Messenger ribonucleoprotein particles in developing sea urchin embryos

Messenger RNA entering polysomes during early development of the sea urchin embryo consists of both oogenetic and newly transcribed sequences. Newly transcribed mRNA enters polysomes rapidly while oogenetic mRNA enters polysomes from a pool of stable, nontranslatable messenger ribonucleoprotein particles (mRNPs) derived from the unfertilized egg. Protein content may relate to differences in the regulation of newly transcribed and oogenetic mRNAs. Oogenetic poly(A)⁺ mRNA was found to be present in both polysomal and subpolysomal fractions of cleavage stage and early blastula stage embryos. This mRNA was found to be present in subpolysomal mRNPs with a density of 1.45 g/cm³ in Cs₂SO₄. Poly(A)⁺ mRNPs released from polysomes of embryos cultured in the presence of actinomycin D sedimented in a broad peak centered at 55 S and contained RNA of 21 S. The density of these particles was sensitive to the method of release; puromycin-released mRNPs had a density of 1.45 g/cm³, while EDTA caused a shift in density to 1.55 g/cm³, indicating a partial loss of protein. The results with newly synthesized mRNAs contrast sharply. Newly transcribed mRNA in subpolysomal mRNPs had a density of 1.55–1.66 g/cm³, a density approaching that of deproteinized RNA. Messenger RNA released from polysomes either by EDTA or puromycin was examined to determine the possible existence of polysomal mRNPs. When [³H]uridine-labeled mRNA was released from late cleavage stage embryo polysomes by either technique, and centrifuged on sucrose gradients, two broad peaks were found. One peak centered at 30 S contained 21 S mRNA while the other at 15 S contained 9 S histone mRNA. When these fractions were fixed with formaldehyde, they banded on Cs₂SO₄ gradients at a density of 1.60–1.66 g/cm³, very similar to that of pure RNA. We conclude that the newly transcribed mRNA may be present in stable mRNPs containing up to 10% protein in either subpolysomal or polysomal fractions. These mRNPs are clearly distinguishable from the protein-rich mRNPs containing oogenetic mRNAs.     

Evidence for the existence of a structural RNA component in the nuclear ribonucleoprotein particles containing heterogeneous RNA

The structural organisation of nuclear ribonucleoprotein particles carrying the HnRNA has been investigated. Experiments are presented which indicate the existence in the RNP particles of two different RNA species: (1) the rapidly labelled HnRNA and (2) stable, low molecular weight RNA, probably located in the interior of the protein moiety, which may serve a structural role for the arrangement of the proteins within the RNP particle.     

Effect of a short term fast on the distribution of cytoplasmic albumin messenger ribonucleic acid in rat liver. Evidence for formation of free albumin messenger ribonucleoprotein particles

Recently, using molecular hybridization techniques with albumin [3H]cDNA, we have determined that in normally fed rats 98% of total liver polyribosomal albumin mRNA sequences are found in membrane-bound polyribosomes (Yap, S. H., Strair, R. K., and Shafritz, D. A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5397-5401). We now observe that a 24- to 30-h withdrawal of food leads to major changes in the amount and subcellular distribution of albumin mRNA molecules. The total amount of cytoplasmic albumin mRNA per liver and concentration of albumin mRNA per unit of membrane-bound polyribosomal RNA are decreased. However, the proportion of albumin mRNA present in the postribosomal supernatant fraction increases dramatically in a short term fast, so that it now represent 60% of total cytoplasmic albumin mRNA sequences. Most of the albumin mRNA sequences in the postribosomal supernatant fraction sediment between 30 S and 50 S. These findings suggest that albumin mRNA is probably stored in the messenger ribonucleoprotein fraction during the fasting state.     

A chicken mRNA similar to heterogeneous nuclear ribonucleoprotein H1

Heterogeneous nuclear ribonucleoproteins are predominantly nuclear RNA-binding proteins that function in a variety of cellular activities. The objective of these experiments was to clone a cDNA for a chicken protein similar to other previously reported heterogeneous ribonucleoproteins for other species. The 5' and 3' ends of the chicken mRNA were cloned using Rapid Amplification of cDNA Ends (RACE). Subsequently, the expression of the mRNA sequence was confirmed via Northern analysis. The deduced amino acid sequence was approximately 86% identical to corresponding regions of human, mouse, or zebrafish proteins similar to heterogeneous nuclear ribonucleoprotein H1. The expression data confirmed the size of the predicted mRNA sequence. The newly identified sequence may be employed in future studies aimed at understanding the role of heterogeneous nuclear ribonucleoproteins in avian species.     

Evidence for the protection of specific RNA sequences in globin messenger ribonucleoprotein particles.

Purified 15 S globin mRNA-protein (mRNP) complexes obtained by EDTA dissociation of duck reticulocytes polyribosomes were digested with the calcium dependant Staphylococcus aureus nuclease (EC 3. 1. 4. 7.). 25% of the globin mRNA sequences were resistant to extensive nuclease digestion as determined by TCA precipitation of the digested 15 S particles labelled in vivo with tritiated uridine. Polyacrylamide gel electrophoresis of the RNA from nuclease digested 15 S particles showed that the protected oligoribonucleotides were distributed into two distinct size classes of 25,000 and 12,000 MW. Comparison between in vitro iodine-labelled 9 S globin mRNA extracted from Staphylococcal nuclease digested 15 S mRNP particles was carried out by fingerprinting. Mapping of T1 ribonuclease digests by high-voltage electrophoresis and homochromatography showed that specific oligoribonucleotides were protected against nuclease attack by proteins of the 15 S mRNP.     

The postnatal methylation of transfer ribonucleic acid in brain. Evidence for the methylation of precursor transfer ribonucleic acid.

Incubation of 3-day-old rat brain with L-[methyl-3H]methionine resulted in the rapid labeling of low-molecular-weight cytoplasmic RNA. Electrophoresis in 15% polyacrylamide gels provided evidence for the methylation of precursor tRNA molecules, and high-performance liquid chromatography demonstrated N2-methylguanine to be the predominant methylated base formed during the first 2 min of labelling.     

The stability, polyadenylic acid content and ribonucleoprotein form of nulcear ribonucleic acid in artichoke.

A nuclear preparation, containing 60-80% of the total tissue DNA and less than 0.5% of the total rRNA, was used to characterize the nuclear RNA species synthesized in cultured artichoke explants. The half-lives of the nuclear RNA species were estimated from first-order-decay analyses to be: hnRNA (heterogeneous nuclear RNA) containing poly(A), 38 min; hnRNA lacking poly(A), 37 min; 2.5 X 10(6)-mol. wt. precursor rRNA, 24 min; 1.4 X 10(6)-mol.wt. precursor rRNA, 58 min; 1.0 X 10(6)-mol.wt. precursor rRNA, 52 min. The shorter half-lives are probably overestimates, owing to the time required for equilibration of the nucleotide-precursor pools. The pathway of rRNA synthesis is considered in terms of these kinetic measurements. The rate of accumulation of cytoplasmic polydisperse RNA suggested that as much as 40% of the hnRNA may be transported to the cytoplasm. The 14-25% of the hnRNA that contained a poly(A) tract had an average molecular size of 0.7 X 10(6) daltons. The poly(A) segment was 40-200 nucleotides long, consisted of at least 95% AMP and accounted for 8-10% of the [32P]orthophosphate incorporated into the poly(A)-containing hnRNA. Ribonucleoprotein particles released from nuclei by sonication, lysis in EDTA or incubation in buffer were analysed by sedimentation through sucrose gradients and by isopycnic centrifugation in gradients of metrizamide and CsCl. More than 50% of the hnRNA remained bound to the chromatin after each treatment. The hnRNA was always associated with protein but the densities of isolated particles suggested that the ratio of protein to RNA was lower than that reported for mammalian cells, The particles separated from chromatin were not enriched for poly(A)-containing hnRNA.