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, 36 (8), 2677-89

Small ncRNA Transcriptome Analysis From Aspergillus Fumigatus Suggests a Novel Mechanism for Regulation of Protein Synthesis

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Small ncRNA Transcriptome Analysis From Aspergillus Fumigatus Suggests a Novel Mechanism for Regulation of Protein Synthesis

Christoph Jöchl et al. Nucleic Acids Res.

Abstract

Small non-protein-coding RNAs (ncRNAs) have systematically been studied in various model organisms from Escherichia coli to Homo sapiens. Here, we analyse the small ncRNA transcriptome from the pathogenic filamentous fungus Aspergillus fumigatus. To that aim, we experimentally screened for ncRNAs, expressed under various growth conditions or during specific developmental stages, by generating a specialized cDNA library from size-selected small RNA species. Our screen revealed 30 novel ncRNA candidates from known ncRNA classes such as small nuclear RNAs (snRNAs) and C/D box-type small nucleolar RNAs (C/D box snoRNAs). Additionally, several candidates for H/ACA box snoRNAs could be predicted by a bioinformatical screen. We also identified 15 candidates for ncRNAs, which could not be assigned to any known ncRNA class. Some of these ncRNA species are developmentally regulated implying a possible novel function in A. fumigatus development. Surprisingly, in addition to full-length tRNAs, we also identified 5'- or 3'-halves of tRNAs, only, which are likely generated by tRNA cleavage within the anti-codon loop. We show that conidiation induces tRNA cleavage resulting in tRNA depletion within conidia. Since conidia represent the resting state of A. fumigatus we propose that conidial tRNA depletion might be a novel mechanism to down-regulate protein synthesis in a filamentous fungus.

Figures

Figure 1.
Figure 1.
(A) Distribution of 3120 cDNA clones from the A. fumigatus expression library encoding ncRNA candidates. The abundance of different ncRNA species is shown as percentage of total clones. (B) Numbers of ncRNA candidates derived from the A. fumigatus cDNA library are indicated in brackets.
Figure 2.
Figure 2.
Northern blot analysis of A. fumigatus snRNAs and selected snoRNA candidates. Designation of clones is indicated on the left. Sizes of ncRNAs, as estimated by comparison with an internal RNA marker, are indicated on the right. Total RNA was isolated from A. fumigatus mycelia grown under seven different conditions and at different time points: AMM, minimal medium; ACM, complete medium; AMM-Fe, iron starvation; AMM-N, nitrogen starvation. Liquid cultures resemble vegetative growth and plate cultures induce conidiogenesis. In-gel ethidium bromide-stained 5.8 S rRNA and 5 S rRNA serve as loading controls. (A) U1-1/U1-2, U5 and U6 snRNAs, respectively. (B) C/D box snoRNAs with predicted targets (C) C/D box snoRNAs without predicted targets.
Figure 3.
Figure 3.
Northern blot analysis of ncRNA candidates from unknown ncRNA classes. Designation of clones is indicated on the left. Sizes of ncRNAs, as estimated by comparison with an internal RNA marker, are indicated on the right. Growth conditions and loading controls (5.8 S rRNA and 5 S rRNA) as described in Figure 2. As an additional loading control, U1 snRNA was included in northern blot analysis.
Figure 4.
Figure 4.
Northern blot analysis of selected nuclear and mitochondrial encoded tRNAs. Total RNA was isolated from A. fumigatus mycelia grown under iron-depleted (AMM-Fe) or iron-repleted conditions. Loading controls (5.8 S rRNA, 5 S rRNA and U1 snRNA) as described in Figure 3.
Figure 5.
Figure 5.
(A) Upper: alignment of cDNA sequences from the cDNA library, representing 5′ and 3′-halves of cytoplasmic tRNAGln. The anti-codon is boxed in red. Northern blot analysis of cytoplasmic tRNAGln (bottom) employing oligonucleotide probes directed against 5′- and 3′-halves of tRNAGln. Northern blot signals correspond to full-length tRNAGln (75 nt) or 5′ or 3′ cleavage products (36 nt or 39 nt), respectively. Lower: total RNAs were isolated from A. fumigatus conidia and from mycelia undergoing conidiogenesis (solid ACM, 6 h, 12 h, 18 h, 24 h, 48 h and 72 h), germination (liquid ACM 6 h) and vegetative growth (liquid ACM 12 h). Conidiogenesis is indicated by expression of the brlA gene. The proposed site of endonucleolytic cleavage within the anti-codon loop is indicated by a red triangle; (B) Comparison of expression levels of most abundant ncRNA species by in-gel ethidium bromide staining or by northern blot analysis from germination to hyphal growth; expression levels of the entire tRNA fraction is indicated by a red arrow; total RNA was isolated from A. fumigatus conidia, germinating conidia (3 h and 6 h) and hyphae (9–21 h), respectively.

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