A single mammalian transcript normally encodes one protein, but the transcript of GNAS (G-protein α-subunit) contains two reading frames and produces two structurally unrelated proteins, XLαs and ALEX. No other confirmed GNAS-like dual-coding transcripts have been reported to date, even though many such candidate genes have been predicted by bioinformatics analysis. In this study, we constructed a series of vectors to test how two protein products were translated from a single transcript in vitro. The length of the ORF (open reading frame), position of the first AUG and the Kozak motif were found to be important factors. These factors, as well as 55-bp NMD (nonsense-mediated mRNA decay) rule, were used in a bioinformatics search for candidate dual-coding transcripts. A total of 1307, 750 and 474 two-ORF-containing transcripts were found in human, mouse and rat, respectively, of which 170, 89 and 70, respectively, were found to be potential dual-coding transcripts. Most transcripts showed low conservation among species. Interestingly, dual-coding transcripts were significantly enriched for transcripts from the zinc-finger protein family, which are usually DNA-binding proteins involved in regulation of the transcription process.
In addition to roles in stress response, heat shock factors (HSFs) play crucial roles in differentiation and development. Heat shock transcription factor 4 (HSF4) deficiency leads to defect in lens epithelial cell (LEC) differentiation and cataract formation. However, the mechanism remains obscure. Here, we identified Src kinase-associated phosphoprotein 2 (SKAP2) as a downstream target of HSF4b and it was highly expressed at the anterior tip of lens elongating fibre cells in vivo. The HSF4-deficient lenses showed reduced SKAP2 expression and defects in actin reorganization. The disassembly of stress fibres and formation of cortical actin fibres are critical for the initiation of LEC differentiation. SKAP2 localized at actin-rich ruffles in human LECs (SRA01/04 cells) and knockdown SKAP2 using RNA interference impaired the disassembly of cellular stress fibres in response to fibroblast growth factor (FGF)-b. Overexpression of SKAP2, but not the N-terminal deletion mutant of SKAP2, induced the actin remodelling. We further found that SKAP2 interacted with the SH2 domain of non-catalytic region of tyrosine kinase adaptor protein 2 (NCK2) via its N-terminus. The complex of SKAP2-NCK2-F-actin accumulated at the leading edge of the lamellipodium, where FGF receptors and focal adhesion were also recruited. These results revealed an essential role for HSF4-mediated SKAP2 expression in the regulation of actin reorganization during lens differentiation, likely through a mechanism that SKAP2 anchors the complex of NCK2/focal adhesion to FGF receptors at the lamellipodium in lens epithelial cells.
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