Developmental changes in the protein and ribonucleic acid components of rat brain messenger ribonucleic acid-protein particles isolated from free polyribosomes by oligo(dT)-cellulose chromatography

Abstract

A study has been made of the developmental changes that occur in the RNA and protein moieties of mRNA-protein particles isolated from newborn and adult rat forebrain free polyribosomes. mRNA-protein particles were isolated by oligo(dT)-cellulose chromatography from salt-washed polyribosomes dissociated by puromycin/0.5 M-KCl treatment as two fractions (E1 and E2) by using Tris/HCl/NaCl eluting buffers containing respectively 25 and 50% (v/v) formamide. Isopycnic centrifugation on CsCl gradients showed that the newborn-derived fractions E1 and E2 has buoyant densities of 1.48--1.50 and 1.41--1.43 g/cm3. Adult-derived E1 and E2 fractions had corresponding values of 1.47 and 1.42 g/cm3. The pooled mRNA-protein particles from the E1 and E2 fractions after deproteinization with proteinase K sedimented with a mean size of approx. 18 S on a sucrose gradient containing 85% formamide with little differences between mRNA molecules from newborn and adult. The mean lengths of the poly(A) segments were similar, being about 130 nucleotides long. Distinct changes were found in the protein composition of the mRNA-protein particles. Fractions E1 and E2 from the newborn contained two major proteins of mol.wts. 74 000 and 52 000 with differences in the relative proportions in each fraction. In contrast, adult fractions E1 and E2 contained predominantly the larger protein. However, the adult fraction E2 contained a more heterogeneous population of minor bands of proteins, including that of mol.wt. 52 000. The findings are discussed briefly in relation to other changes in the developing brain.

Developmental changes in the composition of polyadenylated RNA isolated from free and membrane-bound polyribosomes of the rat forebrain, analysed by translation in vitro

“…However, by assaying the translocational activity of the RNA in the different gradient fractions, the average size of the poly(A)+ RNA could be determined [assuming that the various size classes of brain poly(A)+ RNA are translated with the same efficiency]. Maximal stimulatory activity of the poly(A)+ RNA isolated from both free and mem-brane-bound polyribosomes was associated with RNA of 16-18S; a secondary peak of activity was derived from RNA of 12 S. This is in agreement with other reports of the size of brain poly(A)+ RNA and our own previous studies (Berthold & Lim, 1976b;Elliott et al, 1980). The compositions of translation products from the serial gradient fractions are also shown in Figs.…”

“…Proteins of low molecular weight were also found as translational products of the fractions of larger RNA. The aggregation observed probably involves only a small proportion of the total RNA, which does not substantially influence the overall size distribution of poly(A)+ RNA in the gradient, since the size observed corresponds to that previously found under denaturing conditions with 85% (v/v) formamide (Elliott et al, 1980). Translation products of poly(A)+ RNA: developmental changes…”

“…reported an increased complexity in the protein composition of brain polyribosomal mRNA-protein particles which may be related to translational control mechanisms (Elliott et al, 1980). The brain mRNA population also increases in complexity with age (Grouse et al, 1972), and this probably reflects the differentiation of specific cell types.…”

Phytohormone control of translatable RNA populations in sexual organogenesis of the dioecious plant Mercurialis annua L. (2n = 16)

“…ID). The peak of poly(A) activity was located between the 5 S and the 18 S rRNA peaks, corresponding to values reported for plant (25,36,41) and animal poly(A) RNAs (12,16). That poly(A) RNA did not undergo any great degradation during extraction and analysis is shown by the value of the sedimentation constant.…”

Alterations in the relative amounts of specific mRNA species in the developing human brain in Down's syndrome

“…Protease digestion was performed in situ during re-electrophoresis of the excised proteins as described by Cleveland et al (1977). (Hall & Lim, 1981 (Hall & Lim, 1981) and under denaturing conditions with 85% (v/v) formamide (Elliott et al, 1980). The composition of translation products from the serial gradient fractions is also shown in Fig.…”