Trafficking and/or division: Distinct roles of nucleoporins based on their location within the nuclear pore complex.

The nuclear pore complex (NPC) facilitates the trafficking of proteins and RNA between the nucleus and cytoplasm. The role of nucleoporins (Nups) in transport in the context of the NPC is well established, yet their function in tRNA export has not been fully explored. We selected several nucleoporins from different parts of the NPC to investigate their potential role in tRNA trafficking in Trypanosoma brucei. We show that while all of the nucleoporins studied are essential for cell viability, only TbNup62 and TbNup53a function in tRNA export. In contrast to homologs in yeast TbNup144 and TbNup158, which are part of the inner and outer ring of the NPC, have no role in nuclear tRNA trafficking. Instead, TbNup144 plays a critical role in nuclear division, highlighting the role of nucleoporins beyond nucleocytoplasmic transport. These results suggest that the location of nucleoporins within the NPC is crucial to maintaining various cellular processes.

Genome analysis of five recently described species of the CUG-Ser clade uncovers Candida theae as a new hybrid lineage with pathogenic potential in the Candida parapsilosis species complex.

Candida parapsilosis species complex comprises three important pathogenic species: Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis. The majority of C. orthopsilosis and all C. metapsilosis isolates sequenced thus far are hybrids, and most of the parental lineages remain unidentified. This led to the hypothesis that hybrids with pathogenic potential were formed by the hybridization of non-pathogenic lineages that thrive in the environment. In a search for the missing hybrid parentals, and aiming to get a better understanding of the evolution of the species complex, we sequenced, assembled and analysed the genome of five close relatives isolated from the environment: Candida jiufengensis, Candida pseudojiufengensis, Candida oxycetoniae, Candida margitis and Candida theae. We found that the linear conformation of mitochondrial genomes in Candida species emerged multiple times independently. Furthermore, our analyses discarded the possible involvement of these species in the mentioned hybridizations, but identified C. theae as an additional hybrid in the species complex. Importantly, C. theae was recently associated with a case of infection, and we also uncovered the hybrid nature of this clinical isolate. Altogether, our results reinforce the hypothesis that hybridization is widespread among Candida species, and potentially contributes to the emergence of lineages with opportunistic pathogenic behaviour.

Dynamic queuosine changes in tRNA couple nutrient levels to codon choice in Trypanosoma brucei.

Every type of nucleic acid in cells undergoes programmed chemical post-transcriptional modification. Generally, modification enzymes use substrates derived from intracellular metabolism, one exception is queuine (q)/queuosine (Q), which eukaryotes obtain from their environment; made by bacteria and ultimately taken into eukaryotic cells via currently unknown transport systems. Here, we use a combination of molecular, cell biology and biophysical approaches to show that in Trypanosoma brucei tRNA Q levels change dynamically in response to concentration variations of a sub-set of amino acids in the growth media. Most significant were variations in tyrosine, which at low levels lead to increased Q content for all the natural tRNAs substrates of tRNA-guanine transglycosylase (TGT). Such increase results from longer nuclear dwell time aided by retrograde transport following cytoplasmic splicing. In turn high tyrosine levels lead to rapid decrease in Q content. Importantly, the dynamic changes in Q content of tRNAs have negligible effects on global translation or growth rate but, at least, in the case of tRNATyr it affected codon choice. These observations have implications for the occurrence of other tunable modifications important for 'normal' growth, while connecting the intracellular localization of modification enzymes, metabolites and tRNAs to codon selection and implicitly translational output.

Preferential import of queuosine-modified tRNAs into Trypanosoma brucei mitochondrion is critical for organellar protein synthesis.

Transfer RNAs (tRNAs) are key players in protein synthesis. To be fully active, tRNAs undergo extensive post-transcriptional modifications, including queuosine (Q), a hypermodified 7-deaza-guanosine present in the anticodon of several tRNAs in bacteria and eukarya. Here, molecular and biochemical approaches revealed that in the protozoan parasite Trypanosoma brucei, Q-containing tRNAs have a preference for the U-ending codons for asparagine, aspartate, tyrosine and histidine, analogous to what has been described in other systems. However, since a lack of tRNA genes in T. brucei mitochondria makes it essential to import a complete set from the cytoplasm, we surprisingly found that Q-modified tRNAs are preferentially imported over their unmodified counterparts. In turn, their absence from mitochondria has a pronounced effect on organellar translation and affects function. Although Q modification in T. brucei is globally important for codon selection, it is more so for mitochondrial protein synthesis. These results provide a unique example of the combined regulatory effect of codon usage and wobble modifications on protein synthesis; all driven by tRNA intracellular transport dynamics.

Genome analysis of Candida subhashii reveals its hybrid nature and dual mitochondrial genome conformations.

Candida subhashii belongs to the CUG-Ser clade, a group of phylogenetically closely related yeast species that includes some human opportunistic pathogens, such as Candida albicans. Despite being present in the environment, C. subhashii was initially described as the causative agent of a case of peritonitis. Considering the relevance of whole-genome sequencing and analysis for our understanding of genome evolution and pathogenicity, we sequenced, assembled and annotated the genome of C. subhashii type strain. Our results show that C. subhashii presents a highly heterozygous genome and other signatures that point to a hybrid ancestry. The presence of functional pathways for assimilation of hydroxyaromatic compounds goes in line with the affiliation of this yeast with soil microbial communities involved in lignin decomposition. Furthermore, we observed that different clones of this strain may present circular or linear mitochondrial DNA. Re-sequencing and comparison of strains with differential mitochondrial genome topology revealed five candidate genes potentially associated with this conformational change: MSK1, SSZ1, ALG5, MRPL9 and OYE32.

Selective nuclear export of mRNAs is promoted by DRBD18 in Trypanosoma brucei.

Kinetoplastids, including Trypanosoma brucei, control gene expression primarily at the posttranscriptional level. Nuclear mRNA export is an important, but understudied, step in this process. The general heterodimeric export factors, Mex67/Mtr2, function in the export of mRNAs and tRNAs in T. brucei, but RNA binding proteins (RBPs) that regulate export processes by controlling the dynamics of Mex67/Mtr2 ribonucleoprotein formation or transport have not been identified. Here, we report that DRBD18, an essential and abundant T. brucei RBP, associates with Mex67/Mtr2 in vivo, likely through its direct interaction with Mtr2. DRBD18 downregulation results in partial accumulation of poly(A)+ mRNA in the nucleus, but has no effect on the localization of intron-containing or mature tRNAs. Comprehensive analysis of transcriptomes from whole-cell and cytosol in DRBD18 knockdown parasites demonstrates that depletion of DRBD18 leads to impairment of nuclear export of a subset of mRNAs. CLIP experiments reveal the association of DRBD18 with several of these mRNAs. Moreover, DRBD18 knockdown leads to a partial accumulation of the Mex67/Mtr2 export receptors in the nucleus. Taken together, the current study supports a model in which DRBD18 regulates the selective nuclear export of mRNAs by promoting the mobilization of export competent mRNPs to the cytosol through the nuclear pore complex.

The general mRNA exporters Mex67 and Mtr2 play distinct roles in nuclear export of tRNAs in Trypanosoma brucei.

Transfer RNAs (tRNAs) are central players in protein synthesis, which in Eukarya need to be delivered from the nucleus to the cytoplasm by specific transport receptors, most of which belong to the evolutionarily conserved beta-importin family. Based on the available literature, we identified two candidates, Xpo-t and Xpo-5 for tRNA export in Trypanosoma brucei. However, down-regulation of expression of these genes did not disrupt the export of tRNAs to the cytoplasm. In search of alternative pathways, we tested the mRNA export complex Mex67-Mtr2, for a role in tRNA nuclear export, as described previously in yeast. Down-regulation of either exporter affected the subcellular distribution of tRNAs. However, contrary to yeast, TbMex67 and TbMtr2 accumulated different subsets of tRNAs in the nucleus. While TbMtr2 perturbed the export of all the tRNAs tested, silencing of TbMex67, led to the nuclear accumulation of tRNAs that are typically modified with queuosine. In turn, inhibition of tRNA nuclear export also affected the levels of queuosine modification in tRNAs. Taken together, the results presented demonstrate the dynamic nature of tRNA trafficking in T. brucei and its potential impact not only on the availability of tRNAs for protein synthesis but also on their modification status.

Genome sequence of the opportunistic human pathogen Magnusiomyces capitatus.

The yeast Magnusiomyces capitatus is an opportunistic human pathogen causing rare yet severe infections, especially in patients with hematological malignancies. Here, we report the 20.2 megabase genome sequence of an environmental strain of this species as well as the genome sequences of eight additional isolates from human and animal sources providing an insight into intraspecies variation. The distribution of single-nucleotide variants is indicative of genetic recombination events, supporting evidence for sexual reproduction in this heterothallic yeast. Using RNAseq-aided annotation, we identified genes for 6518 proteins including several expanded families such as kexin proteases and Hsp70 molecular chaperones. Several of these families are potentially associated with the ability of M. capitatus to infect and colonize humans. For the purpose of comparative analysis, we also determined the genome sequence of a closely related yeast, Magnusiomyces ingens. The genome sequences of M. capitatus and M. ingens exhibit many distinct features and represent a basis for further comparative and functional studies.

Correction: The Genomic Aftermath of Hybridization in the Opportunistic Pathogen Candida metapsilosis.

[This corrects the article DOI: 10.1371/journal.pgen.1005626.].

The Genomic Aftermath of Hybridization in the Opportunistic Pathogen Candida metapsilosis.

Candida metapsilosis is a rarely-isolated, opportunistic pathogen that belongs to a clade of pathogenic yeasts known as the C. parapsilosis sensu lato species complex. To gain insight into the recent evolution of C. metapsilosis and the genetic basis of its virulence, we sequenced the genome of 11 clinical isolates from various locations, which we compared to each other and to the available genomes of the two remaining members of the complex: C. orthopsilosis and C. parapsilosis. Unexpectedly, we found compelling genomic evidence that C. metapsilosis is a highly heterozygous hybrid species, with all sequenced clinical strains resulting from the same past hybridization event involving two parental lineages that were approximately 4.5% divergent in sequence. This result indicates that the parental species are non-pathogenic, but that hybridization between them formed a new opportunistic pathogen, C. metapsilosis, that has achieved a worldwide distribution. We show that these hybrids are diploid and we identified strains carrying loci for both alternative mating types, which supports mating as the initial mechanism for hybrid formation. We trace the aftermath of this hybridization at the genomic level, and reconstruct the evolutionary relationships among the different strains. Recombination and introgression -resulting in loss of heterozygosis- between the two subgenomes have been rampant, and includes the partial overwriting of the MTLa mating locus in all strains. Collectively, our results shed light on the recent genomic evolution within the C. parapsilosis sensu lato complex, and argue for a re-definition of species within this clade, with at least five distinct homozygous lineages, some of which having the ability to form hybrids.

Massive programmed translational jumping in mitochondria.

Programmed translational bypassing is a process whereby ribosomes "ignore" a substantial interval of mRNA sequence. Although discovered 25 y ago, the only experimentally confirmed example of this puzzling phenomenon is expression of the bacteriophage T4 gene 60. Bypassing requires translational blockage at a "takeoff codon" immediately upstream of a stop codon followed by a hairpin, which causes peptidyl-tRNA dissociation and reassociation with a matching "landing triplet" 50 nt downstream, where translation resumes. Here, we report 81 translational bypassing elements (byps) in mitochondria of the yeast Magnusiomyces capitatus and demonstrate in three cases, by transcript analysis and proteomics, that byps are retained in mitochondrial mRNAs but not translated. Although mitochondrial byps resemble the bypass sequence in the T4 gene 60, they utilize unused codons instead of stops for translational blockage and have relaxed matching rules for takeoff/landing sites. We detected byp-like sequences also in mtDNAs of several Saccharomycetales, indicating that byps are mobile genetic elements. These byp-like sequences lack bypassing activity and are tolerated when inserted in-frame in variable protein regions. We hypothesize that byp-like elements have the potential to contribute to evolutionary diversification of proteins by adding new domains that allow exploration of new structures and functions.

Mitochondrial genome of the basidiomycetous yeast Jaminaea angkorensis.

Jaminaea angkorensis is an anamorphic basidiomycetous yeast species originally isolated from decaying leaves in Cambodia. Taxonomically, J. angkorensis is affiliated with Microstromatales (Exobasidiomycetes, Ustilaginomycotina, Basidiomycota) and represents a basal phylogenetic lineage of this fungal order. To perform a comparative analysis of J. angkorensis with other basidiomycetes, we determined and analyzed its complete mitochondrial DNA sequence. The mitochondrial genome is represented by 29,999 base pairs long, circular DNA containing 32 % guanine and cytosine residues. Its genetic organization is relatively compact and comprises typical genes for 15 conserved proteins involved in oxidative phosphorylation (atp6, 8, and 9; cob; cox1, 2, and 3; and nad1, 2, 3, 4, 4L, 5, and 6) and translation (rps3), two ribosomal RNAs (rnl and rns) and twenty-two transfer RNAs (trnA-Y). Although the gene content is similar to other basidiomycetes, the gene orders in the examined species exhibit only a limited synteny, reflecting their phylogenetic distances and extensive genome rearrangements. In addition, a comparative analysis of basidiomycete mitochondrial genomes indicates that stop-to-tryptophan reassignment of the UGA codon was accompanied by structural alterations of tRNA-Trp(CCA). These results provide an insight into the evolution of the genetic code in fungal mitochondria.