Global shape mimicry of tRNA within a viral internal ribosome entry site mediates translational reading frame selection

Abstract

The dicistrovirus intergenic region internal ribosome entry site (IRES) adopts a triple-pseudoknotted RNA structure and occupies the core ribosomal E, P, and A sites to directly recruit the ribosome and initiate translation at a non-AUG codon. A subset of dicistrovirus IRESs directs translation in the 0 and +1 frames to produce the viral structural proteins and a +1 overlapping open reading frame called ORFx, respectively. Here we show that specific mutations of two unpaired adenosines located at the core of the threehelical junction of the honey bee dicistrovirus Israeli acute paralysis virus (IAPV) IRES PKI domain can uncouple 0 and +1 frame translation, suggesting that the structure adopts distinct conformations that contribute to 0 or +1 frame translation. Using a reconstituted translation system, we show that ribosomes assembled on mutant IRESs that direct exclusive 0 or +1 frame translation lack reading frame fidelity. Finally, a nuclear magnetic resonance/small-angle X-ray scattering hybrid approach reveals that the PKI domain of the IAPV IRES adopts an RNA structure that resembles a complete tRNA. The tRNA shape-mimicry enables the viral IRES to gain access to the ribosome tRNA-binding sites and form intermolecular contacts with the ribosome that are necessary for initiating IRES translation in a specific reading frame.translation | virus | RNA | ribosome | internal ribosome entry site F idelity of protein synthesis and the transmission of genetic information from mRNA into a nascent protein rely on the accurate selection and maintenance of the translational reading frame. In canonical eukaryotic translation, after recruitment and scanning of ribosomes on an mRNA, the translational reading frame is initially established by methionyl-tRNA i anticodon: codon pairing in the ribosomal P site. Although the mechanisms that specify reading frame selection and maintenance during translation are not completely understood, programmed recoding mechanisms that have been identified in some viral and cellular mRNAs have yielded significant insights into the cis-acting signals that increase coding capacity or allow translation using alternate reading frames (1, 2).We recently demonstrated that a subset of viruses within the Dicistroviridae family harbors an intergenic region internal ribosome entry site (IGR IRES) that can direct translation in alternative reading frames (3), providing an excellent model for studying RNA-ribosome interactions that influence reading frame selection. An IRES is generally a structured RNA element that can recruit the ribosome in a 5′ end-independent manner and without the full complement of canonical translation initiation factors (4, 5). Among the diverse types of IRES elements found in both viral and messenger RNAs, the IGR IRES uses the most streamlined mechanism, dispensing the need for all canonical initiation factors to directly recruit the ribosome and initiate translation at a non-AUG codon (6-8). The IGR IRES adopts an RNA structure comprising two independently folded domains; ps...

Dynamics of IRES-mediated translation

“…Region 3 folds independently and does not make any contacts with the IRES domain in ribosome-bound or unbound states [28,33,41]. Region 3 is dispensable for ribosome binding [27], but is essential for initiation events that occur downstream of 80S ribosome assembly on the IRES [42,43].…”

Topological Structure Determination of RNA Using Small-Angle X-Ray Scattering

“…The E value of riobA RNA is 0.68, indicating that the riboA is compact. Although IRES-PKI also has a three-way junction, an elongated single stranded loop in the RNA forms a pseudoknot at the 3′ end [29, 30]. The E value of IRES-PKI is 0.84, suggesting an extended structure.…”

Commandeering the Ribosome: Lessons Learned from Dicistroviruses about Translation

“…In an extraordinary case of molecular mimicry, the PKI domain adopts a tRNA-like fold to mimic the anticodon stem of a tRNA, thereby enabling access to the ribosome decoding center (A site; Fig. 1B) (7,(9)(10)(11). From here, the IRES undergoes a pseudotranslocation step that involves the movement of the PKI domain to the P site, thereby resulting in delivery of the first aminoacyl-tRNA to the non-AUG codon at the A site, essentially setting the ribosome into an elongation-competent state (Fig.…”

“…As viral genomes are extremely compact, with limited sequence space, viruses have evolved a variety of strategies to expand their coding capacity. Recently, a subset of IGR IRESs have been shown to facilitate the translation of a ϩ1 frame overlapping ORF, termed ORFx, in addition to translation in the 0 frame (10,20). Specific mutations within the tRNA-like PKI domain uncouple 0 and ϩ1 frame translation, which is correlated with distinct, subtle conformations, which may drive reading frame selection (10).…”

“…Recently, a subset of IGR IRESs have been shown to facilitate the translation of a ϩ1 frame overlapping ORF, termed ORFx, in addition to translation in the 0 frame (10,20). Specific mutations within the tRNA-like PKI domain uncouple 0 and ϩ1 frame translation, which is correlated with distinct, subtle conformations, which may drive reading frame selection (10). Unlike most viral RNA signals that shift reading frames of translating ribosomes such as Ϫ1 frameshift signals in retroviruses and coronaviruses, IGR IRES-mediated 0 and ϩ1 frame translation represents a novel translation initiation strategy to increase the coding capacity of a viral genome.…”