RAC protein directs the complete removal of the 3' external transcribed spacer by the Pac1 nuclease

In Schizosaccharomyces pombe, interdependency in rRNA processing is mediated by a large protein complex (RAC) which contains independent binding sites for each of the transcribed spacers. The RAC complex exhibits no nuclease activity but dramatically alters the efficiency and specificity of the Pac1 nuclease, leading to the complete removal of the 3' ETS. Furthermore, the affinity of RAC protein for mutant 3' ETS correlates closely with in vivo effects on rRNA processing, and changes which disrupt RAC protein binding also inhibit Pac1 nuclease cleavage at the 3' end of the 25S rRNA sequence. The observations indicate that, in the presence of the RAC protein/3' ETS complex, cleavage by the RNase III-like homolog is not restricted to the known intermediate sites but also is directed at the 3' end of the 25S rRNA.     

Functional significance of intermediate cleavages in the 3'ETS of the pre-rRNA from Schizosaccharomyces pombe

Pathways for the maturation of ribosomal RNAs are complex with numerous intermediate cleavage sites that are not always conserved closely in the course of evolution. Both in eukaryotes and bacteria genetic analyses and in vitro studies have strongly implicated RNase III-like enzymes in the processing of rRNA precursors. In Schizosacharomyces pombe, for example, the RNase III-like Pac1 nuclease has been shown to cleave the free 3′ETS at two known intermediate sites but, in the presence of RAC protein, the same RNA also is cleaved at the 3′-end of the 25 S rRNA sequence. In this study normal and mutant 3′ETS sequences were digested with the Pac1 enzyme to further evaluate its role in rRNA processing. Accurate cleavage at the known intermediate processing sites was dependent on the integrity of the helical structure at these sites as well as a more distal upper stem region in the conserved extended hairpin structure of the 3′ETS. The cleavage of mutant 3′ETS sequences also generally correlated with the known effects of these mutations on rRNA production, in vivo. One mutant, however, was efficiently processed in vivo but was not a substrate for the Pac1 nuclease, in vitro. In contrast, in the presence of RAC protein, the same RNA remained susceptible to Pac1 nuclease cleavage at the 3′-end of the 25 rRNA sequence, indicating that the removal of the 3′ETS does not require cleavage at the intermediate sites. These results suggest that basic maturation pathways may be less complex than previously reported raising similar questions about other intermediate processing sites, which have been identified by analyses of termini, and/or processing, in vitro.     

A chaperone for ribosome maturation

The nascent pre-rRNA of eukaryotic ribosomes is fully transcribed and assembled into an 80-90 S nucleolar particle before being cleaved into mature ribosomal RNA. The interdependence of steps in the processing of this precursor RNA indicates that RNA processing, at least in part, acts as a quality control mechanism that helps ensure that only functional RNA is incorporated into mature ribosomes. In search of structural components that underlie this interdependence using the Schizosaccharomyces pombe internal transcribed spacer 1 (ITS) as a ligand for affinity chromatography of ITS1-specific proteins, we have isolated a large spliceosome-like protein complex, a ribosome assembly chaperone (RAC) of 20 or more polypeptides (Lalev, A. I., Abeyrathne, P. D., and Nazar, R. N. (2000) J. Mol. Biol. 302, 65-77). When the ITS2 spacer was used in the present study to isolate ITS2-specific proteins, the same proteins were identified consistent with a complex containing multiple specific binding sites. Subsequent competition binding studies indicated that the protein complex actually contains independent binding sites for all four of the transcribed spacers in the pre-rRNA. Because disruption of protein-binding sites in these spacer RNAs is known to severely affect rRNA processing, taken together these results suggest that the RAC complex is a chaperone for ribosome maturation acting as a "rack" on which critical structure is organized.     

Mutational analysis of an essential binding site for the U3 snoRNA in the 5' external transcribed spacer of yeast pre-rRNA.

The small nucleolar RNA U3 is essential for viability in yeast. We have previously shown that U3 can be cross-linked in vivo to the pre-rRNA in the 5' external transcribed spacer (ETS), at +470. This ETS region contains 10 nucleotides of perfect complementarity to U3. In a genetic background where the mutated rDNA is the only transcribed rDNA repeat, the deletion of the 10 nt complementary to U3 is lethal. Cells lacking the U3 complementary sequence in pre-rRNA fail to accumulate 18S rRNA: pre-rRNA processing is inhibited at sites A0 in the 5' ETS, A1 at the 5' end of 18S rRNA and A2 in ITS1. We show here that effects on processing at site A0 are specific for U3 and its associated proteins and are not seen on depletion of other snoRNP components. The deletion of the sequence complementary to U3 in the ETS therefore mimics all the known effects of the depletion of U3 in trans. This indicates that we have identified an essential U3 binding site on pre-rRNA, required in cis for the maturation of 18S rRNA.     

Mutational analysis of an essential binding site for the U3 snoRNA in the 5' external transcribed spacer of yeast pre-rRNA.

The small nucleolar RNA U3 is essential for viability in yeast. We have previously shown that U3 can be cross-linked in vivo to the pre-rRNA in the 5' external transcribed spacer (ETS), at +470. This ETS region contains 10 nucleotides of perfect complementarity to U3. In a genetic background where the mutated rDNA is the only transcribed rDNA repeat, the deletion of the 10 nt complementary to U3 is lethal. Cells lacking the U3 complementary sequence in pre-rRNA fail to accumulate 18S rRNA: pre-rRNA processing is inhibited at sites A0 in the 5' ETS, A1 at the 5' end of 18S rRNA and A2 in ITS1. We show here that effects on processing at site A0 are specific for U3 and its associated proteins and are not seen on depletion of other snoRNP components. The deletion of the sequence complementary to U3 in the ETS therefore mimics all the known effects of the depletion of U3 in trans. This indicates that we have identified an essential U3 binding site on pre-rRNA, required in cis for the maturation of 18S rRNA.     

Yeast Rnt1p is required for cleavage of the pre-ribosomal RNA in the 3' ETS but not the 5' ETS.

We have reexamined the role of yeast RNase III (Rnt1p) in ribosome synthesis. Analysis of pre-rRNA processing in a strain carrying a complete deletion of the RNT1 gene demonstrated that the absence of Rnt1p does not block cleavage at site A0 in the 5' external transcribed spacers (ETS), although the early pre-rRNA cleavages at sites A0, A1, and A2 are kinetically delayed. In contrast, cleavage in the 3' ETS is completely inhibited in the absence of Rnt1p, leading to the synthesis of a reduced level of a 3' extended form of the 25S rRNA. The 3' extended forms of the pre-rRNAs are consistent with the major termination at site T2 (+210). We conclude that Rnt1p is required for cleavage in the 3' ETS but not for cleavage at site A0. The sites of in vivo cleavage in the 3' ETS were mapped by primer extension. Two sites of Rnt1p-dependent cleavage were identified that lie on opposite sides of a predicted stem loop structure, at +14 and +49. These are in good agreement with the consensus Rnt1p cleavage site. Processing of the 3' end of the mature 25S rRNA sequence in wild-type cells was found to occur concomitantly with processing of the 5' end of the 5.8S rRNA, supporting previous proposals that processing in ITS1 and the 3' ETS is coupled.     

Ribosomal External and Internal Transcribed Spacers: Combined Use in the Phylogenetic Analysis of Medicago (Leguminosae)

We have estimated the potential phylogenetic utility of the ribosomal external transcribed spacer (ETS) from the nuclear ribosomal region. The ETS was sequenced from 13 annual Medicago (Fabaceae) species upstream a highly conserved motive which was found among many different organisms. In the genus Medicago, the ETS was found to evolve 1.5 times faster than the internal transcribed spacer and to be 1.5 times more informative. Reduced ribosomal maturation process constraints on ETS are proposed to explain the different evolutionary rates between the two spacers. Maximal phylogenetic resolution and support was obtained when the two spacers were analyzed together. No incongruence between the two spacers was found and ETS appears to be a valuable source of information for solidifying ITS plant phylogeny. The phylogeny obtained in Medicago suggests that none of the three subsections included in the study is monophyletic. Received: 15 April 1997 / Accepted: 29 July 1997     

Phylogenetic Utility of the External Transcribed Spacer (ETS) of 18S–26S rDNA: Congruence of ETS and ITS Trees ofCalycadenia(Compositae)

The 3′ region of the external transcribed spacer (ETS) of 18S–26S nuclear ribosomal DNA was sequenced in 19 representatives ofCalycadenia/Osmadeniaand two outgroup species (Compositae) to assess its utility for phylogeny reconstruction compared to rDNA internal transcribed spacer (ITS) data. Universal primers based on plant, fungal, and animal sequences were designed to amplify the intergenic spacer (IGS) and an angiosperm primer was constructed to sequence the 3′ end of the ETS in members of tribe Heliantheae. Based on these sequences, an internal ETS primer useful across Heliantheaesensu latowas designed to amplify and sequence directly the 3′ ETS region in the study taxa, which were the subjects of an earlier phylogenetic investigation based on ITS sequences. Size variation in the amplified ETS region varied across taxa of Heliantheaesensu latofrom approximately 350 to 700 bp, in part attributable to an approximately 200-bp tandem duplication in a common ancestor ofCalycadenia/Osmadenia.Phylogenetic analysis of the 200-bp subrepeats and examination of apomorphic changes in the duplicated region demonstrate that the subrepeats inCalycadenia/Osmadeniahave evolved divergently. Phylogenetic analyses of the entire amplified ETS region yielded a highly resolved strict consensus tree that is nearly identical in topology to the ITS tree, with strong bootstrap and decay support on most branches. Parsimony analyses of combined ETS and ITS data yielded a strict consensus tree that is better resolved and generally better supported than trees based on either data set analyzed separately. We calculated an approximately 1.3- to 2.4-fold higher rate of sequence evolution by nucleotide substitution in the ETS region studied than in ITS-1 + ITS-2. A similar disparity in the proportion of variable (1.3 ETS:1 ITS) and potentially informative (1.5 ETS:1 ITS) sites was observed for the ingroup. Levels of homoplasy are similar in the ETS and ITS data. We conclude that the ETS holds great promise for augmenting ITS data for phylogenetic studies of young lineages.     

Structure,molecular evolution,and phylogenetic utility of the 5(') region of the external transcribed spacer of 18S-26S rDNA in Lessingia (Compositae,Astereae)

The 18S-26S nuclear rDNA external transcribed spacer (ETS) has recently gained attention as a region that is valuable in phylogenetic analyses of angiosperms primarily because it can supplement nucleotide variation from the widely used and generally shorter internal transcribed spacers (ITS-1 and ITS-2) and thereby improve phylogenetic resolution and clade support in rDNA trees. Subrepeated ETS sequences (often occurring in the 5(') region) can, however, create a challenge for systematists interested in using ETS sequence data for phylogeny reconstruction. We sequenced the 5(')ETS for members of Lessingia (Compositae, Astereae) and close relatives (26 taxa total) to characterize the subrepeat variation across a group of closely related plant lineages and to gain improved understanding of the structure, molecular evolution, and phylogenetic utility of the region. The 5(')ETS region of Lessingia and relatives varied in length from approximately 245 to 1009 bp due to the presence of a variable number of subrepeats (one to eight). We assessed homology of the subrepeats using phylogenetic analysis and concluded that only two of the subrepeats and a portion of a third ( approximately 282 bp in total) were orthologous across Lessingia and could be aligned with confidence and included in further analyses. When the partial 5(')ETS data were combined with 3(')ETS and ITS data in phylogenetic analyses, no additional resolution of relationships among taxa was obtained beyond that found from analysis of 3(')ETS + ITS sequences. Inferred patterns of concerted evolution indicate that homogenization is occurring at a faster rate in the 3(')ETS and ITS regions than in the 5(')ETS region. Additionally, homogenization appears to be acting within but not among subrepeats of the same rDNA array. We conclude that challenges in assessing subrepeat orthology across taxa greatly limit the utility of the 5(')ETS region for phylogenetic analyses among species of Lessingia.     

An evolutionary intra-molecular shift in the preferred U3 snoRNA binding site on pre-ribosomal RNA

Correct docking of U3 small nucleolar RNA (snoRNA) on pre-ribosomal RNA (pre-rRNA) is essential for rRNA processing to produce 18S rRNA. In this report, we have used Xenopus oocytes to characterize the structural requirements of the U3 snoRNA 3′-hinge interaction with region E1 of the external transcribed spacer (ETS) of pre-rRNA. This interaction is crucial for docking to initiate rRNA processing. 18S rRNA production was inhibited when fewer than 6 of the 8 bp of the U3 3′–hinge complex with the ETS could form; moreover, base pairing involving the right side of the 3′-hinge was more important than the left. Increasing the length of the U3 hinge–ETS interaction by 9 bp impaired rRNA processing. Formation of 18S rRNA was also inhibited by swapping the U3 5′- and 3′-hinge interactions with the ETS or by shifting the base pairing of the U3 3′-hinge to the sequence directly adjacent to ETS region E1. However, 18S rRNA production was partially restored by a compensatory shift that allowed the sequence adjacent to the U3 3′-hinge to pair with the eight bases directly adjacent to ETS region E1. The results suggest that the geometry of the U3 snoRNA interaction with the ETS is critical for rRNA processing.     

Molecular evolution and phylogenetic implications of internal transcribed spacer sequences of Ribosomal DNA in Winteraceae

The internal transcribed spacers and the 5.8S coding region of nuclear ribosomal DNA were sequenced and analyzed to address questions of generic relationships in Winteraceae. The molecular data generated a single tree that is congruent with one based on morphological data. The sequences of ITS 1 in the family range from 235 to 252 bases in size and of ITS 2 from 213 to 226 bases. The size of the 5.8S coding region is 164 bases. The range of ITS 1 and ITS 2 sequence divergence between pairs of genera within Winteraceae is relatively low in comparison to other plant families. Two types of ITS 1 and ITS 2 sequences were observed in the same individual for some taxa. Sequence variations between the two arrays are 4.7%–6.3% for ITS 1 and 5.1%–7.0% for ITS 2. Both arrays of sequences, however, generate the same phylogenetic relationships. Rates of nucleotide substitutions for the internal transcribed spacers are 3.2–5.2 × 10^-10 substitution per site per year estimated in ITS 1 and 3.6–5.7 × 10^-10 in ITS 2.     

Phylogenetic utility of the external transcribed spacer (ETS) of 18S-26S rDNA: congruence of ETS and ITS trees of Calycadenia (Compositae)

The 3' region of the external transcribed spacer (ETS) of 18S-26S nuclear ribosomal DNA was sequenced in 19 representatives of Calycadenia/Osmadenia and two outgroup species (Compositae) to assess its utility for phylogeny reconstruction compared to rDNA internal transcribed spacer (ITS) data. Universal primers based on plant, fungal, and animal sequences were designed to amplify the intergenic spacer (IGS) and an angiosperm primer was constructed to sequence the 3' end of the ETS in members of tribe Heliantheae. Based on these sequences, an internal ETS primer useful across Heliantheae sensu lato was designed to amplify and sequence directly the 3' ETS region in the study taxa, which were the subjects of an earlier phylogenetic investigation based on ITS sequences. Size variation in the amplified ETS region varied across taxa of Heliantheae sensu lato from approximately 350 to 700 bp, in part attributable to an approximately 200-bp tandem duplication in a common ancestor of Calycadenia/Osmadenia. Phylogenetic analysis of the 200-bp subrepeats and examination of apomorphic changes in the duplicated region demonstrate that the subrepeats in Calycadenia/Osmadenia have evolved divergently. Phylogenetic analyses of the entire amplified ETS region yielded a highly resolved strict consensus tree that is nearly identical in topology to the ITS tree, with strong bootstrap and decay support on most branches. Parsimony analyses of combined ETS and ITS data yielded a strict consensus tree that is better resolved and generally better supported than trees based on either data set analyzed separately. We calculated an approximately 1.3- to 2.4-fold higher rate of sequence evolution by nucleotide substitution in the ETS region studied than in ITS-1 + ITS-2. A similar disparity in the proportion of variable (1.3 ETS:1 ITS) and potentially informative (1.5 ETS:1 ITS) sites was observed for the ingroup. Levels of homoplasy are similar in the ETS and ITS data. We conclude that the ETS holds great promise for augmenting ITS data for phylogenetic studies of young lineages.     

Nucleotide sequence of the 17S–25S spacer region from rice rDNA

Summary The nucleotide sequence of a spacer region between rice 17S and 25S rRNA genes (rDNAs) has been determined. The coding regions for the mature 17S, 5.8S and 25S rRNAs were identified by sequencing terminal regions of these rRNAs. The first internal transcribed spacer (ITS1), between 17S and 5.8S rDNAs, is 194–195 bp long. The second internal transcribed spacer (ITS2), between 5.8S and 25S rDNAs, is 233 bp long. Both spacers are very rich in G+C, 72.7% for ITS1 and 77.3% for ITS2. The 5.8S rDNA is 163–164 bp long and similar in primary and secondary structures to other eukaryotic 5.8S rDNAs. The 5.8S rDNA is capable of interacting with the 5′ terminal region of 25S rDNA.     

Differentiation of two ovine Babesia based on the ribosomal DNA internal transcribed spacer (ITS) sequences

The first and second internal transcribed spacers (ITS1, ITS2) as well as the intervening 5.8S coding region of the rRNA gene for six Babesia spp. isolated from different geographic origins were characterized. Varying degrees of ITS1 and ITS2 intra- and inter-species sequence polymorphism were found among these isolates. Phylogenetic analysis of the ITS1-5.8S gene-ITS2 region clearly separated the isolates into two clusters. One held an unidentified Babesia sp. transmitted by Hyalomma anatolicum anatolicum. The second held five other isolates, which were considered to be Babesia motasi. Each Babesia species cluster possessed ITS1 and ITS2 of unique size(s) and species specific nucleotide sequences. The results showed that ITS1, ITS2 and the complete ITS1-5.8S-ITS2 region could be used to discriminate these ovine Babesia spp. effectively.     

The evolution of Dactylorhiza (Orchidaceae) allotetraploid complex: insights from nrDNA sequences and cpDNA PCR-RFLP data

Sequence data from a portion of the external transcribed spacer (ETS) and from the internal transcribed spacers (ITS1 and ITS2) of 18S-26S nuclear ribosomal DNA were used together with chloroplast DNA PCR-RFLP data to unravel patterns of allotetraploid speciation within the Western European Dactylorhiza polyploid complex. A maximum likelihood tree based on combined ETS and ITS sequences suggests that the Western European Dactylorhiza allotetraploids have evolved by hybridization between four main diploid lineages. Cloned sequences and the topology of the ITS plus ETS tree indicate that the allotetraploid species D. elata, D. brennensis, and D. sphagnicola have originated from the autotetraploid D. maculata together with the diploid D. incarnata, while D. majalis, D. traunsteineri, and D. angustata seem to have evolved by hybridization between the D. fuchsii s.str and D. incarnata lineages. Finally, the diploid D. saccifera lineage seems to have been involved together with the D. incarnata lineage in the formation of the allotetraploid D. praetermissa. The observed congruence between the chloroplast tree and the ITS/ETS tree suggests a directional evolution of the nrDNA after polyploidization in favor of the maternal genome. Considered together with morphological, biogeographical, and ecological evidence, the molecular analysis leads us to recognize four species within the investigated allotetraploid complex, namely D. majalis, D. praetermissa, D. elata, and D. sphagnicola.