Significance: Recently N ⁶ -methyladenosine (m ⁶ A) methylation has emerged as a biological process with significant impact on cellular functions. However, almost all the research regarding m ⁶ A methylation has been based on mRNAs. In our research, we focus on how m ⁶ A methylation affects microRNA (miRNA) biogenesis in Arabidopsis . In brief, we show that m ⁶ A methylation is necessary to maintain proper levels of mature miRNAs as well as their precursors. m ⁶ A mark affects pri-miRNA secondary structures and affects the recruitment of the Microprocessor to pri-miRNAs. We also demonstrate the interactions of MTA (m ⁶ A writer) with other proteins involved in miRNA biogenesis, namely RNA Polymerase II and TOUGH. Our study provides evidence of the role played by m ⁶ A in plant miRNA biogenesis.

Plants as sessile organisms have developed prompt response mechanisms to react to rapid environmental changes. In addition to the transcriptional regulation of gene expression, microRNAs (miRNAs) are key posttranscriptional regulators of the plant stress response. We show here that the expression levels of many miRNAs were regulated under salt stress conditions. This regulation occurred at the transcriptional and posttranscriptional levels. During salinity stress, the levels of miRNA161 and miRNA173 increased, while the expression of pri-miRNA161 and pri-miRNA173 was down-regulated. Under salt stress conditions, miRNA161 and miRNA173 were stabilized in the cytoplasm, and the expressions of MIR161 and MIR173 were negatively regulated in the nucleus. ARGONAUTE1 (AGO1) participated in both processes. We demonstrated that AGO1 cotranscriptionally controlled the expression of MIR161 and MIR173 in the nucleus. Our results suggests that AGO1 interacts with chromatin at MIR161 and MIR173 loci and causes the disassembly of the transcriptional complex, releasing short and unpolyadenylated transcripts.

MicroRNAs are small RNAs, 20–22 nt long, the main role of which is to downregulate gene expression at the level of mRNAs. MiRNAs are fundamental regulators of plant growth and development in response to internal signals as well as in response to abiotic and biotic factors. Therefore, the deficiency or excess of individual miRNAs is detrimental to particular aspects of a plant’s life. In consequence, the miRNA levels must be appropriately adjusted. To obtain proper expression of each miRNA, their biogenesis is controlled at multiple regulatory layers. Here, we addressed processes discovered to influence miRNA steady-state levels, such as MIR transcription, co-transcriptional pri-miRNA processing (including splicing, polyadenylation, microprocessor assembly and activity) and miRNA-encoded peptides synthesis. MiRNA stability, RISC formation and miRNA export out of the nucleus and out of the plant cell also define the levels of miRNAs in various plant tissues. Moreover, we show the evolutionary conservation of miRNA biogenesis core proteins across the plant kingdom.

SERRATE/ARS2 is a conserved RNA effector protein involved in transcription, processing and export of different types of RNAs. In Arabidopsis, the best-studied function of SERRATE (SE) is to promote miRNA processing. Here, we report that SE interacts with the nuclear exosome targeting (NEXT) complex, comprising the RNA helicase HEN2, the RNA binding protein RBM7 and one of the two zinc-knuckle proteins ZCCHC8A/ZCCHC8B. The identification of common targets of SE and HEN2 by RNA-seq supports the idea that SE cooperates with NEXT for RNA surveillance by the nuclear exosome. Among the RNA targets accumulating in absence of SE or NEXT are miRNA precursors. Loss of NEXT components results in the accumulation of pri-miRNAs without affecting levels of miRNAs, indicating that NEXT is, unlike SE, not required for miRNA processing. As compared to se-2, se-2 hen2-2 double mutants showed increased accumulation of pri-miRNAs, but partially restored levels of mature miRNAs and attenuated developmental defects. We propose that the slow degradation of pri-miRNAs caused by loss of HEN2 compensates for the poor miRNA processing efficiency in se-2 mutants, and that SE regulates miRNA biogenesis through its double contribution in promoting miRNA processing but also pri-miRNA degradation through the recruitment of the NEXT complex.

Developmental epigenetic modifications in plants and animals are mostly reset during gamete formation but some are inherited from the germline. Small RNAs guide these epigenetic modifications but how inherited small RNAs are distinguished in plants and animals is unknown. Pseudouridine (Ψ) is the most abundant RNA modification but has not been explored in small RNAs. Here, we develop assays to detect Ψ in short RNA sequences, demonstrating its presence in mouse and Arabidopsis microRNAs. Germline small RNAs, namely epigenetically activated small interfering RNAs (easiRNAs) in Arabidopsis pollen and Piwi-interacting RNAs in mouse testes, are enriched for Ψ. In pollen, pseudouridylated easiRNAs are transported to sperm cells from the vegetative nucleus, and PAUSED/HEN5 (PSD), the plant homolog of Exportin-t, interacts genetically with Ψ and is required for this transport. We further show that Exportin-t is required for the triploid block: small RNA dosage-dependent seed lethality that is epigenetically inherited from pollen. Thus, Ψ has a conserved role in marking inherited small RNAs in the germline.

The SERRATE (SE) protein is involved in the processing of RNA polymerase II (RNAPII) transcripts. It is associated with different complexes engaged in different aspects of plant RNA metabolism, including assemblies involved in transcription, splicing, polyadenylation, miRNA biogenesis, and RNA degradation. SE stability and interactome properties can be influenced by phosphorylation. SE exhibits an intriguing liquid-liquid phase separation property that may be important in the assembly of different RNA-processing bodies. Therefore, we propose that SE seems to participate in the coordination of different RNA-processing steps and can direct the fate of transcripts, targeting them for processing or degradation when they cannot be properly processed or are synthesized in excess.