miR-mediated gene silencing requires RNAi machinery in <i>C. neoformans</i>.

<p>(A) <i>URA5-miR1/2</i> restored the growth of B4500FOA (<i>ura5</i>) on MIN agar in the RNAi-deficient mutant strains, NE465, NE493, NE473 and NE475, <i>i.e.</i> gene silencing of <i>URA5-miRs</i> that was observed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052734#pone-0052734-g004" target="_blank">Fig. 4</a> did not occur in these mutants. And these transformants failed to grow on plates containing 5-FOA (the panels of MINFOA agar). MIN or MINFOA agar was supplemented with 100 µg/ml hygromycin B for the selection of all transformants of the reporters. The drug was left out for B4500 and B4500FOA. The transformants of miR1-m and miR2-m, together with the wild type B4500 and B4500FOA, served as control. (B) qRT-PCR confirmation of restored expression of <i>URA5</i> in RNAi-deficient mutants, NE465, NE493, NE473 and NE475, which are originally <i>ura5</i> defective strains.</p

Silencing of the reporter gene <i>URA5-miRs</i>.

<p>(A) The upper panels show a slower growth rate of the transformants of <i>URA5-miR1</i> (two transformants was picked in each case, namely, miR1-1 and miR1-2), and <i>URA5-miR2</i> (miR2-1 and miR2-2), than the wild type B4500, and the transformants of miR1-m (<i>i.e.</i> miR1-m1 and m2) and miR2-m, on MIN agar supplemented with 100 µg/ml hygromycin B, no hygromycin for B4500 and B4500FOA. The negative control B4500FOA (<i>ura5</i>) did not grow on MIN. On MINFOA (the bottom panels), the wild type strains and transformants of miR1-m and miR2-m were killed by FOA. Transformants containing miR1 and miR2, as well as <i>ura5</i>⁻ strain B4500FOA grew properly. MIN: minimal medium, and MINFOA: minimal medium with 5-FOA and 50 mg/l uracil. For selection of transformants, 100 µg/ml hygromycin B was added to MIN or MINFOA when needed. (B) Reverse transcription-PCR confirmed the decreased mRNA level of <i>URA5</i> in <i>URA5-miR</i> transformants. <i>URA5</i> mRNA levels in the wild type and in the transformants of miR-ms were close to each other. In each assay, two transformants were picked for double examination. (C) A semi-quantification of <i>URA5</i> mRNA to <i>ACT1</i> mRNA in the strains in (B).</p

Schematic diagram of the construction of reporters in silencing assay.

<p>The upper panel shows the construction of URA5-miRs or URA5-miR-ms. Two pair of primers, URA5-XhoI-S/URA5-miRs-BamHI and URA5-BamHI-S/URA5-XbaI-A, were used to PCR amplify the ORF and the terminator regions of <i>URA5</i>, respectively. Among the primers, URA5-miRs-BamHI harbored the sequence of miRs or miR-ms through primer design. The two PCR fragments were then ligated after digested by <i>BamH</i> I. Similarly, the construction of <i>CLC-miR1/2</i> and <i>CLC-miR1-m/miR2-m</i> was made (the bottom part). The position of miRs or miR-ms is indicated by the boxes. Restriction sites are in small letters. Arrows mark the position and direction of the primers. Start codons and stop codons of <i>URA5</i> and <i>CLC1</i> are underlined. For detailed description, see the section of Materials and Methods.</p

Detection of miRNAs by Northern blotting.

<p>Positive bands for miR1 and miR2 (the left two panels) and their precursors were detected. On the right, rsRNA, a randomly picked small RNA only formed a smear band signal. Probes were the synthesized DNA sequence corresponding to miR1, miR2 or rsRNA. Total RNA was prepared from cultures collected at 18 hr and 36 hr as indicated. EtBr stands for ethium bromide-stained gel showing the 22-nt miRNA bands. Approximately 10 µg of total RNA was equally loaded. The size of the RNA markers is shown on left.</p

Expression of <i>CLC1-miRs</i> determined by qRT-PCR in different strains.

<p>Expression of <i>CLC1-miRs</i> determined by qRT-PCR in different strains.</p

Silencing of the reporter <i>CLC-miRs</i>.

<p>A similar silencing assay was carried out for the reporter <i>CLC-miRs</i>. (A) Melanin-deficient phenotype of the transformants of <i>CLC-miRs</i> (#1 to #3) was observed as expected, suggesting knockdown expression of <i>CLC1</i>. When miRs were mutated, <i>CLC-miR-ms</i> restored melanin production (Second and forth rows from the top). Cells were placed on low-glucose (0.1%) Asn agar with NE and 100 µg/ml hygromycin except for the wild types. (B) Decreased mRNA level of <i>CLC1</i> by miRs silencing determined by reverse transcription-PCR for the strains in (A). Relative abundance of <i>CLC1</i> mRNA verse actin-encoding gene <i>ACT1</i> mRNA was considered. PCR reaction was performed in triplicate.</p

Primers used in this study.

<p>Primers used in this study.</p

Expression of <i>URA5-miRs</i> determined by qRT-PCR in different transformants.

<p>Expression of <i>URA5-miRs</i> determined by qRT-PCR in different transformants.</p

Statistic diagram of small RNAs in <i>C. neoformans</i> B4500.

<p>(A) Frequency of the nucleotides at the 5′ end of the sequenced small RNAs. (B) Genomic distribution of sRNAs in <i>C. neoformans</i>.</p

Structure of the predicted pre-miRNAs of miR1 and miR2.

<p>The sequences of miR1 and miR2 are highlighted by red letters.</p