Abstract
Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels.
Highlights
Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications
We reasoned that polyA tracks, because of their versatility in lengths and sequence composition, can be used as a system to create programmable hypomorphic mutants and regulate gene expression in a wide variety of model organisms (Fig. 1)
The length and the sequence of the polyA track can be manipulated to achieve full range of expression levels, allowing for the generation of an allelic series from the level of a complete knockout to wild type expression for the study of gene function. This method can be used in synthetic biology applications that require precise gene control and modelling of metabolic and signalling networks[14,15]
Summary
Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. For genes that function in multiple cellular/developmental processes and have pleiotropic null mutant phenotypes, it can be difficult to distinguish primary from secondary effects In many of these cases, partial loss of function or hypomorphic mutations can overcome lethality and pleiotropy, allowing later stage cells and organisms to be examined for phenotypic consequences. These difficulties and the importance of hypomorphic alleles have prompted the development of several methods to generate hypomorphic mutations directly in various model organisms[2,3,4,5,6,7,8,9,10,11] These methods again are usually specific to one organism, can have unpredictable alterations in gene activity (separation of function, gain of function), or can change other aspects of regulation that affect interpretation of phenotype, such as spatial-temporal gene expression. Insertion of consecutive adenosine nucleotides into the open reading frame of an mRNA will decrease protein expression by decreasing the efficiency of the translation elongation phase leading to diminished production of protein and mRNA destabilization, and to diminished mRNA levels
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