Human RNase L Employs a Signaling Mechanism to Arrest Translation

To identify small RNA cleaved by RNaseL, we captured intracellular RNA with 2''-3'' cyclic phosphates by ligation an Illumina-compatible adaptor and the RNA ligase RtcB. Overall design: Small RNAs with 2''-3'' cyclic phosphate were ligated to an adaptor with RtcB and converted to Illumina small RNA sequencing libraries. Three independent methods were used to activate RNase L: transfection of poly-IC, a synthetic dsRNA, semi-permeabilization of cells in the presence of 2-5A, and overexpression of RNase L. We also examined basal activity of RNase L by 2''-3'' cyclic phosphate small RNA in WT and RNase L knockout Hap1 cells. Additionally, we showed that the Ire1, an RNase L homologue, does not cleave small RNAs. Last, we show that A549 do not show mRNA decay after prolonged treatment with 2-5A.