Análise de fragmentos de RNA derivados de tRNA em Cryptococcus gattii

Mecanismos de regulação da expressão gênica constituem um ponto chave de células e organismos, sendo fundamentais em diversos aspectos da funcionalidade celular, como quando expostos a estímulos e variações no ambiente. Neste contexto, moléculas de RNA não codificante também desempenham importantes funções. Recentemente, uma nova classe de pequenos RNAs com atividade regulatória pós-transcricional foi descrita em diversas espécies, sendo denominados fragmentos de RNA derivados de tRNAs (tRFs). Embora tRFs possuam uma função de regulação extremamente similar aos microRNAs, existem indícios de que tRFs exercem sua função independentemente da maquinaria celular utilizada pelos microRNAs na via de RNA de interferência (RNAi). As leveduras basidiomicéticas Cryptococcus gattii e Cryptococcus neoformans são os agentes etiológicos da criptococose, doença sistêmica que acomete principalmente pulmões e sistema nervoso central. É conhecido que algumas linhagens de C. gattii não possuem alguns genes envolvidos na via de RNAi, enquanto que C. neoformans, evolutivamente próximo a C. gattii, é proeficiente para esta via. Dessa forma, C. gattii apresenta um grande potencial como modelo de avaliação da independência dos tRFs da maquinaria de RNAi, o que poderia indicar a existência de uma via alternativa de regulação pós-transcricional nesta levedura. Assim, esse projeto teve por objetivo avaliar a presença de tRFs em C. gattii. Empregando metodologias in silico baseadas em dados de alinhamento de bibliotecas de pequenos RNAs, foram identificados 34 tRFs únicos para a linhagem R265 de C. gattii, originados de 23 das 53 sequências únicas de tRNAs preditas no genoma. Dentre estes, estão inclusos tRFs de uma possível nova classe, originada da região 5’leader do transcrito de tRNA primário. Por meio de ensaios de qRT-PCR, foram confirmadas as predições de tRFs representativos, além da identificação de expressão diferencial. Apesar da quantidade de tRNAs que geram tRFs ser extremamente baixa nesta levedura, quando comparada com outros organismos, a identificação de tRFs em uma linhagem de C. gattii que apresenta a inviabilidade da via de RNAi, e especialmente a evidência de expressão diferencial, sugerem a existência de regulação por meio de tRFs de forma independente da via de RNAi.Gene expression regulation mechanisms are a key point for cells and organisms and essential to several aspects of cellular functionality, as those necessary to adapt to stimuli and variations on the environment. In this context, noncoding RNA molecules also have an important role. Recently, a new class of small RNAs with post-transcriptional regulatory activity was described in various species, known as tRNA-derived RNA fragments (tRFs). Although tRFs possess a regulation function extremely similar to microRNAs, there are indications that tRFs exert their functions independently of cellular machinery used by microRNAs in RNA interference pathway (RNAi). The basidiomycetous yeasts Cryptococcus gattii and Cryptococcus neoformans are the etiologic agents of cryptococcosis, a systemic disease which affects mainly the lungs and central nervous system. Some strains of C. gattii do not possess genes involved in RNAi pathway, while the evolutionarily close C. neoformans is proficient in such pathway. Therefore, C. gattii displays a great potential as a model for evaluation of tRFs RNAi machinery independent activity, which could point to an alternative post-transcriptional regulation pathway existence. Therefore, the aim of this work was to evaluate the presence of tRFs in C. gattii R265. Using in silico methodologies based on alignment data from small RNAs libraries, we identified 34 unique tRFs in C. gattii R265 strain, originated from 23 out of the 53 unique sequences of genome predicted tRNAs. Among these are tRFs of a possible new class, derived from the 5’ leader region of primary tRNA transcript. With qRT-PCR assays, we were able to confirm the prediction of representative tRFs, as well as identifying differential expression of some tRFs. Although the number of tRF-producing tRNAs identified was lower compared to other organisms, the tRFs identification in a RNAi pathway-disabled strain of C. gattii, and especially the evidence of differential expression, suggest the existence of a RNAi pathway-independent regulation by the tRFs

sRNAs as possible regulators of retrotransposon activity in Cryptococcus gattii VGII

Background: The absence of Argonaute genes in the fungal pathogen Cryptococcus gattii R265 and other VGII strains indicates that yeasts of this genotype cannot have a functional RNAi pathway, an evolutionarily conserved gene silencing mechanism performed by small RNAs. The success of the R265 strain as a pathogen that caused the Pacific Northwest and Vancouver Island outbreaks may imply that RNAi machinery loss could be beneficial under certain circumstances during evolution. As a result, a hypermutant phenotype would be created with high rates of genome retrotransposition, for instance. This study therefore aimed to evaluate in silicio the effect of retrotransposons and their control mechanisms by small RNAs on genomic stability and synteny loss of C. gattii R265 through retrotransposons sequence comparison and orthology analysis with other 16 C. gattii genomic sequences available. Results: Retrotransposon mining identified a higher sequence count to VGI genotype compared to VGII, VGIII, and VGIV. However, despite the lower retrotransposon number, VGII exhibited increased synteny loss and genome rearrangement events. RNA-Seq analysis indicated highly expressed retrotransposons as well as sRNA production. Conclusions: Genome rearrangement and synteny loss may suggest a greater retrotransposon mobilization caused by RNAi pathway absence, but the effective presence of sRNAs that matches retrotransposon sequences means that an alternative retrotransposon silencing mechanism could be active in genomic integrity maintenance of C. gattii VGII strains

The genetic diversity of “papillomavirome” in bovine teat papilloma lesions

Background: Papillomaviruses are small nonenveloped, circular double-stranded DNA viruses that belong to the Papillomaviridae family. To date, 29 Bos taurus papillomavirus (BPV) types have been described. Studies involving mixed BPV infections have rarely been reported in contrast to human papillomavirus (HPV), which is commonly described in numerous studies showing coinfections. Moreover, previous studies had shown that HPV coinfections increase the risk of carcinogenesis. In the present study, we used rolling-circle amplification followed by a high-throughput sequencing (RCA-HTS) approach in 23 teat papillomas from southern Brazil. Results: Eleven well-characterized BPV types and 14 putative new BPV types were genetically characterized into the Xi, Epsilon and Dyoxipapillomavirus genera according to phylogenetic analysis of the L1 gene, which expands the previous 29 BPV types to 43. Moreover, BPV coinfections were detected in the majority (56.3%) of the papilloma lesions analyzed, suggesting a genetic diverse “papillomavirome” in bovine teat warts. Conclusions: The data generated in this study support the possibility that a wide range of BPV is probably underdetected by conventional molecular detection tools, and that BPV coinfections are underestimated and probably genetic diverse. Additionally, 14 new BPV types were characterized, increasing the knowledge regarding BPV genetic diversity

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

International audienceEvaluating 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 show that the quality of the annotation is sensitive to the quantity of RNA-seq data used and that the best quality is obtained with 5–10 million reads per RNA-seq replicate. We also showed 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

Transcriptional Analysis Allows Genome Reannotation and Reveals that Cryptococcus gattii VGII Undergoes Nutrient Restriction during Infection

Cryptococcus gattii is a human and animal pathogen that infects healthy hosts and caused the Pacific Northwest outbreak of cryptococcosis. The inhalation of infectious propagules can lead to internalization of cryptococcal cells by alveolar macrophages, a niche in which C. gattii cells can survive and proliferate. Although the nutrient composition of macrophages is relatively unknown, the high induction of amino acid transporter genes inside the phagosome indicates a preference for amino acid uptake instead of synthesis. However, the presence of countable errors in the R265 genome annotation indicates significant inhibition of transcriptomic analysis in this hypervirulent strain. Thus, we analyzed RNA-Seq data from in vivo and in vitro cultures of C. gattii R265 to perform the reannotation of the genome. In addition, based on in vivo transcriptomic data, we identified highly expressed genes and pathways of amino acid metabolism that would enable C. gattii to survive and proliferate in vivo. Importantly, we identified high expression in three APC amino acid transporters as well as the GABA permease. The use of amino acids as carbon and nitrogen sources, releasing ammonium and generating carbohydrate metabolism intermediaries, also explains the high expression of components of several degradative pathways, since glucose starvation is an important host defense mechanism