Abstract
Synonymous variations, which are defined as codon substitutions that do not change the encoded amino acid, were previously thought to have no effect on the properties of the synthesized protein(s). However, mounting evidence shows that these “silent” variations can have a significant impact on protein expression and function and should no longer be considered “silent”. Here, the effects of six synonymous and six non-synonymous variations, previously found in the gene of ADAMTS13, the von Willebrand Factor (VWF) cleaving hemostatic protease, have been investigated using a variety of approaches. The ADAMTS13 mRNA and protein expression levels, as well as the conformation and activity of the variants have been compared to that of wild-type ADAMTS13. Interestingly, not only the non-synonymous variants but also the synonymous variants have been found to change the protein expression levels, conformation and function. Bioinformatic analysis of ADAMTS13 mRNA structure, amino acid conservation and codon usage allowed us to establish correlations between mRNA stability, RSCU, and intracellular protein expression. This study demonstrates that variants and more specifically, synonymous variants can have a substantial and definite effect on ADAMTS13 function and that bioinformatic analysis may allow development of predictive tools to identify variants that will have significant effects on the encoded protein.
Highlights
ADAMTS13 (A Disintegrin-like and Metalloprotease with Thrombospondin type-1 repeats, member-13) plays an integral role in vascular hemostasis by cleaving von Willebrand Factor (VWF) within intact blood vessels under shear stress [1]
Synonymous variant 354, independently of the length of the RNA fragment analyzed, was predicted to affect RNA stability to a similar extent, while synonymous variant 420 showed considerably different delta-delta G (DDG) values and trends associated with RNA fragments of different lengths (Figure 1AB)
Analysis of mRNA structure within the coding region is admittedly a yet-to-be developed research area, the predicted changes in DDGs may lead to change in mRNA stability and/or protein expression levels
Summary
ADAMTS13 (A Disintegrin-like and Metalloprotease with Thrombospondin type-1 repeats, member-13) plays an integral role in vascular hemostasis by cleaving von Willebrand Factor (VWF) within intact blood vessels under shear stress [1]. A deficiency in ADAMTS13 activity – either through inactivation by autoantibodies, lack of expression, or a genetic variation affecting function (these primarily include single site codon substitutions) – results in increased VWF thrombogenic potential. Single nucleotide polymorphisms (SNPs), originally defined as single site codon substitutions that occur in .1% of the population, are prevalent and are found across the entire human genome coding sequence, with few exceptions. Synonymous variants have been shown to affect mRNA splicing [5], mRNA stability [11] and/or mRNA secondary structure [12,13,14,15], translation efficiency and kinetics [16,17], protein folding [10,18,19], and protein function [18]
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