BackgroundExpression of the tumor suppressor p16INK4a increases during aging and replicative senescence.Methodology/Principal FindingsHere, we report that the microRNA miR-24 suppresses p16 expression in human diploid fibroblasts and cervical carcinoma cells. Increased p16 expression with replicative senescence was associated with decreased levels of miR-24, a microRNA that was predicted to associate with the p16 mRNA coding and 3′-untranslated regions. Ectopic miR-24 overexpression reduced p16 protein but not p16 mRNA levels. Conversely, introduction of antisense (AS)-miR-24 blocked miR-24 expression and markedly enhanced p16 protein levels, p16 translation, and the production of EGFP-p16 reporter bearing the miR-24 target recognition sites.Conclusions/SignificanceTogether, our results suggest that miR-24 represses the initiation and elongation phases of p16 translation.
Prion diseases are irreversible neurodegenerative disorders caused by the aggregated form of prion protein (PrPSc) derived from the normal form of prion protein (PrPC). Previous studies have reported that shadow of prion protein (Sho) interacts with prion protein (PrP) and accelerates the conversion of PrPC to PrPSc. In addition, genetic polymorphisms of the shadow of the prion protein gene (SPRN) are related to the vulnerability of prion diseases in various hosts. However, to date, polymorphisms and genetic features of the SPRN gene have not been investigated in chickens, which are prion disease-resistant animals. We investigated genetic polymorphisms of the SPRN gene in 2 breeds of chickens, i.e., Dekalb White and Ross, using amplicon sequencing. We analyzed genotype, allele and haplotype frequencies and linkage disequilibrium (LD) among the genetic polymorphisms. In addition, we compared the amino acid sequences of Sho among several prion-related species to identify the unique genetic features of chicken Sho using ClustalW. Furthermore, we evaluated the N-terminal signal peptide and glycosylphosphatidylinositol (GPI)-anchor using SignalP and PredGPI, respectively. Finally, we compared the number of SPRN polymorphisms between prion disease-resistant and prion disease-susceptible animals. We identified 7 novel single nucleotide polymorphisms (SNPs), including 1 synonymous SNP in the open reading frame (ORF) of the chicken SPRN gene. We also found significantly different genotypes, allele frequencies and haplotypes between the 2 chicken breeds. In addition, we found that the interaction regions between Sho and PrP and the NXT glycosylation motif were conserved among all species. Notably, sequence similarity was extremely low in the N-terminal and C-terminal regions between mammals and chickens. Furthermore, we found that chicken Sho was the longest N-terminal signal peptide, and the amino acids of the cutting site of chicken are different from those of mammals. Last, unlike other species investigated, omega-site and signal sequences of the GPI-anchor were not found in chickens. To the best of our knowledge, this is the first report of genetic polymorphisms of the SPRN gene in chickens.
Prion diseases are fatal neurodegenerative disorders characterized by vacuolation and gliosis in the brain. Prion diseases have been reported in several mammals, and genetic polymorphisms of the prion protein gene (PRNP) play an essential role in the vulnerability of prion diseases. However, to date, investigations of PRNP polymorphisms are rare in cats, which are the major host of feline spongiform encephalopathy (FSE). Thus, we investigated the genetic polymorphisms of the cat PRNP gene and analyzed the structural characteristics of the PrP of cats compared to those of dog, prion disease-resistant animal. To investigate the genetic variations of the cat PRNP gene in 208 cats, we performed amplicon sequencing and examined the genotype, allele and haplotype frequencies of cat PRNP polymorphisms. We evaluated the influence of cat PRNP polymorphisms using PolyPhen-2, PANTHER, PROVEAN and AMYCO. In addition, we carried out structural analysis of cat PrP according to the allele of nonsynonymous single nucleotide polymorphism (SNP) (c.457G > A, Glu153Lys) using Swiss-PdbViewer. Finally, we compared the structural differences between cat and canine PrPs for SNPs associated with prion disease resistance in dogs. We identified a total of 15 polymorphisms, including 14 novel SNPs and one insertion/deletion polymorphism (InDel). Among them, Glu153Lys was predicted to affect the structural stability and amyloid propensity of cat PrP. In addition, asparagine at codon 166 of cat PrP was predicted to have longer hydrogen bond than aspartic acid at codon 163 of canine PrP. Furthermore, substitution to dog-specific amino acids in cat PrP showed an increase in structural stability. To the best of our knowledge, this is the first study regarding the structural characteristics of cat PRNP gene.
Prion diseases are fatal infectious neurodegenerative disorders that are induced by misfolded prion protein (PrPSc). Previous studies have reported that the shadow of prion protein (Sho) encoded by the shadow of prion protein gene (SPRN) plays a critical role in stimulating the conversion process of normal PrP (PrPC) into PrPSc, and genetic polymorphisms of the SPRN gene are significantly related to susceptibility to prion diseases. Recent studies have reported that dogs show prion resistance, and there have been several attempts to identify resistance factors to prion diseases in dogs. However, there has been no study of the canine SPRN gene thus far. We investigated genetic polymorphisms of the canine SPRN gene in 201 dogs using amplicon sequencing and compared the number of SPRN polymorphisms among prion-related species. In addition, we performed multiple sequence alignments of the amino acid sequences of Sho among prion-related species by ClustalW and analyzed the 3D structure of Sho using AlphaFold. Furthermore, we assessed the protein–protein interaction of canine PrP with canine Sho carrying wild-type and mutant alleles using HawkDock. We found four novel insertion/deletion polymorphisms of the SPRN gene in 201 dogs and identified a significant difference in the number of SPRN polymorphisms between prion-susceptible and prion-resistant animals. In addition, Sho has two α-helixes linked with the coil. Furthermore, we found different binding complexes and binding free energies between canine Sho and PrP according to SPRN polymorphisms. To the best of our knowledge, this is the first report of canine SPRN polymorphisms.
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