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

Using poly-A + RNA-seq we extended our analysis to the transcriptome. Widely used RNA-seq normalization and differential expression analysis techniques presume that levels of most mRNAs remain unchanged. This assumption would be violated if 2-5AMD inhibited translation by global mRNA decay. To correctly quantify mRNA levels during the course of decay, we supplemented our RNA samples with internal standards Of note, evaluation of RNase L activity in cytosolic cell extracts showed that mRNA decay depends on both, mRNA length and AU content (Fig. S5 ). In S10 cytosolic extracts treated with 2-5A, at the time point when ~60% ACTB mRNA still remains, only 0.1% of FAT1 mRNA and 0.2% of PRKDC mRNAs survive ( Fig. 5C ). The high sensitivity of FAT1 and PRKDC transcripts in the S10 extract is in line with their lower GC content and greater length leading to more net UN^N sites per mRNA. In contrast to these findings, 2-5AMD in live cells shows no dependence on GC . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/484675 doi: bioRxiv preprint content and leads to decay of PRKDC, FAT1, ACTB and most other mRNAs with comparable, within several-fold, kinetics ( Fig. 5A-B ; Discussion). Therefore, cellular mRNA decay agrees with the fast timing of translational inhibition and the loss of global protein synthesis.