Abstract
BackgroundConnexin 43 (Cx43), the most widely expressed gap junction protein, is associated with a number of physiological and pathological conditions. Many functions of Cx43 have been shown to be independent of gap junction formation and only require the expression of Cx43 C-terminal fragments. Recent evidence demonstrated that naturally occurring C-terminal isoforms can be generated via internal translation.FindingsHere, we confirm that C-terminal domains of Cx43, particularly the major 20-kDa isoform, can be independently generated and regulated by internal translation of the same single GJA1 gene transcript that encodes full-length Cx43. Through direct RNA transfection experiments, we provide evidence that internal translation is not due to a bona fide cap-independent IRES-mediated mechanism, as upstream ribosomal scanning or translation is required. In addition to the mTOR pathway, we show for the first time, using both inhibitors and cells from knockout mice, that the Mnk1/2 pathway regulates the translation of the main 20-kDa isoform.ConclusionsInternal translation of the Cx43 transcript occurs but is not cap-independent and requires translation upstream of the internal start codon. In addition to the PI3K/AKT/mTOR pathway, the major 20-kDa isoform is regulated by the Mnk1/2 pathway. Our results have major implications for past and future studies describing gap junction-independent functions of Cx43 in cancer and other pathological conditions. This study provides further clues to the signalling pathways that regulate internal mRNA translation, an emerging mechanism that allows for increased protein diversity and functional complexity from a single mRNA transcript.
Highlights
All other cell lines were obtained from ATCC and cultured as recommended in DMEM with 10% FBS and 1% penicillin/streptomycin
Mission endoribonuclease-prepared siRNA control and esiRNA targeting the entire GJA1 coding region were transfected with RNAiMax according to the recommended protocol (Life Technologies) using 1000ng esiRNA per well in 6-well plates
Hsa-miR-1, has-miR-206 and –ve control miR-mimick were obtained from Ambion (#4464066, Life Technologies) and transfected with RNAiMax to a final concentration of 25 nM
Summary
Cell culture and treatments Primary human keratinocytes and fibroblasts were obtained and cultured as previously described [1]. All other cell lines were obtained from ATCC and cultured as recommended in DMEM with 10% FBS and 1% penicillin/streptomycin. Mission endoribonuclease-prepared siRNA (esiRNA) control and esiRNA targeting the entire GJA1 coding region (obtained from Sigma) were transfected with RNAiMax according to the recommended protocol (Life Technologies) using 1000ng esiRNA per well in 6-well plates.
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