Valeria Yartseva, Carter M. Takacs, Charles E. Vejnar, Miler T. Lee, Antonio J. Giraldez
Gene expression is regulated extensively at the level 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 based on their activity in vivo and uses high-throughput sequencing to provide quantitative measurement of their regulatory function with 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). The RESA platform can be broadly applicable to uncover the regulatory features shaping gene expression and cellular function.
Valeria Yartseva, Carter M. Takacs, Charles E. Vejnar, Miler T. Lee, Antonio J. Giraldez. RESA identifies mRNA-regulatory sequences at high resolution. 2016-12-26. https://doi.org/10.1038/nmeth.4121
The RESA-Bisulfite library was generated from the same zebrafish 3′-UTR fragment pool after partial bisulfite conversion, producing C→U or G→A substitutions across individual reporter molecules. Comparing wild-type and converted-base representation between 2 hpf and 8 hpf embryos identified nucleotide positions and linked bases required for mRNA destabilization or stabilization.
A highly overlapping library of sonicated zebrafish maternal 3′-UTR fragments was placed in a common GFP reporter, transcribed in vitro, and injected into one-cell embryos. RNA-seq coverage at 2 hpf and 8 hpf, with a parallel 430LNA miR-430-inhibition control, was used to call mRNA-destabilizing and stability-promoting regions.
A RESA reporter RNA library was co-injected with FLAG-tagged Ago2 into zebrafish embryos, UV-crosslinked, and immunoprecipitated with anti-FLAG beads. Ago2-bound reporter fragments were compared with the input library to identify RNA regions enriched for Ago2/miR-430-associated binding.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
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