In Vitro Synthesis of Polypeptides of Moderately Large Size by Poly(A)‐Containing Messenger RNA from Postmortem Human Brain and Mouse Brain

Abstract

Studies were undertaken to optimize the conditions for isolation and in vitro translation of poly(A)‐containing mRNA from human postmortem brain. The comparison of several methods for preparation of mRNA from frozen mouse brain indicated that although the yield of mRNA was increased using polysomes prepared in the presence of ribonucleoside vanadyl complexes and subsequently extracted with guanidinium thiocyanate, the translation products were indistinguishable from those synthesized by total cellular RNA directly extracted from tissue with guanidinium thiocyanate. The oligo d(T)‐cellulose‐purified poly(A)‐containing mRNA preparations were translated in vitro in a rabbit reticulocyte lysate in the presence of L‐[35S]methionine. Messenger RNA from frozen mouse brain stimulated protein synthesis from 9‐ to 20‐fold over endogenous mRNA. Over 450 polypeptides were reproducibly synthesized and separated by two‐dimensional polyacrylamide gel electrophoresis (PAGE): size classes up to 130,000 daltons were present. Direct extraction of RNA from frozen human cerebral cortex and cerebellum with guanidinium thiocyanate followed by oligo d(T)‐cellulose chromatography yielded 1.8 μg/g and 2.0 μg/g, respectively, of poly(A)‐containing mRNA; this represents a two‐ to fourfold increase over our earlier results. In the rabbit reticulocyte translation system human brain mRNA stimulated protein synthesis nearly threefold over endogenous mRNA. Compared with earlier studies, the number of newly synthesized polypeptides was increased by 30%. Over 300 species were separated by two‐dimensional PAGE, and size classes up to 130,000 daltons were present, as compared to 70,000 in an earlier report. The polypeptides synthesized by human cerebral cortex and cerebellum were indistinguishable. However, several appeared to be uniquely human when compared with the products synthesized by mouse brain mRNA. The method described for the preparation of postmortem human brain mRNA eliminates the need to prepare polysomes, which are recovered in variable and low yield from the postmortem human brain. The procedure appears applicable to studies on the synthesis of moderately large human brain polypeptides and for investigations of brain protein polymorphism when relatively large numbers of products are required for analysis.

Molecular cloning of amyloid cDNA derived from mRNA of the Alzheimer disease brain: coding and noncoding regions of the fetal precursor mRNA are expressed in the cortex.

“…Irrespective of these observations, we previously demonstrated that poly(A)+ RNA is retained by AD postmortem brains in amounts that are sufficient for functional studies and molecular cloning (16,17,41,42). Thus, similar to the fetal case (9), AD cortical poly(A) + RNA exhibited a doublet at 3.2 and 3.4 kb when hybridized with the amyloid probe.…”

“…Detailed evaluations will be presented elsewhere (16) Preparation of RNA. RNA was prepared from cortex as described (17) with the modifications listed in ref. 18.…”

Biochemical and molecular studies using human autopsy brain tissue

“…1 Adolfsson et al (1979b) Morrison and Kish (1995). mRNAs from frozen autopsy tissue retain sufficient integrity to be used as templates for protein synthesis in vitro Morrison and Griffin 1981;Sajdel-Sulkowska et al 1983Guillemette et al 1986;Johnson et al 1986;Perrett et al 1988;Langstrom et al 1989;Leonard et al 1991). Such translations are highly dependent on the integrity of the 5¢-terminal cap structure and the 3¢ untranslated region (Guhaniyogi and Brewer 2002;Mitchell and Tollervey 2002).…”

Intracellular conversion of thyroxine to triiodothyronine is required for the optimal thermogenic function of brown adipose tissue.

“…The pellets were washed, counted or analyzed by SDS PAGE, and autoradiographed. Polysomal RNA was prepared as described previously (15) in the presence of 10 mM vanadyl ribonucleosides complexes. Polysomes were resuspended in 4 M guanidinium isothiocyanate and RNA was recovered by centrifugation through 5.7 M CsCl (15).…”

“…Polysomal RNA was prepared as described previously (15) in the presence of 10 mM vanadyl ribonucleosides complexes. Polysomes were resuspended in 4 M guanidinium isothiocyanate and RNA was recovered by centrifugation through 5.7 M CsCl (15). Poly A RNA was isolated with a commercial Poly-Ucoated paper, HybondR-mAP (Amersham Corp., Arlington Heights, IL) as described elsewhere (16).…”