The Post-transcriptional Regulations Of Prion Protein Gene By MicroRNAs In Cattle And Buffalo

Prion diseases, also called as transmissible spongiform encephalopathies (TSEs) were detected in a wide range of species, such as bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and variant Creutzfeldt-Jakob disease (vCJD) in humans. All TSEs are characterized by the conversion of the normal host’s cellular prion protein (PrP) into an abnormal protease-resistant isoform (PrPSc) followed by its pathological accumulation in central nervous system (CNS).To date, BSE cases have occurred worldwide in nearly 0.2 millBIn Bos taurus cattle. Contrastively, BSE has never been documented in the domestic buffalo, Bubalus bubalis. Our previous studies have revealed that genetic differences of PRNP and SPRN between cattle and buffalo represent in risk for BSE. Moreover, we analyzed and compared the expression differences between cattle and buffalo at both the transcriptional (PRNP and SPRN) and the post-transcriptional level (PrPc and Sho) in three CNS tissues and three lymphoreticular system (LRS). The results have revealed that buffalo have significantly lower relative expression of PrP in the cerebellum, obex, mesenteric lymph node, and bronchial lymph node tissues, but higher relative expression of Sho in the cerebrum and spleen compared to cattle. Interestingly, PrP expressions did not correlate with corresponding mRNA expression, suggesting that the biological modulations of PrP proteins are at post-translational levels. Therefore, detailed investigation of the post-translational modulation of PrP is key to understand the pathogenesis and biology of prion diseases.An important way of regulating protein expression is the posttranslational regulation by microRNAs (miRNAs). miRNAs have been shown to play roles in many biological processes, such as development, proliferation, differentiation, apoptosis, and diseases. Growing evidence indicates that miRNAs are involved in the susceptibility and pathogenesis of prion diseases. In this study, we investigated the mechanism that the post-translational regulation of PRNP by miRNAs in cattle and buffalo. First, we annotated the PRNP 3’untranslated region (3’UTR) of buffalo by 3’rapid-amplification of cDNA ends (3’RACE) and sequencing. Second, we analyzed and compared the fixed differences of PRNP 3’ UTR between 12 cattle and 14 buffaloes. A total of 144 fixed differences were found between two species, interestingly a UTR-1 (g.67352-67379delâ†'28-bp insertion and g.67387-g.67388TGâ†'CC) and UTR-2(AG insertion) polymorphisms were only found in buffaloes. The two sites were further sequenced in 194 cattle and 162 buffaloes and 192 cattle and 174 buffaloes. The results showed that the frequencies of the UTR-1 andUTR-2 insertions were 80.24% and 96.01% in buffalo, respectively, but no insertion in both two sites was found in cattle. In silico analysis revealed that 17 miRNAs were identified to possibly regulate the two insertion sites of PRNP 3’UTR in buffalo. Functional luciferase-reporter experiments confirmed that 5 miRNAs significantly decreased PRNP expression, including miR-145-5P, miR-328-3P and miR-338-3P for the UTR-1 sequences and miR-125b-5P and miR-331-3P for the UTR-2. Lastly, we investigated the expression levels of the 5 miRNAs in obex tissues in cattle and buffalo. Among of the five miRNAs, the highest expression was miR125b-5p both in cattle and in buffalo. Interestingly, the expression level of miR331-3p in buffalo obex was significantly higher in cattle obex, suggesting that the PRNP expression differences between the two species may be regulated by miR331-3p.These findings may prove insight into better understanding the mechanisms of susceptibility to prion diseases, and prove drug discoveries or development.