A novel structural mechanism of ribosomal stop codon readthrough in VEGF-A

Abstract

The process of ribosomal recoding is generally regulated by an autonomous mRNA signal downstream of stop-codons. While structural studies have provided mechanistic insights into viral systems, no such studies exist in mammalian systems. Here we define a novel structural mechanism for the VEGF-A readthrough system and show that regulation is multifaceted and complex, requiring a multipartite set of RNA elements located at long distances that interact with each other and with hnRNP A2/B1 to synergistically enhance readthrough levels. The Ax-element downstream of the stop codon adopts a unique multistem (SL-Ax1-3) architecture: SL-Ax1 interacts with hnRNP A2/B1, while SL-Ax2 interacts with an RNA element (SL-Au1) located ~500 nt upstream at the start of the coding sequence. SL-Au1 also independently binds to hnRNP A2/B1, which manipulates an equilibrium between alternate structures— from a sequestered bulge towards one that allows for the long-range interaction with SL-Ax2. Overall, our study not only highlights the significance of structural organization of elements within the coding sequence of mRNA, but also provides a functional relevance of the closed-loop mRNA organization in non-canonical translation and suggests complex mechanisms allow for finer integration of many signals for a required output.

Mammalian proteome expansion by stop codon readthrough

“…RNA secondary structure has also been predicted immediately downstream of the stop codon in the case of ACP2 (Loughran et al, 2017). SCR of VEGFA has been recently shown to be driven by long‐range interactions of a multipartite set of RNA elements with each other, and with an RNA‐binding protein called heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 (Wagner, 2019).…”

“…SL‐Ax 1 interacts with hnRNP A2/B1 and SL‐Ax 2 interacts with another stem‐loop structure near the 5′ end of the coding sequence of VEGFA . These interactions are critical for VEGFA SCR as mutations that abrogate stem‐loop formation, or their interactions, reduce or abolish SCR (Wagner, 2019). This study shows the importance of including coding sequence (in addition to the proximal part of the 3′UTR) while screening or testing for SCR.…”

Transcript-specific induction of stop codon readthrough using CRISPR-dCas13 system

“…Therefore, there is a need for a strategy that can induce SCR in a transcriptspecific manner. hnRNPA2/B1, an RNA binding protein, binds downstream of the stop codon (9 nucleotides downstream) and positively regulates the SCR of VEGFA (Eswarappa et al, 2014;Nico et al, 2021). Similarly, let-7a microRNA binds downstream of the stop codon of AGO1 mRNA and enhances its SCR (Singh et al, 2019).…”