<i>milR-1</i> transcription is mediated by a non-conventional Pol III promoter.

<p>A. Northern blot analysis showing the levels of <i>milR-1</i> in the indicated strains. B. qRT-PCR analysis showing the reduction of <i>pri-milR-1</i> levels in the <i>mut1</i> and <i>mut2</i> strains. WT and <i>milR-1^ko</i> served as the positive and negative control, respectively. C. ChIP assays using the c-Myc antibody showing the reduced binding of Myc-RPC7 at the <i>milR-1</i> locus with the mutated TATA-like element. The asterisk indicates <i>P</i> value<0.05. Error bars indicate S.D.</p

Pol III knockdown results in the reduction of milRNA expression.

<p>A. Race tube assays showing the growth rates of the indicated strains in the presence or absence of QA. B. Northern blot analysis showing the levels of <i>rpc5</i>-specific siRNA in the indicated strains. C. qRT-PCR analysis showing the reduction of pri-milR levels when <i>rpc5</i> was silenced. rRNA level was used as the loading control for qRT-PCR. A Pol III-transcribed tRNA was served as a positive control. D. Northern blot showing the levels of <i>milR-1</i> and <i>milR-4</i> small RNAs.</p

Pol III specifically binds to milR loci.

<p>ChIP assay results showing the binding of Pol III to the milR loci. A <i>tRNA</i> and the <i>β-tubulin</i> gene was used as the negative and positive control, respectively. The asterisk indicate <i>P</i> value<0.05. Error bars indicate S.D.</p

The involvement of Pol II in milRNA production.

<p>A. Race tube assays showing the growth rates of the indicated strains in the presence or absence of QA. B. Northern blot analysis showing the levels of <i>rpb5</i>-specific siRNA in the indicated strains. C and D. qRT-PCR analysis results showing the reduction of <i>rpb5 and β-tubulin</i> level in ds<i>rpb5</i> strain in the presence of QA. The asterisks indicate <i>P</i> value<0.05. Error bars indicate S.D. E. Northern blot analysis showing the levels of <i>milR-1</i> and <i>milR-4</i> small RNAs. F. ChIP assay using c-Myc antibody showing the binding of Pol II to milR loci in the Myc-RPB6 strain. A <i>tRNA</i> and the <i>β-tubulin</i> gene was served as the negative control and positive control, respectively.</p

RNA sequencing of poly(A) RNA results showing the presence/absence of Pol II transcripts in the selected milR loci.

<p>Viewing window was set as 2000 nt. The vertical line in each panel indicates the location of the indicated <i>milR</i> gene.</p

RNase Z is required for <i>milR-4</i> processing.

<p>A. A Diagram showing the predicted secondary structure of <i>pri-milR-4</i>. B. RT-PCR analysis, which used a pair of primers indicated in A, showing the presence of transcript that spanning the two alanine tRNAs and <i>milR-4</i>. C. Race tube assays showing the growth rates of the indicated strains in the presence or absence of QA. D. Northern blot analysis showing the levels of <i>rnaseZ</i>-specific siRNA in the indicated strains. E. qRT-PCR analysis showing the reduction of <i>rnaseZ</i> mRNA level in the ds<i>rnaseZ</i> strain in the presence of QA. The asterisk indicates <i>P</i> value<0.05. Error bars indicate S.D. F. Northern blot analysis showing the levels of <i>milR-4</i> milRNAs in the indicated strains in the presence or absence of QA.</p

qPCR primer sequences.

<p>qPCR primer sequences.</p

DNA sequences of <i>milR-1-4</i>.

<p>The sequences of milRNA, milRNA* and the poly T sequences are indicated. TATA-like elements are underlined with single dashed lines. Putative A-boxes and B-boxes are underlined with double and triple dashed lines, respectively. The solid triangles in the <i>milR-1</i> sequence indicate the nucleotides mutated in our promoter analysis.</p