Abstract
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.
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