| Non-coding RNAs(ncRNAs) existing widely in many living organisms are functional RNA molecules, function directly as structural or regulatory RNAs and have significant performance in organisms. Although large and diverse populations of ncRNAs have been extensively studied and well understood in animals and plants, there are a few reports about ncRNAs in fungi. Candida albicans (C. albicans), the major invasive human fungal pathogen, can commonly cause a wide variety of infections and diseases upon immune deficiency. One of the most striking traits of C. albicans is its ability to change the growth morphology from unicellular budding yeast form to filamentous pseudohyphal or hyphal forms. The reversible morphological transition of C. albicans, taking place in response to different environmental stimuli and stresses, is also a process tightly associated with virulence, as the hyphae form is known to play important roles in different kinds of infection processes. In this study, we have obtained hyphal C. albicans cells, and performed high-throughput sequencing (HTS) of small ncRNAs (sncRNAs) isolated from C. albicans yeast or hyphal form cells using Illumina HiseqTM2000sequencing platform. Based on a home-built bioinformatics approach, we have performed a comparative analysis of the C. albicans ncRNAs expression profile, and analyzed the characteristics of these ncRNAs. The following is summarization of our results:1. To obtain C. albicans hyphal cells, we selected two conditions (serum and a growth temperature of37℃) to induce yeast-to-hyphal transition of C. albicans in vitro. Here, we established a stable approach to induce the hyphal morphogenesis of C. albicans. Hyphae were reliably induced from yeast cells of C. albicans SC5314and2.538strains when1×105cells/ml yeast cells were grown in RPMI-1640medium with10%fetal calf serum (FCS),followed by an incubation at37℃in a constant temperature incubator for approximately3h.2. For RNA isolation, yeast and hyphal cells were harvested by centrifugation and followed by an incubation at-80℃overnight. Total RNA was isolated using hot phenol with RNase-free porcelain beads, because RNase-free porcelain beads in a high-speed shaking and eddying incubation at65℃could easily break cell walls of yeast and hyphal cells. The quantity and quality of total RNA characterized by agarose gel electrophoresis assay and Agilent2100bioanalyzer perfectly met the requirements of subsequent experiments.3.18-40nt small RNAs were isolated from SC5314yeast or hyphal cells and2.538yeast or hyphal cells by gel extraction, and then used to prepare sequencing libraries, respectively. The results of our datasets showed that besides18-40nt small RNAs, less than18nt and more than40nt small RNAs were also sequenced, indicating that the high efficiency of gel extraction.4. In this study, we sequenced four small-RNA sequencing libraries by using Illumina HiseqTM2000sequencing platform. Following the process of null and low quality read filtering and adapter removal, about99%high quality clean reads were obtained in each of these C. albicans samples, and a total of128,636,135clean reads were generated for all samples.5. Clean reads were mapped to the reference SC5314Assembly21genome to discard unmapped reads using Bowtie0.12.7with two mismatches. The mapping results showed:about74.8%total clean reads on average for four samples were mapped to rDNA regions of corresponding reference genome, and reads mapped to tDNA regions were about4.5%,8.9%,5.5%and33.0%for four samples, respectively. The additional genome-mappable reads were mainly distributed in the coding gene regions and identified ncRNA regions such as retrotransposons. Only less than6%total clean reads were unmapped to corresponding reference genome.6. The analysis for the length distribution of all genome-mappable reads showed that these sncRNAs were mainly enriched in19-34nt and had the highest abundance at the size of22nt in four C. albicans samples. Additionally, sncRNA reads from hyphal cells of strain SC5314and strain2.538were slightly more abundant than that from their respective yeast cells in the26-34nt length region, suggesting that some kinds of sncRNAs distributed in26-34nt were probably correlated with the growth of C. albicans hyphae, and might play potential roles in the morphological regulation.7. For the sncRNAs derived from known ncRNAs, most of them were located in the retrotransposon regions, and most enriched in22nt RNA beginning with A or U, characterized by our home-build bioinformatics approach. This distribution pattern was consistent with the characteristic of previously identified small RNAs in strain SC5314, suggesting the high reliability of our sequencing data.8. The analysis for rRNA-and tRNA-derived sncRNAs showed that both rRNA-and tRNA-derived sncRNAs were mainly distributed in the19-36nt length region. SncRNAs derived from rRNAs had a peak at23nt. while tRNA-derived sncRNAs had the highest abundance at34nt in both strain SC5314and2.538. 9. Based on our small-RNA sequencing data, the microRNA cadidates in C. albicans were predicted by the microRNA predict software miRDeep2with the default parameters. The results showed:2microRNA candidates were predicted in SC5314yeast cell sample, only1microRNA candidate was predicted in SC5314hyphal cell sample,8microRNA candidates were predicted in2.538yeast cell sample, and6microRNA candidates were predidcted in2.538hyphal cell sample.Surprisingly, among these microRNA candidates, there was1microRNA candidate existing in all of these samples.10. In order to discover sncRNAs associated with the morphological plasticity of C. albicans, the differences in gene expression of all of known ncRNAs were determined by bioinformatic analysis of our small-RNA sequencing data using Cufflinks. For strain SC5314and strain2.538, we found21and60ncRNA genes with significant difference in gene expression between yeast and hyphal cells, respectively. Interestinnly, there were17ncRNA genes associated with the hyhpal morphogenesis existing in both strain SC5314and strain2.538. |
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