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 whether 2-5AMD disrupts assembly of cap binding initiation complexes, we pulled down the cap-binding initiation factor eIF4E and examined its association with . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 the key partner factors eIF4A and eIF4G that together form the eIF4F complex. This tripartite complex was readily identified using the pulldown and remained unchanged by 2-5AMD (Fig. 2C ). Total RNA profiling by NanoChip revealed that eIF4E additionally pulled down the 40S ribosomal subunit both in naïve cells and in cells with activated 2-5AMD, suggesting normal loading of the small subunit. As expected, 18S and 28S rRNAs were degraded during 2-5AMD and exhibited the characteristic pattern of RNase L activity (Fig. 2D ). The 18S rRNA from the 40S subunit pulled down with eIF4E following 2-5AMD was cleaved as in the input rRNA. Thus, binding of the core components of the translation initiation complex is not disrupted during 2-5AMD.