Rapid RNase L–driven arrest of protein synthesis in the dsRNA response without degradation of translation machinery

Abstract

Mammalian cells respond to double-stranded RNA (dsRNA) by activating a translation-inhibiting endoribonuclease, RNase L. Consensus in the field indicates that RNase L arrests protein synthesis by degrading ribosomal RNAs (rRNAs) and messenger RNAs (mRNAs). However, here we provide evidence for a different and far more efficient mechanism. By sequencing abundant RNA fragments generated by RNase L in human cells, we identify site-specific cleavage of two groups of noncoding RNAs: Y-RNAs, whose function is poorly understood, and cytosolic tRNAs, which are essential for translation. Quantitative analysis of human RNA cleavage versus nascent protein synthesis in lung carcinoma cells shows that RNase L stops global translation when tRNAs, as well as rRNAs and mRNAs, are still intact. Therefore, RNase L does not have to degrade the translation machinery to stop protein synthesis. Our data point to a rapid mechanism that transforms a subtle RNA cleavage into a cell-wide translation arrest.

Real-time 2-5A kinetics suggest that interferons β and λ evade global arrest of translation by RNase L

“…S8 A-C). RNase L −/− cells exhibited a delayed and incomplete translational attenuation involving eIF2α phosphorylation, presumably due to PKR (5). Translational arrest ahead of ISG induction was also present in HeLa cells (SI Appendix, Fig.…”

“…To determine whether cellular 2-5A dynamics correspond to a rapid arrest of translation by RNase L, we measured nascent protein synthesis by puromycin pulse labeling in WT and RNase L −/− A549 cells (5). Treatment of WT, but not of RNase L −/− , cells with poly-IC halted global translation before ISG induction ( Fig.…”

“…Inhibition of protein synthesis arises in part from activation of the dsRNA-dependent protein kinase R (PKR) and in part from signaling by conserved small RNAs that contain 2′,5′-linked oligoadenylates (2-5A) (1)(2)(3)(4). The action of 2-5A is sufficient for the arrest of translation independent of PKR, and at least in some cell lines, 2-5A is the main cause of translational arrest (5).…”

The role of RNA modifications in the regulation of tRNA cleavage

“…In vitro transcribed tRNA His is susceptible to cleavage at each of these three sites in contrast to physiological tRNA His , which is only cleaved at position 37. The authors of this study found that modification of G34 to Q34 provides specificity that guides cleavage at the physiological site (Figs and ). RNase L activation is accompanied by a dramatic decrease in protein synthesis.…”

“…Emerging data show that human cells may also target tRNA for bulk cleavage in response to viral infections. In this instance, cleavage occurs at position 37 of the mature tRNA and predominantly occurs on tRNA His , although cleavage also occurs on tRNA Pro and various Y‐RNA . Mammalian cells have an intricate network of sensors monitoring double‐stranded RNA (dsRNA), a hallmark of viral infection.…”