Iron-mediated degradation of ribosomes under oxidative stress is attenuated by manganese

Protein biosynthesis is fundamental to cellular life and requires the efficient functioning of the translational machinery. At the center of this machinery is the ribosome, a ribonucleoprotein complex that depends heavily on Mg2+ for structure. Recent work has indicated that other metal cations can substitute for Mg2+, raising questions about the role different metals may play in the maintenance of the ribosome under oxidative stress conditions. Here, we assess ribosomal integrity following oxidative stress both in vitro and in cells to elucidate details of the interactions between Fe2+ and the ribosome and identify Mn2+ as a factor capable of attenuating oxidant-induced Fe2+-mediated degradation of rRNA. We report that Fe2+ promotes degradation of all rRNA species of the yeast ribosome and that it is bound directly to RNA molecules. Furthermore, we demonstrate that Mn2+ competes with Fe2+ for rRNA-binding sites and that protection of ribosomes from Fe2+-mediated rRNA hydrolysis correlates with the restoration of cell viability. Our data, therefore, suggest a relationship between these two transition metals in controlling ribosome stability under oxidative stress.

Nucleolar Structure and Function in Trypanosomatid Protozoa

The nucleolus is the conspicuous nuclear body where ribosomal RNA genes are transcribed by RNA polymerase I, pre-ribosomal RNA is processed, and ribosomal subunits are assembled. Other important functions have been attributed to the nucleolus over the years. Here we review the current knowledge about the structure and function of the nucleolus in the trypanosomatid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania ssp., which represent one of the earliest branching lineages among the eukaryotes. These protozoan parasites present a single nucleolus that is preserved throughout the closed nuclear division, and that seems to lack fibrillar centers. Trypanosomatids possess a relatively low number of rRNA genes, which encode rRNA molecules that contain large expansion segments, including several that are trypanosomatid-specific. Notably, the large subunit rRNA (28S-type) is fragmented into two large and four small rRNA species. Hence, compared to other organisms, the rRNA primary transcript requires additional processing steps in trypanosomatids. Accordingly, this group of parasites contains the highest number ever reported of snoRNAs that participate in rRNA processing. The number of modified rRNA nucleotides in trypanosomatids is also higher than in other organisms. Regarding the structure and biogenesis of the ribosomes, recent cryo-electron microscopy analyses have revealed several trypanosomatid-specific features that are discussed here. Additional functions of the nucleolus in trypanosomatids are also reviewed.

Knock-Down of a Novel snoRNA in Tetrahymena Reveals a Dual Role in 5.8S rRNA Processing and Generation of a 26S rRNA Fragment

In eukaryotes, 18S, 5.8S, and 28S rRNAs are transcribed as precursor molecules that undergo extensive modification and nucleolytic processing to form the mature rRNA species. Central in the process are the small nucleolar RNAs (snoRNAs). The majority of snoRNAs guide site specific chemical modifications but a few are involved in defining pre-rRNA cleavages. Here, we describe an unusual snoRNA (TtnuCD32) belonging to the box C/D subgroup from the ciliate Tetrahymena thermophila. We show that TtnuCD32 is unlikely to function as a modification guide snoRNA and that it is critical for cell viability. Cell lines with genetic knock-down of TtnuCD32 were impaired in growth and displayed two novel and apparently unrelated phenotypes. The most prominent phenotype is the accumulation of processing intermediates of 5.8S rRNA. The second phenotype is the decrease in abundance of a ~100 nt 26S rRNA fragment of unknown function. Sequence analysis demonstrated that TtnuCD32 share features with the essential snoRNA U14 but an alternative candidate (TtnuCD25) was more closely related to other U14 sequences. This, together with the fact that the observed rRNA processing phenotypes were not similar to what has been observed in U14 depleted cells, suggests that TtnuCD32 is a U14 homolog that has gained novel functions.

Trigonocephalotrema (Digenea : Haplosplanchnidae), a new genus for trematodes parasitising fishes of two Indo-West Pacific acanthurid genera

The Great Barrier Reef is the largest coral reef ecosystem on the planet and supports a diverse community of marine fishes, as well as the organisms that parasitise them. Although the digenetic trematodes that parasitise fishes of the Great Barrier Reef have been studied for over a century, the species richness and diversity of many trematode lineages is yet to be explored. Trigonocephalotrema, gen. nov. is proposed to accommodate three new species, Trigonocephalotrema euclidi, sp. nov., T. hipparchi, sp. nov. and T. sohcahtoa, sp. nov., parasitic in fishes of Naso Lacepède and Zebrasoma Swainson (Acanthuridae) in the tropical Pacific. Species of Trigonocephalotrema are characterised with morphological and molecular data (18S rRNA, ITS2 and 28S rRNA). Species of Trigonocephalotrema are morphologically distinguished from all other haplosplanchnid lineages by having terminal, triangular, plate-like oral suckers. With the inclusion of the new molecular data, Bayesian inference and maximum likelihood analyses of the Haplosplanchnidae Poche, 1926 recovered identical tree topologies and demonstrated Trigonocephalotrema as a well-supported monophyletic group. Although species of Trigonocephalotrema are differentiated from all other haplosplanchnid lineages on the basis of morphology, species within the genus are morphologically cryptic; thus, accurate species identification will require inclusion of host and molecular data. Species of Trigonocephalotrema cannot be assigned to a recognised subfamily within the Haplosplanchnidae using either morphological or molecular data and would require the erection of a new subfamily to accommodate them. However, we find little value in the use of subfamilies within the Haplosplanchnidae, given that there are so few taxa in the family, and herein propose that their use be avoided.

The human RNA-binding protein RBFA promotes the maturation of the mitochondrial ribosome

Accurate assembly and maturation of human mitochondrial ribosomes is essential for synthesis of the 13 polypeptides encoded by the mitochondrial genome. This process requires the correct integration of 80 proteins, 1 mt (mitochondrial)-tRNA and 2 mt-rRNA species, the latter being post-transcriptionally modified at many sites. Here, we report that human ribosome-binding factor A (RBFA) is a mitochondrial RNA-binding protein that exerts crucial roles in mitoribosome biogenesis. Unlike its bacterial orthologue, RBFA associates mainly with helices 44 and 45 of the 12S rRNA in the mitoribosomal small subunit to promote dimethylation of two highly conserved consecutive adenines. Characterization of RBFA-depleted cells indicates that this dimethylation is not a prerequisite for assembly of the small ribosomal subunit. However, the RBFA-facilitated modification is necessary for completing mt-rRNA maturation and regulating association of the small and large subunits to form a functional monosome implicating RBFA in the quality control of mitoribosome formation.

Decreased Expression of Stable RNA Can Alleviate the Lethality Associated with RNase E Deficiency in Escherichia coli

ABSTRACT The endoribonuclease RNase E participates in mRNA degradation, rRNA processing, and tRNA maturation in Escherichia coli, but the precise reasons for its essentiality are unclear and much debated. The enzyme is most active on RNA substrates with a 5′-terminal monophosphate, which is sensed by a domain in the enzyme that includes residue R169; E. coli also possesses a 5′-pyrophosphohydrolase, RppH, that catalyzes conversion of 5′-terminal triphosphate to 5′-terminal monophosphate on RNAs. Although the C-terminal half (CTH), beyond residue approximately 500, of RNase E is dispensable for viability, deletion of the CTH is lethal when combined with an R169Q mutation or with deletion of rppH. In this work, we show that both these lethalities can be rescued in derivatives in which four or five of the seven rrn operons in the genome have been deleted. We hypothesize that the reduced stable RNA levels under these conditions minimize the need of RNase E to process them, thereby allowing for its diversion for mRNA degradation. In support of this hypothesis, we have found that other conditions that are known to reduce stable RNA levels also suppress one or both lethalities: (i) alterations in relA and spoT, which are expected to lead to increased basal ppGpp levels; (ii) stringent rpoB mutations, which mimic high intracellular ppGpp levels; and (iii) overexpression of DksA. Lethality suppression by these perturbations was RNase R dependent. Our work therefore suggests that its actions on the various substrates (mRNA, rRNA, and tRNA) jointly contribute to the essentiality of RNase E in E. coli. IMPORTANCE The endoribonuclease RNase E is essential for viability in many Gram-negative bacteria, including Escherichia coli. Different explanations have been offered for its essentiality, including its roles in global mRNA degradation or in the processing of several tRNA and rRNA species. Our work suggests that, rather than its role in the processing of any one particular substrate, its distributed functions on all the different substrates (mRNA, rRNA, and tRNA) are responsible for the essentiality of RNase E in E. coli.

Human mitochondrial ribosomes can switch their structural RNA composition

The recent developments in cryo-EM have revolutionized our access to previously refractory structures. In particular, such studies of mammalian mitoribosomes have confirmed the absence of any 5S rRNA species and revealed the unexpected presence of a mitochondrially encoded tRNA (mt-tRNA) that usurps this position. Although the cryo-EM structures resolved the conundrum of whether mammalian mitoribosomes contain a 5S rRNA, they introduced a new dilemma: Why do human and porcine mitoribosomes integrate contrasting mt-tRNAs? Human mitoribosomes have been shown to integrate mt-tRNAVal compared with the porcine use of mt-tRNAPhe. We have explored this observation further. Our studies examine whether a range of mt-tRNAs are used by different mammals, or whether the mt-tRNA selection is strictly limited to only these two species of the 22 tRNAs encoded by the mitochondrial genome (mtDNA); whether there is tissue-specific variation within a single organism; and what happens to the human mitoribosome when levels of the mt-tRNAVal are depleted. Our data demonstrate that only mt-tRNAVal or mt-tRNAPhe are found in the mitoribosomes of five different mammals, each mammal favors the same mt-tRNA in all tissue types, and strikingly, when steady-state levels of mt-tRNAVal are reduced, human mitoribosome biogenesis displays an adaptive response by switching to the incorporation of mt-tRNAPhe to generate translationally competent machinery.

The complete structure of the cucumber (Cucumis sativus L.) chloroplast genome: Its composition and comparative analysis

The complete nucleotide sequence of the cucumber (C. sativus L. var. Borszczagowski) chloroplast genome has been determined. The genome is composed of 155,293 bp containing a pair of inverted repeats of 25,191 bp, which are separated by two single-copy regions, a small 18,222-bp one and a large 86,688-bp one. The chloroplast genome of cucumber contains 130 known genes, including 89 protein-coding genes, 8 ribosomal RNA genes (4 rRNA species), and 37 tRNA genes (30 tRNA species), with 18 of them located in the inverted repeat region. Of these genes, 16 contain one intron, and two genes and one ycf contain 2 introns. Twenty-one small inversions that form stem-loop structures, ranging from 18 to 49 bp, have been identified. Eight of them show similarity to those of other species, while eight seem to be cucumber specific. Detailed comparisons of ycf2 and ycf15, and the overall structure to other chloroplast genomes were performed.

Comprehensive Mutational Analysis of Yeast DEXD/H Box RNA Helicases Required for Small Ribosomal Subunit Synthesis

ABSTRACT The 17 putative RNA helicases required for pre-rRNA processing are predicted to play a crucial role in ribosome biogenesis by driving structural rearrangements within preribosomes. To better understand the function of these proteins, we have generated a battery of mutations in five putative RNA helicases involved in 18S rRNA synthesis and analyzed their effects on cell growth and pre-rRNA processing. Our results define functionally important residues within conserved motifs and demonstrate that lethal mutations in predicted ATP binding-hydrolysis motifs often confer a dominant negative phenotype in vivo when overexpressed in a wild-type background. We show that dominant negative mutants delay processing of the 35S pre-rRNA and cause accumulation of pre-rRNA species that normally have low steady-state levels. Our combined results establish that not all conserved domains function identically in each protein, suggesting that the RNA helicases may have distinct biochemical properties and diverse roles in ribosome biogenesis.