TY - JOUR
T1 - HnRNPA1/UP1 Unfolds KRAS G-Quadruplexes and Feeds a Regulatory Axis Controlling Gene Expression
AU - Ferino, Annalisa
AU - Marquevielle, Julien
AU - Choudhary, Himanshi
AU - Cinque, Giorgio
AU - Robert, Coralie
AU - Bourdoncle, Anne
AU - Picco, Raffaella
AU - Mergny, Jean Louis
AU - Salgado, Gilmar F.
AU - Xodo, Luigi E.
N1 - Publisher Copyright:
© 2021 American Chemical Society.
PY - 2021/12/14
Y1 - 2021/12/14
N2 - Recent studies have proven that the genetic landscape of pancreatic cancer is dominated by the KRAS oncogene. Its transcription is controlled by a G-rich motif (called 32R) located immediately upstream of the TSS. 32R may fold into a G-quadruplex (G4) in equilibrium between two G4 conformers: G9T (TM = 61.2 °C) and G25T (TM = 54.7 °C). We found that both G4s bind to hnRNPA1 and its proteolytic fragment UP1, promoting several contacts with the RRM protein domains. 1D NMR analysis of DNA imino protons shows that, upon binding to UP1, G25T is readily unfolded at both 5′ and 3′ tetrads, while G9T is only partially unfolded. The impact of hnRNPA1 on KRAS expression was determined by comparing Panc-1 cells with two Panc-1 knockout cell lines in which hnRNPA1 was deleted by the CRISPR/Cas9 technology. The results showed that the expression of KRAS is inhibited in the knockout cell lines, indicating that hnRNPA1 is essential for the transcription of KRAS. In addition, the knockout cell lines, compared to normal Panc-1 cells, show a dramatic decrease in cell growth and capacity of colony formation. Pull-down and Western blot experiments indicate that conformer G25T is a better platform than conformer G9T for the assembly of the transcription preinitiation complex with PARP1, Ku70, MAZ, and hnRNPA1. Together, our data prove that hnRNPA1, being a key transcription factor for the activation of KRAS, can be a new therapeutic target for the rational design of anticancer strategies.
AB - Recent studies have proven that the genetic landscape of pancreatic cancer is dominated by the KRAS oncogene. Its transcription is controlled by a G-rich motif (called 32R) located immediately upstream of the TSS. 32R may fold into a G-quadruplex (G4) in equilibrium between two G4 conformers: G9T (TM = 61.2 °C) and G25T (TM = 54.7 °C). We found that both G4s bind to hnRNPA1 and its proteolytic fragment UP1, promoting several contacts with the RRM protein domains. 1D NMR analysis of DNA imino protons shows that, upon binding to UP1, G25T is readily unfolded at both 5′ and 3′ tetrads, while G9T is only partially unfolded. The impact of hnRNPA1 on KRAS expression was determined by comparing Panc-1 cells with two Panc-1 knockout cell lines in which hnRNPA1 was deleted by the CRISPR/Cas9 technology. The results showed that the expression of KRAS is inhibited in the knockout cell lines, indicating that hnRNPA1 is essential for the transcription of KRAS. In addition, the knockout cell lines, compared to normal Panc-1 cells, show a dramatic decrease in cell growth and capacity of colony formation. Pull-down and Western blot experiments indicate that conformer G25T is a better platform than conformer G9T for the assembly of the transcription preinitiation complex with PARP1, Ku70, MAZ, and hnRNPA1. Together, our data prove that hnRNPA1, being a key transcription factor for the activation of KRAS, can be a new therapeutic target for the rational design of anticancer strategies.
U2 - 10.1021/acsomega.1c05538
DO - 10.1021/acsomega.1c05538
M3 - Article
AN - SCOPUS:85120901094
SN - 2470-1343
VL - 6
SP - 34092
EP - 34106
JO - ACS Omega
JF - ACS Omega
IS - 49
ER -
