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 sodiumcoupled 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.Initiation of translation on the majority of the cellular mRNAs is 5Ј cap dependent. This process relies on the recruitment of the 43S initiation complex to the cap structure at the 5Ј end of the mRNA promoted by cap-binding initiation factor eIF4F to form 48S initiation complex and subsequent scanning of the 48S complex across the 5Ј untranslated region (UTR) in search of the initiator AUG codon. This major initiation pathway is widely believed to account for most cellular mRNA translation (19,20). On the other hand, a minor cap-independent initiation pathway relying on the presence of internal ribosome entry sites (IRESs) has been described for a subset of viral mRNAs (for a recent review, see reference 15). Viral IRES-dependent translation has been shown to rely on both canonical and noncanonical interactions between IRES elements, canonical eukaryotic initiation factors (eIFs), IRES trans-acting factors (ITAFs; largely considered to act as noncanonical initiation factors), and 40S ribosomal subunits. As a rule, viral IRES-mediated translation does not require the cap-binding initiation factor eIF4E and in general has reduced requirements for other members of the eIF4F factor/complex (specifically, intact eIF4G) (15, 19). It has been further shown that certain cellular mRNAs may rely on similar translation initiation mechanism (21,23,24). Reports on the IRES elements in cellular mRNAs are emerging on a continuing basis; however, the mechanism(s) of their activation and function remains less well understood.The best-studied viral IRES elements can be assigned (based on their structure and requirements for the canonical initiation factors) to four different types. Type 1 (e.g., poliovirus) and type 2 (e.g., encephalomyocarditis v...

Insights into the Mechanism of Ribosomal Incorporation of Mammalian L13a Protein during Ribosome Biogenesis

“…6). The importance of L13a for rRNA methylation in the 90S preribosome is consistent with our previous finding that L13a is a component of the fibrillarin-containing C/D box snoRNP complex responsible for methyltransferase activity (15). Another demonstrated function of L13a occurs outside the ribosome.…”

“…In contrast to many other ribosomal proteins of both the small and large ribosomal subunits (48-54), L13a does not affect ribosome biogenesis and maturation or global protein synthesis (14). We have, however, previously demonstrated that L13a is required for rRNA methylation, one of the rRNA modifications that takes place during ribosome biogenesis (15). Here, we confirm that L13a is required for rRNA methylation and demonstrate that this occurs within the 90S preribosome (Fig.…”

“…We also showed that RNA interference (RNAi)-mediated depletion of L13a does not affect rRNA processing and assembly of mature 80S ribosomes and polyribosomes (14) or their primary function, protein synthesis. These studies did, however, identify a critical role for L13a in rRNA methylation, being required for translation of several cellular internal ribosome entry site (IRES)-containing mRNAs (14,15). Therefore, while the ribosomal protein L13a appears to be dispensable for the canonical function of ribosomes in protein synthesis, it is important for promoting IRES-dependent translation of particular sets of mRNAs via an effect on rRNA methylation and for mediating translational suppression of GAIT element-containing mRNAs following IFN-␥-induced signaling.…”

Translational Control in Cancer Etiology

“…It would be interesting to assess whether rRNA pseudouridylation regulates, at the translational level, the expression of mRNAs containing a PRF signal, perhaps in a cell-and/or tissue-specific manner. An emerging role for rRNA modifications in regulating specific modes of translation has recently been highlighted by findings that methylation of rRNA is also implicated in IRES-dependent translation of specific cellular mRNAs Belin et al 2009;Basu et al 2011). Furthermore, loss of rRNA methylation at specific sites in zebrafish leads to severe developmental defects (HigaNakamine et al 2012).…”

Translation factors and ribosomal proteins control tumor onset and progression: how?

“…The importance of rRNA modifications is also supported by the finding that rRNA methylation is important for IRES-dependent translation of specific mRNAs. 132,133 The more general model proposed by Ruggero 17 , therefore, is that altered ribosomes can induce tumorigenesis because of specific changes in the translation pattern.…”