Stop Codon Readthrough in VEGF-a Is Regulated by Synergistic Activity of a Complex Tripartite Signal

Abstract

Translational recoding via frameshifting or stop-codon readthrough is generally regulated by a structured mRNA signal downstream of the slippery sequence or stop-codon, respectively. The strength of this signal can be enhanced or attenuated by neighboring sequences, and in some rare cases, the signal is known to interact with sequences hundreds of nucleotides downstream in the 3’ region to regulate the level of recoding. In contrast, here we show that recoding of the VEGF-A mRNA is regulated by three distinct elements that can each independently support stop codon readthrough: one, the recently discovered Ax-element downstream of the stop codon; and two other elements, Au1 and Au2 in the coding region ~500 and ~250 nucleotides upstream of the stop codon, respectively. The Ax-element folds into three stem loops (SL-Ax1-3) while the Au elements are predicted to fold into single stem loops. Furthermore, we also show that the trans-acting protein hnRNP A2/B1 independently interacts with SL-Au1 and SL-Ax1 to confer efficient readthrough. While the Ax-element individually allows for only 0.2% readthrough, and each Au element for 0.25% readthrough levels, together they allow for a robust 2% readthrough level. Given that structural manipulations of SL-Au1 at the 5’ end the coding sequences led to a 16% readthrough activity of the stop codon at the 3’ end, the observed synergistic activity is potentially indicative of long-range interactions. Our study thus highlights both the control of absolute readthrough levels through strategic structures and the functional importance of maintenance of 5’ - 3’ end interactions during the process of active translation.