Cotranslational association of mRNA encoding subunits of heteromeric ion channels

Abstract

Oligomers of homomeric voltage-gated potassium channels associate early in biogenesis as the nascent proteins emerge from the polysome. Less is known about how proteins emerging from different polysomes associate to form hetero-oligomeric channels. Here, we report that alternate mRNA transcripts encoding human ether-à-go-go-related gene (hERG) 1a and 1b subunits, which assemble to produce ion channels mediating cardiac repolarization, are physically associated during translation. We show that shRNA specifically targeting either hERG 1a or 1b transcripts reduced levels of both transcripts, but only when they were coexpressed heterologously. Both transcripts could be copurified with an Ab against the nascent hERG 1a N terminus. This interaction occurred even when translation of 1b was prevented, indicating the transcripts associate independent of their encoded proteins. The association was also demonstrated in cardiomyocytes, where levels of both hERG transcripts were reduced by either 1a or 1b shRNA, but native KCNE1 and ryanodine receptor 2 (RYR2) transcripts were unaffected. Changes in protein levels and membrane currents mirrored changes in transcript levels, indicating the targeted transcripts were undergoing translation. The physical association of transcripts encoding different subunits provides the spatial proximity required for nascent proteins to interact during biogenesis, and may represent a general mechanism facilitating assembly of heteromeric protein complexes involved in a range of biological processes.KCNH2 | hERG | cotranslational assembly | microtranslatome | hERG 1a/1b

Tail-anchored Protein Insertion in Mammals

“…Very recently, a phenomenon similar to the one observed here was reported for the two subunits of a heteromeric K ϩ channel expressed in cardiomyocytes, where silencing of one subunit caused a parallel decrease in the transcript for the other one (47). In that study, transcript stability was not investigated, but very interestingly, it was found that the two subunit mRNAs are physically associated and that this association is not dependent on the presence of the nascent-encoded proteins.…”

Potassium channels in the heart: structure, function and regulation

“…For example, subunit expression can be regulated by co‐translational association with RNA (Liu et al . ).…”

“…The mechanisms that determine the stoichiometric balance of different subunits are poorly understood, are often not recapitulated in heterologous expression systems, and may not be specified by amino acid sequence alone. J Physiol 595.7 For example, subunit expression can be regulated by co-translational association with RNA (Liu et al 2016).…”

Transmural gradients in ion channel and auxiliary subunit expression

“…2). Some potassium channels can begin to dimerize cotranslationally, before the subunits are released from the ribosome (Liu et al, 2016; Lu et al, 2001; Phartiyal et al, 2007; Robinson and Deutsch, 2005; Tu et al, 2000). The amino terminal domains of these channels, such as the T1 tetramerization domain of Kv channels, make the assembly process considerably faster and presumably also more efficient (Lu et al, 2001).…”

“…Multiple channel subunits can be translated from the same mRNA molecule in the polyribosome complex resulting in a high local concentration of identical subunits. This will tend to favor the assembly of homo-dimers, especially for those channels that can begin dimerization before the subunits are released from the ribosome (Liu et al, 2016; Lu et al, 2001; Phartiyal et al, 2007; Robinson and Deutsch, 2005; Tu et al, 2000). Block of homo-dimerization by the binding of auxiliary subunits could facilitate the formation of heteromultimers.…”