The control of mRNA production, translation and turnover in suspended and reattached anchorage-dependent fibroblasts

Abstract

Strictly anchorage-dependent 3T6 fibroblasts require contact with a solid surface for growth and for normal macromolecular metabolism. Denial of surface contact or anchorage by suspension in methylcellulose leads to dramatic changes in mRNA metabolism and protein synthesis. These are reversed when cells contact a surface, even when spreading is prevented by agents such as concanavalin A. The responses to suspension appear to be well ordered regulatory events. While the synthesis of hn [heterogenous nuclear] RNA remains constant, the production of mRNA is reduced 5 fold within a few hours under suspension conditions, suggesting a possible post-transcriptional control mechanism. Normally turning over mRNA is stabilized; the breakdown rate is reduced to match the lowered rate of message production so that the total amount of mRNA in the suspended fibroblasts remains constant. Protein synthesis declines slowly but extensively, and amounts to only 15% of control cells after 72 h of suspension. The rate of protein synthesis is clearly not regulated by the amount of mRNA present, but rather by its availability. This is shown by the 6-7 fold increased of protein synthesis which occurs within 4 h after the reattachment of suspended cells to a solid substrate. mRNA production remains low during this period and cannot account for the resumption of protein synthesis. The recovery utilizes preexisting mRNA that was translationally inactive, and this mRNA, once returned to translation, is engaged on full-sized polyribosomes, indicating the absence of a lesion in polypeptide initiation. At least 1 major protein, of about 43,000 dalton MW, shows greatly enhanced synthesis during recovery. This polypeptide comigrates with actin in 2-dimensional gel electrophoresis and is extensively associated with the cytoskeleton. Its enhanced synthesis following cell reattachment may represent a modulation of a morphology-related gene.