Denaturing CLIP, dCLIP, Pipeline Identifies Discrete RNA Footprints on Chromatin-Associated Proteins and Reveals that CBX7 Targets 3' UTRs to Regulate mRNA Expression.

Cell Systems
Michael RosenbergJeannie T Lee

Abstract

Interaction networks between chromatin complexes and long noncoding RNAs have become a recurrent theme in epigenetic regulation. However, technical limitations have precluded identification of RNA binding motifs for chromatin-associated proteins. Here, we add a denaturation step to UV-crosslink RNA immunoprecipitation (dCLIP) and apply dCLIP to mouse and human chromobox homolog 7 (CBX7), an RNA binding subunit of Polycomb repressive complex 1 (PRC1). In both species, CBX7 predominantly binds 3' UTRs of messenger RNAs. CBX7 binds with a median RNA "footprint" of 171-183 nucleotides, the small size of which facilitates motif identification by bioinformatics. We find four families of consensus RNA motifs in mouse, and independent analysis of human CBX7 dCLIP data identifies similar motifs. Their mutation abolishes CBX7 binding in vitro. Pharmacological intervention with antisense oligonucleotides paradoxically increases CBX7 binding and enhances gene expression. These data support the utility of dCLIP and reveal an unexpected functional interaction between CBX7 and the 3' UTRs of mRNA.

Citations

Feb 15, 2018·Endocrine-related Cancer·Carolyn M Klinge
Apr 12, 2019·The Biochemical Journal·Emily J ShieldsRoberto Bonasio
May 18, 2019·Quantitative Biology·Molly M HanniganDonny D Licatalosi
May 12, 2019·Nucleic Acids Research·Chenyu Lin, Wayne O Miles
Jan 8, 2020·Nucleic Acids Research·Yunqing LinDong Wang
Nov 29, 2020·Nature Communications·He FangChristine M Disteche
Jan 6, 2021·Nature Structural & Molecular Biology·Michael RosenbergJeannie T Lee
Apr 17, 2021·Wiley Interdisciplinary Reviews. RNA·Jackson B TrotmanJ Mauro Calabrese
Sep 9, 2019·Methods : a Companion to Methods in Enzymology·Tino KösterDorothee Staiger

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