A dynamic RNA loop in an IRES affects multiple steps of elongation factor-mediated translation initiation

Abstract

Internal ribosome entry sites (IRESs) are powerful model systems to understand how the translation machinery can be manipulated by structured RNAs and for exploring inherent features of ribosome function. The intergenic region (IGR) IRESs from the Dicistroviridae family of viruses are structured RNAs that bind directly to the ribosome and initiate translation by co-opting the translation elongation cycle. These IRESs require an RNA pseudoknot that mimics a codon-anticodon interaction and contains a conformationally dynamic loop. We explored the role of this loop and found that both the length and sequence are essential for translation in different types of IGR IRESs and from diverse viruses. We found that loop 3 affects two discrete elongation factor-dependent steps in the IRES initiation mechanism. Our results show how the IRES directs multiple steps after 80S ribosome placement and highlights the often underappreciated significance of discrete conformationally dynamic elements within the context of structured RNAs.DOI: http://dx.doi.org/10.7554/eLife.08146.001

Dynamics of IRES-mediated translation

“…Toeprint analysis of initiation by IRES mutants with a shortened VRL indicates that the loss of translational activity is due to an inability to pseudotranslocate, while toeprints for the sequence mutants are consistent with several rounds of translocation. However, the toeprints of the translocated complexes with the VRL sequence mutant IRES had lower band intensity compared with wild-type IRES translocated complexes, suggesting that subtle changes in translocation efficiency are responsible for the decreased translational activity of the VRL sequence mutants [64].…”

“…For the CrPV IRES, and other IGR IRESs, both the 0 and þ1 frames are translated, with the 0 frame being disproportionally favoured [43,61,62]. The dynamics of region 3 of the IRES are critical for positioning PKI in productive conformations within the ribosome, frame selection and translocation [43,63,64]. PKI can adopt conformations that mimic classical and hybrid tRNA states during IRES initiation [45,55]; however, the molecular mimicry is imperfect.…”

Molecular analysis of the factorless internal ribosome entry site in Cricket Paralysis virus infection

“…The conserved L1.1 loop of the IGR IRES interacts with the L1 stalk of the 60S ribosomal subunit, which is reminiscent of interactions of the L1 stalk with an E-site deacylated tRNA1230. The variable loop region (VLR) in the PKI domain, which is reported to facilitate ribosome positioning and eEF2-mediated pseudotranslocation, interacts with the β-hairpin loop of uS7 and helix 23 of 18S rRNA in the E site, suggesting that the VLR has a stabilizing role as the IRES translocates through the ribosome253132. In addition to the ribosome, the IGR IRES may interact with eEF2 to facilitate its movement through the ribosome31.…”

“…The variable loop region (VLR) in the PKI domain, which is reported to facilitate ribosome positioning and eEF2-mediated pseudotranslocation, interacts with the β-hairpin loop of uS7 and helix 23 of 18S rRNA in the E site, suggesting that the VLR has a stabilizing role as the IRES translocates through the ribosome253132. In addition to the ribosome, the IGR IRES may interact with eEF2 to facilitate its movement through the ribosome31. IGR IRES recruitment of the ribosome is inhibited by depleting pseudouridylation of rRNA, suggesting that specific rRNA modifications can affect IRES function33.…”

Commandeering the Ribosome: Lessons Learned from Dicistroviruses about Translation

“…1B). Indeed, a recent report showed that specific mutations in the variable loop region (VLR) of the IRES may drive the pseudotranslocation step specifically (16). Thus, how the IGR IRES manipulates the ribosome offers insights into the core mechanics of translation such as ribosome-tRNA dynamics during translocation.…”