Requirement of rRNA Methylation for 80S Ribosome Assembly on a Cohort of Cellular Internal Ribosome Entry Sites

Abstract

Protein syntheses mediated by cellular and viral internal ribosome entry sites (IRESs) are believed to have many features in common. Distinct mechanisms for ribosome recruitment and preinitiation complex assembly between the two processes have not been identified thus far. Here we show that the methylation status of rRNA differentially influenced the mechanism of 80S complex formation on IRES elements from the cellular sodium-coupled neutral amino acid transporter 2 (SNAT2) versus the hepatitis C virus mRNA. Translation initiation involves the assembly of the 48S preinitiation complex, followed by joining of the 60S ribosomal subunit and formation of the 80S complex. Abrogation of rRNA methylation did not affect the 48S complex but resulted in impairment of 80S complex assembly on the cellular, but not the viral, IRESs tested. Impairment of 80S complex assembly on the amino acid transporter SNAT2 IRES was rescued by purified 60S subunits containing fully methylated rRNA. We found that rRNA methylation did not affect the activity of any of the viral IRESs tested but affected the activity of numerous cellular IRESs. This work reveals a novel mechanism operating on a cohort of cellular IRESs that involves rRNA methylation for proper 80S complex assembly and efficient translation initiation.

ACKNOWLEDGMENTS

This work was supported by NIH HL79164 and American Heart Association (AHA) G.I.A0855555D to B.M., NIH RO1-DK53307 and DK60596 to M.H., AI 059267 to S.B., AHA S.D.G0730120N to A.A.K., an AHA postdoctoral fellowship to S.C., an AHA predoctoral fellowship to P.D., and a predoctoral training fellowship to the Center for Lung Biology (NIH T32HL076125 to J.A.).

We sincerely thank Francesca Gaccioli for providing some critical reagents.