Biology subjects

Janbon, G.

Publications and source records attributed to Janbon, G..

10 recordsLinked to original sources

bioRxiv2025

The Lsm1-7 complex marginally affects unstable mRNA degradation in yeast

Withdrawal StatementThe authors have withdrawn this manuscript because additional controls did not confirm the involvement of LSM1 in NMD. Our initial conclusions were based on wrong assumptions about the used degron system and the required controls. RNA sequencing, phenotype and RT-qPCR results are affected, but other results presented in the manuscript remain valid. We apologize to the colleagues who based any of their work or conclusion on that part of the preprint. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

molecular biology↗

bioRxiv2025

Comprehensive mapping of the 5' and 3' untranslated regions of Aspergillus fumigatus reveals new insights into gene regulation

In the twenty years since the first genome sequencing of Aspergillus fumigatus, the field has seen an explosion in both the number of sequenced genomes and our molecular understanding of this ubiquitous human fungal pathogen. Despite an improved knowledge of the A. fumigatus genome, we still know little about the transcriptome, with key regulatory sequences like the untranslated regions of mRNA based only on in silico predictions and bulk-RNA-seq. Here, we provide an improved description of 5' and 3' untranslated regions of A. fumigatus poly(A)-enriched RNA through experimental mapping of transcription start sites and polyadenylation sites using 5' and 3' End-Seq. We assigned high-quality 5' ends to 2,747 genes (average length 126 nt), 3' ends to 7,079 genes (average length 268 nt), and improved our understanding of the regulatory landscape of A. fumigatus gene expression. We leveraged the refined 5' UTRs to identify upstream open reading frames and binding sites for important RNA binding proteins like the translational regulator Ssd1 and the 3' UTRs to define binding sites for PUF proteins known to contribute to mRNA localization and regulation. Although a single isoform typically dominated expression, we observed 148 instances of alternative start sites and 1,675 alternative stop sites. Interestingly, we detected multiple examples of premature transcriptional termination, including the first evidence for promoter-proximal premature transcriptional termination in a member of the Eurotiomycetes. Ultimately, we provide a resource to the Aspergillus community and an accurate starting point for unravelling the complexities of gene regulation in an important human pathogen.

microbiology↗

bioRxiv2023

The Cryptocococcus extracellular vesicle transcriptome

Extracellular vesicles (EVs) are now recognized as key players in the biology of numerous organisms, including pathogenic fungi. However, studying EVs in these organisms remains challenging. The recent implementation of new protocols to purify EVs in the pathogenic yeast Cryptococcus neoformans has resulted in a more detailed description of their structure and protein composition. Although a few publications describing RNA molecules associated with EVs have already been published, we reasoned that these new protocols would be beneficial for gaining a deeper understanding of the EV transcriptome. We thus purified EVs and confirmed that some RNAs were associated with these EV extracts. Iodixanol gradient analyses also revealed that these RNAs co-sedimented with EVs. We then sequenced these RNAs in parallel with RNAs extracted from the very cells producing these EVs using different types of sequencing libraries. Our data confirm the presence of siRNAs and tRFs associated with EVs, some of which are enriched. We also identified some snoRNAs, which in Cryptococcus are mostly borne by coding gene or lncRNA introns.

microbiology↗

bioRxiv2023

Tur1 regulates alternative TSS usage in Cryptococcus

Alternative transcription start site (TSS) usage regulation has been identified as a major means of gene expression regulation in metazoans. However, in fungi, its impact remains elusive as its study has thus far been restricted to model yeasts. Here, we first re-analysed TSS-seq data to define genuine TSS clusters in two species of pathogenic Cryptococcus. We identified two types of TSS clusters associated with specific DNA sequence motifs. Our analysis also revealed that alternative TSS usage regulation in response to environmental cues is widespread in Cryptococcus, altering gene expression and protein targeting. Importantly, we performed a forward genetic screen to identify a unique transcription factor (TF) named Tur1, which regulates alternative TSS (altTSS) usage genome-wide when cells switch from exponential phase to stationary phase. ChiP-Seq and DamID-Seq analyses suggest that at some loci the role of Tur1 might be direct. Tur1 has been previously shown to be essential for virulence in C. neoformans. We demonstrated here that a tur1{Delta} mutant strain is more sensitive to superoxide stress and phagocytosed more efficiently by macrophages than the wild-type (WT) strain.

molecular biology↗

bioRxiv2023

Extracellular vesicles production regulates fluconazole resistance in Cryptococcus neoformans

Resistance to fluconazole (FLC), the most widely used antifungal drug, is typically achieved by altering the azole drug target and/or drug efflux pumps. Recent reports have suggested a link between vesicular trafficking and antifungal resistance. Here, we identified novel Cryptococcus neoformans regulators of extracellular vesicle (EV) biogenesis that impact FLC resistance. In particular, the transcription factor Hap2 does not affect the expression of the drug target or efflux pumps, yet it impacts the cellular sterol profile. Subinhibitory FLC concentrations also downregulate EV production. Moreover, in vitro spontaneous FLC-resistant colonies showed altered EV production, and the acquisition of FLC resistance was associated with decreased EV production in clinical isolates. Finally, the reversion of FLC resistance was associated with increased EV production. These data suggest a model in which fungal cells can regulate EV production in place of regulating the drug target gene expression as a first line of defense against antifungal assault in this fungal pathogen. IMPORTANCEExtracellular vesicles (EVs) are membrane-enveloped particles that are released by cells into the extracellular space. Fungal EVs can mediate community interactions and biofilm formation but thier functions remain poorly understood. Here, we report the identification of the first regulators of EV production in the major fungal pathogen Cryptococcus neoformans. Surprisingly, we uncover a novel role of EVs in modulating antifungal drug resistance. Disruption of EV production was associated with altered lipid composition and changes in fluconazole susceptibility. Spontaneous azole-resistant mutants were deficient in EV production, while loss of resistance restored initial EV production levels. These findings were recapitulated in C. neoformans clinical isolates, indicating that azole resistance and EV production are coregulated in diverse strains. Our study reveals a new mechanism of drug resistance in which cells adapt to azole stress by modulating EV production.

microbiology↗

bioRxiv2021

POPULATION GENOMIC ANALYSIS OF CRYPTOCOCCUS BRAZILIAN STRAINS REVEALS AN AFRICAN TYPE SUBCLADES DISTRIBUTION

The genomes of a large number of Cryptococcus neoformans isolates have been sequenced and analyzed in recent years. These genomes have been used to understand the global population structure of this opportunistic pathogen. However, only a small number of South American isolates have been considered in these studies, and the population structure of C. neoformans in this part of the world remains elusive. Here, we analyzed the genomic sequences of 53 Brazilian Cryptococcus isolates and deciphered the C. neoformans population structure in this country. Our data reveal an African-like structure that suggested repeated intercontinental transports from Africa to South America. We also identified a mutator phenotype in one VNBII Brazilian isolate, exemplifying how fast-evolving isolates can shape the Cryptococcus population structure. Finally, phenotypic analyses revealed wide diversity but not lineage specificity in the expression of classical virulence traits within the set of isolates.

genomics↗

bioRxiv2020

Application of an optimized annotation pipeline to the Cryptococcus deuterogattii genome reveals dynamic primary metabolic gene clusters and genomic impact of RNAi loss

Evaluating the quality of a de novo annotation of a complex fungal genome based on RNA-seq data remains a challenge. In this study, we sequentially optimized a Cufflinks-CodingQuary based bioinformatics pipeline fed with RNA-seq data using the manually annotated model pathogenic yeasts Cryptococcus neoformans and Cryptococcus deneoformans as test cases. Our results demonstrate that the quality of the annotation is sensitive to the quantity of RNA-seq data used and that the best quality is obtained with 5 to 10 million reads per RNA-seq replicate. We also demonstrated that the number of introns predicted is an excellent a priori indicator of the quality of the final de novo annotation. We then used this pipeline to annotate the genome of the RNAi-deficient species Cryptococcus deuterogattii strain R265 using RNA-seq data. Dynamic transcriptome analysis revealed that intron retention is more prominent in C. deuterogattii than in the other RNAi-proficient species C. neoformans and C. deneoformans. In contrast, we observed that antisense transcription was not higher in C. deuterogattii than in the two other Cryptococcus species. Comparative gene content analysis identified 21 clusters enriched in transcription factors and transporters that have been lost. Interestingly, analysis of the subtelomeric regions in these three annotated species identified a similar gene enrichment, reminiscent of the structure of primary metabolic clusters. Our data suggest that there is active exchange between subtelomeric regions, and that other chromosomal regions might participate in adaptive diversification of Cryptococcus metabolite assimilation potential.

microbiology↗

bioRxiv2020

New insights into Cryptococcus extracellular vesicles suggest a new structural model and an antifungal vaccine strategy

Whereas extracellular vesicle (EV) research has become commonplace in different biomedical fields, this field of research is still in its infancy in mycology. Here we provide a robust set of data regarding the structural and compositional aspects of EVs isolated from the fungal pathogenic species Cryptococcus neoformans, C. deneoformans and C. deuterogattii. Using cutting-edge methodological approaches including cryogenic electron microscopy and cryogenic electron tomography, proteomics, and flow cytometry, we revisited cryptococcal EV features and suggest a new EV structural model, in which the vesicular lipid bilayer is covered by mannoprotein-based fibrillar decoration, bearing the capsule polysaccharide as its outer layer. About 10% of the EV population is devoid of fibrillar decoration, adding another aspect to EV diversity. By analyzing EV protein cargo from the three species, we characterized the typical Cryptococcus EV proteome. It contains several membrane-bound protein families, including some Tsh proteins bearing a SUR7/PalI motif. The presence of known protective antigens on the surface of Cryptococcus EVs, resembling the morphology of encapsulated virus structures, suggested their potential as a vaccine. Indeed, mice immunized with EVs obtained from an acapsular C. neoformans mutant strain rendered a strong antibody response in mice and significantly prolonged their survival upon C. neoformans infection.

microbiology↗

bioRxiv2019

Mating-type specific divergent RPL22 paralogs are essential, differentially regulated, and control aspects of sexual reproduction in Cryptococcus neoformans

The MAT locus of Cryptococcus neoformans has a bipolar organization characterized by an unusually large structure, spanning over 100 kb. MAT genes have been characterized by functional genetics as being involved in sexual reproduction and virulence. However, classical gene replacement failed to achieve mutants for five MAT genes (RPL22, RPO41, MYO2, PRT1, RPL39), indicating that they are likely essential. In the present study, targeted gene replacement was performed in a diploid strain for both the and a alleles of the ribosomal genes RPL22 and RPL39. Mendelian analysis of the progeny confirmed that both RPL22 and RPL39 are essential for viability. Ectopic integration of the RPL22 allele of opposite MAT identity in the heterozygous RPL22a/rpl22{Delta} or RPL22/rpl22a{Delta} mutant strains failed to complement their essential phenotype. Evidence suggests that this is due to differential expression of the RPL22 genes, and an RNAi-dependent mechanism that contributes to control RPL22a expression. Furthermore, via CRISPR/Cas9 technology the RPL22 alleles were exchanged in haploid MAT and MATa strains of C. neoformans. These RPL22 exchange strains displayed morphological and genetic defects during bilateral mating. These results contribute to elucidate functions of C. neoformans essential mating type genes that may constitute a type of imprinting system to promote inheritance of nuclei of both mating types.

genetics↗

bioRxiv2019

Start codon context controls translation initiation in the fungal kingdom

Eukaryotic protein synthesis initiates at a start codon defined by an AUG and its surrounding Kozak sequence context, but studies of S. cerevisiae suggest this context is of little importance in fungi. We tested this concept in two pathogenic Cryptococcus species by genome-wide mapping of translation and of mRNA 5 and 3 ends. We observed that upstream open reading frames (uORFs) are a major contributor to translation repression, that uORF use depends on the Kozak sequence context of its start codon, and that uORFs with strong contexts promote nonsense-mediated mRNA decay. Numerous Cryptococcus mRNAs encode predicted dual-localized proteins, including many aminoacyl-tRNA synthetases, in which a leaky AUG start codon is followed by a strong Kozak context in-frame AUG, separated by mitochondrial-targeting sequence. Further analysis shows that such dual-localization is also predicted to be common in Neurospora crassa. Kozak-controlled regulation is correlated with insertions in translational initiation factors in fidelity-determining regions that contact the initiator tRNA. Thus, start codon context is a signal that programs the expression and structures of proteins in fungi.

genomics↗