RNA-RNA Interactions Enable Specific Targeting of Noncoding RNAs to Nascent Pre-mRNAs and Chromatin Sites
Abstract
Intermolecular RNA-RNA interactions are used by many noncoding RNAs (ncRNAs) to achieve their diverse functions. To identify these contacts, we developed a method based on RNA antisense purification to systematically map RNA-RNA interactions (RAP-RNA) and applied it to investigate two ncRNAs implicated in RNA processing: U1 small nuclear RNA, a component of the spliceosome, and Malat1, a large ncRNA that localizes to nuclear speckles. U1 and Malat1 interact with nascent transcripts through distinct targeting mechanisms. Using differential crosslinking, we confirmed that U1 directly hybridizes to 5′ splice sites and 5′ splice site motifs throughout introns and found that Malat1 interacts with pre-mRNAs indirectly through protein intermediates. Interactions with nascent pre-mRNAs cause U1 and Malat1 to localize proximally to chromatin at active genes, demonstrating that ncRNAs can use RNA-RNA interactions to target specific pre-mRNAs and genomic sites. RAP-RNA is sensitive to lower abundance RNAs as well, making it generally applicable for investigating ncRNAs.
Additional Information
© 2014 Elsevier Inc. Received: March 19, 2014. Revised: June 20, 2014. Accepted: August 18, 2014. Published: September 25, 2014. We thank Amy Pandya-Jones for chromatin fractionation protocols; Xuebing Wu for assistance with scoring 50ss motif matches; Tarjei Mikkelsen for assistance with oligonucleotide synthesis; Phil Sharp, Jason West, and Chris Davis for helpful discussions; and Leslie Gaffney for assistance with figures. This work was supported by the Fannie and John Hertz Foundation (J.M.E.), National Defense Science and Engineering Graduate Fellowship (J.M.E.), NIH Director's Early Independence Award DP5OD012190 (M.G.), Edward Mallincrodt Jr. Foundation (M.G.), Searle Scholars program (M.G.), the Broad Institute of MIT and Harvard (E.S.L. and M.G.), and the California Institute of Technology (M.G.). J.M.E., E.S.L., and M.G. are inventors on a patent application from the Broad Institute that covers the selective purification of RNA bound molecular complexes in cells. Author Contributions: All authors designed experiments and analyses and approved the final manuscript. J.M.E. and K.S. performed experiments. J.M.E. performed computational analyses. J.M.E., P.M., A.Y.C., K.S., A.S., C.S., and M.G. developed experimental protocols. J.M.E., P.R., S.R.G., M.G., and E.S.L. developed analytical methods and tools. J.M.E. and M.G. conceived and designed the study. J.M.E., M.G., and E.S.L. wrote the manuscript.Attached Files
Accepted Version - nihms625961.pdf
Supplemental Material - mmc1.xlsx
Supplemental Material - mmc2.xlsx
Supplemental Material - mmc3.xlsx
Supplemental Material - mmc4.xlsx
Supplemental Material - mmc5.xlsx
Supplemental Material - mmc6.pdf
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Additional details
- PMCID
- PMC4177037
- Eprint ID
- 50169
- DOI
- 10.1016/j.cell.2014.08.018
- Resolver ID
- CaltechAUTHORS:20141002-105821728
- Fannie and John Hertz Foundation
- National Defense Science and Engineering Graduate (NDSEG) Fellowship
- NIH
- DP5OD012190
- Edward Mallincrodt Jr. Foundation
- Searle Scholars Program
- Broad Institute of MIT and Harvard
- Caltech
- Created
-
2014-10-02Created from EPrint's datestamp field
- Updated
-
2021-11-10Created from EPrint's last_modified field