Fixed differences in the 3′UTR of buffalo PRNP gene provide binding sites for miRNAs post-transcriptional regulation

Bovine spongiform encephalopathy, a member of transmissible spongiform encephalopathies, has not been reported in buffaloes, Bubalus bubalis. Prion protein (PrP), encoded by the prion protein gene (PRNP), is fundamental in the pathogenesis of transmissible spongiform encephalopathies. We previously showed that cattle express more PrP proteins but lower PRNP mRNA than buffaloes in several pivotal tissues like the obex. Therefore, we sought to establish whether genetic variability in PRNP 3′UTR, mediated by miRNA down-regulation, causes PrP expression differences between cattle and buffaloes. We annotated the 3′UTR of buffalo PRNP gene by 3′RACE experiment. A total of 92 fixed differences in the complete 3′UTR (∼ 3 kb) were identified between 13 cattle and 13 buffaloes. Resequencing of UTR-C (g.786-1436) and UTR-B (g.778-1456) fragments confirmed that all mutations except g.1022T in cattle are fixed differences between 147 cattle and 146 buffaloes. In addition, analysis of the variation of ΔG between cattle and buffalo sequences reveals four remarkable differences. Two buffalo-specific insertion sites (a 28-bp insertion and an AG insertion in buffalo 3′UTR of PRNP g.970-997 and g. 1088-1089, respectively) and two mutants (g. 1007-1008 TG→CC) create compatible binding sites for miRNAs in buffalo 3′UTR. This was validated through luciferase reporter assays which demonstrated that miR-125b-5p, miR-132-3p, miR-145-5p, miR-331-3p, and miR-338-3p directly act on the fixed difference sites in buffalo 3′UTR. Additional expressional analyses show that these five miRNAs are coexpressed with PRNP in bovine obex tissues. Our study reveals a miRNAs regulated mechanism explaining the differences in prion expression between cattle and buffalo.