Structure of the ribosome-bound cricket paralysis virus IRES RNA

Abstract

Internal ribosome entry sites (IRESs) facilitate an alternative, end-independent pathway of translation initiation. A particular family of dicistroviral IRESs can assemble elongation-competent 80S ribosomal complexes in the absence of canonical initiation factors and initiator transfer RNA. We present here a cryo-EM reconstruction of a dicistroviral IRES bound to the 80S ribosome. The resolution of the cryo-EM reconstruction, in the subnanometer range, allowed the molecular structure of the complete IRES in its active, ribosome-bound state to be solved. The structure, harboring three pseudoknot-containing domains, each with a specific functional role, shows how defined elements of the IRES emerge from a compactly folded core and interact with the key ribosomal components that form the A, P and E sites, where tRNAs normally bind. Our results exemplify the molecular strategy for recruitment of an IRES and reveal the dynamic features necessary for internal initiation.Initiation of protein synthesis is an essential phase of protein synthesis and a key regulatory step in gene expression 1,2 . In eukaryotes, the canonical 5¢ cap-dependent pathway is facilitated and orchestrated by approximately 11 translation initiation factors. However, IRES RNAs can functionally substitute for initiation factors and facilitate the alternative pathway of internal initiation 3,4 . IRESs are present in 5¢ untranslated regions (UTRs) of many viral RNAs and are efficient tools to hijack the translational apparatus of the host during viral infection. They are also used by a subset of cellular messenger RNAsfor example, several proto-oncogenes [3][4][5] . In this context, they act as regulatory tools and are used to initiate translation during cellular stress or other periods when overall global translation is compromised.The molecular mechanisms of initiation by IRES RNAs are largely unknown. IRES RNAs fall into different classes that are distinguished by their structure and dependence on different sets of canonical initiation factors and IRES trans-acting factors 3-6 . The simplest mechanism of initiation is used by the intergenic IRESs of dicistroviruses, such as the cricket paralysis virus (CrPV). This family of IRESs does not require any initiation factor or even initiator tRNA in order to assemble elongation-competent 80S ribosomes 7-10 . According to biochemical studies, the IRES binds directly to the ribosomal 40S subunit and sets the translational reading frame by positioning the first codon into the ribosomal A site. This is highly unusual, because canonical initiation starts from the P site. Moreover, like the hepatitis C virus IRES 11 , the CrPV IRES actively manipulates the conformation of the translational machinery, suggesting that the IRES acts like an RNA-based translation factor 12 .A detailed knowledge of the CrPV IRES structure, especially in the ribosome-bound state, is a prerequisite for understanding the mechanism of internal initiation without initiation factors. The low resolution of the previous cryo-EM maps limit...

Structure of the full-length HCV IRES in solution

“…We showed here that it is an articulated molecule in which structured elements undergo large reorientations relative to each other 16,18,28 . By comparison, IRES of group I found in the intergenic region of Dicistroviridae assemble ribosome in the absence of canonical initiation factors and Met-tRNA and undergo only local rearrangements upon formation of the initiation complex [51][52][53] . The structure of group I IRES is composed of two well-structured regions for which atomic structures have been obtained 54,55 .…”

New insights into internal ribosome entry site elements relevant for viral gene expression

“…This tight relationship suggests that the RNA structure of IRES elements regulates translation efficiency. On the other hand, structural studies performed on the HCV IRES and the IGR of members of the family Dicistroviridae have shown the capacity of these IRES elements to be accommodated in the interface of the ribosomal subunits (Spahn et al, 2001;Boehringer et al, 2005;Schuler et al, 2006;Pfingsten et al, 2006). Although the IGR and the HCV IRES elements exhibit different structural organization (Kieft et al, 2002;Rijnbrand et al, 2004;Jan & Sarnow, 2002;Nishiyama et al, 2003) and their binding sites in the ribosomal subunit are different, similar conformational changes are induced in the 40S ribosomal subunit (Spahn et al, 2004).…”

“…In marked difference to any other IRES described to date, the IGRs of dicistroviruses assemble initiation complexes in the absence of any eIFs (Table 1) (Spahn et al, 2004;Wilson et al, 2000a). This RNA sequence establishes direct contacts with ribosomal proteins (Schuler et al, 2006). Mapping of the PSIV IGR interactions with ribosomal proteins revealed that the strongest signal was S25 (Nishiyama et al, 2007), a protein located near S5 that has been shown to interact with this IGR (Pfingsten et al, 2006).…”

Cryo-EM image alignment based on nonuniform fast Fourier transform

“…The problem of 2-D alignment is defined as: given two images f and g, we wish to find two translation parameters x and y and one rotation angle θ which minimize the least square discrepancy: (4) where D is the region of interest (for example, a disk with diameter d 0 ), u is vector [u x u y 1] T containing the coordinates, and T is the transformation matrix defining rotation and translations of image [2]. We designate f the reference (or template) image and g the subject (aligned) image.…”

“…The deterioration of results due to interpolation errors is customarily minimized by oversampling of EM micrographs with respect to the target resolution. For example, to determine the 7.3 Å structure of the 70S ribosome, it was necessary to use a pixel size of 1.22 Å [4]. If we consider that the usable information in this structure extends to ~4.8 Å, as estimated by the Fourier Shell Correlation method [5], according to the Shannon sampling theorem the necessary pixel size should be 2.4 Å.…”