Cryptic prokaryotic promoters explain instability of recombinant neuronal sodium channels in bacteria

Jean-Marc Dekeyser,Christopher H Thompson,Alfred L George

doi:10.1016/j.jbc.2021.100298

Jean-Marc Dekeyser, Christopher H Thompson + Show 1 more

Open Access

https://doi.org/10.1016/j.jbc.2021.100298

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Abstract

Mutations in genes encoding the human-brain-expressed voltage-gated sodium (NaV) channels NaV1.1, NaV1.2, and NaV1.6 are associated with a variety of human diseases including epilepsy, autism spectrum disorder, familial migraine, and other neurodevelopmental disorders. A major obstacle hindering investigations of the functional consequences of brain NaV channel mutations is an unexplained instability of the corresponding recombinant complementary DNA (cDNA) when propagated in commonly used bacterial strains manifested by high spontaneous rates of mutation. Here, using a combination of in silico analysis, random and site-directed mutagenesis, we investigated the cause for instability of human NaV1.1 cDNA. We identified nucleotide sequences within the NaV1.1 coding region that resemble prokaryotic promoter-like elements, which are presumed to drive transcription of translationally toxic mRNAs in bacteria as the cause of the instability. We further demonstrated that mutations disrupting these elements mitigate the instability. Extending these observations, we generated full-length human NaV1.1, NaV1.2, and NaV1.6 plasmids using one or two introns that interrupt the latent reading frames along with a minimum number of silent nucleotide changes that achieved stable propagation in bacteria. Expression of the stabilized sequences in cultured mammalian cells resulted in functional NaV channels with properties that matched their parental constructs. Our findings explain a widely observed instability of recombinant neuronal human NaV channels, and we describe re-engineered plasmids that attenuate this problem.

Highlights

Introduction of the cassette3 stabilizing mutations into the full-length NaV1.1 complementary DNA (cDNA) did not stabilize the entire plasmid, and we suspected the existence of other cryptic promoter-like elements elsewhere in the sequence
We investigated causes of the bacterial instability of a recombinant human NaV1.1 cDNA (NCBI accession NM_001165963) by determining the stability of individual cloned subfragments (Fig. 1A)
The mutation disrupted a putative -35 box of a predicted Sigma 70 factor promoter-like sequence (Fig. 2B). These findings suggested that stop codons in the first two clones truncated a toxic reading frame translated from mRNA transcribed by the promoter-like sequence that was disrupted by the nonsynonymous mutation in the third clone

Summary

Introduction

Introduction of the cassette3 stabilizing mutations into the full-length NaV1.1 cDNA did not stabilize the entire plasmid, and we suspected the existence of other cryptic promoter-like elements elsewhere in the sequence. We investigated causes of the bacterial instability of a recombinant human NaV1.1 cDNA (NCBI accession NM_001165963) by determining the stability of individual cloned subfragments (Fig. 1A).

Results

Conclusion