of LIN28B-bound mRNAs reveals features of target recognition and regulation.
abstract: The conserved human LIN28 RNA-binding proteins function in development, maintenance of pluripotency and oncogenesis. We used PAR-CLIP and a newly developed variant of this method, iDo-PAR-CLIP, to identify LIN28B targets as well as sites bound by the individual RNA-binding domains of LIN28B in the human transcriptome at nucleotide resolution. The position of target binding sites reflected the known structural relative orientation of individual LIN28B-binding domains, validating iDo-PAR-CLIP. Our data suggest that LIN28B directly interacts with most expressed mRNAs and members of the let-7 microRNA family. The Lin28-binding motif detected in pre-let-7 was enriched in mRNA sequences bound by LIN28B. Upon LIN28B knockdown, cell proliferation and the cell cycle were strongly impaired. Quantitative shotgun proteomics of LIN28B depleted cells revealed significant reduction of protein synthesis from its RNA targets. Computational analyses provided evidence that the strength of protein synthesis reduction correlated with the location of LIN28B binding sites within target transcripts.
authors.affiliation: Systems Biology of Gene Regulatory Elements; Max-Delbrück-Center for Molecular Medicine; Berlin, Germany.
authors.forename: Robin
authors.initials: R
authors.lastname: Graf
authors.forename: Mathias
authors.initials: M
authors.lastname: Munschauer
authors.forename: Guido
authors.initials: G
authors.lastname: Mastrobuoni
authors.forename: Florian
authors.initials: F
authors.lastname: Mayr
authors.forename: Udo
authors.initials: U
authors.lastname: Heinemann
authors.forename: Stefan
authors.initials: S
authors.lastname: Kempa
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Rajewsky
authors.forename: Markus
authors.initials: M
authors.lastname: Landthaler
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D059372:Nucleotide Motifs; D056510:Position-Specific Scoring Matrices; D011485:Protein Binding; D014176:Protein Biosynthesis; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C516326:LIN28B protein, human; D035683:MicroRNAs; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C487931:mirnlet7 microRNA, human
keywords: CLIP; RNA-binding protein; post-transcriptional regulation; stem cell