Abstract
Purpose :
mRNAs for opsin (RHO) are nonuniformly distributed in highly polarized photoreceptors and abundant within the inner segment of the nucleus, where they can be efficiently translated and trafficked to the phototransduction environment of the outer segment (Brann and Scott, FEBS 1986; 200(2):275-8); no RHO mRNA is distributed toward the synapse. A unidirectional transport may infer a trafficking signal within the RHO mRNA. We investigated properties of 5’ untranslated regions (5’UTRs) of a group (N=23) of rod opsin mRNAs from vertebrate species by examining homology, accessibility, & presence of known signal elements.
Methods :
Using Clustal Omega, we aligned sequences (seq) to identify areas of preserved homology across phylogeny. Multiparameter prediction of RNA accessibility (mppRNA), developed in Sullivan lab, was used to identify sites of 2D accessibility in RHO 5’UTRs (N=23); this process used vector outputs from MFold, SFold, and Oligowalk algorithms input to a bioinformatics model. G-quadruplex regions (GQRs) were identified by QGRS. Statistical analysis was done in Origin.
Results :
We found substantial seq homology across the RHO 5’UTRs (mean seq % identity = 81.40% +/- 13.74%). Areas of homology aligned with areas of highly predicted 2D accessibility. ANOVA of the 5’UTRs with human showed 16 seqs with insignificant differences of the means (p>0.05), indicating similar accessibility. 6 phylogenetically distant seqs showed significant differences of the means (p<0.05). GQRs were found in 15 out of 23 seqs and aligned with areas of predicted accessibility and homology. The region of conserved accessibility is substantial (~40 nt), which could be a platform for target knockdown by post-transcriptional gene silencing agents.
Conclusions :
These outcomes indicate that 5’UTRs may be a significant factor in the efficient and abundant expression of RHO in rod photoreceptors. Evolutionarily conserved homology implies function. Accessible regions and GQRs may support interaction with molecular partners (e.g., proteins, other nucleic acids) to support biological roles such as mRNA trafficking or stability. Experimental measures are needed to test these hypotheses. Accessible conserved regions may also support testing of unified knockdown therapeutics (e.g., ribozyme, RNAi, antisense) across multiple animal model species; RHO is a target for autosomal dominant RP and AMD.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.