Determine the effects of METTL16 on global translation efficiency via ribosome profiling (Ribo-seq)

METTL16 is a member of methyltransferase like (METTL) family. Unlike well-studied METTL3 and METTL14, we found a much higher percentage of METTL16 is localized in the cytosol. The subcellular distribution holds the ability to potentiate translation efficiency. Via Far-western blotting and Co-Immunoprecipitation (Co-IP) assays, we have identified the direct interactions between METTL16 and eukaryotic initiation factor 3 (eIF3) a and b. Via cross-linking immunoprecipitation and qPCR (CLIP-qPCR), we have discovered the direct associations between METTL16 and rRNAs. The METTL16-eIF3a/b and METTL16-rRNAs interactions induce the binding between eIF3 and 18S rRNA, promote the formation of 43S preinitiation complex, and eventually expedite translation initiation, the rate-limiting step of translation. To determine the exact effects of METTL16 on translation efficiency, we performed ribosome profiling (Ribo-seq) with HEK293T upon CRISPR-Cas9-induced METTL16 knockout. To guarantee the repeatability and avoid any potential off-target effects, we included 3 distint sgRNAs against METTL16. The Cas9 single clone of HEK293T cells was infected with lentivirus sgRNAs against METTL16 and the cells were collected at 70-80% confluency after selection with blasticidin and puromycin for at least 1 week. The samples were subjected to ribosome profiling and both the input RNA and footprint RNAs were used for sequencing. Three sgRNAs against METTL16 groups and one sgNS group were included.