Beta-actin mRNA-binding proteins associated with the cytoskeletal framework.
Association of mRNA with the cytoskeletal framework (CSK) is thought to play a strategic role in the placement of mRNA in the cytoplasm. However, the molecular determinants underlying mRNA/CSK association are completely unknown. To begin addressing this issue, we have employed a binding assay to identify proteins of the CSK compartment of NIH 3T3 cells that bind in-vitro-transcribed 32P-labelled beta-actin mRNA with high affinity. Three proteins, of approximate molecular masses 27, 50 and 97 kDa, were observed to exhibit strong binding. Binding to these proteins took place at physiological salt concentration and withstood washing in 0.5 M salt. Furthermore, binding was unaffected by heparin but was inhibited by unlabelled beta-actin mRNA. Treatment of isolated CSKs with the microfilament-severing agent DNase I abolished all beta-actin mRNA-binding activities, thus suggesting a possible association of beta-actin mRNA with the microfilament network in situ. Removal of the 3' untranslated region (UTR) significantly reduced beta-actin mRNA binding to all three CSK proteins but removal of the 5' UTR mainly affected binding to the 97-kDa species and that to a lesser extent. beta-Tubulin mRNA bound to the same three CSK proteins as did beta-actin mRNA, but with considerably less avidity. In contrast, vimentin mRNA strongly recognized these CSK proteins, and further bound to a group of smaller proteins (< 29 kDa). As beta-actin mRNA, beta-tubulin mRNA and vimentin mRNA have been observed to occupy separate cytoplasmic locales, the proteins detected here may be operative both in binding mRNAs to the CSK in situ, as well as in localizing mRNA in the cytoplasm.
Sharpless
K
K
Department of Physiology, University of Connecticut Health Center, Farmington.
Biegel
D
D
Yang
T
T
Pachter
J S
JS
D016475:3T3 Cells; D000199:Actins / Q000235:genetics*; D000818:Animals; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003599:Cytoskeleton / Q000737:chemistry; D003850:Deoxyribonuclease I / Q000494:pharmacology; D051379:Mice; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000032:analysis / Q000378:metabolism*
D000199:Actins; D003598:Cytoskeletal Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D003850:Deoxyribonuclease I