Institute of Biochemistry; University of Giessen; Giessen, Germany.
Kharkevich Institute for Information Transmission Problems; Russian Academy of Sciences; Moscow, Russia; Department of Bioengineering and Bioinformatics; Lomonosov Moscow State University; Moscow, Russia.
RNA Biol. 2014;11(2):146-55. doi: 10.4161/rna.27991. Epub 2014 Feb 7.
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.
异质核核糖核蛋白 L (hnRNP L) 是一种多功能 RNA 结合蛋白，参与许多不同的过程，如转录、翻译和 RNA 稳定性的调节。我们之前已经将 hnRNP L 鉴定为可变剪接的全局调节剂，与 CA 重复和 CA 丰富的 RNA 元件结合。有趣的是，hnRNP L 可以激活和抑制可变外显子的剪接，但 hnRNP L 介导的剪接调节的精确机制仍不清楚。为了在全基因组水平上分析 hnRNP L 的活性，我们进行了单个核苷酸分辨率的交联免疫沉淀结合深度测序 (iCLIP-Seq)。iCLIP 交联位点的序列分析显示 C/A 基序显著富集，这与早期通过 SELEX 方法获得的体外结合共识完全一致，表明体内 hnRNP L 结合靶标主要由蛋白质的 RNA 结合活性决定。hnRNP L 结合的全基因组图谱显示，该蛋白优先结合内含子和 3'UTR。此外，hnRNP L 的位置依赖性剪接调节也得到了证明：当结合在可变外显子上游的内含子时，该蛋白抑制剪接，而当结合在下游内含子时，该蛋白激活剪接。这些发现为长期存在的 hnRNP L 介导的剪接调节差异问题提供了线索。最后，关于 3'UTR 结合，hnRNP L 结合优先与预测的 microRNA 靶位点重叠，表明 hnRNP L 与 microRNA 结合之间存在全局竞争。hnRNP L 对预测靶 3'UTR 的一部分进行了翻译调控的验证。
