SRA

The viral non-coding RNA HSUR2 regulates apoptosis by recruiting host microRNAs to host mRNAs encoding apoptotic factors. The mechanism whereby HSUR2 targets specific host mRNAs is unknown. Here we developed iRICC to identify sequences mediating RNA-RNA interactions in vivo between a single RNA of interest and its binding RNAs. Sequences that mediate HSUR2-mRNA interactions identified by iRICC-seq were functionally validated by mutational analyses. Unlike other small regulatory non-coding RNAs, HSUR2 does not contain a “seed” region responsible for interaction with most target mRNAs, but rather uses different sequences to bind to different targets. Our findings describe a mode of interaction that provides HSUR2 with flexibility both in target recognition and in the selection of trans-acting miRNAs that are recruited for target mRNA repression. Overall design: Polyadenylated (polyA+) RNA was selected and hybridized to an antisense DNA oligonucleotide complementary to full-length HSUR2 (anti-H2 DNA) followed by limited single-strand S1 endonuclease digestion to fragment polyA+ RNA while maintaining HSUR2 integrity. After removal of anti-H2 DNA by DNAse I digestion, samples were split into two samples and hybridization with anti-H2 DNA was repeated in one (Control) sample, followed by digestion of both samples with RNAse H, which resulted in removal of HSUR2 only from the Control sample. Following a second digestion with DNAse I, HSUR2 and crosslinked RNAs were purified in both samples under stringent conditions with a biotinylated RNA oligonucleotide antisense to HSUR2. High-throughput sequencing libraries were prepared for both samples using random primers. HSUR2 target mRNAs were identified by comparison of pull downs from HSUR2 samples with Control samples in three independent replicate experiments