RESUMEN
Lowering expression of prion protein (PrP) is a well-validated therapeutic strategy in prion disease, but additional modalities are urgently needed. In other diseases, small molecules have proven capable of modulating pre-mRNA splicing, sometimes by forcing inclusion of cryptic exons that reduce gene expression. Here, we characterize a cryptic exon located in human PRNP's sole intron and evaluate its potential to reduce PrP expression through incorporation into the 5' untranslated region. This exon is homologous to exon 2 in nonprimate species but contains a start codon that would yield an upstream open reading frame with a stop codon prior to a splice site if included in PRNP mRNA, potentially downregulating PrP expression through translational repression or nonsense-mediated decay. We establish a minigene transfection system and test a panel of splice site alterations, identifying mutants that reduce PrP expression by as much as 78%. Our findings nominate a new therapeutic target for lowering PrP.
Asunto(s)
Exones , Proteínas Priónicas , Sitios de Empalme de ARN , Humanos , Proteínas Priónicas/metabolismo , Proteínas Priónicas/genética , Empalme del ARN , Intrones , Regulación de la Expresión Génica , Animales , Priones/metabolismo , Priones/genética , Enfermedades por Prión/metabolismo , Enfermedades por Prión/genética , Regiones no Traducidas 5'RESUMEN
Lowering expression of prion protein (PrP) is a well-validated therapeutic strategy in prion disease, but additional modalities are urgently needed. In other diseases, small molecules have proven capable of modulating pre-mRNA splicing, sometimes by forcing inclusion of cryptic exons that reduce gene expression. Here, we characterize a cryptic exon located in human PRNP's sole intron and evaluate its potential to reduce PrP expression through incorporation into the 5' untranslated region (5'UTR). This exon is homologous to exon 2 in non-primate species, but contains a start codon that would yield an upstream open reading frame (uORF) with a stop codon prior to a splice site if included in PRNP mRNA, potentially downregulating PrP expression through translational repression or nonsense-mediated decay. We establish a minigene transfection system and test a panel of splice site alterations, identifying mutants that reduce PrP expression by as much as 78%. Our findings nominate a new therapeutic target for lowering PrP.