Ribosomal RNAs are tolerant toward genetic insertions: evolutionary origin of the expansion segments

Ribosomal RNAs (rRNAs), assisted by ribosomal proteins, form the basic structure of the ribosome, and play critical roles in protein synthesis. Compared to prokaryotic ribosomes, eukaryotic ribosomes contain elongated rRNAs with several expansion segments and larger numbers of ribosomal proteins. To investigate architectural evolution and functional capability of rRNAs, we employed a Tn5 transposon system to develop a systematic genetic insertion of an RNA segment 31 nt in length into Escherichia coli rRNAs. From the plasmid library harboring a single rRNA operon containing random insertions, we isolated surviving clones bearing rRNAs with functional insertions that enabled rescue of the E. coli strain (Δ7rrn) in which all chromosomal rRNA operons were depleted. We identified 51 sites with functional insertions, 16 sites in 16S rRNA and 35 sites in 23S rRNA, revealing the architecture of E. coli rRNAs to be substantially flexible. Most of the insertion sites show clear tendency to coincide with the regions of the expansion segments found in eukaryotic rRNAs, implying that eukaryotic rRNAs evolved from prokaryotic rRNAs suffering genetic insertions and selections.     

Composite structure of the chloroplast 23 S ribosomal RNA genes of Chlamydomonas reinhardii: Evolutionary and functional implications

The chloroplast ribosomal unit of Chlamydomonas reinhardii displays two features which are not shared by other chloroplast ribosomal units. These include the presence of an intron in the 23 S ribosomal RNA gene and of two small genes coding for 3 S and 7 S rRNA in the spacer between the 16 S and 23 S rRNA genes (Rochaix & Malnoë, 1978). Sequencing of the 7 S and 3 S rRNAs as well as their genes and neighbouring regions has shown that: (1) the 7 S and 3 S rRNA genes are 282 and 47 base-pairs long, respectively, and are separated by a 23 base-pair A + T-rich spacer. (2) A sequence microheterogeneity exists within the 3 S RNA genes. (3) The sequences of the 7 S and 3 S rRNAs are homologous to the 5′ termini of prokaryotic and other chloroplast 23 S rRNAs, indicating that the C. reinhardii counterparts of 23 S rRNA have a composite structure. (4) The sequences of the 7 S and 3 S rRNAs are related to that of cytoplasmic 5.8 S rRNA, suggesting that these RNAs may perform similar functions in the ribosome. (5) Partial nucleotide sequence complementarity is observed between the 5′ ends of the 7 S and 3 S RNAs on one hand and the 23 S rRNA sequences which flank the ribosomal intron on the other. These data are compatible with the idea that these small rRNAs may play a role in the processing of the 23 S rRNA precursor.     

RNA end-labeling and RNA ligase activities can produce a circular rRNA in whole cell extracts from trypanosomes.

We have found two enzymatic activities in whole cell extracts from Trypanosoma brucei; an end-labeling reaction involving a single uridine at the 3' end of ribosomal RNAs (rRNAs) and an RNA ligase activity joining 5' monophosphates to 3' hydroxyl groups. The RNA ligase acts upon one of the small rRNAs (180 nucleotides) from the trypanosome ribosomal repeat unit, forming a circular RNA. The specific circularization of this small rRNA is probably dependent on the secondary structure of the molecule and is not detectable in vivo.     

RNA synthesis in whole cells and protoplasts of centaurea: a comparison

Protoplasts enzymically isolated from suspension cultures of Centaurea cyanus L. incorporate radioactive precursors into RNA with kinetics similar to that of whole cells. There are differences, however, in several other aspects of RNA metabolism. The proportion of total RNA that contains poly(A) sequences (25 to 30%) is similar in both freshly isolated protoplasts and whole cells after a 20-minute pulse with [³H]adenosine. After a 4-hour pulse, however, poly(A)-containing RNA makes up 30% of the total RNA in protoplasts whereas it drops to 8% in whole cells. There appears to be a faulty processing of ribosomal precursor into the mature ribosomal species, as the precursor seems to accumulate to higher levels relative to the mature 18S and 25S rRNAs in protoplasts as compared to whole cells. Additional differences are seen in the size distributions of poly(A)-containing RNA, although the length of the poly(A) segment is similar in both protoplasts and whole cells. Within 24 hours protoplasts appear to have resumed a pattern of RNA synthesis similar to that of whole cells.     

Similarity of the primary sequences of ribosomal RNAs isolated from vegetative and developing cells of Dictyostelium discoideum

Differentiating cell aggregates of Dictyostelium discoideum exhibit a pattern of rRNA metabolism quite different from that observed in the single-celled vegetative amoebas of this organism. We have examined whether the differences are related to a requirement for the production of new types of ribosomal RNA during development. Oligonucleotide maps and supplementary sequence data for 25 S, 17 S, and 5 S rRNAs from vegetative and developing cells have revealed no detectable alterations in primary sequence distinguishing any species of rRNA in developing cells from its vegetative cell counterpart.     

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.     

Characterization of the complete mitochondrial genome of Drawida gisti (Metagynophora,Moniligastridae) and comparison with other Metagynophora species

Here, the complete mitochondrial genome (mitogenome) of Drawida gisti was sequenced and compared with the mitogenomes of other Metagynophora species. The circular mitogenome was 14,648 bp in length and contained two ribosomal RNA genes (rRNAs), 13 protein-coding genes (PCGs), and 22 transfer RNA genes (tRNAs). The types of constitutive genes and the direction of the coding strand that appeared in Drawida mitogenome were identical to those observed in other Metagynophora species, except for a missing lengthy non-coding region. The conservative relationships between Drawida species were supported by the overall analyses of 13 PCGs, two rRNAs, and 22 tRNAs. A comparison of the Metagynophora mitogenomes revealed that the ATP8 gene possessed the highest polymorphism among the 13 PCGs and two rRNAs. Phylogenetic analysis suggested that the Moniligastridae contained Drawida, which is a primitive Metagynophora group. Our study provides a step forward toward elucidating the evolutionary linkages within Drawida and even Metagynophora.     

RNomics in Archaea reveals a further link between splicing of archaeal introns and rRNA processing

The bulge–helix–bulge (BHB) motif recognised by the archaeal splicing endonuclease is also found in the long processing stems of archaeal rRNA precursors in which it is cleaved to generate pre-16S and pre-23S rRNAs. We show that in two species, Archaeoglobus fulgidus and Sulfolobus solfataricus, representatives from the two major archaeal kingdoms Euryarchaeota and Crenarchaeota, respectively, the pre-rRNA spacers cleaved at the BHB motifs surrounding pre-16S and pre-23S rRNAs subsequently become ligated. In addition, we present evidence that this is accompanied by circularisation of ribosomal pre-16S and pre-23S rRNAs in both species. These data reveal a further link between intron splicing and pre-rRNA processing in Archaea, which might reflect a common evolutionary origin of the two processes. One spliced RNA species designated 16S-D RNA, resulting from religation at the BHB motif of 16S pre-rRNA, is a highly abundant and stable RNA which folds into a three-stem structure interrupted by two single-stranded regions as assessed by chemical probing. It spans a region of the pre-rRNA 5′ external transcribed spacer exhibiting a highly conserved folding pattern in Archaea. Surprisingly, 16S-D RNA contains structural motifs found in archaeal C/D box small RNAs and binds to the L7Ae protein, a core component of archaeal C/D box RNPs. This supports the notion that it might have an important but still unknown role in pre-rRNA biogenesis or might even target RNA molecules other than rRNA.     

DNA-dependent in vitro synthesis of ribosomal proteins,protein elongation factors,and RNA polymerase subunit α: Inhibition by ppGpp

We have previously shown that the synthesis of ribosomal proteins (r proteins) in E. coli cells is under stringent control (Dennis and Nomura, 1974). Since guanosine tetraphosphate (ppGpp) had been implicated in stringent control, we examied the effects of ppGpp on the in vitro synthesis of r proteins directed by DNA from transducing phage λfus3 and λrif^d18. λfus3 carries genes for protein elongation factors EF-Tu and EF-G, and RNA polymerase subunit α, in addition to genes for approximately 27 r proteins. λrif^d18 carries genes for EF-Tu, RNA polymerase subunits β and β^I, and a set of rRNAs, in addition to genes for approximately five r proteins. We have shown that low concentrations of ppGpp (0.2–0.3 mM) specifically inhibit DNA-dependent r protein synthesis in this system, and that this inhibition takes place directly, rather than as a consequence of the inhibition of rRNA synthesis by ppGpp. In addition, we have also shown that ppGpp inhibits the synthesis of EF-G, EF-Tu, and RNA polymerase subunit α, as well as rRNAs.     

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.