Genomic determinants of the efficiency of internal ribosomal entry sites of viral and cellular origin

Abstract

Variation in cellular gene expression levels has been shown to be inherited. Expression is controlled at transcriptional and post-transcriptional levels. Internal ribosome entry sites (IRES) are used by viruses to bypass inhibition of cap-dependent translation, and by eukaryotic cells to control translation under conditions when protein synthesis is inhibited. We aimed at identifying genomic determinants of variability in IRES-mediated translation of viral [Encephalomyocarditis virus (EMCV)] and cellular IRES [X-linked inhibitor-of-apoptosis (XIAP) and c-myc]. Bicistronic lentiviral constructs expressing two fluorescent reporters were used to transduce laboratory and B lymphoblastoid cell lines [15 CEPH pedigrees (n = 205) and 50 unrelated individuals]. IRES efficiency varied according to cell type and among individuals. Control of IRES activity has a significant genetic component (h(2) of 0.47 and 0.36 for EMCV and XIAP, respectively). Quantitative linkage analysis identified a suggestive locus (LOD 2.35) on chromosome 18q21.2, and genome-wide association analysis revealed of a cluster of SNPs on chromosome 3, intronic to the FHIT gene, marginally associated (P = 5.9E-7) with XIAP IRES function. This study illustrates the in vitro generation of intermediate phenotypes by using cell lines for the evaluation of genetic determinants of control of elements such as IRES.

Figure 1.

IRES activity in cell lines of diverse origins. IRES activity was estimated as the ratio between eGFP expression (second cistron) and mRFP (first cistron) in the transduced population and normalized by values from mock transduced cells. Each panel is representative for one of two independent experiments performed in triplicate. Bars, SEM; a.u., arbitrary units.

Figure 2.

GFP expression requires an intact XIAP IRES. (A) PCR amplification using primers SG-1667 and SG-1669 on DNA constructs containing an intact XIAP IRES or a spliced XIAP IRES. (B) Fluorescence microscopy showing the expression of mRFP and eGFP in cells transfected with the construct containing an intact XIAP IRES or a spliced XIAP IRES. (C) PCR amplification using primers SG-1667 and MA.pr-216 (XIAP IRES specific) on DNA constructs (left panel) and donor cDNAs (right panel), showing the presence of an intact XIAP IRES in all cell lines.

Figure 3.

EMCV and XIAP IRES activity. Distribution of IRES activity values for EMCV (A), and XIAP (B) in transduced CEPH cell lines.

Figure 4.

Genome scan and association analyses. (A) Genome-wide linkage analysis results in 15 CEPH families. Horizontal lines show significance thresholds as determined by multiple permutations. Dashed line shows 90% and dotted line 95% significance threshold, respectively. (B) Genome-wide association analysis using the 2.2 million HapMap SNPs.