The HLA-C locus is distinct relative to the other classical HLA class I loci in that it has relatively limited polymorphism1, lower expression on the cell surface2, 3, and more extensive ligand–receptor interactions with killer-cell immunoglobulin-like receptors4. A single nucleotide polymorphism (SNP) 35 kb upstream of HLA-C (rs9264942; termed −35) associates with control of HIV5, 6, 7, and with levels of HLA-C messenger RNA transcripts8 and cell-surface expression7, but the mechanism underlying its varied expression is unknown. We proposed that the −35 SNP is not the causal variant for differential HLA-C expression, but rather is marking another polymorphism that directly affects levels of HLA-C7. Here we show that variation within the 3′ untranslated region (UTR) of HLA-C regulates binding of the microRNA hsa-miR-148 to its target site, resulting in relatively low surface expression of alleles that bind this microRNA and high expression of HLA-C alleles that escape post-transcriptional regulation. The 3′ UTR variant associates strongly with control of HIV, potentially adding to the effects of genetic variation encoding the peptide-binding region of the HLA class I loci. Variation in HLA-C expression adds another layer of diversity to this highly polymorphic locus that must be considered when deciphering the function of these molecules in health and disease.
MicroRNAs (miRNAs) are a class of non-protein-coding RNAs that are estimated to regulate 30% of all genes in animals9 by binding to specific sites in the 3′ UTR, resulting in post-transcriptional repression, cleavage or destabilization10, 11, 12. The 3′ UTR of the HLA-C gene is predicted to be a target for 26 distinct human miRNAs using three miRNA-target-prediction programs (Supplementary Fig. 1), of which three (miR-148a and miR-148b, which bind the same target site, and miR-657) were shown to have the greatest likelihood of binding. We sequenced the 3′ UTRs of the common HLA-C alleles (Supplementary Fig. 2) and show that the two binding sites of these three miRNAs are polymorphic (Supplementary Fig. 3a). The binding site for miR-148a/miR-148b contains a single base pair insertion/deletion at position 263 downstream of the HLA-C stop codon (rs67384697G representing the insertion (263ins) and rs67384697− representing the deletion (263del)) along with other precisely linked variants (259C/T, 261T/C, 266C/T). These variants are likely to impose a restriction in miR-148a/miR-148b binding, as prediction algorithms indicate that the binding of these miRNAs to the alleles marked by 263ins (for example, Cw*0702, a low-expression allotype) is more stable than to alleles with 263del (for example, Cw*0602, a high-expression allotype) (Supplementary Fig. 3b). Similarly, alleles with 307C within the miR-657 target site are predicted to be better targets of miR-657 than those with 307T (Supplementary Fig. 4). Thus, variation in the 3′ UTR of HLA-C may influence the interaction between these miRNAs and their putative binding sites in an allele-specific manner, potentially leading to differential levels of HLA-C allotype expression.
To test directly whether the variation in the HLA-C 3′ UTR affects levels of protein expression, the full-length 3′ UTRs containing intact miR-148a/miR-148b- and miR-657-binding sites (that is, 263ins and 307C, respectively; Cw*0702, Cw*0303, Cw*0401, Cw*0701) and disrupted binding sites (that is, 263del and 307T, respectively; Cw*0602, Cw*0802, Cw*1203, Cw*1502) were each cloned downstream of the luciferase gene in a pGL3 reporter construct (Fig. 1a). The constructs were then transfected into HLA class I negative B721.221 cells, and the level of luciferase activity was measured (fold increase of relative light units). Although the Cw*0602 3′ UTR repressed luciferase activity as compared to the control containing no 3′ UTR, the constructs containing intact miRNA-binding sites (that is, 263ins and 307C; Cw*0702, Cw*0303, Cw*0401, Cw*0701) produced significantly lower luciferase activity relative to the construct containing the 3′ UTR of Cw*0602, which contains 263del and 307T (Fig. 1b). However, 3′ UTRs from other alleles with the 263del and 307T variants (Cw*0802, Cw*1203, Cw*1502) did not show significant variation in luciferase activity as compared to Cw*0602 (Fig. 1b). Psicheck2 reporter constructs containing 3′ UTRs of Cw*0602 also produced significantly higher luciferase activity as compared to those with Cw*0702 3′ UTR (Supplementary Fig. 5a), indicating that this effect was reproducible in a distinct reporter construct. Further, pGL3 constructs containing 3′ UTRs of Cw*0602 and Cw*0702 in three additional cell lines showed the same pattern as that seen in B721.221 cells, indicating a consistent difference of these 3′ UTRs in the regulation of HLA-C expression that is independent of cell type (Supplementary Fig. 5b–e). Thus, HLA-C 3′ UTR alleles characterized by variation at positions 263 and 307 within miRNA-binding regions differentially regulate gene expression.
To test directly whether the variation in the HLA-C 3′ UTR affects levels of protein expression, the full-length 3′ UTRs containing intact miR-148a/miR-148b- and miR-657-binding sites (that is, 263ins and 307C, respectively; Cw*0702, Cw*0303, Cw*0401, Cw*0701) and disrupted binding sites (that is, 263del and 307T, respectively; Cw*0602, Cw*0802, Cw*1203, Cw*1502) were each cloned downstream of the luciferase gene in a pGL3 reporter construct (Fig. 1a). The constructs were then transfected into HLA class I negative B721.221 cells, and the level of luciferase activity was measured (fold increase of relative light units). Although the Cw*0602 3′ UTR repressed luciferase activity as compared to the control containing no 3′ UTR, the constructs containing intact miRNA-binding sites (that is, 263ins and 307C; Cw*0702, Cw*0303, Cw*0401, Cw*0701) produced significantly lower luciferase activity relative to the construct containing the 3′ UTR of Cw*0602, which contains 263del and 307T (Fig. 1b). However, 3′ UTRs from other alleles with the 263del and 307T variants (Cw*0802, Cw*1203, Cw*1502) did not show significant variation in luciferase activity as compared to Cw*0602 (Fig. 1b). Psicheck2 reporter constructs containing 3′ UTRs of Cw*0602 also produced significantly higher luciferase activity as compared to those with Cw*0702 3′ UTR (Supplementary Fig. 5a), indicating that this effect was reproducible in a distinct reporter construct. Further, pGL3 constructs containing 3′ UTRs of Cw*0602 and Cw*0702 in three additional cell lines showed the same pattern as that seen in B721.221 cells, indicating a consistent difference of these 3′ UTRs in the regulation of HLA-C expression that is independent of cell type (Supplementary Fig. 5b–e). Thus, HLA-C 3′ UTR alleles characterized by variation at positions 263 and 307 within miRNA-binding regions differentially regulate gene expression.
Full article: http://www.nature.com/nature/journal/v472/n7344/full/nature09914.html
