Globin mRNA sequences in polyadenylated and nonpolyadenylated nuclear precursor-messenger RNA from avian erythroblasts

Nuclear RNA from immature duck erythrocytes was fractionated into polyadenylated and nonpolyadenylated fractions, and globin mRNA sequences were determined by hybridization to DNA complementary to globin mRNA.80–90% of labeled nuclear RNA is found to be nonpolyadenylated, and 70–80% of the globin mRNA sequences present in the nucleus are found in nonpolyadenylated molecules. These data suggest that polyadenylation does not specifically select for globin mRNA sequences.The nonpolyadenylated globin mRNA sequences present in the nucleus are found mostly in molecules of small size, close to the size of polyribosomal globin mRNA, suggesting that polyadenylation is a later event in globin mRNA formation.     

Changes in total RNA, polyadenylated RNA, and actin mRNA during meiotic maturation of mouse oocytes

The total RNA content of mouse oocytes, as measured by ethidium bromide fluorescence, was found to decrease by 19% during meiotic maturation (ovulated eggs contain 19% less RNA than full-grown oocytes). Consistent with these results, prelabeled stable RNA of full-grown oocytes decreased by about 20% during in vitro maturation. Polyadenylated RNA represented 19% of total prelabeled RNA in full-grown oocytes and 10% in oocytes matured in vitro, confirming previous results on in vivo prepared material. To distinguish between deadenylation and degradation for one mRNA, the amount and state of adenylation of actin mRNA was examined using Northern blots of oocyte RNA probed with a nick-translated beta-actin cloned chicken cDNA. The results showed that the amount of actin mRNA remained similar during maturation, but its molecular weight decreased slightly. Experiments in which RNA was treated with oligo(dT) and RNase H demonstrated that the actin mRNA was deadenylated during maturation, when actin synthesis is known to decline. These results indicate that the previously defined loss of bulk RNA and changes in the state of adenylation of mRNA during the first 11/2 days of embryogenesis actually begin during the 12 hr of meiotic maturation preceding fertilization.     

Ribosomal RNA processing and an RNase R family member in chloroplasts of Arabidopsis

An Arabidopsis mutant rnr1, which has a defect in the basic genetic system in chloroplasts, was isolated using the screening of the high chlorophyll fluorescence phenotype. Whereas chlorophyll fluorescence and immunoblot studies showed the mutant had reduced activities of photosystems I and II, molecular characterization of the mutant suggested that a T-DNA insertion impaired the expression of a gene encoding a RNase R family member with a targeting signal to chloroplasts. Since RNase R family members have a 3–5 exoribonuclease activity, we examined the RNA profile in chloroplasts. In rnr1 the intercistronic cleavage between 23S and 4.5S rRNA was impaired, and a significant reduction in rRNA in chloroplasts was found, suggesting that RNR1 functions in the maturation of chloroplast rRNA. The present results suggest that defects in the genetic system in chloroplasts cause high chlorophyll fluorescence, pale green leaf, and marked reduction in the growth rate, whereas the levels of some chloroplast RNA were higher in rnr1 than in the wild-type.     

Preparation of chlamydomonas chloroplasts for the in vitro import of polypeptide precursors

To study the import of polypeptide precursors we have adapted and compared two procedures for the isolation of competent chloroplasts from the green unicellular alga, Chlamydomonas reinhardtii: silicasol gradient centrifugation and elutriation. The chloroplasts actively import the precursor of the small subunit of ribulose bisphosphate carboxylase-oxygenase in vitro.     

RNA synthesis and coding capacity of polyadenylated and nonpolyadenylated mRNA from cultures of differentiating Drosophila melanogaster myoblasts

We have investigated the synthesis and coding capacity of RNA isolated from cultures of differentiating Drosophila embryonic muscle cells. We find that following muscle cell fusion, the sedimentation profile of newly synthesized polyadenylated RNA becomes somewhat lighter. In vitro translation products analyzed by two-dimensional gel electrophoresis indicate that the coding capacity of translatable myogenic mRNA changes during differentiation. A group of several muscle-specific proteins (including the contractile proteins) is translated only from mRNA isolated after the initiation of fusion. This pattern coincides with proteins synthesized in vivo during differentiation. Additionally, we find that polyadenylated and nonpolyadenylated myogenic mRNA from a given developmental stage in culture have extremely similar coding potentials.     

Stability of polyadenylated RNA in differentiating myogenic cells

Three independent methods of measurement showed that cytoplasmic polyadenylated RNA from the differentiating myogenic cell line L8 consists of two main populations with regard to stability, one with a half-life of less than 4 h and the other with a half-life of 17--54 h. Similar results were obtained in the presence and absence of actinomycin D. During the fusion of mononucleated myoblasts into multinucleated fibers, there was an increase in both the steady-state pool of the more stable polyadenylated RNA and the proportion of stable polyadenylated RNA synthesized in pulse labelling.     

Alterations in polyadenylated RNA during pollen maturation and germination

Developmental changes in poly(A)-bearing RNA in male tobacco gametophyte were examined by sedimentation analysis and by hybridization with³H-poly(U). The results indicate that the transition of microspore undergoing postmeiotic division to mature pollen is accompanied by characteristic changes in RNA and poly(A) content and the size of poly(A)⁺RNA. The volume of pollen grain increases about 2times, total RNA per grain from 34 to 230 pg and poly(A) from 22 to 450 fg, which together with the estimated increase in the number average size of poly(A)⁺RNA from 700 to 2 100 nucleotides suggests an approx. rise of RNA containing poly(A) from 0.3 to 2.7% of total RNA. Size distribution of the populations of polyadenylated RNAs shows progressive formation of species with a higher molecular mass and differentiation of the pollen-characteristic pattern with main sedimentation maxima close to 12S, 19S and 26S. This pattern remains almost unchanged during 8 h of pollen tube growth and is also found in polysomes formed at the beginning of germination. The amount of poly(A) decreases gradually after the onset of soaking at a rate of slightly more than 1 % per h within 24 h of pollen cultivation. As a whole, the results demonstrate that in the course of pollen maturation a specific population of polyadenylated mRNAs is formed which persists as stored mRNA in quiescent pollen and is used as template during-pollen tube formation.     

Metabolism of polyadenylated mRNA in growing human lymphocytes.

The kinetics of degradation of newly synthesized, cytoplasmic polyadenylated RNA have been examined in normal human lymphocytes stimulated to grow with phytohemagglutinin. A single class of poly(A)-bearing RNA was identified with a half-life of approximately 50 h. In the presence of actinomycin D, the half-life was 5 to 6 h, and virtually no decay of pulse-labeled material was detectable after 6 h of chase incubation with cordycepin. These findings contrast sharply with data obtained from other growing human cells used as controls: polyadenylated mRNA in MOLT-4 cells, a cultured line of T lymphocytes, had a half-life of 2 h in the presence of actinomycin D. The stability of poly(A)-containing RNA in stimulated lymphocytes from normal donors is therefore not simply a manifestation of cell proliferation. In normal resting lymphocytes, Berger and Copper [(1975) Proc. Natl. Acad. Sci. U.S. 72, 3873--3877] reported the existence of 2 classes of polyadenylated mRNA with half-lives of under an hour and greater than 20 h, respectively. Since short-lived poly(A)-bearing mRNA is absent from mitogen-stimulated lymphocytes, the data suggest that stabilization of previously labile poly(A)-bearing RNA is one of many carefully regulated processes accompanying growth induction in normal lymphoid cells.     

Complexity and characterization of polyadenylated RNA in the mouse brain

The complexity of nuclear RNA, poly(A)hnRNA, poly(A)mRNA, and total poly(A)RNA from mouse brain has been measured by saturation hybridization with nonrepeated DNA. These DNA populations were complementary, respectively, to 21, 13.5, 3.8, and 13.3% of the DNA. From the RNA Cot required to achieve half-sturation, it was estimated that about 2.5–3% of the mass of total nuclear RNA constituted most of the complexity. Similarly, complexity driver molecules constituted 6–7% of the mass of the poly(A)hnRNA. 75–80% of the poly(A)mRNA diversity is contained in an estimated 4–5% of the mass of this mRNA. Poly(A)hnRNA constituted about 20% of the mass of nuclear RNA and was comprised of molecules which sedimented in DMSO-sucrose gradients largely between 16S and 60S. The number average size of poly(A)hnRNA determined by sedimentation, electron microscopy, or poly(A) content was 4200–4800 nucleotides. Poly(A)mRNA constituted about 2% of the total polysomal RNA, and the number average size was 1100–1400 nucleotides. The complexity of whole cell poly(A)RNA, which contains both poly(A)hnRNA and poly(A)mRNA populations, was the same as poly(A)hnRNA. This implies that cytoplasmic polyadenylation does not occur to any apparent qualitative extent and that poly(A)mRNA is a subset of the poly(A)hnRNA population. The complexity of poly(A)hnRNA and poly(A)mRNA in kilobases was 5 × 10⁵ and 1.4 × 10⁵, respectively. DNA which hybridized with poly(A)mRNA renatures in the presence of excess total DNA at the same rate as nonrepetitive tracer DNA. Hence saturation values are due to hybridization with nonrepeated DNA and are therefore a direct measure of the sequence complexity of poly(A)mRNA. These results indicate that the nonrepeated sequence complexity of the poly(A)mRNA population is equal to about one fourth that observed for poly(A)hnRNA.     

RNA processing: splicing and the cytoplasmic localisation of mRNA

An unexpected link has been discovered between pre-mRNA splicing in the nucleus and mRNA localisation in the cytoplasm. The new findings suggest that recruitment of the Mago Nashi and Y14 proteins upon splicing of oskar mRNA is an essential step in the localisation of the RNA to the posterior pole of the Drosophila oocyte.     

Diversity and abundance of polyadenylated RNA from Achlya ambisexualis

The diversity, abundance, and DNA sequence representation of poly(adenylic acid) containing RNA derived from cells of Achlya ambisexualis cultured in defined and undefined media have been determined. The kinetics of hybridization of polyadenylated RNA with complementary DNA were the same for both culture conditions and revealed the presence of three frequency classes containing 29, 220, and 3000 different sequences of an average length of 1150 nucleotides. Complexity estimates derived from experiments in which polyadenylated RNA was hybridized to unique sequence DNA were in good agreement with these results. The kinetics of hybridization of complementary DNA with an excess of nuclear DNA indicate that approximately 10% of the RNA is transcribed from reiterated DNA sequences while the remainder is transcribed from single copy sequences.     

Regional distribution of polyadenylated mRNA in Xenopus laevis embryos

Xenopus laevis embryos were dissected into dorsal and ventral regions in post-gastrula stages. Polyadenylated and nonpolyadenylated ribonucleic acids were separated on oligo (dT) cellulose and translated in vitro. The radioactivity incorporated into the translation products directed by polyadenylated and nonpolyadenylated messenger ribonucleic acids shows that in the dorsal region most proteins are synthesized on polyadenylated messenger ribonucleic acid templates in all the stages, while in the ventral region the major templates seem to be, until the neural fold stage, nonpolyadenylated messenger ribonucleic acids. Later the polyadenylated messenger ribonucleic acid activity there too increases.