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HITS-CLIP yields genome-wide insights into brain alternative RNA processing

聚腺苷酸 生物 RNA剪接 RNA结合蛋白 选择性拼接 计算生物学 核糖核酸 遗传学 基因组 非翻译区 基因 信使核糖核酸
作者
Donny D. Licatalosi,Aldo Mele,John J. Fak,Jernej Ule,Melis Kayikci,Sung Wook,Tyson A. Clark,Anthony Schweitzer,John E. Blume,Xuning Wang,Jennifer C. Darnell,Robert B. Darnell
出处
期刊:Nature [Springer Nature]
卷期号:456 (7221): 464-469 被引量:1319
标识
DOI:10.1038/nature07488
摘要

Protein–RNA interactions have critical roles in all aspects of gene expression. However, applying biochemical methods to understand such interactions in living tissues has been challenging. Here we develop a genome-wide means of mapping protein–RNA binding sites in vivo, by high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). HITS-CLIP analysis of the neuron-specific splicing factor Nova revealed extremely reproducible RNA-binding maps in multiple mouse brains. These maps provide genome-wide in vivo biochemical footprints confirming the previous prediction that the position of Nova binding determines the outcome of alternative splicing; moreover, they are sufficiently powerful to predict Nova action de novo. HITS-CLIP revealed a large number of Nova–RNA interactions in 3′ untranslated regions, leading to the discovery that Nova regulates alternative polyadenylation in the brain. HITS-CLIP, therefore, provides a robust, unbiased means to identify functional protein–RNA interactions in vivo. When the human genome was decoded, the lower than expected number of genes prompted renewed interest in alternative splicing — a mechanism by which more than one protein is made from a single gene. Licatalosi et al. have developed an unbiased, genome-wide method to characterize RNA–protein binding interactions in living tissue, and demonstrate its potential by applying it to the mammalian brain. They characterize the binding sites of the neuronal alternative splicing regulator, Nova, and make the unexpected discovery that it may have an additional function in regulating alternative polyadenylation. In a separate study, Wang et al. used deep sequencing of mRNAs to study alternative splicing in various human tissues and cancers. By mapping sequence reads to splice junctions, they show that alternative splicing is essentially universal in human multi-exon genes. They also show that alternative splicing is mechanistically linked to mRNA polyadenylation. Recent studies have indicated that a cell's proteome is significantly larger than the number of protein-coding genes due to extensive alternative splicing. This study describes an unbiased, genome-wide method to characterize RNA-protein binding interactions in vivo. The binding sites of the neuron-specific splicing factor Nova are characterized with the unexpected result that Nova may have an additional function in regulating alternative polyadenylation as well.
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