Antisense transcription from stress-responsive transcription factors fine-tunes the cold response in Arabidopsis

Transcription of antisense long noncoding RNAs (lncRNAs) occurs pervasively across eukaryotic genomes. Only a few antisense lncRNAs have been characterized and shown to control biological processes, albeit with idiosyncratic regulatory mechanisms. Thus, we largely lack knowledge about the general role of antisense transcription in eukaryotic organisms. Here, we characterized genes with antisense transcription initiating close to the Poly(A) signal (PAS genes) in Arabidopsis (Arabidopsis thaliana). We compared plant native elongation transcript sequencing (plaNET-seq) with RNA sequencing (RNA-seq) during short-term cold exposure and detected massive differences between the response in active transcription and steady-state levels of PAS gene-derived mRNAs. The cold-induced expression of transcription factors B-BOX DOMAIN PROTEIN28 (BBX28) and C2H2-TYPE ZINC FINGER FAMILY PROTEIN5 (ZAT5) was detected by plaNET-seq, while their steady-state level was only slightly altered due to high mRNA turnover. Knockdown of BBX28 and ZAT5 or of their respective antisense transcripts severely compromised plant freezing tolerance. Decreased antisense transcript expression levels resulted in a reduced cold response of BBX28 and ZAT5, revealing a positive regulatory role of both antisense transcripts. This study expands the known repertoire of noncoding transcripts. It highlights that native transcription approaches can complement steady state RNA techniques to identify biologically relevant players in stress responses. Overall design: WT (Col-0) Arabidopsis seedlings (12 day old) were subjected to cold (4°C) for 12h after which RNA was isolated and sent for sequencing.

反义长链非编码RNA（long noncoding RNAs, lncRNAs）的转录在真核基因组中广泛发生。目前仅少数反义长链非编码RNA得到功能鉴定，且被证实可调控生物学过程，但其调控机制具有独特性。因此，我们对反义转录在真核生物中的普遍作用仍知之甚少。本研究对拟南芥（Arabidopsis thaliana）中反义转录起始于多聚腺苷酸化信号（Poly(A) signal）附近的基因（下称PAS基因）进行了功能鉴定。本研究对比了短期低温胁迫下的植物原生延伸转录组测序（plant native elongation transcript sequencing, plaNET-seq）与RNA测序（RNA sequencing, RNA-seq）数据，发现PAS基因来源的mRNA的活跃转录响应与稳态水平之间存在显著差异。plaNET-seq检测到转录因子B盒结构域蛋白28（B-BOX DOMAIN PROTEIN28, BBX28）与C2H2型锌指蛋白家族5（C2H2-TYPE ZINC FINGER FAMILY PROTEIN5, ZAT5）的冷诱导表达，但由于mRNA周转速率较高，二者的稳态水平仅发生轻微变化。敲低BBX28、ZAT5或其各自的反义转录本，会严重削弱植物的耐寒性。反义转录本表达水平的降低会导致BBX28与ZAT5的低温响应减弱，这表明两种反义转录本均发挥正向调控作用。本研究拓展了已知的非编码转录本库，并指出原生转录组测序方法可作为稳态RNA分析技术的补充，用于鉴定胁迫响应中具有生物学功能的关键作用因子。整体实验设计：将生长12天的野生型（Col-0）拟南芥幼苗置于4℃低温环境处理12小时后，提取RNA并进行测序。