Sixteen discrete RNA components in the cytoplasmic ribosome of Euglena gracilis

We have isolated cytoplasmic ribosomes from Euglena gracilis and characterized the RNA components of these particles. We show here that instead of the four rRNAs (17-19 S, 25-28 S, 5.8 S and 5 S) found in typical eukaryotic ribosomes, Euglena cytoplasmic ribosomes contain 16 RNA components. Three of these Euglena rRNAs are the structural equivalents of the 17-19 S, 5.8 S and 5 S rRNAs of other eukaryotes. However, the equivalent of 25-28 S rRNA is found in Euglena as 13 separate RNA species. We demonstrate that together with 5 S and 5.8 S rRNA, these 13 RNAs are all components of the large ribosomal subunit, while a 19 S RNA is the sole RNA component of the small ribosomal subunit. Two of the 13 pieces of 25-28 S rRNA are not tightly bound to the large ribosomal subunit and are released at low (0 to 0.1 mM) magnesium ion concentrations. We present here the complete primary sequences of each of the 14 RNA components (including 5.8 S rRNA) of Euglena large subunit rRNA. Sequence comparisons and secondary structure modeling indicate that these 14 RNAs exist as a non-covalent network that together must perform the functions attributed to the covalently continuous, high molecular weight, large subunit rRNA from other systems.     

Comparison of cytoplasmic and nuclear poly(A)-containing RNA sequences in Xenopus liver cells.

Poly(A)-containing RNAs from cytoplasm and nuclei of adult Xenopus liver cells are compared. After denaturation of the RNA by dimethysulfoxide the average molecule of nuclear poly(A)-containing RNA has a sedimentation value of 28 S whereas the cytoplasmic poly(A)-containing RNA sediments slightly ahead of 18 S. To compare the complexity of cytoplasmic and nuclear poly(A)-containing RNA, complementary DNA (cDNA) transcribed on either cytoplasmic or nuclear RNA is hybridized to the RNA used as a template. The hybridization kinetics suggest a higher complexity of the nuclear RNA compared to the cytoplasmic fraction. Direct evidence of a higher complexity of nuclear poly(A)-containing RNA is shown by the fact that 30% of the nuclear cDNA fails to hybridize with cytoplasmic poly(A)-containing RNA. An attempt to isolate a specific probe for this nucleus-restricted poly(A)-containing RNA reveals that more than 10(4) different nuclear RNA sequences adjacent to the poly(A) do not get into the cytoplasm. We conclude that a poly(A) on a nuclear RNA does not ensure the transport of the adjacent sequence to the cytoplasm.     

Localization of specific rDNA spacer sequences to the mouse L-cell nucleolar matrix.

Mouse L-cell nucleoli were isolated from sonicated nuclei by centrifugation and extensively treated with pancreatic DNase or micrococcal nuclease to obtain "core nucleoli." Core nucleoli still contained the precursors to rRNA and about 1% of the total nuclear DNA, which remained tightly bound even after the removal of some chromatin proteins with 2 M NaCl. The core nucleolar DNA electrophoresed in a series of discrete bands, 20 to about 200 base pairs in length. Hybridization tests with specific DNA probes showed that the DNA was devoid of sequences complementary to mouse satellite, mouse Alu-like, and 5S RNA sequences. It also lacked sequences coding for cytoplasmic rRNA species, since it did not hybridize to the 18S to 28S portion of rDNA in Northern blot analyses and none of it was protected by hybridization to a 100-fold excess of total cytoplasmic RNA in S1 nuclease assays. However, the core nucleolar DNA did hybridize to nontranscribed and external transcribed spacer rDNA sequences. We infer that specific portions of rDNA are protected from DNase action by a tight association with nucleolar structural proteins.     

Sequence and secondary structure of mouse 28S rRNA 5''terminal domain. Organisation of the 5.8S-28S rRNA complex.

We present the sequence of the 5' terminal 585 nucleotides of mouse 28S rRNA as inferred from the DNA sequence of a cloned gene fragment. The comparison of mouse 28S rRNA sequence with its yeast homolog, the only known complete sequence of eukaryotic nucleus-encoded large rRNA (see ref. 1, 2) reveals the strong conservation of two large stretches which are interspersed with completely divergent sequences. These two blocks of homology span the two segments which have been recently proposed to participate directly in the 5.8S-large rRNA complex in yeast (see ref. 1) through base-pairing with both termini of 5.8S rRNA. The validity of the proposed structural model for 5.8S-28S rRNA complex in eukaryotes is strongly supported by comparative analysis of mouse and yeast sequences: despite a number of mutations in 28S and 5.8S rRNA sequences in interacting regions, the secondary structure that can be proposed for mouse complex is perfectly identical with yeast's, with all the 41 base-pairings between the two molecules maintained through 11 pairs of compensatory base changes. The other regions of the mouse 28S rRNA 5'terminal domain, which have extensively diverged in primary sequence, can nevertheless be folded in a secondary structure pattern highly reminiscent of their yeast' homolog. A minor revision is proposed for mouse 5.8S rRNA sequence.     

Mouse ubiquitous B2 repeat in polysomal and cytoplasmic poly(A)+RNAs: uniderectional orientation and 3''-end localization.

A cDNA library in pBR322 was prepared with cytoplasmic poly(A)+RNA from mouse liver cells. From 1 to 1.5% of clones hybridized to either B1 or B2 ubiquitous repetitive sequences. Several clones hybridizing to a B2 repeat were partially sequenced. The full-length B2 sequence was found at the 3'-end of abundant 20S poly(A)+RNA (designated as B2+mRNAx) within the non-coding part of it. B2+mRNAx is concentrated in mouse liver polysomes and absent from cytoplasm of Ehrlich carcinoma cells. The B2 sequence seems to be located at the 3'-end of some other mRNAs as well. To determine the orientation of the B2 sequence in different RNAs, its two strands were labeled, electrophoretically separated, and used for hybridization with Northern blotts containing nuclear, cytoplasmic and polysomal RNAs. In nuclear RNA, the B2 sequence is present in both orientations; in polysomal and cytoplasmic poly(A)+RNAs, only one ("canonical") strand of it can be detected. Low molecular weight poly(A)+B2+RNA [1] also contains the same strand of the B2 element. The conclusion has been drawn that only one its strand can survive the processing. This strand contains promoter-like sequences and AATAAA blocks. The latter can be used in some cases by the cell as mRNA polyadenylation signals.     

Mobilization of newly synthesized RNAs into polysomes inXenopus laevis embryos

Summary The mobilization of newly synthesized 18S and 28S rRNAs, 4S RNA and poly(A)⁺ RNA into polysomes was studied in isolated cells ofXenopus laevis embryos between cleavage and neurula stages. Throughout these stages, 4S RNA and poly(A)⁺ RNA were mobilized immediately following their appearance in the cytoplasm. 18S rRNA however, stayed in the ribosomal subunit fraction for about 30 min until the 28S rRNA appeared, when the two rRNAs were mobilized together at an equimolar ratio. This mobilization, at a 1:1 molar ratio, appeared to be realized at initiation monome formation. Thus, the efficiency of the mobilization of two newly synthesized rRNAs, shortly after their arrival at the cytoplasm, differed considerably but difference disappeared once steady state was reached.The contribution of newly synthesized 18S and 28S rRNAs to polysomes remains small throughout early development. around 3% of newly synthesized 4S RNA is polysomal which is the same distribution observed for unlabeled 4S RNA. Less than 10% of the newly synthesized cytoplasmic poly(A)⁺ RNA was mobilized into polysomes during cleavage, but in later stages the proportion increased to around 20%–25%. These results show that newly synthesized RNAs are utilized for protein synthesis at characteristic rates soon after they are synthesized during early embryonic development. On the basis of the data presented here and elsewhere we discuss quantitative aspects of the utilization of newly synthesized and maternal RNAs during early embryogenesis.     

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.     

Structure and metabolism of adenovirus RNAs containing sequences from the fiber gene

The body of adenovirus fiber messenger RNA is specified by viral r-strand co-ordinates 86.2 to 91.2. Since this mRNA is transcribed from the major late promoter at map position 16, nuclear precursors to the mRNA could be as large as 84% of the length of the 35,000 nucleotide genome. This study identified and characterized polyadenylated nuclear RNAs that contain fiber sequences and therefore are possible processing intermediates. These nuclear RNAs were characterized by hybridization of [³H]RNA preparations and by electron microscopy of RNA-DNA hybrids. Three size classes of RNAs containing fiber sequences were identified: (1) a 22 S species maps from 86.2 to 90.3. This RNA has essentially the same co-ordinates as fiber mRNA. (2) Two 28 S species have co-ordinates of 80.1 to 90.4 and 85.9 to 96.9, respectively. Thus one species has a 5′ terminus coincident with that of the mRNA body, and one has a 3′ terminus coincident with that of the 3′ end of the mRNA body. The polyadenylated terminus at 96.9 does not coincide with the 3′ end of any known mRNA. (3) There are at least two 35 S species. The 3′ end of one species is coincident with that of fiber mRNA. The 3′ terminus of the second RNA is at approximately 96.9.The labeling kinetics of each of these polyadenylated nuclear RNAs were investigated. In continuous label experiments, the two 35 S RNAs and the 85.9 to 96.9 28 S RNA became uniformly labeled in approximately 60 minutes. The 22 S RNA and the 80.1 to 90.4 28 S species continued to accumulate for at least several hours. These results are consistent with a precursor function for the 35 S RNAs and the 85.9 to 96.9 28 S species. The structures of the putative precursors imply that processing of the 3′ end is not a prerequisite for 5′ cleavage.     

Hybridizable sequences between cytoplasmic ribosomal RNAs and 3 micron circular DNAs of Saccharomyces cerevisiae and Torulopsis glabrata.

We have shown that 2.8 and 3.1 micron circular DNA molecules, previously reported to be present in Saccharomyces cerevisiae and Torulopsis glabrata respectively, contain sequences hybridizing to cytoplasmic ribosomal RNAs. In S. cerevisiae the 2.8 micron circular DNA appears to be identical to the rDNA repeating unit from nuclear DNA, both in length (approximately 9000 base pairs) and in the location of the 25, 18 and 5.8S rRNA sequences on the large HindIII fragment (6500 bp) and the presence of the 5S rRNA sequence on the small HindIII fragment. The 3.1 micron molecule from T. glabrata is approximately 2000 base pairs longer than the S. cerevisiae molecule and in addition, one of the HindIII sites lies within the region hybridizing to 25, 18 and 5.8S rRNAs. In S. cerevisiae the 4-5 copies of the 2.8 micron circular DNA molecules per cell, which have an extra-nuclear location, do not appear to be essential for cell viability as in one strain they were undetectable.     

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.     

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.     

Localization and structure of endonuclease cleavage sites involved in the processing of the rat 32S precursor to ribosomal RNA.

The initial endonuclease cleavage site in 32 S pre-rRNA (precursor to rRNA) is located within the rate rDNA sequence by S1-nuclease protection mapping of purified nucleolar 28 S rRNA and 12 S pre-rRNA. The heterogeneous 5'- and 3'-termini of these rRNA abut and map within two CTC motifs in tSi2 (internal transcribed spacer 2) located at 50-65 and 4-20 base-pairs upstream from the homogeneous 5'-end of the 28 S rRNA gene. These results show that multiple endonuclease cleavages occur at CUC sites in tSi2 to generate 28 S rRNA and 12 S pre-rRNA with heterogeneous 5'- and 3'-termini, respectively. These molecules have to be processed further to yield mature 28 S and 5.8 S rRNA. Thermal-denaturation studies revealed that the base-pairing association in the 12 S pre-rRNA:28 S rRNA complex is markedly stronger than that in the 5.8 S:28 S rRNA complex. The sequence of about one-quarter (1322 base-pairs) of the 5'-part of the rat 28 S rDNA was determined. A computer search reveals the possibility that the cleavage sites in the CUC motifs are single-stranded, flanked by strongly base-paired GC tracts, involving tSi2 and 28 S rRNA sequences. The subsequent nuclease cleavages, generating the termini of mature rRNA, seem to be directed by secondary-structure interactions between 5.8 S and 28 S rRNA segments in pre-rRNA. An analysis for base-pairing among evolutionarily conserved sequences in 32 S pre-rRNA suggests that the cleavages yielding mature 5.8 S and 28 S rRNA are directed by base-pairing between (i) the 3'-terminus of 5.8 S rRNA and the 5'-terminus of 28 S rRNA and (ii) the 5'-terminus of 5.8 S rRNA and internal sequences in domain I of 28 S rRNA. A general model for primary- and secondary-structure interactions in pre-rRNA processing is proposed, and its implications for ribosome biogenesis in eukaryotes are briefly discussed.     

Intermolecular base-paired interaction between complementary sequences present near the 3'' ends of 5S rRNA and 18S (16S) rRNA might be involved in the reversible association of ribosomal subunits.

Highly conserved sequences present at an identical position near the 3' ends of eukaryotic and prokaryotic 5S rRNAs are complementary to the 5' strand of the m2(6)A hairpin structure near the 3' ends of 18S rRNA and 16S rRNA, respectively. The extent of base-pairing and the calculated stabilities of the hybrids that can be constructed between 5S rRNAs and the small ribosomal subunit RNAs are greater than most, if not all, RNA-RNA interactions that have been implicated in protein synthesis. The existence of complementary sequences in 5S rRNA and small ribosomal subunit RNA, along with the previous observation that there is very efficient and selective hybridization in vitro between 5S and 18S rRNA, suggests that base-pairing between 5S rRNA in the large ribosomal subunit and 18S (16S) rRNA in the small ribosomal subunit might be involved in the reversible association of ribosomal subunits. Structural and functional evidence supporting this hypothesis is discussed.     

Evidence for a Competitive-Displacement Model for the initiation of protein synthesis involving the intermolecular hybridization of 5 S rRNA, 18 S rRNA and mRNA.

We have previously shown that a 5'-terminal region of mouse 5 S rRNA can base-pair in vitro with two distinct regions of 18 S rRNA. Further analysis reveals that these 5 S rRNA-complementary sequences in 18 S rRNA also exhibit complementarity to the Kozak consensus sequence surrounding the mRNA translational start site. To test the possibility that these 2 regions in 18 S rRNA may be involved in mRNA binding and translational initiation, we have tested, using an in vitro translation system, the effects of DNA oligonucleotides complementary to these 18 S rRNA sequences on protein synthesis. Results show that an oligonucleotide complementary to one 18 S rRNA region does inhibit translation at the step of initiation. We propose a Competitive-Displacement Model for the initiation of translation involving the intermolecular base-pairing of 5 S rRNA, 18 S rRNA and mRNA.