The hepatitis C virus 3′-untranslated region or a poly(A) tract promote efficient translation subsequent to the initiation phase

Enhancement of eukaryotic messenger RNA (mRNA) translation initiation by the 3′ poly(A) tail is mediated through interaction of poly(A)-binding protein with eukaryotic initiation factor (eIF) 4G, bridging the 5′ terminal cap structure. In contrast to cellular mRNA, translation of the uncapped, non-polyadenylated hepatitis C virus (HCV) genome occurs independently of eIF4G and a role for 3′-untranslated sequences in modifying HCV gene expression is controversial. Utilizing cell-based and in vitro translation assays, we show that the HCV 3′-untranslated region (UTR) or a 3′ poly(A) tract of sufficient length interchangeably stimulate translation dependent upon the HCV internal ribosomal entry site (IRES). However, in contrast to cap-dependent translation, the rate of initiation at the HCV IRES was unaffected by 3′-untranslated sequences. Analysis of post-initiation events revealed that the 3′ poly(A) tract and HCV 3′-UTR improve translation efficiency by enabling termination and possibly ribosome recycling for successive rounds of translation

IFNL3 mRNA structure is remodeled by a functional non-coding polymorphism associated with hepatitis C virus clearance

Polymorphisms near the interferon lambda 3 (IFNL3) gene strongly predict clearance of hepatitis C virus (HCV) infection. We analyzed a variant (rs4803217 G/T) located within the IFNL3 mRNA 3′ untranslated region (UTR); the G allele (protective allele) is associated with elevated therapeutic HCV clearance. We show that the IFNL3 3′ UTR represses mRNA translation and the rs4803217 allele modulates the extent of translational regulation. We analyzed the structures of IFNL3 variant mRNAs at nucleotide resolution by SHAPE-MaP. The rs4803217 G allele mRNA forms well-defined 3′ UTR structure while the T allele mRNA is more dynamic. The observed differences between alleles are among the largest possible RNA structural alterations that can be induced by a single nucleotide change and transform the UTR from a single well-defined conformation to one with multiple dynamic interconverting structures. These data illustrate that non-coding genetic variants can have significant functional effects by impacting RNA structure

Gemin5 proteolysis reveals a novel motif to identify L protease targets

Translation of picornavirus RNA is governed by the internal ribosome entry site (IRES) element, directing the synthesis of a single polyprotein. Processing of the polyprotein is performed by viral proteases that also recognize as substrates host factors. Among these substrates are translation initiation factors and RNA-binding proteins whose cleavage is responsible for inactivation of cellular gene expression. Foot-and-mouth disease virus (FMDV) encodes two proteases, Lpro and 3Cpro. Widespread definition of Lpro targets suffers from the lack of a sufficient number of characterized substrates. Here, we report the proteolysis of the IRES-binding protein Gemin5 in FMDV-infected cells, but not in cells infected by other picornaviruses. Proteolysis was specifically associated with expression of Lpro, yielding two stable products, p85 and p57. In silico search of putative L targets within Gemin5 identified two sequences whose potential recognition was in agreement with proteolysis products observed in infected cells. Mutational analysis revealed a novel Lpro target sequence that included the RKAR motif. Confirming this result, the Fas-ligand Daxx, was proteolysed in FMDV-infected and Lpro-expressing cells. This protein carries a RRLR motif whose substitution to EELR abrogated Lpro recognition. Thus, the sequence (R)(R/K)(L/A)(R) defines a novel motif to identify putative targets of Lpro in host factors

Identification of Gemin5 as a Novel 7-Methylguanosine Cap-Binding Protein

A unique attribute of RNA molecules synthesized by RNA polymerase II is the presence of a 7-methylguanosine (m(7)G) cap structure added co-transcriptionally to the 5' end. Through its association with trans-acting effector proteins, the m(7)G cap participates in multiple aspects of RNA metabolism including localization, translation and decay. However, at present relatively few eukaryotic proteins have been identified as factors capable of direct association with m(7)G.Employing an unbiased proteomic approach, we identified gemin5, a component of the survival of motor neuron (SMN) complex, as a factor capable of direct and specific interaction with the m(7)G cap. Gemin5 was readily purified by cap-affinity chromatography in contrast to other SMN complex proteins. Investigating the underlying basis for this observation, we found that purified gemin5 associates with m(7)G-linked sepharose in the absence of detectable eIF4E, and specifically crosslinks to radiolabeled cap structure after UV irradiation. Deletion analysis revealed that an intact set of WD repeat domains located in the N-terminal half of gemin5 are required for cap-binding. Moreover, using structural modeling and site-directed mutagenesis, we identified two proximal aromatic residues located within the WD repeat region that significantly impact m(7)G association.This study rigorously identifies gemin5 as a novel cap-binding protein and describes an unprecedented role for WD repeat domains in m(7)G recognition. The findings presented here will facilitate understanding of gemin5's role in the metabolism of non-coding snRNAs and perhaps other RNA pol II transcripts

Causes and Consequences of Flavivirus RNA Methylation

Mosquito-borne flaviviruses are important human pathogens that represent global threats to human health. The genomes of these positive-strand RNA viruses have been shown to be substrates of both viral and cellular methyltransferases. N7-methylation of the 5′ cap structure is essential for infection whereas 2′-O-methylation of the penultimate nucleotide is required for evasion of host innate immunity. N6-methylation of internal adenosine nucleotides has also been shown to impact flavivirus infection. Here, I summarize recent progress made in understanding roles for methylation in the flavivirus life-cycle and discuss relevant emerging hypotheses

Mapping of determinants within gemin5 required for association with m⁷G-sepharose.

<p>(A) Multiple C-terminal truncations and a single N-terminal truncation of FLAG-tagged gemin5 were evaluated by cap-affinity chromatography. Deletion sites within gemin5 are indicated above by amino acid number. (B) FLAG-tagged gemin5, gemin5(Δ25) variant, and Ago2 expression constructs were transfected into 293T cells along with pcDNA3 alone (M). Input samples used for co-IP assays were analyzed by detection of gemin3 and over-expressed FLAG-tagged proteins (left). Each lysate was subjected to IP using α-FLAG antibody and precipitate samples were analyzed for the presence of gemin3 and gemin4. Positions of heavy (H) and light (L) antibody chains are indicated.</p

Gemin5 mutations that affect m⁷G interaction reduce association with U1 snRNA.

<p>(A) Gemin5-FLAG and the indicated variants were transiently expressed in 293T cells and then immunoprecipitated with α-FLAG antibody or negative control mouse IgG. A 10% fraction of IP samples along with input samples were subjected to α-FLAG western blot. The asterisk indicates a cross-reactive band that serves as a loading control. The remaining IPs were used for RNA extraction. (B) RT-qPCR was performed on extracted RNAs for measurements of U1 and U6 snRNA levels in positive and negative IP samples for each gemin5 variant. Error bars indicate values for standard deviation.</p

Identification of amino acid residues that affect m⁷G recognition by gemin5.

<p>(A) PHYRE analysis was used to model the gemin5 WD repeat domains onto the structure of actin-interacting protein 1 (AIP-1; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007030#s4" target="_blank">Materials and Methods</a>). The backbone of the AIP-1 structure, consisting of two β-propellers, is shown with indicated locations of amino acids highlighted in yellow. A 90° x-axis rotation of the left structure is shown at right. Arrows indicate positions of W286 (red), F304 (yellow), F338 (blue) and the N-terminal 25 amino acids of AIP-1 (green). Note that the N-terminus of AIP-1 forms a β-sheet in the last WD repeat domain of the second β-propeller before looping into the first β-propeller to form another β-sheet. PHYRE analysis predicts only the second β-sheet in gemin5. AIP-1 structures were visualized using Cn3D <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007030#pone.0007030-Keene1" target="_blank">[59]</a>. (B) Alignments of gemin5 sequences from selected vertebrate species is shown. Bold letters in the human sequence indicate uniform conservation and positions of residues 286, 304 and 338 are indicated. (C) Cap-binding assays were performed with wild-type gemin5-FLAG and variants as in previous figures. Input (i), supernatant (s), and precipitate (ppt) samples are indicated.</p

Gemin5 isolated by cap-affinity is not bound to the SMN complex.

<p>(A) Schematic representation of gemin5 and eIF4E indicating locations of WD repeats, the putative coiled coil domain, and epitope tags. (B) 293T cells were co-transfected with expression constructs encoding FLAG-tagged gemin5 and myc-tagged eIF4E. Cell lysate was derived 24 hours later and applied to cap-sepharose 4B or sepharose 4B. Tagged proteins were detected with α-FLAG and α-myc antibodies. (C) Purification of proteins by cap-affinity was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007030#pone-0007030-g001" target="_blank">figure 1A</a> and levels of endogenous gemin3–5 were examined in input, supernatant and precipitate samples.</p