RAM is upregulated during T cell activation and is required for RNA cap formation and gene expression.

On T cell activation, upregulation of gene expression produces the protein required for the differentiation and proliferation of effector cell populations. RAM (RNMT-Activating Mini protein/RAMAC/Fam103a1), the cofactor of the RNA cap methyltransferase RNMT (RNA guanosine N-7 cap methyltransferase), is upregulated following activation. Formation of the RNA cap protects RNA during synthesis and guides RNA processing and translation. Using conditional gene deletion, we found that Ram expression stabilizes RNMT protein in T cells and is required for its upregulation on activation. When the Ram gene is deleted in naïve T cells, there are major impacts on activation-induced RNA cap formation and gene expression. Activated T cell proliferation is dependent on increased ribosome production; in Ram knockout T cells, activation-induced expression of ribosomal protein genes and snoRNAs is most severely reduced. Consistent with these changes, Ram deletion resulted in reduced protein synthesis, and reduced growth and proliferation of CD4 T cells. Deletion of Ram results in a similar but milder phenotype to Rnmt deletion, supporting the role of RAM as a RNMT cofactor.

m6A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5' splice site.

Alternative splicing of messenger RNAs is associated with the evolution of developmentally complex eukaryotes. Splicing is mediated by the spliceosome, and docking of the pre-mRNA 5' splice site into the spliceosome active site depends upon pairing with the conserved ACAGA sequence of U6 snRNA. In some species, including humans, the central adenosine of the ACAGA box is modified by N6 methylation, but the role of this m6A modification is poorly understood. Here, we show that m6A modified U6 snRNA determines the accuracy and efficiency of splicing. We reveal that the conserved methyltransferase, FIONA1, is required for Arabidopsis U6 snRNA m6A modification. Arabidopsis fio1 mutants show disrupted patterns of splicing that can be explained by the sequence composition of 5' splice sites and cooperative roles for U5 and U6 snRNA in splice site selection. U6 snRNA m6A influences 3' splice site usage. We generalise these findings to reveal two major classes of 5' splice site in diverse eukaryotes, which display anti-correlated interaction potential with U5 snRNA loop 1 and the U6 snRNA ACAGA box. We conclude that U6 snRNA m6A modification contributes to the selection of degenerate 5' splice sites crucial to alternative splicing.

All the information necessary to build the proteins that perform the biological processes required for life is encoded in the DNA of an organism. Making these proteins requires the DNA sequence of a gene to be transcribed into a 'messenger RNA' (mRNA), which is then processed into a final, mature form. This blueprint is then translated to assemble the corresponding protein. When an mRNA is processed, segments of the sequence that do not code for protein are removed and the remaining coding sequences are joined together in the right order. An intricate molecular machine known as the spliceosome controls this mechanism by recognising the 'splice sites' where coding and non-coding sequences meet. Depending on external conditions, the spliceosome can 'pick-and-mix' the coding sequences to create different processed mRNAs (and therefore proteins) from a single gene. This alternative splicing mechanism is often used to regulate when certain biological processes take place based on environmental cues; for example, the splicing of genes which control the timing of plant flowering is sensitive to ambient temperatures. To investigate this mechanism, Parker et al. focused on Arabidopsis thaliana, a plant that blooms later when temperatures are low. This precise timing partly relies on a gene whose mRNA is efficiently spliced in the cold, resulting in an active form of its protein that blocks blooming. Parker et al. grew and screened many A. thaliana plants to find individuals that could flower early in the cold, in which splicing of this gene was disrupted. A mutant fitting these criteria was identified and subjected to further investigation, which revealed that it could not produce FIONA1. In non-mutant plants, this enzyme chemically modifies one of the components of the spliceosome, a small nuclear RNA known as U6. Parker et al found that there are two types of splice site ­ one more likely to interact with U6 and another that preferentially interacts with another small nuclear RNA, U5. When FIONA1 is inactive (such as in the mutant identified by Parker et al.), splice sites that tend to strongly interact with U5 are selected. However, when the enzyme is active, splice sites that tend to bind with the chemically modified U6 are used instead. Further work by Parker et al. showed that these two types of splice sites ('preferring' either U5 or U6) are found in equal proportions in the genomes of many species, including humans. This suggests that Parker et al. have uncovered an essential feature of how genomes are organised and splicing is controlled.

Chromosome evolution and the genetic basis of agronomically important traits in greater yam.

The nutrient-rich tubers of the greater yam, Dioscorea alata L., provide food and income security for millions of people around the world. Despite its global importance, however, greater yam remains an orphan crop. Here, we address this resource gap by presenting a highly contiguous chromosome-scale genome assembly of D. alata combined with a dense genetic map derived from African breeding populations. The genome sequence reveals an ancient allotetraploidization in the Dioscorea lineage, followed by extensive genome-wide reorganization. Using the genomic tools, we find quantitative trait loci for resistance to anthracnose, a damaging fungal pathogen of yam, and several tuber quality traits. Genomic analysis of breeding lines reveals both extensive inbreeding as well as regions of extensive heterozygosity that may represent interspecific introgression during domestication. These tools and insights will enable yam breeders to unlock the potential of this staple crop and take full advantage of its adaptability to varied environments.

Widespread premature transcription termination of Arabidopsis thaliana NLR genes by the spen protein FPA.

Genes involved in disease resistance are some of the fastest evolving and most diverse components of genomes. Large numbers of nucleotide-binding, leucine-rich repeat (NLR) genes are found in plant genomes and are required for disease resistance. However, NLRs can trigger autoimmunity, disrupt beneficial microbiota or reduce fitness. It is therefore crucial to understand how NLRs are controlled. Here, we show that the RNA-binding protein FPA mediates widespread premature cleavage and polyadenylation of NLR transcripts, thereby controlling their functional expression and impacting immunity. Using long-read Nanopore direct RNA sequencing, we resolved the complexity of NLR transcript processing and gene annotation. Our results uncover a co-transcriptional layer of NLR control with implications for understanding the regulatory and evolutionary dynamics of NLRs in the immune responses of plants.

2passtools: two-pass alignment using machine-learning-filtered splice junctions increases the accuracy of intron detection in long-read RNA sequencing.

Transcription of eukaryotic genomes involves complex alternative processing of RNAs. Sequencing of full-length RNAs using long reads reveals the true complexity of processing. However, the relatively high error rates of long-read sequencing technologies can reduce the accuracy of intron identification. Here we apply alignment metrics and machine-learning-derived sequence information to filter spurious splice junctions from long-read alignments and use the remaining junctions to guide realignment in a two-pass approach. This method, available in the software package 2passtools ( https://github.com/bartongroup/2passtools ), improves the accuracy of spliced alignment and transcriptome assembly for species both with and without existing high-quality annotations.

Nanopore direct RNA sequencing maps the complexity of Arabidopsis mRNA processing and m6A modification.

Understanding genome organization and gene regulation requires insight into RNA transcription, processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA methylation (m6A). Here we show that m6A can be mapped in full-length mRNAs transcriptome-wide and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, poly(A) site choice and poly(A) tail length. Loss of m6A from 3' untranslated regions is associated with decreased relative transcript abundance and defective RNA 3' end formation. A functional consequence of disrupted m6A is a lengthening of the circadian period. We conclude that nanopore direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying this approach to less well-studied species could transform our understanding of what their genomes encode.

miRNA Detection by Stem-Loop RT-qPCR in Studying microRNA Biogenesis and microRNA Responsiveness to Abiotic Stresses.

This chapter is devoted to a PCR-based method for analyzing the expression level of mature miRNAs which utilizes the TaqMan® technology. Stem-loop RT-qPCR requires preparation of separate cDNA templates for each analyzed miRNA as reverse transcription occurs in the presence of a miRNA-specific stem-loop reverse primer. In quantitative analysis, SYBR® Green is not used but the more sensitive TaqMan® probe that on 5' end contains a covalently attached fluorophore and on 3' quencher. When quencher and fluorophore are spatially separated due to nucleolytic DNA polymerase activity, the signal is released and quantified. This section provides a detailed and comprehensive protocol allowing for the successful analysis of mature miRNA levels in analyzed sample. Reverse transcription combined with classic real-time PCR as well as ddPCR™ (Droplet Digital™ PCR) will be presented.

A stable tRNA-like molecule is generated from the long noncoding RNA GUT15 in Arabidopsis.

The Arabidopsis GUT15 RNA belongs to a class of noncoding RNAs that are expressed from the intergenic regions of protein-coding genes. We show that the RNA polymerase II transcribed GUT15 transcript serves as a precursor for two stable RNA species, a tRNA-like molecule and GUT15-tRF-F5, which are both encoded by the final intron in the GUT15 gene. The GUT15-encoded tRNA-like molecule cannot be autonomously transcribed by RNA polymerase III. However, this molecule contains a CCA motif, suggesting that it may enter the tRNA maturation pathway. The GUT15-encoded tRNA-like sequence has an inhibiting effect on the splicing of its host intron. Moreover, we demonstrate that the canonical tRNA genes nested within introns do not affect the splicing patterns of their host protein-coding transcripts.

Active 5' splice sites regulate the biogenesis efficiency of Arabidopsis microRNAs derived from intron-containing genes.

Arabidopsis, miR402 that is encoded within the first intron of a protein-coding gene At1g77230, is induced by heat stress. Its upregulation correlates with splicing inhibition and intronic proximal polyA site selection. It suggests that miR402 is not processed from an intron, but rather from a shorter transcript after selection of the proximal polyA site within this intron. Recently, introns and active 5' splice sites (5'ss') have been shown to stimulate the accumulation of miRNAs encoded within the first exons of intron-containing MIR genes. In contrast, we have observed the opposite effect of splicing inhibition on intronic miR402 production. Transient expression experiments performed in tobacco leaves revealed a significant accumulation of the intronic mature miR402 when the 5'ss of the miR402-hosting intron was inactivated. In contrast, when the miR402 stem-loop structure was moved into the first exon, mutation of the first-intron 5'ss resulted in a decrease in the miRNA level. Thus, the 5'ss controls the efficiency of miRNA biogenesis. We also show that the SERRATE protein (a key component of the plant microprocessor) colocalizes and interacts with several U1 snRNP auxiliary proteins. We postulate that SERRATE-spliceosome connections have a direct effect on miRNA maturation.

Posttranscriptional coordination of splicing and miRNA biogenesis in plants.

MicroRNAs (miRNAs) are short, single-stranded, noncoding RNAs that play a crucial role in basic physiological and morphological processes and in response to various stresses in eukaryotic organisms. However, the miRNA biogenesis, which is based on the action of complex protein machinery, varies between plants and animals, with the differences largely concerning the location of the process, the protein composition of the microprocessor, the mechanism of miRNA action on mRNA target, and the miRNA gene (MIR) structure. Roughly half of known Arabidopsis MIRs contain introns, and 29 miRNAs are encoded within the introns of host genes. Selection of alternative transcription start sites, alternative splice sites (SSs), and polyadenylation sites has been identified within miRNA primary transcripts (pri-miRNAs), and such variety is essential for the production and fine-tuning of miRNA levels. For example, the posttranscriptional processing of intron-containing pri-miRNAs involves the action of additional RNA metabolism machineries, such as the spliceosome and polyadenylation machinery, and to a large extent is based on direct communication between SERRATE (one of the core components of the plant microprocessor) and U1 snRNP auxiliary proteins. Moreover, the position of the miRNA stem-loop structure relative to the closest active 5'SS is essential for the miRNA production efficiency. Indeed, it is highly probable that this pre-miRNA location affects recruitment of the microprocessor to pri-miRNAs and therefore influences miRNA maturation and target mRNA regulation. Such complicated crosstalk between several machineries is important for a proper miRNA-connected response to biotic and abiotic stresses, ensuring plant survival in a changing environment. WIREs RNA 2017, 8:e1403. doi: 10.1002/wrna.1403 For further resources related to this article, please visit the WIREs website.

Arabidopsis thaliana microRNA162 level is posttranscriptionally regulated via splicing and polyadenylation site selection.

Arabidopsis microRNA162 (miRNA162) level regulation was studied under abiotic stresses, such as drought and salinity. The TaqMan® microRNA assay proved that A. thaliana miRNA162 level was elevated under these stresses, confirming its salt and drought responsiveness. The promoter region analyses of A. thaliana miRNA162a and b genes (MIR162a and MIR162b) identified numerous salinity and drought responsive elements. However, our results indicated that Arabidopsis MIR162a was presumably the main locus responsible for the mature ath-miRNA162 accumulation under the stresses tested, and the MIR162b was generally rather weakly expressed, both in control and under the stress conditions. The MIR162a structure was confirmed to be complex and the pri-miRNA162a hairpin structure was shown to span an alternative exon and an intron. The MIR162a transcription generated a few pri-miRNA162a splicing isoforms that could be functional and non-functional. Upon drought and salinity stresses, the regulation of the pri-miRNA162a alternative splicing pattern revealed an increase of a functional pri-miR162a isoform and a preferential distal polyA site selection under the stress conditions. Apart from the potential transcriptional regulation of the miRNA genes (MIRs) expression, the data obtained point to an essential role of posttranscriptional regulation of Arabidopsis microRNA162 level.

mirEX 2.0 - an integrated environment for expression profiling of plant microRNAs.

BACKGROUND: MicroRNAs are the key post-transcriptional regulators of gene expression in development and stress responses. Thus, precisely quantifying the level of each particular microRNA is of utmost importance when studying the biology of any organism. DESCRIPTION: The mirEX 2.0 web portal ( http://www.combio.pl/mirex ) provides a comprehensive platform for the exploration of microRNA expression data based on quantitative Real Time PCR and NGS sequencing experiments, covering various developmental stages, from wild-type to mutant plants. The portal includes mature and pri-miRNA expression levels detected in three plant species (Arabidopsis thaliana, Hordeum vulgare and Pellia endiviifolia), and in A. thaliana miRNA biogenesis pathway mutants. In total, the database contains information about the expression of 461 miRNAs representing 268 families. The data can be explored through the use of advanced web tools, including (i) a graphical query builder system allowing a combination of any given species, developmental stages and tissues, (ii) a modular presentation of the results in the form of thematic windows, and (iii) a number of user-friendly utilities such as a community-building discussion system and extensive tutorial documentation (e.g., tooltips, exemplary videos and presentations). All data contained within the mirEX 2.0 database can be downloaded for use in further applications in a context-based way from the result windows or from a dedicated web page. CONCLUSIONS: The mirEX 2.0 portal provides the plant research community with easily accessible data and powerful tools for application in multi-conditioned analyses of miRNA expression from important plant species in different biological and developmental backgrounds.

Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses.

Arabidopsis microRNA expression regulation was studied in a wide array of abiotic stresses such as drought, heat, salinity, copper excess/deficiency, cadmium excess, and sulfur deficiency. A home-built RT-qPCR mirEX platform for the amplification of 289 Arabidopsis microRNA transcripts was used to study their response to abiotic stresses. Small RNA sequencing, Northern hybridization, and TaqMan® microRNA assays were performed to study the abundance of mature microRNAs. A broad response on the level of primary miRNAs (pri-miRNAs) was observed. However, stress response at the level of mature microRNAs was rather confined. The data presented show that in most instances, the level of a particular mature miRNA could not be predicted based on the level of its pri-miRNA. This points to an essential role of posttranscriptional regulation of microRNA expression. New Arabidopsis microRNAs responsive to abiotic stresses were discovered. Four microRNAs: miR319a/b, miR319b.2, and miR400 have been found to be responsive to several abiotic stresses and thus can be regarded as general stress-responsive microRNA species.