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Abstract MicroRNAs (miRNAs) play a key role in regulating gene expression and their biogenesis is precisely controlled through modulating the activity of microprocessor. Here, we report that CWC15, a spliceosome-associated protein, acts as a positive regulator of miRNA biogenesis. CWC15 binds the promoters of genes encoding miRNAs (MIRs), promotes their activity, and increases the occupancy of DNA-dependent RNA polymerases at MIR promoters, suggesting that CWC15 positively regulates the transcription of primary miRNA transcripts (pri-miRNAs). In addition, CWC15 interacts with Serrate (SE) and HYL1, two key components of microprocessor, and is required for efficient pri-miRNA processing and the HYL1-pri-miRNA interaction. Moreover, CWC15 interacts with the 20 S proteasome and PRP4KA, facilitating SE phosphorylation by PRP4KA, and subsequent non-functional SE degradation by the 20 S proteasome. These data reveal that CWC15 ensures optimal miRNA biogenesis by maintaining proper SE levels and by modulating pri-miRNA levels. Taken together, this study uncovers the role of a conserved splicing-related protein in miRNA biogenesis. 

Abstract MicroRNAs (miRNAs) are important regulators of genes expression. Their levels are precisely controlled through modulating the activity of the microprocesser complex (MC). Here, we report that JANUS, a homology of the conserved U2 snRNP assembly factor in yeast and human, is required for miRNA accumulation. JANUS associates with MC components Dicer-like 1 (DCL1) and SERRATE (SE) and directly binds the stem-loop of pri-miRNAs. In a hypomorphic janus mutant, the activity of DCL1, the numbers of MC, and the interaction of primary miRNA transcript (pri-miRNAs) with MC are reduced. These data suggest that JANUS promotes the assembly and activity of MC through its interaction with MC and/or pri-miRNAs. In addition, JANUS modulates the transcription of some pri-miRNAs as it binds the promoter of pri-miRNAs and facilitates Pol II occupancy of at their promoters. Moreover, global splicing defects are detected in janus. Taken together, our study reveals a novel role of a conserved splicing factor in miRNA biogenesis. 

Abstract It is challenging to identify the smallest microexons (≤15-nt) due to their small size. Consequently, these microexons are often misannotated or missed entirely during genome annotation. Here, we develop a pipeline to accurately identify 2,398 small microexons in 10 diverse plant species using 990 RNA-seq datasets, and most of them have not been annotated in the reference genomes. Analysis reveals that microexons tend to have increased detained flanking introns that require post-transcriptional splicing after polyadenylation. Examination of 45 conserved microexon clusters demonstrates that microexons and associated gene structures can be traced back to the origin of land plants. Based on these clusters, we develop an algorithm to genome-wide model coding microexons in 132 plants and find that microexons provide a strong phylogenetic signal for plant organismal relationships. Microexon modeling reveals diverse evolutionary trajectories, involving microexon gain and loss and alternative splicing. Our work provides a comprehensive view of microexons in plants. 

Abstract A number of crop wild relatives can tolerate extreme stress to a degree outside the range observed in their domesticated relatives. However, it is unclear whether or how the molecular mechanisms employed by these species can be translated to domesticated crops. Paspalum (Paspalum vaginatum) is a self-incompatible and multiply stress-tolerant wild relative of maize and sorghum. Here, we describe the sequencing and pseudomolecule level assembly of a vegetatively propagated accession ofP. vaginatum. Phylogenetic analysis based on 6,151 single-copy syntenic orthologues conserved in 6 related grass species places paspalum as an outgroup of the maize-sorghum clade. In parallel metabolic experiments, paspalum, but neither maize nor sorghum, exhibits a significant increase in trehalose when grown under nutrient-deficit conditions. Inducing trehalose accumulation in maize, imitating the metabolic phenotype of paspalum, results in autophagy dependent increases in biomass accumulation. 

Summary Pre‐mRNA splicing is an essential step for the regulation of gene expression. In order to specifically capture splicing variants in plants for genome‐wide association studies (GWAS), we developed a software tool to quantify and visualise Variations of Splicing in Population (VaSP).VaSP can quantify splicing variants from short‐read RNA‐seq datasets and discover genotype‐specific splicing (GSS) events, which can be used to prioritise causal pre‐mRNA splicing events in GWAS. We applied our method to an RNA‐seq dataset with 328 samples from 82 genotypes from a rice diversity panel exposed to optimal and saline growing conditions.In total, 764 significant GSS events were identified in salt stress conditions. GSS events were used as markers for a GWAS with the shoot Na⁺accumulation, which identified six GSS events in five genes significantly associated with the shoot Na⁺content. Two of these genes,OsNUC1andOsRAD23emerged as top candidate genes with splice variants that exhibited significant divergence between the variants for shoot growth under salt stress conditions.VaSP is a versatile tool for alternative splicing analysis in plants and a powerful tool for prioritising candidate causal pre‐mRNA splicing and corresponding genomic variations in GWAS. 

Summary MicroRNAs (miRNAs) are essential regulators of gene expression in metazoans and plants. In plants, most miRNAs are generated from primary miRNA transcripts (pri‐miRNAs), which are processed by the Dicer‐like 1 (DCL1) complex along with accessory proteins.Serrate‐Associated Protein 1 (SEAP1), a conserved splicing‐related protein, has been studied in human and yeast. However, the functions of SEAP1 in plants remain elusive.Lack ofSEAP1results in embryo lethality and knockdown ofSEAP1by an artificial miRNA (amiR^SEAP1) causes pleiotropic developmental defects and reduction in miRNA accumulation. SEAP1 associates with the DCL1 complex, and may promote the interaction of the DCL1 complexes with pri‐miRNAs. SEAP1 also enhances pri‐miRNA accumulation, but does not affect pri‐miRNA transcription, suggesting it may indirectly or directly stabilize pri‐miRNAs. In addition, SEAP1 affects the splicing of some pri‐miRNAs and intron retention of messenger RNAs at global levels.Our findings uncover both conserved and novel functions of SEAP1 in plants. Besides the role as a splicing factor, SEPA1 may promote miRNA biogenesis by positively modulating pri‐miRNA splicing, processing and/or stability. 

Abstract Rice, an important food resource, is highly sensitive to salt stress, which is directly related to food security. Although many studies have identified physiological mechanisms that confer tolerance to the osmotic effects of salinity, the link between rice genotype and salt tolerance is not very clear yet. Association of gene co‐expression network and rice phenotypic data under stress has penitential to identify stress‐responsive genes, but there is no standard method to associate stress phenotype with gene co‐expression network. A novel method for integration of gene co‐expression network and stress phenotype data was developed to conduct a system analysis to link genotype to phenotype. We applied aLASSO‐based method to the gene co‐expression network of rice with salt stress to discover key genes and their interactions for salt tolerance‐related phenotypes. Submodules in gene modules identified from the co‐expression network were selected by theLASSOregression, which establishes a linear relationship between gene expression profiles and physiological responses, that is, sodium/potassium condenses under salt stress. Genes in these submodules have functions related to ion transport, osmotic adjustment, and oxidative tolerance. We argued that these genes in submodules are biologically meaningful and useful for studies on rice salt tolerance. This method can be applied to other studies to efficiently and reliably integrate co‐expression network and phenotypic data.