RESA Identifies mRNA-regulatory Sequences at High Resolution

Nat Methods. 2017 Feb;14(2):201-207. doi: 10.1038/nmeth.4121. Epub 2016 Dec 26.

Abstract

Gene expression is extensively regulated at the levels of mRNA stability, localization and translation. However, decoding functional RNA-regulatory features remains a limitation to understanding post-transcriptional regulation in vivo. Here, we developed RNA-element selection assay (RESA), a method that selects RNA elements on the basis of their activity in vivo and uses high-throughput sequencing to provide a quantitative measurement of their regulatory functions at near-nucleotide resolution. We implemented RESA to identify sequence elements modulating mRNA stability during zebrafish embryogenesis. RESA provides a sensitive and quantitative measure of microRNA activity in vivo and also identifies novel regulatory sequences. To uncover specific sequence requirements within regulatory elements, we developed a bisulfite-mediated nucleotide-conversion strategy for large-scale mutational analysis (RESA-bisulfite). Finally, we used the versatile RESA platform to map candidate protein-RNA interactions in vivo (RESA-CLIP).

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions
  • Animals
  • Embryo, Nonmammalian
  • Genetic Techniques*
  • High-Throughput Nucleotide Sequencing / methods*
  • Immunoprecipitation
  • RNA Stability
  • RNA, Messenger* / genetics
  • Regulatory Sequences, Nucleic Acid*
  • Sulfites
  • Zebrafish / embryology

Substances

  • 3' Untranslated Regions
  • RNA, Messenger
  • Sulfites
  • hydrogen sulfite