title: Crosslinking-immunoprecipitation (iCLIP) analysis reveals global regulatory roles of hnRNP L.
abstract: Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is a multifunctional RNA-binding protein that is involved in many different processes, such as regulation of transcription, translation, and RNA stability. We have previously characterized hnRNP L as a global regulator of alternative splicing, binding to CA-repeat, and CA-rich RNA elements. Interestingly, hnRNP L can both activate and repress splicing of alternative exons, but the precise mechanism of hnRNP L-mediated splicing regulation remained unclear. To analyze activities of hnRNP L on a genome-wide level, we performed individual-nucleotide resolution crosslinking-immunoprecipitation in combination with deep-sequencing (iCLIP-Seq). Sequence analysis of the iCLIP crosslink sites showed significant enrichment of C/A motifs, which perfectly agrees with the in vitro binding consensus obtained earlier by a SELEX approach, indicating that in vivo hnRNP L binding targets are mainly determined by the RNA-binding activity of the protein. Genome-wide mapping of hnRNP L binding revealed that the protein preferably binds to introns and 3' UTR. Additionally, position-dependent splicing regulation by hnRNP L was demonstrated: The protein represses splicing when bound to intronic regions upstream of alternative exons, and in contrast, activates splicing when bound to the downstream intron. These findings shed light on the longstanding question of differential hnRNP L-mediated splicing regulation. Finally, regarding 3' UTR binding, hnRNP L binding preferentially overlaps with predicted microRNA target sites, indicating global competition between hnRNP L and microRNA binding. Translational regulation by hnRNP L was validated for a subset of predicted target 3'UTRs.
authors: Institute of Biochemistry; University of Giessen; Giessen, Germany.
authors: Oliver
authors: O
authors: Rossbach
authors: Institute of Biochemistry; University of Giessen; Giessen, Germany.
authors: Lee-Hsueh
authors: LH
authors: Hung
authors: Kharkevich Institute for Information Transmission Problems; Russian Academy of Sciences; Moscow, Russia; Department of Bioengineering and Bioinformatics; Lomonosov Moscow State University; Moscow, Russia.
authors: Ekaterina
authors: E
authors: Khrameeva
authors: Institute of Biochemistry; University of Giessen; Giessen, Germany.
authors: Silke
authors: S
authors: Schreiner
authors: Institute of Molecular Biology (IMB); Mainz, Germany; Institute of Neurology; University College London; London, United Kingdom.
authors: Julian
authors: J
authors: König
authors: Faculty of Computer and Information Science; University of Ljubljana; Ljubljana, Slovenia.
authors: Tomaž
authors: T
authors: Curk
authors: Faculty of Computer and Information Science; University of Ljubljana; Ljubljana, Slovenia.
authors: Blaž
authors: B
authors: Zupan
authors: Institute of Neurology; University College London; London, United Kingdom.
authors: Jernej
authors: J
authors: Ule
authors: Kharkevich Institute for Information Transmission Problems; Russian Academy of Sciences; Moscow, Russia; Department of Bioengineering and Bioinformatics; Lomonosov Moscow State University; Moscow, Russia.
authors: Mikhail S
authors: MS
authors: Gelfand
authors: Institute of Biochemistry; University of Giessen; Giessen, Germany.
authors: Albrecht
authors: A
authors: Bindereif
mesh_terms: D020413:3' Untranslated Regions; D017398:Alternative Splicing; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D053263:Gene Regulatory Networks; D015894:Genome, Human; D006367:HeLa Cells; D034664:Heterogeneous-Nuclear Ribonucleoprotein L / Q000235:genetics / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D047468:Immunoprecipitation; D007438:Introns; D035683:MicroRNAs / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*
chemical_list: D020413:3' Untranslated Regions; D034664:Heterogeneous-Nuclear Ribonucleoprotein L; D035683:MicroRNAs; D012333:RNA, Messenger
keywords: CLIP; hnRNP L; microRNA; splicing regulation