Alternative polyadenylation signals regulate HLA-A surface expression

Genomic variations in the 5′ and 3′ untranslated region (UTR) have been shown to influence Human leukocyte antigen (HLA) class I expression, the strength of immune responses and disease outcomes beyond peptide binding. Sequencing of the 3′UTR of common HLA-A alleles indicated a presence of three polyadenylation signals (PAS). The proximal and the middle PAS are conserved whereas the distal PAS is disrupted in some alleles. Using 3′ rapid amplification of cDNA ends (3′RACE), we confirmed expression of two distinct forms of HLA-A 3′UTR that use the proximal or the distal PAS and differ in length by 100 base pairs. HLA-A alleles varied in the usage of PAS, with some alleles using only the proximal, and others using both the PAS to differing degrees. The short and long 3′UTRs cloned in luciferase vectors and expressed in the human T cell line-Jurkat showed similar mRNA expression levels. However, the long 3′UTR conferred lower luciferase expression as compared to the short form, indicating translation inhibition of the long 3′UTR. RNA affinity pulldown followed by mass-spectrometric analysis indicated enrichment of an RNA-binding protein (RBP), Syncrip, among proteins bound to the long 3′UTR. Only the long form of cellular HLA-A 3′UTR co-precipitated with Syncrip. The siRNA depletion of Syncrip increased surface expression of HLA-A alleles with the long 3′UTR, whereas an allele expressing only the short form was unaffected. Further, specific blocking of the proximal 3′UTR reduced surface expression without a decrease in mRNA expression. These data demonstrate allele-specific variation in the PAS usage by HLA-A modulated its expression, which adds another layer of regulation and variation to the expression of HLA-A.