Analysis of Messenger RNA Coding for S100 Protein in the Mammalian Brain

Abstract

S100 protein is a brain-specific protein which is absent at birth and first appears in rabbit brain 2-3 days after birth. To determine how the synthesis of this brain-specific protein is regulated, mRNA was isolated from brain polysomes and assayed for S100 protein mRNA activity by in vitro translation in a heterologous cell-free system and immunoprecipitation of released polypeptides with rabbit anti-S100 protein antiserum. S100 protein mRNA was detected primarily in small polysomes containing five to eight ribosomes, and virtually no S100 protein mRNA was present in polysomes containing more than eight ribosomes. S100 protein mRNA was not detected in brain polysomes at stages prior to the induction of synthesis of S100 protein, i.e., in fetal brain or in 1-day neonates. The amount of S100 protein mRNA in polysomes of the cerebral cortex and cerebellum was measured to see if it correlated with the level of S100 protein in the two regions of adult brain. The cerebellum, which contained three to four times the level of S100 protein in the cerebral cortex, contained four times more S100 protein mRNA.

Synthesis of S100 Protein on Free and Membrane‐Bound Polysomes of the Rabbit Brain

“…somes isolated from the cerebral hemispheres. This is in agreement with the regional distribution of SlOO protein in the rodent brain (Hershman et al, 1971;Cicero et al, 1972;Labourdette and Mandel, 1978) and our previous work on the analysis of mRNA coding for S 100 protein in various regions of the rabbit brain (Mahony and Brown, 1980).…”

“…The involvement of an NH,-terminal hydrophobic peptide, which directs the nascent polypeptide chain translation complex to the rough endoplasmic reticulum before being cleaved off, is unlikely, since this mechanism would require the biosynthesis of S 100 protein in the form of a precursor, subsequently processed to the mature product. In fact, a precursor of the protein has never been detected when poly(A+)mRNA from brain was translated in cell-free systems derived from rabbit reticulocytes or wheat embryo (Mahony et al, 1976;Mahony and Brown, 1980;Marks et al, 1980b). On the other hand, examination of the amino acid sequence of SlOO protein reveals two distinct internal clusters of amino acids, which can be evaluated for a potential role as a binding peptide for attaching the nascent polypeptide chain to the rough endoplasmic reticulum .…”

“…The cell-free synthesis of SlOO protein has been reported in reconstituted homologous systems derived from rat brain and in heterologous systems programmed with mRNA or polysomes from the brains of several mammalian species (Zomzely-Neurath e t al., 1972;Amaldi et al, 1973;Mahony et al, 1976;Marks et al, 1980b;Mahony and Brown, 1980;reviewed by Zomzely-Neurath and Walker, 1980). In a previous study we have analyzed mRNA coding for S 100 protein during the development of the rabbit brain by means of in vitro translation in a wheat embryo extract (Mahony and Brown, 1980). These results indicated that t h e mRNA for SlOO protein was not present in brain polysomes a t stages prior to the induction of synthesis of SlOO protein, i.e., in fetal brain or in 1day-old neonates.…”