Analysis of intergenic spacer transcripts suggests 'read-around' transcription of the extrachromosomal circular rDNA in Euglena gracilis.

We report here the sequence of the 1743 bp intergenic spacer (IGS) that separates the 3'-end of the large subunit ribosomal RNA (rRNA) gene from the 5'-end of the small subunit (SSU) rRNA gene in the circular, extrachromosomal ribosomal DNA (rDNA) of Euglena gracilis. The IGS contains a 277 nt stretch of sequence that is related to a sequence found in ITS 1, an internal transcribed spacer between the SSU and 5.8S rRNA genes. Primer extension analysis of IGS transcripts identified three abundant reverse transcriptase stops that may be analogous to the transcription initiation site (TIS) and two processing sites (A' and A0) that are found in this region in other eukaryotes. Features that could influence processing at these sites include an imperfect palindrome near site A0 and a sequence near site A' that could potentially base pair with U3 small nucleolar RNA. Our identification of the TIS (verified by mung bean nuclease analysis) is considered tentative because we also detected low-abundance transcripts upstream of this site throughout the entire IGS. This result suggests the possibility of 'read-around' transcription, i.e. transcription that proceeds multiple times around the rDNA circle without termination.

The 28S-18S rDNA intergenic spacer from Crithidia fasciculata: repeated sequences, length heterogeneity, putative processing sites and potential interactions between U3 small nucleolar RNA and the ribosomal RNA precursor.

In Crithidia fasciculata, the ribosomal RNA (rRNA) gene repeats range in size from approximately 11 to 12 kb. This length heterogeneity is localized to a region of the intergenic spacer (IGS) that contains tandemly repeated copies of a 19mer sequence. The IGS also contains four copies of an approximately 55 nt repeat that has an internal inverted repeat and is also present in the IGS of Leishmania species. We have mapped the C.fasciculata transcription initiation site as well as two other reverse transcriptase stop sites that may be analogous to the A0 and A' pre-rRNA processing sites within the 5' external transcribed spacer (ETS) of other eukaryotes. Features that could influence processing at these sites include two stretches of conserved primary sequence and three secondary structure elements present in the 5' ETS. We also characterized the C.fasciculata U3 snoRNA, which has the potential for base-pairing with pre-rRNA sequences. Finally, we demonstrate that biosynthesis of large subunit rRNA in both C. fasciculata and Trypanosoma brucei involves 3'-terminal addition of three A residues that are not present in the corresponding DNA sequences.

Phylogenetic analysis of molluscan mitochondrial LSU rDNA sequences and secondary structures.

Mollusks are an extraordinarily diverse group of animals with an estimated 200,000 species, second only to the phylum Arthropoda. We conducted a comparative analysis of complete mitochondrial ribosomal large subunit sequences (LSU) of a chiton, two bivalves, six gastropods, and a cephalopod. In addition, we determined secondary structure models for each of them. Comparative analyses of nucleotide variation revealed substantial length variation among the taxa, with stylommatophoran gastropods possessing the shortest lengths. Phylogenetic analyses of the nucleotide sequence data supported the monophyly of Albinaria, Euhadra herklotsi + Cepaea nemoralis, Stylommatophora, Cerithioidea, and when only transversions are included, the Bivalvia. The phylogenetic limits of the mitochondrial LSU rRNA gene within mollusks appear to be up to 400 million years, although this estimate will have to be tested further with additional taxa. Our most novel finding was the discovery of phylogenetic signal in the secondary structure of rRNA of mollusks. The absence of entire stem/loop structures in Domains II, III, and V can be viewed as three shared derived characters uniting the stylommatophoran gastropods. The absence of the aforementioned stem/loop structure explains much of the observed length variation of the mitochondrial LSU rRNA found within mollusks. The distribution of these unique secondary structure characters within mollusks should be examined.

Complete sequence of the mitochondrial genome of Tetrahymena pyriformis and comparison with Paramecium aurelia mitochondrial DNA.

We report the complete nucleotide sequence of the Tetrahymena pyriformis mitochondrial genome and a comparison of its gene content and organization with that of Paramecium aurelia mtDNA. T. pyriformis mtDNA is a linear molecule of 47,172 bp (78.7 % A+T) excluding telomeric sequences (identical tandem repeats of 31 bp at each end of the genome). In addition to genes encoding the previously described bipartite small and large subunit rRNAs, the T. pyriformis mitochondrial genome contains 21 protein-coding genes that are clearly homologous to genes of defined function in other mtDNAs, including one (yejR) that specifies a component of a cytochrome c biogenesis pathway. As well, T. pyriformis mtDNA contains 22 open reading frames of unknown function larger than 60 codons, potentially specifying proteins ranging in size from 74 to 1386 amino acid residues. A total of 13 of these open reading frames ("ciliate-specific") are found in P. aurelia mtDNA, whereas the remaining nine appear to be unique to T. pyriformis; however, of the latter, five are positionally equivalent and of similar size in the two ciliate mitochondrial genomes, suggesting they may also be homologous, even though this is not evident from sequence comparisons. Only eight tRNA genes encoding seven distinct tRNAs are found in T. pyriformis mtDNA, formally confirming a long-standing proposal that most T. pyriformis mitochondrial tRNAs are nucleus-encoded species imported from the cytosol. Atypical features of mitochondrial gene organization and expression in T. pyriformis mtDNA include split and rearranged large subunit rRNA genes, as well as a split nad1 gene (encoding subunit 1 of NADH dehydrogenase of respiratory complex I) whose two segments are located on and transcribed from opposite strands, as is also the case in P. aurelia. Gene content and arrangement are very similar in T. pyriformis and P. aurelia mtDNAs, the two differing by a limited number of duplication, inversion and rearrangement events. Phylogenetic analyses using concatenated sequences of several mtDNA-encoded proteins provide high bootstrap support for the monophyly of alveolates (ciliates, dinoflagellates and apicomplexans) and slime molds.

Structural conservation and variation among U5 small nuclear RNAs from trypanosomatid protozoa.

U5 snRNAs in trypanosomatid protozoa do not contain the trimethylguanosine cap structures that are often targeted in snRNA isolation procedures. As a result, the trypanosomatids are not well represented in the database of available U5 snRNA sequences. We have isolated and determined the sequence of the U5 snRNA from Crithidia fasciculata. Comparison with previously published trypanosomatid U5 snRNA sequences allows us to deduce the pattern of structural conservation and variation among these very divergent snRNA molecules.

Spliced leader-associated RNA from Crithidia fasciculata contains a structure resembling stem/loop II of U1 snRNA.

In contrast to earlier proposals, recent evidence suggests that trans-spliceosomes in trypanosomatid protozoa may contain a homolog of U1 small nuclear (sn) RNA (Schnare, M.N. and Gray, M.W. (1999) J. Biol. Chem. 274, 23,691-23,694). However, the candidate trypanosomatid U1 snRNA is unconventional because it lacks the highly conserved stem/loop II present in all other U1 snRNAs. Trypanosomatids also possess a unique spliced leader-associated (SLA) RNA of unknown function. We present the complete sequence of the SLA RNA from Crithidia fasciculata and propose that it may contribute a U1 snRNA-like stem/loop II to the trans-spliceosome.

A candidate U1 small nuclear RNA for trypanosomatid protozoa.

In trypanosomatid protozoa, all mRNAs obtain identical 5'-ends by trans-splicing of the 5'-terminal 39 nucleotides of a small spliced leader RNA to appropriate acceptor sites in pre-mRNA. Although this process involves spliceosomal small nuclear (sn) RNAs, it is thought that trypanosomatids do not contain a homolog of the cis-spliceosomal U1 snRNA. We show here that a trypanosomatid protozoon, Crithidia fasciculata, contains a novel small RNA that displays several features characteristic of a U1 snRNA, including (i) a methylguanosine cap and additional 5'-terminal modifications, (ii) a potential binding site for common core proteins that are present in other trans-spliceosomal ribonucleoproteins, (iii) a U1-like 5'-terminal sequence, and (iv) a U1-like stem/loop I structure. Because trypanosomatid pre-mRNAs do not appear to contain cis-spliced introns, we argue that this previously unrecognized RNA species is a good candidate to be a trans-spliceosomal U1 snRNA.

U1 small nuclear RNA and spliceosomal introns in Euglena gracilis.

In the flagellated protozoon Euglena gracilis, characterized nuclear genes harbor atypical introns that usually are flanked by short repeats, adopt complex secondary structures in pre-mRNA, and do not obey the GT-AG rule of conventional cis-spliced introns. In the nuclear fibrillarin gene of E. gracilis, we have identified three spliceosomal-type introns that have GT-AG consensus borders. Furthermore, we have isolated a small RNA from E. gracilis and propose, on the basis of primary and secondary structure comparisons, that it is a homolog of U1 small nuclear RNA, an essential component of the cis-spliceosome in higher eukaryotes. Conserved sequences at the 5' splice sites of the fibrillarin introns can potentially base pair with Euglena U1 small nuclear RNA. Our observations demonstrate that spliceosomal GT-AG cis-splicing occurs in Euglena, in addition to the nonconventional cis-splicing and spliced leader trans-splicing previously recognized in this early diverging unicellular eukaryote.

Molecular characterization of U3 small nucleolar RNA from the early diverging protist, Euglena gracilis.

U3 small nucleolar RNA (snoRNA) has been isolated from Euglena gracilis, an early diverging protist, and its primary sequence determined. Although this 180-nucleotide-long RNA is considerably smaller than its homolog in vertebrate animals, it contains the conserved sequence blocks (boxes A, Ao, B, C and D) characteristic of U3 snoRNAs from other organisms. A secondary structure can be modelled that displays many of the salient features found in published core structures of vertebrate, yeast and trypanosome U3 snoRNAs. The functional significance of this proposed secondary structure is discussed in relation to the role E. gracilis U3 snoRNA may have in pre-rRNA processing in this organism. Multiple expressed species of E. gracilis U3 snoRNA were found to differ in nucleotide sequence at a number of positions; some of these differences alter pairing in the proposed secondary structure. Analysis of E. gracilis genomic DNA revealed a complex pattern of U3-hybridizing sequences that parallels the multiplicity of expressed species of U3 snoRNA revealed by transcript analysis.

Comprehensive comparison of structural characteristics in eukaryotic cytoplasmic large subunit (23 S-like) ribosomal RNA.

Comparative modeling of secondary structure is a proven approach to predicting higher order structural elements in homologous RNA molecules. Here we present the results of a comprehensive comparison of newly modeled or refined secondary structures for the cytoplasmic large subunit (23 S-like) rRNA of eukaryotes. This analysis, which covers a broad phylogenetic spectrum within the eukaryotic lineage, has defined regions that differ widely in their degree of structural conservation, ranging from a core of primary sequence and secondary structure that is virtually invariant, to highly variable regions. New comparative information allows us to propose structures for many of the variable regions that had not been modeled before, and rigorously to confirm or refine variable region structures previously proposed by us or others. The present analysis also serves to identify phylogenetically informative features of primary and secondary structure that characterize these models of eukaryotic cytoplasmic 23 S-like rRNA. Finally, the work summarized here provides a basis for experimental studies designed both to test further the validity of the proposed secondary structures and to explore structure-function relationships.

RNA: RNA interactions in the large subunit ribosomal RNA of Euglena gracilis.

In Euglena gracilis, the cytoplasmic large subunit (LSU) rRNA is composed of 14 discrete small RNA species that must somehow interact in the functional ribosome. We have isolated native complexes of Euglena rRNA and show here that the largest of these complexes contains eight of the 14 LSU rRNA species. Several of these small rRNA species are able to associate in vitro to reform an isolated domain of LSU rRNA structure.

Primary sequence and post-transcriptional modification pattern of an unusual mitochondrial tRNA(Met) from Tetrahymena pyriformis.

In a previous investigation of the rDNA region in Tetrahymena pyriformis mitochondrial DNA, we identified a putative tRNA(Met) gene [Heinonen et al. (1987) J. Biol. Chem. 262, 2879-2887]. On the basis of Northern hybridization analyses, we suggested that this gene is expressed, even though the resulting tRNA would be unusually small and have an atypical dihydrouridine stem-loop domain. We report here the complete nucleotide sequence and post-transcriptional modification pattern of this tRNA(Met), confirming its predicted primary structure and supporting the view that this structurally aberrant species functions in translation in T. pyriformis mitochondria.

Sequence and organization of a 7.2 kb region of wheat mitochondrial DNA containing the large subunit (26S) rRNA gene.

We report the sequence of a 7.2 kilobase pair DNA fragment containing a copy of the wheat mitochondrial gene (rrn26) that encodes the mitochondrial large-subunit ribosomal RNA (26S rRNA). The mature 26S rRNA was determined by direct RNA sequencing to be 3467 nucleotides long, and to share a 5'-terminal pentanucleotide (5'-AUCAU), thought to be important in post-transcriptional processing, with the wheat mitochondrial small-subunit (18S) rRNA. Two other prominent features of the sequence were noted. First, upstream of rrn26 are located two tandem copies of a 70 base pair element containing a putative mitochondrial promoter motif (TCGTATAAAAA). Second, downstream of rrn26 is a sequence element that, if transcribed, would produce an RNA with a secondary structure resembling that of tRNAs but differing sufficiently from the latter structure to preclude any transcript from functioning normally in translation. These upstream and downstream sequence elements may play a role in the expression of rrn26 in wheat mitochondria.

Six group I introns and three internal transcribed spacers in the chloroplast large subunit ribosomal RNA gene of the green alga Chlamydomonas eugametos.

The chloroplast large subunit rRNA gene of Chlamydomonas eugametos and its 5' flanking region encoding tRNA(Ile) (GAU) and tRNA(Ala) (UGC) have been sequenced. The DNA sequence data along with the results of a detailed RNA analysis disclosed two unusual features of this green algal large subunit rRNA gene: (1) the presence of six group I introns (CeLSU.1-CeLSU.6) whose insertion positions have not been described previously, and (2) the presence of three short internal transcribed spacers that are post-transcriptionally excised to yield four rRNA species of 280, 52, 810 and 1720 nucleotides, positioned in this order (5' to 3') in the primary transcript. Together, these RNA species can assume a secondary structure that is almost identical to that proposed for the 23 S rRNA of Escherichia coli. All three internal transcribed spacers map to variable regions of primary sequence and/or potential secondary structure, whereas all six introns lie within highly conserved regions. The first three introns are inserted within the sequence encoding the 810 nucleotide rRNA species and map within domain II of the large subunit rRNA structure; the remaining introns, found in the sequence encoding the 1720 nucleotide rRNA species, lie within either domain IV or V, as is the case for all other large subunit rDNA introns that have been documented to date. CeLSU.5 and CeLSU.6 each contain a long open reading frame (ORF) of more than 200 codons. While the CeLSU.6 ORF is not related to any known ORFs, the CeLSU.5 ORF belongs to a family of ORFs that have been identified in Podospora and Neurospora mitochondrial group I introns. The finding that a polymorphic marker showing unidirectional gene conversion during crosses between C. eugametos and Chlamydomonas moewusii is located within the CeLSU.5 ORF makes it likely that this intron is a mobile element and that its ORF encodes a site-specific endonuclease promoting the transfer of the intron DNA sequence.

Sequence heterogeneity in the duplicate large subunit ribosomal RNA genes of Tetrahymena pyriformis mitochondrial DNA.

In the ciliated protozoan, Tetrahymena pyriformis, the mitochondrial large subunit ribosomal RNA (LSU rRNA) is discontinuous, consisting of two discrete RNA species: a 280-nucleotide LSU alpha (constituting the 5'-portion) and a 2315-nucleotide LSU beta (corresponding to the remaining 3'-portion of this rRNA). The T. pyriformis mitochondrial genome contains two copies of the LSU alpha.beta gene complex, and we have previously provided evidence that both copies are transcribed (Heinonen, T. Y. K., Schnare, M. N., Young, P. G., and Gray, M. W. (1987) J. Biol. Chem. 262, 2879-2887). We now report the complete sequences of the two copies of the LSU alpha.beta gene complex. These are not identical, but differ at 5 out of the 2595 positions by single nucleotide substitutions in one sequence relative to the other. In the secondary structure model we propose here, two of these differences are located in base-paired regions of the LSU rRNA; however, they do not interrupt the complementary interactions in these helices. The other three differences occur in single-stranded regions of the secondary structure. The base substitutions documented here are not localized to those regions of LSU rRNA that are the most highly conserved in global phylogenetic comparisons, and therefore it seems unlikely that they are of fundamental functional significance. Whether they might exert more subtle effects on ribosome function remains to be determined.

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.