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

Marisa D Ruehle,Haibo Zhang,Yuanwei Chen,Somdeb Mitra,Jeffrey S Kieft,Barry S Cooperman,Ryan M Sheridan,Ruben L Gonzalez

doi:10.7554/elife.08146

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.

Highlights

A vital step in infection by viruses is translation of the viral ribonucleic acid (RNA)
We assessed the functional importance of loop 3 in intergenic region (IGR) internal ribosome entry sites (IRESs)-driven translation using a dual luciferase (LUC) reporter construct in rabbit reticulocyte lysate (RRL) (Figure 1F)
We made several mutants (Table 1): (1) we shortened loop 3 by three nucleotides, reasoning this would reduce flexibility that may be important for function (43 mutants); (2) noting the loops’ high adenosine content, we replaced several adenosines with guanosines (G-rich mutants); (3) because sequence alignment from various IRESs suggested the presence of conserved bases in loop 3 (Figure 1—figure supplement 1B) (Au et al, 2012), we replaced a single conserved adenosine with a guanosine in the highly active Israeli Acute Paralysis Virus (IAPV) IRES

Summary

Introduction

A vital step in infection by viruses is translation of the viral RNA. Many RNA viruses initiate translation using internal ribosome entry sites (IRESs), which are cis-acting RNA elements that recruit the host cell’s translation machinery in a cap- and end-independent fashion (Filbin and Kieft, 2009; Doudna and Sarnow, 2007; Plank and Kieft, 2012). Current mechanistic models for how the IGR IRESs operate in eukaryotes suggest that after the IGR IRES assembles an 80S ribosome, eukaryotic elongation factor (eEF) 2 catalyzes an initial pseudotranslocation event (translocation without peptide bond formation) which positions the first codon of the open reading frame in the A site (Figure 1A) (Fernandez et al, 2014; Koh et al, 2014; Zhu et al, 2011).

Methods

Results

Conclusion