Repression-free utrophin-A 5'UTR variants

Abstract

Mutation in the dystrophin gene results Duchenne Muscular Dystrophy (DMD), an X-linked fatal neuromuscular disorder. Dystrophin deficiency can be compensated by upregulation of utrophin, an autosomal homologue of dystrophin. But the expression of utrophin in adults is restricted to myotendinous and neuromuscular junctions. Therefore utrophin upregulation throughout the muscle fiber can only be achieved if we understand regulatory mechanisms behind its expression. Utrophin-A 5'UTR mediated repression of translation was reported earlier. In this article, we present evidences of two transcript variants of utrophin-A that do not confer repression to the downstream reporter ORF in mouse myoblast C2C12 cells. These repression-free variants may be targeted for utrophin upregulation.

Keywords: 5′RACE; DMD; Utrophin.

Figure 1

Transcription Start Sites in utrophin A genomic region. (A) Schematic presentation of TSS. A and A´ are two splice variants of utrophin transcript produced from the utrophin-A promoter. The numbers after TSS represent distance of different TSS in nucleotide from start codon. (B) Positions of TSS in genomic sequence. The white arrow indicates the previously reported TSS and the grey arrow indicates the new TSS of utrophin A and A′. (C) Existence of TSS downstream of already reported TSS 490A. Utrophin-A mRNA copy number was determined with qPCR using different primer pairs. Amplicon positions are shown by black lines (X, Y, Z) in Figure 1 (A). Copy number normalized against utrophin-A coding region was plotted as mean ± SD (n=6 independent experiments). Student’s t test was used to analyze the data. The asterisk denotes P<0.0001. Difference present between copy number corresponding to amplicon X and Y confirmed subsistence of TSS between denoted amplicons. Z represents utrophin coding region amplicon used for normalisation. (D) Schematic presentation of reporter mRNAs. The utrophin-A 5′UTRs found in the 5′RACE experiment was placed at the upstream of firefly luciferase ORF in pGL3. In vitro transcribed capped, A₅₀ tailed reporter mRNA with 5′UTRs were transfected in C2C12 cells followed by estimation of reporter activity. Renilla reporter mRNA was used as transfection control. (E) Regulatory roles of different utrophin-A 5′UTRs. Two repression-free variants were identified. Results presented as mean ±SD (n=6 independent experiments). Student’s t test was done to analyze the data. The asterisk denotes the statistically significant (P< 0.0001) difference