TY - JOUR
T1 - A novel protein-RNA binding assay: functional interactions of the foot-and-mouth disease virus internal ribosome entry site with cellular proteins
AU - Stassinopoulos, I. A.
AU - Belsham, Graham
PY - 2001
Y1 - 2001
N2 - Translation initiation on foot-and-mouth disease virus (FMDV) RNA occurs by a cap-independent mechanism directed by a highly structured element (similar to 435 nt) termed an internal ribosome entry site (IRES). A functional assay to identify proteins that bind to the FMDV IRES and are necessary for FMDV IRES-mediated translation initiation has been developed. In vitro-transcribed polyadenylated RNAs corresponding to the whole or part of the FMDV IRES were immobilized on oligo-dT Dynabeads and used to deplete rabbit reticulocyte lysate (RRL) of IRES-binding proteins. Translation initiation factors elF4G, elF4A, and elF4B bound to the 3' domain of the FMDV IRES. Depletion of elF4G from RRL by this region of the FMDV IRES correlated with the loss of translational capacity of the RRL for capped, uncapped, and FMDV IRES-dependent mRNAs. However, this depleted RRL still supported hepatitis C virus IRES-directed translation. Poly (rC) binding protein-2 bound to the central domain of the FMDV IRES, but depletion of RRL with this IRES domain had no effect on FMDV IRES-directed translation initiation.
AB - Translation initiation on foot-and-mouth disease virus (FMDV) RNA occurs by a cap-independent mechanism directed by a highly structured element (similar to 435 nt) termed an internal ribosome entry site (IRES). A functional assay to identify proteins that bind to the FMDV IRES and are necessary for FMDV IRES-mediated translation initiation has been developed. In vitro-transcribed polyadenylated RNAs corresponding to the whole or part of the FMDV IRES were immobilized on oligo-dT Dynabeads and used to deplete rabbit reticulocyte lysate (RRL) of IRES-binding proteins. Translation initiation factors elF4G, elF4A, and elF4B bound to the 3' domain of the FMDV IRES. Depletion of elF4G from RRL by this region of the FMDV IRES correlated with the loss of translational capacity of the RRL for capped, uncapped, and FMDV IRES-dependent mRNAs. However, this depleted RRL still supported hepatitis C virus IRES-directed translation. Poly (rC) binding protein-2 bound to the central domain of the FMDV IRES, but depletion of RRL with this IRES domain had no effect on FMDV IRES-directed translation initiation.
U2 - 10.1017/S1355838201001170
DO - 10.1017/S1355838201001170
M3 - Journal article
SN - 1355-8382
VL - 7
SP - 114
EP - 122
JO - R N A
JF - R N A
IS - 1
ER -