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

2-5AMD does not interfere with the initiation step. To further define the mechanism, we analyzed the hallmark substrate of RNase L, the ribosome, and examined whether 2-5AMD inhibits global translation by directly affecting the ribosomal translation activity. 18S and 28S rRNAs are both cleaved by RNase L (Fig. 2D ) Malathi et al., 2007) . Although a high-resolution structure of the . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 human 80S ribosome has become available (Khatter et al., 2015) , mechanistic understanding of how rRNA cleavage by 2-5AMD could affect the ribosome is critically limited by the absence of reliable mapping of the cleavage sites. Based on primer extension analysis it has been proposed that RNase L cleaves nucleotides 4032, and to a smaller extent 4031, in 28S rRNA (Iordanov et al., 2000) . In contrast, subsequent RNA-seq analyses of cleaved rRNAs captured using Arabidopsis tRNA ligase did not detect cleavage at either of the 28S rRNA sites, but observed predominantly RNase Lindependent 18S/28S rRNA background cleavage events (Cooper et al., 2014b) .