Effectiveness of near-cognate amino acid insertion for KCNJ13 nonsense suppression

Abstract

Purpose :
We have recently found that a substitution of nt 295 in exon 2 region of KCNJ13 gene, coding region c.158G>A, results in a non-sense mutation at aa position 53 (W53X). This gene encodes an inwardly rectifying potassium channel (Kir7.1) which is functional in retinal pigment epithelium (RPE). The truncated protein product (W53X) is non-functional and cause Leber Congenital Amaurosis type 16 (LCA16). Using a selection of read-through drugs that insert a near-cognate amino acid at the mutation site, we have previously shown functional rescue of Kir7.1 protein in both heterologous expression system and patient derived iPSC-RPE cells. In this study, we used site-directed mutagenesis to determine the effectiveness of nonsense suppression by near-cognate tRNAs on Kir7.1 function[.

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Methods :
We performed site-directed mutagenesis to generate Tyr (TAC), Ser (TCG), Glu (GAG), and Gln (CAG) at position 53 in GFP-fused Kir7.1 ORF carrying a TAG mutation. The clones were subjected to Sanger sequencing and plasmid purification. We used HEK293T cells for transient expression of the mutant proteins. Protein localization was determined by both Confocal microscopy and Western blot. Kir7.1 current measurement using whole-cell patch-clamp of GFP expressing cells is underway.

Results : GFP-positive HEK-293 cells revealed which cells expressed the plasmids. Imaging cells live after labeling for the nucleus and membrane, showed clear membrane localization of protein in whichTyr, Ser, and Glu had been substituted Gln at 53 position showed non membrane localization similar to the W53X truncated protein product. Whereas a full length protein product of 67 kDa band was detected for Tyr, Ser, and Glu, only a shorter 35 kDa band for Gln was observed. Preliminary measurements of Kir7.1 current confirm that Gln is a non-functional channel.

Conclusions :
Read-through of W53X with drugs in both heterologous expression as well as patient derived iPSC-RPE is probably due to the insertion of a wild-type or near cognate amino acid. We further demonstrate that Tyr, Ser, or Glu at position 53 are preferred over Gln to produce a full length protein product with no trafficking defect. Gln does not form a full length protein product consistent with a favored nonstandard Watson-Crick mispairing (U-G) referring to UAG present at the A-site of the ribosome.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

Figure: Insertion of near-cognate aminoacids for W53X non-sense mutation.