Recently, the number of cryptic species known has increased considerably, showing that species diversity has in many cases been underestimated in the past. Parmelia sulcata is a widely distributed species and one of the most common taxa in temperate Europe. The first intra-specific molecular studies on P. sulcata showed an unexpectedly high genetic variability. In the present work, we study the biodiversity of this taxon including specimens from four continents and using three molecular markers (nuITS, nuIGS rDNA, and partial β-tubulin gene). Two monophyletic groups of P. sulcata were encountered; one of these is epitypified as P. sulcata s. str and the other one is segregated as the new cryptic species P. encryptata sp. nov. Issues surrounding the lectotypification of Parmelia sulcata have also been elucidated.

We examined three species of diminutive Porphyra,Porphyra suborbiculata Kjellman from the North Pacific, Porphyra lilliputiana W. A. Nelson, G. A. Knight et M. W. Hawkes from the South Pacific, and Porphyra carolinensis Coll et J. Cox from the western North Atlantic. These taxa were compared in terms of morphology, habitat data and sequence haplotypes of nuclear small subunit rDNA (SSU) and internal transcribed spacers of the nuclear rDNA cistron (ITS). These three species have similar morphologies and growth habits, and share very similar type descriptions and habitat records. Haplotype variation was found within the 11 samples of P. lilliputiana we examined and within P. suborbiculata samples from two locations, but the single P. carolinensis haplotype (from collections from two separate locations) was identical to one found in several widespread P. lilliputiana samples. Unrooted phylogenetic trees based on sequence data do not support any of the three species as being a monophyletic group. We conclude that these three taxa represent a single species with the oldest name P. suborbiculata having nomenclatural priority. It is likely that P. suborbiculata has recently been introduced to the western Atlantic from the Pacific region.

In vivo selection was used to improve the activity of the Tetrahymena pre-rRNA self-splicing intron in the context of heterologous exons. The intron was engineered into a kanamycin nucleotidyltransferase gene, with the pairing between intron bases and the 5′ and 3′ splice sites maintained. The initial construct failed to confer kanamycin resistance on Escherichia coli, although the pre-mRNA was active in splicing in vitro. Random mutation libraries were constructed to identify active intron variants in E. coli. All the active mutants sequenced contained mutations disrupting a base-paired region above the paired region P1 (referred to as the P1 extension region or P1ex) that involves the very 5′ end of the intron. Subsequent site-directed mutagenesis confirmed that these P1ex mutations are responsible and sufficient to activate the intron splicing in E. coli. Thus, it appears that too strong of a secondary structure in the P1ex element can be inhibitory to splicing in vivo. In vitro splicing assays demonstrated that two P1ex mutant constructs splice six to eight times faster than the designed construct at 40 μM GTP concentration. The relative reaction rates of the mutant constructs compared to the original design are further increased at a lower GTP concentration. Possible mechanisms by which the disrupted P1ex structure could influence splicing rates are discussed. This study emphasizes the value of using libraries of random mutations to improve the activity of ribozymes in heterologous contexts in vivo.

Self-splicing of the Tetrahymena pre-rRNA is inhibited by a conserved rRNA hairpin P(−1) upstream of the 5′ splice site. P(−1) inhibits self-splicing by competing with formation of the P1 splice site helix. Here we show that the P(−1) hairpin also enhances dissociation of the spliced products, which was monitored by native gel electrophoresis. Mutations that stabilize the rRNA hairpin increase the rate of dissociation approximately 10-fold, from 0.5 min−1 for the wild-type RNA to ∼4 min−1 at 30 °C. Conversely, mutations or oligonucleotides that inhibit refolding of the exons and that stabilize the P1 helix decrease the rate of product release. The results suggest that refolding of products can be used to stimulate the turnover of ribozyme-catalyzed reactions. In the pre-rRNA, this conformational change helps shift the equilibrium of self-splicing toward the mature rRNA.

Large ribozymes typically require very long times to refold into their active conformation in vitro, because the RNA is easily trapped in metastable misfolded structures. Theoretical models show that the probability of misfolding is reduced when local and long-range interactions in the RNA are balanced. Using the folding kinetics of the Tetrahymena ribozyme as an example, we propose that folding rates are maximized when the free energies of forming independent domains are similar to each other. A prediction is that the folding pathway of the ribozyme can be reversed by inverting the relative stability of the tertiary domains. This result suggests strategies for optimizing ribozyme sequences for therapeutics and structural studies.

NaSSU1 is a complex nuclear group I intron found in several species of Naegleria, consisting of a large self-splicing group I ribozyme (NaGIR2), which itself is interrupted by a small, group I-like ribozyme (NaGIR1) and an open reading frame (ORF) coding for a homing endonuclease. The GIR1 ribozyme cleaves in vitro transcripts of NaSSU1 at two internal processing sites about 400 nt downstream of the 5′ end of the intron, proximal to the endonuclease ORF. Here we demonstrate that self-cleavage of the excised intron also occurs in vivo in Naegleria gruberi, generating an ORF-containing RNA that possesses a short leader with a sequence element likely to be involved in gene expression. To assess the functional significance of self-cleavage, we constructed a genetic system in Saccharomyces cerevisiae. First, a mutant yeast strain was selected with a mutation in all the rRNA genes, rendering the rDNA resistant to cleavage by the Naegleria endonuclease. Active endonuclease, which is otherwise lethal, could be expressed readily in these cells. Endonuclease activity also could be detected in extracts of yeast harboring plasmids in which the endonuclease ORF was embedded in its native context in the intron. Analysis of the RNA from these yeast cells showed that the excised intron RNA was processed as in N. gruberi. A mutant intron constructed to prevent self-cleavage of the RNA failed to express endonuclease activity. These results support the hypothesis that the NaGIR1-catalyzed self-cleavage of the intron RNA is a key event in expression of the endonuclease.

Divalent metal ions are essential for the folding and catalytic activities of many RNAs. A commonly employed biochemical technique to identify metal-binding sites in RNA is the rescue of RP α-phosphorothioate (PS) interference by the addition of soft divalent metal ions. To access the ability of such experiments to accurately identify metal-ion coordinations within a complex RNA fold, we report metal-rescue results from the Tetrahymena group I intron P4–P6 domain, where the location and coordination of five divalent metal ions have been determined by X-ray crystallography [J.H. Cate et al., Nat Struct Biol, 1997, 4:553]. We used a native gel mobility-shift to assay for P4–P6 folding in the presence of various divalent metal ions, and found that even moderate concentrations of Mn2+ (≥0.5 mM) can rescue PS interference at sites that do not coordinate metal ions within the P4–P6 crystal structure. To control for such effects, 2′-deoxynucleotide interference was used to titrate the Mn2+ concentration to a level that produces metal-ion-specific rescue (0.3 mM). This concentration of Mn2+ specifically rescued four of the six metal-dependent phosphorothioate effects within the RNA domain, including PS interference resulting from outer-sphere coordination to the metals. Both sites that were not specifically rescued make inner-sphere metal-ion coordinations. Cd2+ and Zn2+ afforded rescue at a smaller subset of the six metal-specific PS sites, though again phosphates making outer-sphere coordinations to metal ions were rescued preferentially. These data on P4–P6 domain folding reinforce the need for caution when interpreting metal-rescue experiments.

Group I introns constitute excellent systems for analyzing the relationship between RNA tertiary folding and catalysis. Within a hierarchical framework interpretation of RNA folding, secondary structure motifs subtend RNA three-dimensional (3D) architecture. Thus, mutations in two-dimensional motifs are expected to have effects different from those disrupting 3D contacts. Using UV spectroscopy, we have studied the influence of nucleotide substitutions, in both secondary and tertiary structure elements, on the thermal stability of the tertiary folding of the bacteriophage T4 td group I intron. Further, we present a quantitative analysis of the relationship between the splicing efficiency in vivo and the stability of the intron structure as monitored by UV melting curves. We conclude that the stability of the tertiary structure of a group I intron as measured by UV melting is generally a good indication of its ability to splice in vivo.

The 3′ splice site of group I introns is defined, in part, by base pairs between the intron core and residues just upstream of the splice site, referred to as P9.0. We have studied the specificity imparted by P9.0 using the well-characterized L–21 ScaI ribozyme from Tetrahymena by adding residues to the 5′ end of the guanosine (G) that functions as a nucleophile in the oligonucleotide cleavage reaction: CCCUCUA5 (S) + NNG [lhard ][rharu ] CCCUCU + NNGA5. UCG, predicted to form two base pairs in P9.0, reacts with a (kcat/KM) value ∼10-fold greater than G, consistent with previous results. Altering the bases that form P9.0 in both the trinucleotide G analog and the ribozyme affects the specificity in the manner predicted for base-pairing. Strikingly, oligonucleotides incapable of forming P9.0 react ∼10-fold more slowly than G, for which the mispaired residues are simply absent. The observed specificity is consistent with a model in which the P9.0 site is sterically restricted such that an energetic penalty, not present for G, must be overcome by G analogs with 5′ extensions. Shortening S to include only one residue 3′ of the cleavage site (CCCUCUA) eliminates this penalty and uniformly enhances the reactions of matched and mismatched oligonucleotides relative to guanosine. These results suggest that the 3′ portion of S occupies the P9.0 site, sterically interfering with binding of G analogs with 5′ extensions. Similar steric effects may more generally allow structured RNAs to avoid formation of incorrect contacts, thereby helping to avoid kinetic traps during folding and enhancing cooperative formation of the correct structure.

The aminoglycoside antibiotic neomycin B inhibits translation in prokaryotes and interferes with RNA–protein interactions in HIV both in vivo and in vitro. Hitherto, inhibition of ribozyme catalysis has only been observed in vitro. We therefore monitored the activity of neomycin B and several other aminoglycoside antibiotics on splicing of the T4 phage thymidylate synthase (td) intron in vivo. All antibiotics tested inhibited splicing, even chloramphenicol, which does not inhibit splicing in vitro. Splicing of the td intron in vivo requires translation for proper folding of the pre-mRNA. In the absence of translation, two interactions between sequences in the upstream exon and the 5′ and 3′ splice sites trap the pre-mRNA in splicing-incompetent conformations. Their disruption by mutations rendered splicing less dependent on translation and also less sensitive to neomycin B. Intron splicing was affected by neither neomycin B nor gentamicin in Escherichia coli strains carrying antibiotic-resistance genes that modify the ribosomal RNA. Taken together, this demonstrates that in vivo splicing of td intron is not directly inhibited by aminoglycosides, but rather indirectly by their interference with translation. This was further confirmed by assaying splicing of the Tetrahymena group I intron, which is inserted in the E. coli 23 S rRNA and, thus, not translated. Furthermore, neomycin B, paromomycin, and streptomycin enhanced missplicing in antibiotic-sensitive strains. Missplicing is caused by an alternative structural element containing a cryptic 5′ splice site, which serves as a substrate for the ribozyme. Our results demonstrate that aminoglycoside antibiotics display different effects on ribozymes in vivo and in vitro.

Self-splicing of the Tetrahymena group I intron is attenuated by an rRNA stem-loop in the 5′ exon, which competes with formation of the P1 splice site helix. The equilibrium between the P1 and P(−1) stem-loops is influenced by rRNA sequences upstream and downstream of the intron. To investigate the mechanism of this conformational switch, internal deletions and point mutations were introduced in the second rRNA stem-loop upstream of the 5′ splice site. Nuclease protection, native gel electrophoresis, and self-splicing results show that this helix is important for maintaining self-splicing activity. Co-axial base stacking of adjacent helices in the 5′ exon is proposed to enable exchange between inactive and active conformations of the pre-rRNA.

P5abc domain of Tetrahymena LSU intron functions as an activator that is not essential for but enhances the activity of the ribozyme either when present in cis or when added in trans. This domain contains three regions (A-rich bulge, L5b, and L5c) that have been demonstrated to interact with the rest of the intron. Although these regions are presumably important for efficient activation, the role of each element is not understood in the mechanism of activation. We employed circularly permuted introns and examined the roles of each element. The results show that each of the three elements can activate the intron independently. We also found that a correlation between the activation by P5abc and the physical affinity of P5abc to the intron exists.

Adenosines are present at a disproportionately high frequency within several RNA structural motifs. To explore the importance of individual adenosine functional groups for group I intron activity, we performed Nucleotide Analog Interference Mapping (NAIM) with a collection of adenosine analogues. This paper reports the synthesis, transcriptional incorporation, and the observed interference pattern throughout the Tetrahymena group I intron for eight adenosine derivatives tagged with an α-phosphorothioate linkage for use in NAIM. All of the analogues were accurately incorporated into the transcript as an A. The sites that interfere with the 3′-exon ligation reaction of the Tetrahymena intron are coincident with the sites of phylogenetic conservation, yet the interference patterns for each analogue are different. These interference data provide several biochemical constraints that improve our understanding of the Tetrahymena ribozyme structure. For example, the data support an essential A-platform within the J6/6a region, major groove packing of the P3 and P7 helices, minor groove packing of the P3 and J4/5 helices, and an axial model for binding of the guanosine cofactor. The data also identify several essential functional groups within a highly conserved single-stranded region in the core of the intron (J8/7). At four sites in the intron, interference was observed with 2′-fluoro A, but not with 2′-deoxy A. Based upon comparison with the P4-P6 crystal structure, this may provide a biochemical signature for nucleotide positions where the ribose sugar adopts an essential C2′-endo conformation. In other cases where there is interference with 2′-deoxy A, the presence or absence of 2′-fluoro A interference helps to establish whether the 2′-OH acts as a hydrogen bond donor or acceptor. Mapping of the Tetrahymena intron establishes a basis set of information that will allow these reagents to be used with confidence in systems that are less well understood.

A new category of self-splicing group I introns with conserved structural organization and function is found among the eukaryotic microorganisms Didymium and Naegleria. These complex rDNA introns contain two distinct ribozymes with different functions: a regular group I splicing-ribozyme and a small internal group I-like ribozyme (GIR1), probably involved in protein expression. GIR1 was found to cleave at two internal sites in an obligate sequential order. Both sites are located 3′ of the catalytic core. GIR1-catalyzed transesterification reactions could not be detected. We have compared all available GIR1 sequences and propose a common RNA secondary structure resembling that of group I splicing-ribozymes, but with some important differences. The GIR1s lack most peripheral sequence components, as well as a P1 segment, and, at approximately 160–190 nt, they are the smallest functional group I ribozymes known from nature. All GIR1s were found to contain a novel 6-bp pseudoknot (P15) within their catalytic core region. Experimental support of the proposed structure was obtained from the Didymium GIR1 by RNA structure probing and site-directed mutagenesis. Three-dimensional modeling indicates a compactly folded ribozyme with the functionally essential P15 exposed in the cleft between the two principal domains P3–P8 and P4–P6.