Dicer-Dependent Biogenesis of Small RNAs and Evidence for MicroRNA-Like RNAs in the Penicillin Producing Fungus Penicillium chrysogenum

Abstract

MicroRNAs (miRNAs) are non-coding small RNAs (sRNAs) that regulate gene expression in a wide range of eukaryotes. In this study, we analyzed regulatory sRNAs in Penicillium chrysogenum, the industrial producer of the β-lactam antibiotic penicillin. To identify sRNAs and microRNA-like RNAs (milRNAs) on a global approach, two sRNA sequencing libraries were constructed. One library was created with pooled total RNA, obtained from twelve differently grown cultures (RNA Mix), and the other with total RNA from a single submerged cultivation (∆ku70FRT2). Illumina sequencing of both RNA libraries produced 84,322,825 mapped reads. To distinguish between Dicer-dependent and independent sRNA formation, we further constructed two single dicer gene mutants (∆dcl2 and ∆dcl1) and a dicer double mutant (∆dcl2∆dcl1) and analyzed an sRNA library from the Dicer-deficient double-mutant. We identified 661 Dicer-dependent loci and in silico prediction revealed 34 milRNAs. Northern blot hybridization of two milRNAs provided evidence for mature milRNAs that are processed either in a complete or partial Dicer-dependent manner from an RNA precursor. Identified milRNAs share typical characteristics of previously discovered fungal milRNAs, like a strong preference for a 5' uracil and the typical length distribution. The detection of potential milRNA target sites in the genome suggests that milRNAs might play a role in posttranscriptional gene regulation. Our data will further increase our knowledge of sRNA dependent gene regulation processes, which is an important prerequisite to develop more effective strategies for improving industrial fermentations with P. chrysogenum.

Fig 1. Chromosomal distribution of small RNAs and sRNA-producing loci.

Pie graphs for total reads (A) and unique reads (B) are showing the relative abundance of sRNAs located in tRNAs, rRNAs, intergenic, exonic and intronic regions in ∆ku70FRT2. Alignments of sRNA-producing loci of ∆ku70FRT2 (C) and ∆dcl2∆dcl1 (D) show that the number of sRNAs that map to both DNA strands of one feature have strongly increased and that the fraction of sRNA loci that align to exonic regions in sense orientation has decreased substantially in ∆dcl2∆dcl1 compared to ∆ku70FRT2.

Fig 2. Length distribution of P. chrysogenum small RNA population.

Length distribution of mapping sRNA reads for the datasets of total (A) and unique (B) reads obtained from three different samples. Frequency of the 5'-nucleotide of the unique reads of ∆ku70FRT2 (C) and ∆dcl2∆dcl1 (D) in dependency of their read length.

Fig 3. Characterization of Dicer-dependent small RNAs.

(A) Strain-specific and overrepresented unique reads in ∆ku70FRT2 compared to ∆dcl2∆dcl1 and vice versa. (B) Nucleotide preference and size distribution of Dicer-dependent small RNAs. (C) Pie graphs of the relative abundance of Dicer-dependent sRNAs and (D) Dicer-dependent sRNA-producing loci in accordance to their strand bias.

Fig 4. Accumulation of sRNAs along representative coding-sequences.

(A) Normalized read count (TPTM: transcripts per ten million) of ∆ku70FRT2 (grey graph) and ∆dcl2∆dcl1 (black graph) of two representative Dicer-dependent coding regions, the Copia13-like transposable element Pc17g00440 and the putative DNA-binding protein Pc12g14660. (B) Dicer-independent sRNA accumulation for the coding region of the putative cell-wall protein Pc20g06530 and for a histidine tRNA-gene cluster. To ensure a faultless representation of Dicer-independent reads the graphs for ∆dcl2∆dcl1 were slightly moved to the right.

Fig 5. Validation and expression analysis of (A) milR-1 and (B) milR-21.

Total RNA from strain P2niaD18, the recipient ∆ku70FRT2 as well as Dicer single and double mutant strains were used for polyacrylamide gel electrophoresis and northern blot analysis. Mature milRNAs (milRs) and milRNA precursors (pre-milRs) were detected with revers complement ³²P-labled DNA probes. Below, loading controls of the total RNA, stained with ethidium bromide (EtBr), and the predicted secondary structures of milRNA precursors are given. On the secondary structures, milRNA sequences are highlighted in red and arrows indicate the expected Dicer cleavage sites.