Journal
ISCIENCE
Volume 23, Issue 7, Pages -Publisher
CELL PRESS
DOI: 10.1016/j.isci.2020.101318
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Funding
- MSTP [T32GM007288, F30CA214009, RO1GM57829]
- Howard Hughes Medical Institute [R01DA037721]
- National Institutes of Health [R01NS083085]
- NIH Common Fund 4D Nucleome Program [U01DA047729]
- NIH [P30CA013330]
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Both UV-cross-linking and immunoprecipitation (CLIP) and RNA editing (TRIBE) can identify the targets of RNA-binding proteins. To evaluate false-positives of CLIP and TRIBE, endogenous beta-actin mRNA was tagged with MS2 stem loops, making it the only bona fide target mRNA for the MS2 capsid protein (MCP). CLIP and TRIBE detected beta-actin, albeit with false-positives. False-positive CLIP signals were attrib-uted to nonspecific antibody interactions. In contrast, putative false-positive TRIBE targets were genes spatially proximal to the 0-actin gene. MCP-ADAR edited nearby nascent transcripts consistent with interchromosomal contacts observed in Hi-C. The identification of nascent contacts implies RNA regulatory proteins (e.g., splicing factors) associated with multiple nascent transcripts, forming domains of post-transcriptional activity. Repeating these results with an integrated inducible MS2 reporter indicated that MS2-TRIBE can be applied to a broad array of cells and transcripts to study spatial organization and nuclear RNA regulation.
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