Institute for Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, D-14476, Potsdam-Golm, Germany.
Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung, Corrensstrasse 3, D-06466 Seeland, OT, Gatersleben, Germany.
Plant J. 2019 Aug;99(4):655-672. doi: 10.1111/tpj.14348. Epub 2019 May 30.
RNA-based processes play key roles in the regulation of eukaryotic gene expression. This includes both the processing of pre-mRNAs into mature mRNAs ready for translation and RNA-based silencing processes, such as RNA-directed DNA methylation (RdDM). Polyadenylation of pre-mRNAs is one important step in their processing and is carried out by three functionally specialized canonical nuclear poly(A) polymerases in Arabidopsis thaliana. Null mutations in one of these, termed PAPS1, result in a male gametophytic defect. Using a fluorescence-labelling strategy, we have characterized this defect in more detail using RNA and small-RNA sequencing. In addition to global defects in the expression of pollen-differentiation genes, paps1 null-mutant pollen shows a strong overaccumulation of transposable element (TE) transcripts, yet a depletion of 21- and particularly 24-nucleotide-long short interfering RNAs (siRNAs) and microRNAs (miRNAs) targeting the corresponding TEs. Double-mutant analyses support a specific functional interaction between PAPS1 and components of the RdDM pathway, as evident from strong synergistic phenotypes in mutant combinations involving paps1, but not paps2 paps4, mutations. In particular, the double-mutant of paps1 and rna-dependent rna polymerase 6 (rdr6) shows a synergistic developmental phenotype disrupting the formation of the transmitting tract in the female gynoecium. Thus, our findings in A. thaliana uncover a potentially general link between canonical poly(A) polymerases as components of mRNA processing and RdDM, reflecting an analogous interaction in fission yeast.
RNA 为基础的过程在真核生物基因表达的调控中起着关键作用。这包括将前体 mRNA 加工成成熟的 mRNAs 以供翻译，以及 RNA 为基础的沉默过程，如 RNA 指导的 DNA 甲基化（RdDM）。前体 mRNA 的多聚腺苷酸化是其加工的一个重要步骤，在拟南芥中由三种功能上专门化的经典核多(A)聚合酶执行。这些酶中的一种，称为 PAPS1，的 null 突变导致雄性配子体缺陷。我们使用荧光标记策略，通过 RNA 和小 RNA 测序更详细地描述了这种缺陷。除了花粉分化基因表达的全局缺陷外，paps1 突变体花粉还表现出转座因子（TE）转录物的强烈过度积累，但 21 核苷酸和特别是 24 核苷酸长的针对相应 TE 的短干扰 RNA（siRNA）和 microRNA（miRNA）的耗尽。双突变分析支持 PAPS1 与 RdDM 途径的成分之间存在特定的功能相互作用，这从涉及 paps1 但不涉及 paps2 paps4 突变的突变体组合中的强烈协同表型中可以明显看出。特别是，paps1 和 RNA 依赖性 RNA 聚合酶 6（rdr6）的双突变体表现出协同的发育表型，破坏了雌性雌蕊中传递道的形成。因此，我们在拟南芥中的发现揭示了经典多(A)聚合酶作为 mRNA 加工成分与 RdDM 之间的潜在普遍联系，反映了裂殖酵母中类似的相互作用。
