Synthesis and transport of various RNA species in developing embryos of Xenopus laevis.

Embryonic cells of Xenopus laevis were labeled for varying lengths of time, and their nuclear and cytoplasmic RNAs were analyzed, with the following results. (1) The synthesis of small nuclear RNAs (snRNAs) is detected from blastula stage on. (2) The initiation of 4 S and 5 S RNA syntheses occurs at blastula stage. However, while the former is transported into the cytoplasm immediately after its synthesis, the latter remains within the nucleus, until its transport starts later, concomitantly with that of 28 S rRNA. (3) As soon as “blastula” cells start to synthesize 40 S rRNA precursor at 5th hr of cultivation, 18 S rRNA is transported first; the transport of 28 S rRNA begins 2 hr later. (4) On a per-cell basis, poly(A)-RNA is synthesized in blastula stage at a much higher rate than in the later stages. About one-third of the total blastula poly(A)-RNA, and about one-fifth in the case of tailbud cells, is transported quickly into the cytoplasm. Then, it appears that the RNAs which are synthesized at early embryonic stages are transported to the cytoplasm without delays, except for 5 S RNA and snRNAs.     

Mitochondrial poly(A) RNA synthesis during early sea urchin development

The synthesis of mitochondrial messenger RNA during early sea urchin development was examined. Oligo(dT) chromatography and electrophoresis on aqueous or formamide gels of mitochondrial RNA from pulse-labeled embryos showed the presence of eight distinct poly(A)-containing RNA species, ranging in size from 9 to 22 S. Nuclease digestion of these RNAs revealed poly(A) sequences of 4 S size. Using sea urchin anucleate fragments, we were able to demonstrate that all eight messenger RNAs are transcribed from mitochondrial DNA, rather than being transcribed from nuclear DNA and imported into the mitochondria.There was no change in the electrophoretic profile of the eight poly(A) RNAs when embryos were pulsed with [³H]uridine at various times after fertilization. Neither was there any change in the incorporation of [³H]uridine into these species or in the percentage of total newly synthesized mitochondrial RNA that contains poly(A) sequences as development progresses. Even though these RNAs appear to be transcribed at a constant rate throughout early development, they were not detected in mitochondrial polysomes until 18 hr after fertilization.     

Ribosomal RNAs synthesized by isolated squid nerves and ganglia differ from native ribosomal RNAs

The large rRNA of the squid comprises two chains that may be dissociated by heating at 65 degrees C. A single chain constitutes the small rRNA. Surprisingly, the RNAs synthesized by dissected squid fin nerves and stellate nerves and ganglia differed in size from native rRNAs and did not manifest thermal instability. Nonetheless, they resembled native rRNAs in relative abundance, subcellular distribution, lack of poly(A), and metabolic stability. In addition, newly synthesized RNA was localized in nerve and glial cells, as shown by autoradiographic analysis, and was assembled into 80S ribosomes, which supported the synthesis of neuron-specific neurofilament proteins. Following incubation of nerves and ganglia for >10 h, native rRNAs started to disappear, while two major newly synthesized RNAs progressively accumulated. As a result, after 20 h, native rRNAs were substituted by the two novel RNAs. With use of 32P-cDNA synthesized from the latter RNAs as a probe, the novel RNAs demonstrated a considerable degree of homology with native rRNA in northern analysis. Taken together, the data suggest that in dissected squid nerves and ganglia, the synthesis of native rRNAs is gradually terminated while two novel rRNAs are being synthesized, presumably as a correlate of reactive gliosis and/or neuronal degeneration/regeneration.     

Relative amounts of newly synthesized poly(A)+ and poly(A)- messenger RNA during development of Xenopus laevis

The relative amounts of newly synthesized poly(A)⁺ and poly(A)^? mRNA have been determined in developing embryos of the frog Xenopus laevis. Polysomal RNA was isolated and fractionated into poly(A)⁺ and poly(A)^? RNA fractions with oligo(dT)-cellulose. In normal embryos the newly synthesized polysomal poly(A)⁺ RNA has a heterodisperse size distribution as expected of mRNA. The labeled poly(A)^? RNA of polysomes is composed mainly of rRNA and 4S RNA. The amount of poly(A)^? mRNA in this fraction cannot be quantitated because it represents a very small proportion of the labeled poly(A)^? RNA. By using the anucleolate mutants of Xenopus which do not synthesize rRNA, it is possible to estimate the percentage of mRNA which contains poly(A) and lacks poly(A). All labeled polysomal RNA larger than 4S RNA which does not bind to oligo(dT)-cellulose in the anucleolate mutants is considered presumptive poly(A)^? mRNA. The results indicate that about 80% of the mRNA lacks a poly(A) segment long enough to bind to oligo(dT). The poly(A)⁺ and poly(A)^? mRNA populations have a similar size distribution with a modal molecular weight of about 7 × 10⁵. The poly(A) segment of poly(A)⁺ mRNA is about 125 nucleotides long. Analysis of the poly(A)^? mRNA fraction has shown that it lacks poly(A)₁₂₅.     

Alteration in cellular RNAs during the in vitro lifespan of cultured human diploid fibroblasts.

An abrupt concommitant increase in total cellular RNA and protein was observed as cultured human diploid fibroblasts entered the senescent phase of their in vitro lifespan. DNA content remained stable from early to final passages. Fractionation of cellular RNAs by polyacrylamide gel electrophoresis demonstrated an increase in both 28S and 18S ribosomal and 4S transfer RNAs in these senescent cells. Separation of poly(A) RNA (mRNA) by oligo(dT)-cellulose chromatography suggests an increase in this group of RNAs. However, the ratios of 28S to 18S rRNAs, tRNA to rRNA, and mRNA to total cellular RNA were not significantly different in cells before and after senescence, indicating that the overall increases in total cellular RNA was not due to an accumulation of a single RNA class.     

Very long-lived messenger RNA in ovaries of Xenopus laevis

It is possible to label with radioactivity newly synthesized ovarian RNA after intraperitoneal injection of [³H]guanosine and [³H]uridine into immature Xenopus laevis, if ovaries in which only previtellogenic stage 1 oocytes are present. Following the amount of radioactivity in the ovarian pool of acid-soluble precursors indicates a complete clearance of acid-soluble radioactivity within 15–20 days after injection. Incorporation of radioactivity into total RNA (which is almost exclusively 4 and 5S RNAs at this stage) and poly(A)⁺ RNA ceases between 15 and 20 days after injection, but the total amount of radioactivity in these RNA fractions does not decline appreciably over the next 18 months. During this time, the ovary grows and develops since stage 6 oocytes eventually appear and there is a 10- to 20-fold increase in total RNA content, which changes in composition from almost exclusively (95%) 4 and 5S RNAs to mainly (75%) 18 and 28S RNAs. Thus, despite continued growth and development, radioactive RNA molecules synthesized during previtellogenesis survive for lengths of time commensurate with the length of oogenesis (1–2 years). Although very limited (<7%) reincorporation of radioactivity into RNA is detected, it cannot alone account for the stability of the label in poly(A)⁺ RNA. These results are interpreted as indicative of synthesis during previtellogenesis of tRNA, 5SrRNA, and messenger RNA molecules which are very long-lived.     

Electrophoretic analysis of newly synthesized and stored maternal RNA during oogenesis ofCalliphora erythrocephala (Dipt.)

Summary Ovaries ofC. erythrocephala synthesize large amounts of poly(A)⁺ and poly(A)^– RNA during early and middle stages of oogenesis as shown by labelling with³H-uridine in vivo. After incubation for 1 h, a striking difference in the electrophoretic pattern of newly synthesized labelled poly(A)⁺ RNA and the poly(A)⁺ RNA present in sufficient amounts for optical density measurements (steady state poly(A)⁺ RNA) was observed. During early and mid-oogenesis, in the poly(A)^– RNA fraction, 4S predominantly mature rRNA, 5S RNA and tRNA were labelled. These fractions were no longer synthesized during late oogenesis, whereas poly(A)⁺ RNA was labelled continously During oogenesis stage specific differences in the size distribution of newly synthesized and steady state poly(A)⁺ RNA were not obvious. However, different sizes of labelled poly(A)⁺ RNA species were detected in 0–2h old preblastoderm embryos, after injection of³H-uridine into females either 3–4 days (stage 3–4 of oogenesis) or 24 h before oviposition (stage 5–6 of oogenesis). This difference in RNA synthesis was related to the presence of active nurse cell nuclei. The poly(A)⁺ RNA fraction represents about 2–3% of the total RNA in both ovaries and freshly laid eggs as judged by measurements of optical density and radioactivity bound to oligo(dT). The length of poly(A)-segments in ovarian poly(A)⁺ RNA varied from about 30 to 200 nucleotides.     

Defective structural RNA processing in relapsing-remitting multiple sclerosis

BackgroundSurveillance of integrity of the basic elements of the cell including DNA, RNA, and proteins is a critical element of cellular physiology. Mechanisms of surveillance of DNA and protein integrity are well understood. Surveillance of structural RNAs making up the vast majority of RNA in a cell is less well understood. Here, we sought to explore integrity of processing of structural RNAs in relapsing remitting multiple sclerosis (RRMS) and other inflammatory diseases.ResultsWe employed mononuclear cells obtained from subjects with RRMS and cell lines. We used quantitative-PCR and whole genome RNA sequencing to define defects in structural RNA surveillance and siRNAs to deplete target proteins. We report profound defects in surveillance of structural RNAs in RRMS exemplified by elevated levels of poly(A) + Y1-RNA, poly(A) + 18S rRNA and 28S rRNAs, elevated levels of misprocessed 18S and 28S rRNAs and levels of the U-class of small nuclear RNAs. Multiple sclerosis is also associated with genome-wide defects in mRNA splicing. Ro60 and La proteins, which exist in ribonucleoprotein particles and play different roles in quality control of structural RNAs, are also deficient in RRMS. In cell lines, silencing of the genes encoding Ro60 and La proteins gives rise to these same defects in surveillance of structural RNAs.ConclusionsOur results establish that profound defects in structural RNA surveillance exist in RRMS and establish a causal link between Ro60 and La proteins and integrity of structural RNAs.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0629-x) contains supplementary material, which is available to authorized users.     

A comparison of sequence complexity of nuclear and polysomal poly(A)+ RNA from different developmental stages ofXenopus laevis

Summary Nuclear poly(A)⁺ and polysomal poly(A)⁺ RNA were isolated from gastrula and early tadpole stages of the amphibianXenopus laevis. Complementary DNA was synthesized from all RNA preparations. Hybridization reactions revealed that at least all abundant and probably most of the less frequent nuclear and polysomal poly(A)⁺ RNA species present at the gastrula stage are also present at the early tadpole stage. On the other hand, there are nuclear RNA sequences at the latter stage which appear, if at all, only at lower concentrations at the gastrula stage. The polysomal poly(A)⁺ RNA hybridization reactions suggest the existence of polysomal poly(A)⁺ RNA sequences at early tadpole stages which are not present in the corresponding gastrula stage RNA.By cDNA hybridization with poly(A)^– RNA it could be shown that most of the poly(A)⁺ containing RNA sequences transcribed into cDNA were also present within the poly(A)^– RNA. It was estimated, that these sequences are 10 fold more abundant within the poly(A)^– polysomal RNA and 3–6 more abundant within the poly(A)^– nuclear RNA as compared to the poly(A)⁺ RNAs.     

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.     

Effect of 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole on ribonucleotide metabolism and accumulation of mitochondrial RNA and low-molecular-weight cytoplasmic RNA in HeLa cells

These results provide additional information on the selective inhibition of RNA synthesis by 5,6-dichloro-1-β-d-ribofuranosyl benzimidazole (DRB). DRB only slightly inhibited the poly(A⁺) RNA and ribosomal RNA in the mitochondria (maximal inhibition was ~25%) but severely inhibited the poly(A⁺) RNA in the postmitochondrial supernatant (~95%) and the poly(A⁺) RNA associated with the cytoplasmic membranes (~80%). Separation of the cytoplasmic low-molecular-weight RNAs showed that DRB inhibited the 5.8 S rRNA, a product of RNA polymerase I, by ~95% while there was only a slight inhibition of the 4 S RNAs (~20%) and 5 S RNA (<5%), products of RNA polymerase III. DRB severely inhibited the appearance in the cytoplasm of 28 S rRNA (~95%) and 18 S rRNA (~80%). These results, along with other recent reports (31–34), may suggest that DRB most severely inhibits RNAs that are extensively processed and/or transcribed from genes that contain extensive intervening sequences. These experiments also indicate that the mechanism of DRB inhibition does not involve alterations in ribonucleotide metabolism. DRB did not affect the phosphorylation of any ribonucleotides to triphosphates or the cellular conversion of [³H]uridine to UTP. Also, the size of the UTP and ATP pools in DRB-treated cells was equal to or greater than those in control cells through a period of 240 min. Significant amounts of DRB triphosphate could not be detected in DRB-treated cells suggesting that this may not be the inhibitory form of DRB. Measurements of the specific activity of the UTP pool allowed direct measurements of the accumulation of picomoles of the individual RNAs in the presence of DRB.     

Axoplasmic RNA species synthesized in the isolated squid giant axon

Isolated squid stellate nerves and giant fiber lobes were incubated for 8 hr in Millipore filtered sea water containing [³H]uridine. The electrophoretic patterns of radioactive RNA purified from the axoplasm of the giant axon and from the giant fiber lobe (cell bodies of the giant axon) demonstrated the presence of RNA species with mobilities corresponding to tRNA and rRNA. The presence of labeled rRNAs was confirmed by the behavior of the large rRNA component (31S) which, in the squid, readily dissociates into its two constituent moyeties (17S and 20S). Comparable results were obtained with the axonal sheath and the stellate nerve. In all the electrophoretic patterns, additional species of radioactive RNA migrated between the 4S and the 20S markers, i.e. with mobilities corresponding to presumptive mRNAs. Chromatographic analysis of the purified RNAs on oligo(dT)cellulose indicated the presence of labeled poly(A)⁺ RNA in all tissue samples. Radioactive poly(A)⁺ RNA represented approximately 1% of the total labeled RNA in the axoplasm, axonal sheath and stellate nerve, but more than 2% in the giant fiber lobe. The labeled poly(A)⁺ RNAs of the giant fibre lobe showed a prevalence of larger species in comparison to the axonal sheath and stellate nerve. In conclusion, the axoplasmic RNAs synthesized by the isolated squid giant axon appear to include all the major classes of axoplasmic RNAs, that is rRNA, tRNA and mRNA.Special Issue dedicated to Prof. Holger Hydén.     

Use of a cloned library for the study of abundant poly(A)+RNA during Xenopus laevis development

Over 200 cloned sequences from recombinant DNA libraries prepared from Xenopus laevis embryonic poly(A)⁺RNA have been analyzed by colony hybridization with [³²P]cDNA prepared from poly(A)⁺RNA from several stages of development. The period of early embryogenesis extending through the beginning of gastrulation (stage 10) is marked by the relative constancy of the abundant poly(A)⁺RNA population. Between the gastrula and tailbud stages (stage 24) there is a dramatic change in the pattern of abundant poly(A)⁺RNA species; the new pattern remains fairly constant for at least 2 days of development to the late prefeeding tadpole stages (stage 41). We have also compared nonpolysomal and polysomal poly(A)⁺RNA populations at two different stages. In stage 10 (early gastrula) postribosomal (free ribonucleoprotein) and polysomal poly(A)⁺RNA populations partly overlap; however, many cloned sequences occur in quite different concentrations in one fraction or the other. Among the sequences that are predominantly nonpolysomal at gastrula few become predominantly polysomal at tailbud stages. Thus, we have no evidence for a major recruitment of abundant nonpolysomal RNAs into polysomes with progressing development. We rather observe a general pattern in which a cloned sequence that is nonpolysomal in one stage of development tends to be nonpolysomal (if detectable at all) in other stages as well.     

Appearance of newly formed mRNA and rRNA as ribonucleoprotein-particles in the cytoplasmic subribosomal fraction of pea embryos

Incorporation studies with ³H-uridine or ³H-adenosine showedthat germinating pea embryos synthesize all types of poly A(+)RNA, rRNA and 4–5S RNA at the early stage of germination.After the pulse labeling for 30 min, only heterodisperse RNAand 4–5S RNA appeared in the cytoplasm as labeled RNAspecies. At this time the radioactivity was associated withcytoplasmic structures heavier than 80S and RNP particles of68–70S, 52–55S, 36–38S and 20–22S whichare presumed to be free mRNP particles in plants. When the pulse-labeledembryos were incubated for a further 60 min in an isotope-freemedium, the labeled 17S and 25S rRNA emerged in the cytoplasm,together with labeled heterodisperse and 4–5S RNAs. Moreradioactivity accumulated in the regions of the polysome, 62–65Sand 38–42S particles. The results of analysis of RNAsextracted from the whole cytoplasm, polysome or subribosomalfractions indicated that small subunits of newly formed ribosomesappear more rapidly in the cytoplasm than new large subunits,which accumulate for a while as free particles in the cytoplasmthen are incorporated into polysomes. The actino-mycin treatmentwhich caused preferential inhibition of rRNA synthesis reducedthe accumulation of free, newly formed ribosome subunits andpartially permitted detection of the presumed mRNP particlesin the subribosomal region even after the chase treatment. (Received June 28, 1976; )     

Complementarity of sequences in low molecular weight RNAs to regions of messenger and ribosomal RNAs.

Total low molecular weight nuclear RNAs of mouse ascites cells have been labeled in vitro and used as probes to search for complementary sequences contained in nuclear or cytoplasmic RNA. From a subset of hybridizing lmw RNAs, two major species of 58,000 and 35,000 mol. wt. have been identified as mouse 5 and 5.8S ribosomal RNA. Mouse 5 and 5.8S rRNA hybridize not only to 18 and 28S rRNA, respectively, but also to nuclear and cytoplasmic poly(A+) RNA. Northern blot analysis and oligo-dT cellulose chromatography have confirmed the intermolecular base-pairing of these two small rRNA sequences to total poly(A+) RNA as well as to purified rabbit globin mRNA. 5 and 5.8S rRNA also hybridize with positive (coding) but not negative (noncoding) strands of viral RNA. Temperature melting experiments have demonstrated that their hybrid stability with mRNA sequences is comparable to that observed for the 5S:18S and 5.8S:28S hybrids. The functional significance of 5 and 5.8S rRNA base-pairing with mRNAs and larger rRNAs is unknown, but these interactions could play important coordinating roles in ribosome structure, subunit interaction, and mRNA binding during translation.     

Isolation and characterization of the RNA of membrane-bound ribosomes in Dictyostelium discoideum

The RNA of membrane-bound ribosomes, isolated from Dictyostelium discoideum, represented 13 to 16% of the total ribosomal RNA (rRNA) present throughout growth and development. Membrane-bound ribosomes were released by treatment with sodium deoxycholate and Brij 58. There were no obvious differences in size and base composition between RNAs derived from membrane-bound or free ribosomes. The 17S membrane-bound rRNA and free rRNAs appeared to have similar methyl contents. However, the 25S membrane-bound rRNA contained about 16 to 20% fewer methyl groups than the 17S membrane-bound rRNA and free rRNAs. Free rRNAs turned over rapidly during early development but not during the disaggregation and reaggregation processes. Membrane-bound rRNAs showed very little turnover during the early stages of morphogenesis, but showed rapid turnover during the late stages of development; this class of rRNAs did not turn over during early stages of reaggregation but turned over rapidly during later stages of reaggregation.     

Action of ribonuclease upon the polysomes of cultured mouse cells

Summary Pancreatic ribonuclease (RNase) and³H-uridine were used to study certain compositional and ontogenetic features of the polysomes of strain L mouse cells. Growing cells were exposed to the radioactive nucleoside,³H-uridine, for brief defined periods, and the sensitivity of the polysomes to digestion by RNase was determined. The RNase-resistant RNA of polysomes is shown to be primarily ribosomal, and the RNase-sensitive material formed during brief pulse labeling studies is largely messenger RNA. Actinomycin D inhibition of RNA synthesis was used to confirm this identification. The technique described here was used to investigate the effects of hydrocortisone on polysome formation. The hormone (10⁻⁶ m) lessens the extent of the nucleoside incorporation into polysomal and total RNA and delays the appearance of newly synthesized 18 S and 28 S rRNA into cytoplasmic polysomes. This work was partially supported by grants from the United States Public Health Service (GM 10866), from the National Science Foundation (GB 13924), and from The University of Kansas General Research Fund.     

Poliovirus genome RNA hybridizes specifically to higher eukaryotic rRNAs.

The RNA genome of poliovirus hybridizes to 28S and 18S rRNAs of higher eukaryotes under stringent conditions. The hybridization detected by Northern blot analyses is specific since little or no signal was detected for yeast or prokaryotic rRNAs or other major cellular RNAs. Southern blot analysis of DNA clones of mouse rRNA genes leads us to conclude that several regions of 28S rRNA, and at least one region in 18S rRNA, are involved in the hybridization to polio RNA, and that G/C regions are not responsible for this phenomenon. We have precisely mapped one of these hybridizing regions in both molecules. Computer analysis confirms that extensive intermolecular base-pairing (81 out of 104 contiguous bases in the rRNA strand) could be responsible for this one particular site of interaction (polio genome, bases 5075-5250; 28S rRNA, bases 1097-1200). We discuss the possible functional and/or evolutionary significance of this novel type of interaction.