In vivo RNA structural probing of uracil and guanine base-pairing by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

Many biological functions performed by RNAs arise from their in vivo structures. The structure of the same RNA can differ in vitro and in vivo owing in part to the influence of molecules ranging from protons to secondary metabolites to proteins. Chemical reagents that modify the Watson-Crick (WC) face of unprotected RNA bases report on the absence of base-pairing and so are of value to determining structures adopted by RNAs. Reagents have thus been sought that can report on the native RNA structures that prevail in living cells. Dimethyl sulfate (DMS) and glyoxal penetrate cell membranes and inform on RNA secondary structure in vivo through modification of adenine (A), cytosine (C), and guanine (G) bases. Uracil (U) bases, however, have thus far eluded characterization in vivo. Herein, we show that the water-soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is capable of modifying the WC face of U and G in vivo, favoring the former nucleobase by a factor of ∼1.5, and doing so in the eukaryote rice, as well as in the Gram-negative bacterium Escherichia coli While both EDC and glyoxal target Gs, EDC reacts with Gs in their typical neutral state, while glyoxal requires Gs to populate the rare anionic state. EDC may thus be more generally useful; however, comparison of the reactivity of EDC and glyoxal may allow the identification of Gs with perturbed pKas in vivo and genome-wide. Overall, use of EDC with DMS allows in vivo probing of the base-pairing status of all four RNA bases.

RNA degradation paths in a 12-subunit nuclear exosome complex.

The eukaryotic exosome is a conserved RNA-degrading complex that functions in RNA surveillance, turnover and processing. How the same machinery can either completely degrade or precisely trim RNA substrates has long remained unexplained. Here we report the crystal structures of a yeast nuclear exosome containing the 9-subunit core, the 3'-5' RNases Rrp44 and Rrp6, and the obligate Rrp6-binding partner Rrp47 in complex with different RNAs. The combined structural and biochemical data of this 12-subunit complex reveal how a single-stranded RNA can reach the Rrp44 or Rrp6 active sites directly or can bind Rrp6 and be threaded via the central channel towards the distal RNase Rrp44. When a bulky RNA is stalled at the entrance of the channel, Rrp6-Rrp47 swings open. The results suggest how the same molecular machine can coordinate processive degradation and partial trimming in an RNA-dependent manner by a concerted swinging mechanism of the two RNase subunits.

A Novel Model for the RNase MRP-Induced Switch between the Formation of Different Forms of 5.8S rRNA.

Processing of the RNA polymerase I pre-rRNA transcript into the mature 18S, 5.8S, and 25S rRNAs requires removing the "spacer" sequences. The canonical pathway for the removal of the ITS1 spacer involves cleavages at the 3' end of 18S rRNA and at two sites inside ITS1. The process can generate either a long or a short 5.8S rRNA that differs in the number of ITS1 nucleotides retained at the 5.8S 5' end. Here we document a novel pathway to the long 5.8S, which bypasses cleavage within ITS1. Instead, the entire ITS1 is degraded from its 5' end by exonuclease Xrn1. Mutations in RNase MRP increase the accumulation of long relative to short 5.8S rRNA. Traditionally this is attributed to a decreased rate of RNase MRP cleavage at its target in ITS1, called A3. However, results from this work show that the MRP-induced switch between long and short 5.8S rRNA formation occurs even when the A3 site is deleted. Based on this and our published data, we propose that the link between RNase MRP and 5.8S 5' end formation involves RNase MRP cleavage at unknown sites elsewhere in pre-rRNA or in RNA molecules other than pre-rRNA.

Tracking fluctuation hotspots on the yeast ribosome through the elongation cycle.

Chemical modification was used to quantitatively determine the flexibility of nearly the entire rRNA component of the yeast ribosome through 8 discrete stages of translational elongation, revealing novel observations at the gross and fine-scales. These include (i) the bulk transfer of energy through the intersubunit bridges from the large to the small subunit after peptidyltransfer, (ii) differences in the interaction of the sarcin ricin loop with the two elongation factors and (iii) networked information exchange pathways that may functionally facilitate intra- and intersubunit coordination, including the 5.8S rRNA. These analyses reveal hot spots of fluctuations that set the stage for large-scale conformational changes essential for translocation and enable the first molecular dynamics simulation of an 80S complex. Comprehensive datasets of rRNA base flexibilities provide a unique resource to the structural biology community that can be computationally mined to complement ongoing research toward the goal of understanding the dynamic ribosome.

Molecular phylogeny of Candidula (Geomitridae) land snails inferred from mitochondrial and nuclear markers reveals the polyphyly of the genus.

The genus Candidula (Geomitridae), consisting of 28 species in Western Europe as currently described, has a disjunct distribution in the Iberian Peninsula, Italy, the Balkans, the Aegean Islands, and one species on the Canary Islands. Although the genus is seemingly well defined by characters of the reproductive system, the relationships within the genus are still unclear and some authors have indicated a possible subgeneric division based on the internal morphology of the dart sac. Despite substantial phylogenetic incongruence, we present a well-resolved molecular phylogeny of Candidula based on two mitochondrial genes (COI and 16S rRNA), the nuclear rDNA region (5.8S rNRA + ITS2 + 28S rRNA) and seven additional nuclear DNA regions developed specifically for this genus (60SL13, 60SL17, 60SL7, RPL14, 40SS6, 60SL9, 60SL13a), in total 5595 bp. Six reciprocally monophyletic entities including Candidula species were recovered, grouping into two major clades. The incorporation of additional geomitrid genera allowed us to unequivocally demonstrate the polyphyly of the genus Candidula. One major clade grouped species from southern France and Italy with the widely distributed species C. unifasciata. The second major clade grouped all the species from the Iberian Peninsula, including C. intersecta and C. gigaxii. Candidula ultima from the Canary Islands was recovered as separated lineage within the latter clade and related to African taxa. The six monophyla were defined as six new genera belonging to different tribes within the Helicellinae. Thus, we could show that similar structures of the stimulatory apparatus of the genital system in different taxa do not necessarily indicate a close phylogenetic relationship in the Geomitridae. More genera of the family are needed to clarify their evolutionary relationships, and to fully understand the evolution of the stimulatory apparatus of the genital system within the Geomitridae.

Has1 regulates consecutive maturation and processing steps for assembly of 60S ribosomal subunits.

Ribosome biogenesis requires ∼200 assembly factors in Saccharomyces cerevisiae. The pre-ribosomal RNA (rRNA) processing defects associated with depletion of most of these factors have been characterized. However, how assembly factors drive the construction of ribonucleoprotein neighborhoods and how structural rearrangements are coupled to pre-rRNA processing are not understood. Here, we reveal ATP-independent and ATP-dependent roles of the Has1 DEAD-box RNA helicase in consecutive pre-rRNA processing and maturation steps for construction of 60S ribosomal subunits. Has1 associates with pre-60S ribosomes in an ATP-independent manner. Has1 binding triggers exonucleolytic trimming of 27SA3 pre-rRNA to generate the 5' end of 5.8S rRNA and drives incorporation of ribosomal protein L17 with domain I of 5.8S/25S rRNA. ATP-dependent activity of Has1 promotes stable association of additional domain I ribosomal proteins that surround the polypeptide exit tunnel, which are required for downstream processing of 27SB pre-rRNA. Furthermore, in the absence of Has1, aberrant 27S pre-rRNAs are targeted for irreversible turnover. Thus, our data support a model in which Has1 helps to establish domain I architecture to prevent pre-rRNA turnover and couples domain I folding with consecutive pre-rRNA processing steps.

Fasciola hepatica - where is 28S ribosomal RNA?

Advanced molecular biology techniques are currently used to develop new effective strategies against fasciolosis. Assessment of the quality of extracted total RNA is an important step prior to commencing many molecular biology methods such as transcriptomics. However, RNA quality assessment is complicated for some organisms, including Fasciola hepatica, by the absence of a 28S rRNA peak/band, when assessed with modern protocols. In this study, electrophoretic profiles of F. hepatica ribosomal RNAs were evaluated using microfluidics capillary based and conventional non-denaturing gel electrophoresis methods. An important modification to recommended protocols, the exclusion of heat-denaturation step, in the microfluidics capillary based electrophoresis is critical to visualise the expected 28S rRNA and obtain an RNA integrity number (RIN). The intensity of the 28S rRNA band is reduced by the effect of non-denaturing gel electrophoresis.

Molecular phylogenetic and zoospore ultrastructural analyses of Chytridium olla establish the limits of a monophyletic Chytridiales.

Chytridium olla A. Braun, the first described chytrid and an obligate algal parasite, is the type for the genus and thus the foundation of family Chytridiaceae, order Chytridiales, class Chytridiomycetes and phylum Chytridiomycota. Chytridium olla was isolated in coculture with its host, Oedogonium capilliforme. DNA was extracted from the coculture, and 18S, 28S and ITS1-5.8S-ITS2 rDNA were amplified with universal fungal primers. Free swimming zoospores and zoospores in mature sporangia were examined with electron microscopy. Molecular analyses placed C. olla in a clade in Chytridiales with isolates of Chytridium lagenaria and Phlyctochytrium planicorne. Ultrastructural analysis revealed C. olla to have a Group II-type zoospore, previously described for Chytridium lagenaria and Phlyctochytrium planicorne. On the basis of zoospore ultrastructure, family Chytridiaceae is emended to include the type of Chytridium and other species with a Group II-type zoospore, and the new family Chytriomycetaceae is delineated to include members of Chytridiales with a Group I-type zoospore.

Alternaria hungarica sp. nov., a minor foliar pathogen of wheat in Hungary.

During routine wheat disease surveys in Hungary in 2007 Alternaria was isolated from leaf samples collected in Debrecen. Macro- and micro-morphological examinations and ITS sequence analyses indicated that the isolates represented a new Alternaria species, which we described as A. hungarica. The usually solitary conidia of A. hungarica resemble those of A. mouchaccae and A. molesta. However growth and sporulation pattern are more like those of A. geniostomatis and A. soliaridae. Phylogenetic analysis of ITS sequences indicated that this new species can be distinguished from all other examined Alternaria and Embellisia species. Pathogenicity tests indicated that A. hungarica can be considered a minor pathogen of wheat.

Nectria eustromatica sp. nov., an exceptional species with a hypocreaceous stroma.

A new species with remarkable morphology, Nectria eustromatica, is described, based on morphology of the teleomorph and anamorph, ecology and molecular phylogenetic analyses. Nectria eustromatica is characterized by sphaeroid perithecia immersed in pseudoparenchymatous stromata formed singly or collectively on a subiculum. Despite its deviating teleomorph morphology, it is placed within Nectria sensu stricto in phylogenetic analyses of a combined dataset of LSU, ITS, rpb2 and tef1 sequences with high internal support. Nectria eustromatica has been collected specifically on Hippocrepis (Coronilla) emerus in southern Europe. The anamorph of N. eustromatica shares morphological traits with the genera Stilbella and Tubercularia but produces non-phialidic macroconidia in addition to phialoconidia.

Trichoderma amazonicum, a new endophytic species on Hevea brasiliensis and H. guianensis from the Amazon basin.

A new species of Trichoderma (teleomorph Hypocrea, Ascomycota, Sordariomycetes, Hypocreales, Hypocreaceae), T. amazonicum, endophytic on the living sapwood and leaves of Hevea spp. trees is described. Trichoderma amazonicum is distinguished from closely related species in the Harzianum clade (e.g. Hypocrea alni, H. brunneoviridis, H. epimyces, H. parepimyces, T. aggressivum, T. harzianum, T. pleuroticola and T. pleuroti) by morphological and ecological characteristics and phylogenetic analysis of three loci (ITS nrDNA, tef1 and rpb2). The closest relatives of this species are the facultatively fungicolous species T. pleuroticola and T. pleuroti.

Pseudotripoconidium, a new anamorph genus connected to Orbilia.

A new anamorphic fungus is described based on four isolates from ascospores of Orbilia aff. luteorubella. This fungus differs from previously known Orbilia anamorphs in producing inversely pyramidal, unicellular conidia with several protuberances at their distal end. Conidia produce 1-7 prominent denticles that emerge from a node at the conidiophore apex. Conidiogenesis is holoblastic. Because phylogenetic analysis indicated greater than 90% ITS sequence similarities among the four isolates they are treated here as a single species. In the sequence analysis of the internal transcribed spacer region (ITS) these isolates and other sequences identified as O. aff. luteorubella were nested within Orbilia and formed a clade with 99% bootstrap support. This clade is separated from nematode-trapping species of Orbilia. Based on both morphological and molecular analyses, we propose a new genus, Pseudotripoconidium.

Leptographium tereforme sp. nov. and other Ophiostomatales isolated from the root-feeding bark beetle Hylurgus ligniperda in California.

The redhaired pine bark beetle Hylurgus ligniperda (F.) is native to Europe but was discovered in Los Angeles, California, in 2003. This root-and stump-feeding beetle is a common vector of Ophiostomatales, which are potential tree pathogens or causes of blue stain of conifer sapwood. In this study Ophiostomatales were isolated on a cycloheximide-amended medium from 118 adult H. ligniperda collected from infested logs of Pinus halepensis and P. pinea at two sites in California. In total eight species of Ophiostomatales were identified and seven species that occasionally were isolated were unidentified. The most frequently isolated species were Ophiostoma ips and Grosmannia galeiforme, which were isolated respectively from 31% and 23% of the 118 beetles. The other species isolated included O. piceae (isolated from 9% of the beetles), O. querci (8%) and Leptographium tereforme sp. nov. (6%). Grosmannia huntii, L. serpens, three Sporothrix species, O. floccosum, O. stenoceras, two unidentified Hyalorhinocladiella sp. and a sterile fungus each were isolated from fewer then 5% of beetles. Most of the identified species already were known in USA and have been found in association with H. ligniperda in other countries. However the new species, L. tereforme, and G. galeiforme were recorded from USA for the first time, and this is the first report of L. serpens from western North America.

Single origin and subsequent diversification of central Andean endemic Umbilicaria species.

We studied an Andean endemic group of species of the lichen-forming fungal genus Umbilicaria from the subalpine and low-alpine zone, with their biogeographic center in Bolivia and Peru. A number of species and varieties have been described from this element, but apparent instability in several morphological traits has made it difficult to precisely delimit taxa. Based on DNA sequences of nuclear ITS, LSU and mitochondrial SSU from extensive collections from Argentina, Bolivia, Chile, Colombia, Ecuador and Peru, we present here a molecular phylogenetic analysis of this Andean endemic element within genus Umbilicaria. All analyses (MP, ML and Bayesian) support a single origin for the element and a division into two major groups characterized by different apothecium types: the Umbilicaria dichroa group and U. calvescens group. Taxa U. krempelhuberi, U. peruviana and U. subcalvescens are nested withinn U. calvescens and are treated as conspecific with the latter species. The endemic element shares a most recent common ancestor with the Umbilicaria vellea group, which has a worldwide distribution and contains several asexually reproducing (sorediate) species. Independent reversals to sexual reproduction might explain the evolution of two types of apothecia in this monophyletic endemic lineage. A number of cosmopolitan, mostly high-alpine, species of Umbilicaria also present in the central Andes are related only remotely to the endemic element and do not exhibit speciation into endemics. Because the An-dean element dominates the Umbilicaria habitats of the low- and subalpine zones we propose that the founder colonized the Andes at a time when the mountains had not yet reached their current elevation while the high-alpine species arrived more recently.

Phylogeny of Pilobolaceae.

The three genera traditionally classified as Pilobolaceae have been identified on the basis of morphological characteristics. In the absence of distinctive morphological differences phylogenetic techniques have proven to be superior for developing phylogenies. Molecular techniques have been used primarily for studies of higher fungi; there are few investigations of the Zygomycota using genetic sequences for classification. DNA sequences coding for three regions of rRNA were used to investigate phylogenetic relationships of the three genera traditionally considered within the Pilobolaceae. Evidence indicates that Pilaira should be removed from Pilobolaceae and the family redescribed. Sporangiospore size is the morphological characteristic that most closely correlates with rDNA clades of phylogenetic trees. This study demonstrates that traditional morphological characteristics alone are not adequate to differentiate species of Pilobolus.

Population genetic structure of the seed pathogen Pyrenophora semeniperda on Bromus tectorum in western North America.

We examined genetic variation in the ascomycete pathogen Pyrenophora semeniperda cultured from seeds of the invasive grass Bromus tectorum in the Intermountain West of North America. We sequenced the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA genome in 417 monoconidial cultures collected from 20 sites in Washington, Idaho, Utah and Colorado, USA. ITS sequence diversity was surprisingly high; 12 unique haplotypes were identified, averaging 1.3% pairwise sequence divergence. All sites had at least two haplotypes present, and three sites had seven or more. One haplotype composed 60% of the isolates and occurred at all 20 locations; the remaining haplotypes generally occurred at low frequencies within sites but at multiple sites throughout the region. Sites in Washington and Idaho were more diverse than those in Utah and Colorado, averaging two more haplotypes and 67% more pairwise differences among haplotypes at a site. Analysis of molecular variance (AMOVA) indicated that more than 80% of the genetic variation was found within sampling locations, while 7-11% of the variation can be attributed to differences between northern (Washington and Idaho) and southern (Utah and Colorado) populations. The wide distribution of even uncommon haplotypes among sampling sites and weak correlations between genetic and geographic distances among populations (< 0.2) suggested that these populations recently were established from a common source. We hypothesize that the strains of P. semeniperda infecting B. tectorum in western North America probably arrived with the invasive grass from its native Eurasian range.

Babesia bovis: transcriptional analysis of rRNA gene unit expression.

The complex life cycle of Babesia bovis includes erythrocytic stages in the bovine host and other stages occurring inside its common tick vector Rhipicephalus microplus. In related apicomplexa, changing environmental conditions affect the expression of ribosomal RNA, but it remained unknown whether the polymorphic A, B, and C rRNA coding units of B. bovis are differentially expressed. Northern blot analysis confirmed that polymorphic regions in the B. bovis 18s and ITS-2 rRNA coding units are transcribed. Then, rRNA transcript expression profiles were compared by analyzing cDNA libraries generated from total RNA extracted from in vitro cultured parasites, B. bovis infected cattle, R. microplus larvae and egg sources. The 18s and ITS-2 expression profiles indicate that rRNA unit B is almost exclusively expressed in cultured parasites while units A, B, and C are co-transcribed in the in vivo total RNA sources. Collectively, the data indicate that differential transcription of rRNA occurs in B.bovis, depending on the life stage of the parasite and on the environment.

Fungal growth on anion surfactant medium.

Before the present study, no fungi using anion surfactant as a nutrient had been identified, although some fungi were known to use nonion surfactant. Washing water collected from 63 washing machines was inoculated onto 0.1% LAS (Sodium dodecyl benzenesulfonate) anion surfactant media to identify fungi that can feed on anion-surfactant. Small dark colonies of fungi were found on several of the Petri-dishes from 12 days after inoculation. These were identified as Cladophialophora boppii and Exophiala spinifera using morphological features and rDNA data. A number of the isolates of C. boppii specifically were recognized as using anion surfactant as a nutrient. The growth characteristics of the two fungal species were examined on surfactant media of three kinds. Apart from anion surfactant, the fungi were also able to grow on nonion surfactant and on soap. The application of these fungi for environmental cleansing after detergent pollution is also discussed.

Cryo-EM structure of an early precursor of large ribosomal subunit reveals a half-assembled intermediate.

Assembly of eukaryotic ribosome is a complicated and dynamic process that involves a series of intermediates. It is unknown how the highly intertwined structure of 60S large ribosomal subunits is established. Here, we report the structure of an early nucleolar pre-60S ribosome determined by cryo-electron microscopy at 3.7 Å resolution, revealing a half-assembled subunit. Domains I, II and VI of 25S/5.8S rRNA pack tightly into a native-like substructure, but domains III, IV and V are not assembled. The structure contains 12 assembly factors and 19 ribosomal proteins, many of which are required for early processing of large subunit rRNA. The Brx1-Ebp2 complex would interfere with the assembly of domains IV and V. Rpf1, Mak16, Nsa1 and Rrp1 form a cluster that consolidates the joining of domains I and II. Our structure reveals a key intermediate on the path to establishing the global architecture of 60S subunits.

A highly efficient electrophoretic method for discrimination between two Neoscytalidium species using a specific fungal internal transcribed spacer (ITS) fragment.

Neoscytalidium (or N.) dimidiatum and N. novaehollandiae are two aggressive plant pathogenic species that affect several agricultural crops. Early detection and identification of these fungi are of critical importance to bring about the effective minimization to the threat they pose to the infected plants. Herein, two species of Neoscytalidium were rapidly discriminated by utilizing the rRNA internal transcribed (ITS4-5.8S-ITS5) PCR primers. A total of 100 isolates of Neoscytalidium species, which were isolated from Iraqi canker-infected fig trees, were included in this study. Two discrete electrophoretic PCR bands were observed in Neoscytalidium isolates-A-variants were about 546 bp, while B-variants were about 993 bp in length. The comprehensive phylogenetic analysis of both DNA variants revealed that A-variants resided between N. novaehollandiae and N. hyalinum, while B-variants were closely related to N. dimidiatum. Furthermore, the highly specific re-constructed tree of both electrophoretic variants demonstrated that B-variants share a high similarity with N. novaehollandiae. Additionally, the secondary structures for both variants were predicted computationally to reveal the structural patterns that each variant follows. In conclusion, a small rRNA locus comprising 22 nucleotides that differs in the two variants is potentially responsible for this species-specific classification. The main divergence in the amplified loci led to the classification of these fungal variants into two main species, namely N. dimidiatum and N. novaehollandiae, demonstrating that the amplification by ITS4-ITS5 rRNA fragment is a beneficial strategy that can be employed for the assessment of Neoscytalidium diversity in the natural ecosystems.