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.

mRNA adenosine methylase (MTA) deposits m6A on pri-miRNAs to modulate miRNA biogenesis in Arabidopsis thaliana.

In Arabidopsis thaliana, the METTL3 homolog, mRNA adenosine methylase (MTA) introduces N6-methyladenosine (m6A) into various coding and noncoding RNAs of the plant transcriptome. Here, we show that an MTA-deficient mutant (mta) has decreased levels of microRNAs (miRNAs) but accumulates primary miRNA transcripts (pri-miRNAs). Moreover, pri-miRNAs are methylated by MTA, and RNA structure probing analysis reveals a decrease in secondary structure within stem-loop regions of these transcripts in mta mutant plants. We demonstrate interaction between MTA and both RNA Polymerase II and TOUGH (TGH), a plant protein needed for early steps of miRNA biogenesis. Both MTA and TGH are necessary for efficient colocalization of the Microprocessor components Dicer-like 1 (DCL1) and Hyponastic Leaves 1 (HYL1) with RNA Polymerase II. We propose that secondary structure of miRNA precursors induced by their MTA-dependent m6A methylation status, together with direct interactions between MTA and TGH, influence the recruitment of Microprocessor to plant pri-miRNAs. Therefore, the lack of MTA in mta mutant plants disturbs pri-miRNA processing and leads to the decrease in miRNA accumulation. Furthermore, our findings reveal that reduced miR393b levels likely contributes to the impaired auxin response phenotypes of mta mutant plants.

N6-methyladenosine (m6A): Revisiting the Old with Focus on New, an Arabidopsis thaliana Centered Review.

N6-methyladenosine (m6A) is known to occur in plant and animal messenger RNAs (mRNAs) since the 1970s. However, the scope and function of this modification remained un-explored till very recently. Since the beginning of this decade, owing to major technological breakthroughs, the interest in m6A has peaked again. Similar to animal mRNAs, plant mRNAs are also m6A methylated, within a specific sequence motif which is conserved across these kingdoms. m6A has been found to be pivotal for plant development and necessary for processes ranging from seed germination to floral development. A wide range of proteins involved in methylation of adenosine have been identified alongside proteins that remove or identify m6A. This review aims to put together the current knowledge regarding m6A in Arabidopsis thaliana.

MicroRNA biogenesis: Epigenetic modifications as another layer of complexity in the microRNA expression regulation.

Since their discovery, microRNAs have led to a huge shift in our understanding of the regulation of key biological processes. The discovery of epigenetic modifications that affect microRNA expression has added another layer of complexity to the already tightly controlled regulatory machinery. Modifications like uridylation, adenylation and RNA editing have been shown to have variable effects on miRNA biogenesis and action. Methylation of the N6 adenosine has been studied extensively in mRNA. Presence of the N6-methyl-adenosine (m6A) mark and its critical importance in miRNA biogenesis in animals adds to our understanding of the regulatory mechanisms, while its effect on miRNA biogenesis in plants is yet to be understood.