Elsevier

Methods

Volume 91, December 2015, Pages 3-12
Methods

RNA–protein interaction methods to study viral IRES elements

https://doi.org/10.1016/j.ymeth.2015.06.023Get rights and content

Highlights

  • Functional analysis of viral IRES elements.

  • Analysis of RNA-binding proteins interacting with viral IRES elements.

  • Structural analysis of viral IRES elements by SHAPE and DMS probing.

  • Approaches to study the impact of RNA-binding proteins on IRES RNA structure.

Abstract

Translation control often takes place through the mRNA untranslated regions, involving direct interactions with RNA-binding proteins (RBPs). Internal ribosome entry site elements (IRESs) are cis-acting RNA regions that promote translation initiation using a cap-independent mechanism. A subset of positive-strand RNA viruses harbor IRESs as a strategy to ensure efficient viral protein synthesis. IRESs are organized in modular structural domains with a division of functions. However, viral IRESs vary in nucleotide sequence, secondary RNA structure, and transacting factor requirements. Therefore, in-depth studies are needed to understand how distinct types of viral IRESs perform their function. In this review we describe methods to isolate and identify RNA-binding proteins important for IRES activity, and to study the impact of RNA structure and RNA–protein interactions on IRES activity.

Introduction

Viral RNAs have evolved mechanisms to initiate translation different from cellular mRNAs. Most cellular mRNAs initiate protein synthesis using a mechanism that depends on the recognition of the m7G(5′)ppp(5′)N structure (termed cap) located at the 5′ end of mRNAs (reviewed in [1]). In contrast, the picornavirus genomic RNA initiate translation internally through a cis-acting region designated internal ribosome entry site (IRES) element, using a cap-independent mechanism [2], [3]. Moreover, IRESs also drive translation initiation in hepatitis C virus (HCV), pestiviruses, dicistroviruses, retroviruses, and some plant RNA viruses [4], [5], [6].

IRESs differ in primary sequence, RNA structure and trans-acting factors requirement, hence promoting internal initiation throughout distinct mechanisms. The intergenic region (IGR) of dicistroviruses can assemble a preinitiation 48S complex in the absence of any initiation factor [7]. The IGR consists of a three-pseudoknoted (PKI-III) RNA structure [8] in which PKI mimics a tRNA/mRNA interaction in the decoding center of the 40S ribosomal subunit [9]. The HCV-like IRESs, also present in pestiviruses and some picornaviruses [10], [11], represent the next level of complexity. These IRESs require eIF3 and the ternary complex (eIF2-GTP-metRNAi) to assemble 48S initiation complexes in reconstitution assays [12]. The RNA structure of the HCV IRES is organized in conformationally flexible domains, designated II, III, and IV. Each domain performs a different function during internal initiation of translation. Domain III binds the 40S ribosomal subunit and eIF3; domain IV harbors the AUG initiation codon; and domain II is involved in eIF2-catalyzed GTP hydrolysis and 60S subunit joining. In addition, domain II contacts the ribosomal proteins RPS5 and RPS25, stabilizing the ribosome in a single conformation leading to translation initiation [13], [14]. Concerning the interaction of viral IRESs with ribosomal proteins, recent evidences have shown that depletion of RPS25 in mammalian cells affects HCV and IGR IRES activity, and to a lower extent, picornavirus IRES function [15].

The highest level of complexity is so far found in picornavirus IRESs. These elements are diverse in primary sequence and secondary RNA structure, being classified into five different types [16]. Functionally related picornavirus IRESs harbor a common RNA structure core and sequence motifs maintained by evolutionary conserved covariant substitutions [17]. Assembly of 48S initiation complexes into IRESs of entero-, cardio-, and aphthovirus requires eIF4G, eIF4A, eIF1, eIF3 and the ternary complex. In addition, host proteins designated IRES-transacting factors (ITAFs) contribute to IRES activity [18], [19]. ITAFs are RNA-binding proteins (RBPs) also involved in splicing, RNA transport, or RNA stability [20], which generally do not contribute to the canonical cap-dependent translation initiation mechanism. Cleavage of host factors in picornavirus-infected cells by viral proteases (L, 2A and 3C) disrupts cap-dependent protein synthesis, in addition to affect transcription, nucleo-cytoplasmic transport, and RNA granules composition. This adverse situation for cellular gene expression, however, does not compromise IRES-dependent translation. Instead, all picornavirus RNAs evade translation inhibition and hijack the translation machinery taking advantage of host factor proteolysis products that, in some cases, activate IRES function [21].

Generally, IRES activity is determined as the expression of reporter genes from dicistronic or monocistronic reporter RNAs. In the first case, the expression of the second reporter gene is normalized to the protein expressed from the first cistron, which monitors cap-dependent translation. In the second case, protein synthesis driven by the IRESs should be normalized to the expression from another RNA, transfected in parallel. Additionally, when the expression is monitored from transfected plasmids, the constructs should be carefully analyzed for the absence of cryptic promoters and potential splicing events [22], [23]. To measure IRES activity under conditions of cap-dependent inhibition, cells are transfected with the IRES reporter plasmids and a plasmid expressing the L protease of aphthovirus or the 2A protease of enterovirus [24], [25]. Both 2A and L proteases induce the cleavage of eIF4G, among other host factors, inhibiting cap-dependent translation but allowing viral IRES-dependent translation. Alternative approaches to analyze IRES activity rely on viral cDNA clones within which parts of the genome can be modified. This approach allows determining protein synthesis within the viral RNA context [26], in addition to analyze potential effects of viral proteins and untranslated regions on IRES activity [27].

Section snippets

RNA-affinity methods to study IRES-protein interactions

IRES function depends on the interaction with host factors [17], [20]. In turn, these factors interact within the cell with other proteins, which can influence IRES activity by indirect interactions. RNA-affinity methods to isolate and identify RBPs are crucial in RNA biology research. Identifying the interaction of proteins with RNA, as well as with other proteins, has been critical for the understanding of networks controlling gene expression. A variety of assays have been developed to

Analysis of sequence covariation and identification of conserved motifs

Evolutionary conserved motifs determine the RNA structure organization of viral IRESs [50]. Conserved sequences essential for RNA function can be identified by alignment of sequences retrieved from GenBank using Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Duplicates and incomplete sequences must be removed and each sequence is given a unique identifier. FASTA-formatted sequences are aligned using CLUSTALW (http://www-ebi.ac.uk/Tools/clustaw2/index.html). RNA secondary structure features

Concluding remarks

Here we have described distinct approaches for studying viral IRES function, IRES-protein interaction and structural organization using in vitro and in vivo methodologies. It is worth mentioning that complementary in vitro approaches, such as reconstitution of translation competent complexes using purified factors, are powerful in elucidating the mechanism used by IRESs to recruit the translation machinery. However, analyzing the factors that impact on IRES function in vivo is critical to

Acknowledgements

We are indebted to J. Ramajo, D. Piñeiro, and N. Fernandez for insightful contributions to the laboratory work. We also thank J.J. Garcia-Berlanga for the kind gift of TAP expression plasmids, and C. Gutierrez for comments on the manuscript. This work was supported by grants BFU2011-25437 and CSD2009-00080 from MINECO (Ministerio de Economia y Competitividad), and by an Institutional grant from Fundación Ramón Areces.

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