RNA binding proteins (RBPs) interact with RNA through a wide variety of primary sequence motifs and RNA structural elements to control all processing steps. RNA can act as a carrier of information from the nucleus to the cytoplasm in the processing of protein-coding genes, as a regulatory molecule that can control gene expression, and even as an extracellular signal to coordinate trans-generational inheritance. Further, our quantification of both mRNA and other element association will enable further research to identify novel roles of RBPs in regulating RNA processing.
This work reveals novel insights into RNA biology by integrated analysis of eCLIP profiling of 150 RBPs with distinct functions. Finally, we show that eCLIP peak co-occurrences across RBPs enable the discovery of novel co-interacting RBPs. Analysis of spliceosomal RBPs indicates that eCLIP resolves AQR association after intronic lariat formation, enabling identification of branch points with single-nucleotide resolution, and provides genome-wide validation for a branch point-based scanning model for 3′ splice site recognition. Quantification of enrichment for repetitive and abundant multicopy elements reveals 70% of RBPs have enrichment for non-mRNA element classes, enables identification of novel ribosomal RNA processing factors and sites, and suggests that association with retrotransposable elements reflects multiple RBP mechanisms of action. Our analysis of 223 eCLIP datasets reveals a range of binding modalities, including highly resolved positioning around splicing signals and mRNA untranslated regions that associate with distinct RBP functions.
The development of enhanced CLIP (eCLIP) enabled the mapping of targets for 150 RBPs in K562 and HepG2, creating a unique resource of RBP interactomes profiled with a standardized methodology in the same cell types. Crosslinking and immunoprecipitation (CLIP) methods enable mapping RBP targets transcriptome-wide, but methodological differences present challenges to large-scale analysis across datasets. A critical step in uncovering rules of RNA processing is to study the in vivo regulatory networks of RNA binding proteins (RBPs).
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