The nuclear cap-binding protein complex (CBC) participates in 5′ splice site selection of introns that are proximal to the mRNA cap. However, it is not known whether CBC has a role in alternative splicing. Using an RT–PCR alternative splicing panel, we analysed 435 alternative splicing events in Arabidopsis thaliana genes, encoding mainly transcription factors, splicing factors and stress-related proteins. Splicing profiles were determined in wild type plants, the cbp20 and cbp80(abh1) single mutants and the cbp20/80 double mutant. The alternative splicing events included alternative 5′ and 3′ splice site selection, exon skipping and intron retention. Significant changes in the ratios of alternative splicing isoforms were found in 101 genes. Of these, 41% were common to all three CBC mutants and 15% were observed only in the double mutant. The cbp80(abh1) and cbp20/80 mutants had many more changes in alternative splicing in common than did cbp20 and cbp20/80 suggesting that CBP80 plays a more significant role in alternative splicing than CBP20, probably being a platform for interactions with other splicing factors. Cap-binding proteins and the CBC are therefore directly involved in alternative splicing of some Arabidopsis genes and in most cases influenced alternative splicing of the first intron, particularly at the 5′ splice site.

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.

Abstract 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.

Seed dormancy is adopted by plants to optimize their reproductive strategy. The DOG1 (DELAY OF GERMINATION 1) gene is the main QTL controlling this trait in Arabidopsis (Bentsink et al., 2006) and therefore is extensively regulated. This includes the alternative polyadenylation (APA) of DOG1 mRNA (Cyrek et al., 2016) and an antisense transcript, asDOG1, which in cis suppresses DOG1 expression during seed maturation (Fedak et al., 2016). As with many antisense transcripts (Mellor et al., 2016; Rosa et al., 2016), asDOG1 originates from close to the transcription termination site of the sense gene. This raises the question of how this proximity affects antisense promoter activity.

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m 6 A, one of the most abundant mRNA modifications, has been associated with various metabolic processes in plants. Here we show that m 6 A also plays a role in miRNA biogenesis in Arabidopsis thaliana. Significant reductions in plant m 6 A/MTA levels results in lower accumulation of miRNAs whereas pri-miRNA levels tend to be higher in such plants. m 6 A-IP Seq and MTA-GFP RIP were used to show that many pri-miRNAs are m 6 A methylated and are bound by MTA, further demonstrating that pri-miRNAs can also be substrates for m 6 A methylation by MTA. We report that MTA interacts with RNA Pol II, supporting the assumption that m 6 A methylation is a co-transcriptional process, and also identify TGH, a known miRNA biogenesis related protein, as a novel protein that interacts with MTA. Finally, reduced levels of 2 miR393b may partially explain the strong auxin insensitivity seen in Arabidopsis plants with reduced m 6 A levels.Introduction: N 6 -methyladenosine (m 6 A), one of the most abundant mRNA modifications in eukaryotic cells can regulate eukaryote gene expression at multiple post-and cotranscriptional levels. m 6 A methylation in animal mRNAs is associated with several biological processes, ranging from cancer 1 , viral infections 2,3 to cell development 4,5 with the underpinning mechanisms including m 6 A regulated pre-mRNA splicing patterns, mRNA export, mRNA stability and changes in translational efficiency 6 . A group of proteins that collectively form the RNA methylation "writer" complex have been characterized and are well conserved between plants and animals. The mammalian m 6 A methyltranserase complex consists of Methyltransferase Like 3 (METTL3) 7 , Methyltransferase Like 14 (METTL14) 8 , Wilms' Tumour1-Associating Protein (WTAP) 9 , VIRMA (KIAA1429) 10 , RNA-binding motif protein 15 (RBM15) 11 and Zinc Finger CCCH-Type Containing 13 (ZC3H13) 12,13 . While METTL3 has been identified as the catalytic protein in this complex 7 , auxiliary proteins provide specificity and/or help with proper localization of the complex 6 . The m 6 A mark can be recognized by various "readers", the best characterized of which belong to the YT521-B homology (YTH) domain family [14][15][16][17] . The modification can also be removed from transcripts by "erasers", which in humans include fat mass and obesity-associated protein (FTO) 18 and α-ketoglutaratedependent dioxygenase alkB homolog 5 (ALKBH5) 19 .In Arabidopsis thaliana, the presence of m 6 A was first reported in 2008 and was shown to be dependent upon the activity of mRNA adenosine methylase (MTA) [homolog of human METTL3], the catalytic component of Arabidopsis m 6 A methyltransferase complex 20 . FKBP12 interacting protein 37 kDa (FIP37, homolog of WTAP) was the first identified methyltransferase

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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.