Concerted 2-5A-Mediated mRNA Decay and Transcription Reprogram Protein Synthesis in dsRNA Response

Sneha Rath; Eliza Prangley; Jesse Donovan; Kaitlin Demarest; Yigal Meir; Ned Wingreen; Alexei Korennykh

To test this prediction, we directly assessed the translation activity of the cleaved ribosomes in rabbit reticulocyte lysate (RRL). We depleted this lysate of ribosomes by centrifugation (Fig. S3B ) and re-supplied intact ribosomes purified from RRL, A549 cells, or cleaved ribosomes from dsRNA-transfected human cells (Fig. 3C ). Using capped luciferase mRNA translation as the readout, we assessed the activity of each ribosome type. In ribosome-depleted RRL, luciferase translation was absent, suggesting that we obtained a suitable assay. Addition of either rabbit ribosomes from nucleasetreated RRL, intact human ribosomes, or human ribosomes with rRNA degraded by 2-5AMD, readily supported translation. We observed the same specific activity for intact and cleaved ribosomes, which we reproduced over a range of ribosomal concentrations to exclude saturation effects (Fig. 3C ). Therefore, 2-5AMD does not functionally damage human ribosomes even after a nearly complete degradation of full-length rRNA (Fig. 3C, last lane) . In agreement with this observation, the single-exponential decay kinetics for rRNA lags behind the kinetics of translational shutdown (Fig. 3D ). Our data and the previously reported disconnect between rRNA cleavage and translation together indicate that the loss of global translation during 2-5AMD involves a process physically distinct from rRNA degradation.