Dahlmann Tim A, Kück Ulrich
Christian Doppler Laboratory for "Fungal Biotechnology", Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Universitätsstr. 150, D-44780 Bochum, Germany.
PLoS One. 2015 May 8;10(5):e0125989. doi: 10.1371/journal.pone.0125989. eCollection 2015.
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.
微小RNA（miRNA）是一类非编码小RNA（sRNA），可在多种真核生物中调节基因表达。在本研究中，我们分析了产β-内酰胺抗生素青霉素的工业生产菌产黄青霉中的调节性sRNA。为了从全局角度鉴定sRNA和微小RNA样RNA（milRNA），构建了两个sRNA测序文库。一个文库是用从十二种不同生长培养物中获得的混合总RNA构建的（RNA Mix），另一个文库是用来自单一深层培养的总RNA构建的（∆ku70FRT2）。对这两个RNA文库进行Illumina测序产生了84,322,825条比对读数。为了区分依赖于Dicer和不依赖于Dicer的sRNA形成，我们进一步构建了两个单Dicer基因突变体（∆dcl2和∆dcl1）和一个Dicer双突变体（∆dcl2∆dcl1），并分析了来自Dicer缺陷双突变体的sRNA文库。我们鉴定出661个依赖于Dicer的位点，并且通过计算机预测揭示了34个milRNA。对两个milRNA进行Northern印迹杂交，为成熟的milRNA提供了证据，这些成熟的milRNA是以完全或部分依赖于Dicer的方式从RNA前体加工而来的。鉴定出的milRNA具有先前发现的真菌milRNA的典型特征，例如对5'尿嘧啶的强烈偏好和典型的长度分布。在基因组中检测到潜在的milRNA靶位点表明milRNA可能在转录后基因调控中发挥作用。我们的数据将进一步增加我们对依赖于sRNA的基因调控过程的了解，这是开发更有效的策略以改善产黄青霉工业发酵的重要前提。
