U6 snRNA m6A modification is required for accurate and efficient splicing of C. elegans and human pre-mRNAs.

pre-mRNA splicing is a critical feature of eukaryotic gene expression. Both cis- and trans-splicing rely on accurately recognising splice site sequences by spliceosomal U snRNAs and associated proteins. Spliceosomal snRNAs carry multiple RNA modifications with the potential to affect different stages of pre-mRNA splicing. Here, we show that the conserved U6 snRNA m6A methyltransferase METT-10 is required for accurate and efficient cis- and trans-splicing of C. elegans pre-mRNAs. The absence of METT-10 in C. elegans and METTL16 in humans primarily leads to alternative splicing at 5' splice sites with an adenosine at +4 position. In addition, METT-10 is required for splicing of weak 3' cis- and trans-splice sites. We identified a significant overlap between METT-10 and the conserved splicing factor SNRNP27K in regulating 5' splice sites with +4A. Finally, we show that editing endogenous 5' splice site +4A positions to +4U restores splicing to wild-type positions in a mett-10 mutant background, supporting a direct role for U6 snRNA m6A modification in 5' splice site recognition. We conclude that the U6 snRNA m6A modification is important for accurate and efficient pre-mRNA splicing.

How well do RNA-Seq differential gene expression tools perform in a complex eukaryote? A case study in Arabidopsis thaliana.

MOTIVATION: RNA-seq experiments are usually carried out in three or fewer replicates. In order to work well with so few samples, differential gene expression (DGE) tools typically assume the form of the underlying gene expression distribution. In this paper, the statistical properties of gene expression from RNA-seq are investigated in the complex eukaryote, Arabidopsis thaliana, extending and generalizing the results of previous work in the simple eukaryote Saccharomyces cerevisiae. RESULTS: We show that, consistent with the results in S.cerevisiae, more gene expression measurements in A.thaliana are consistent with being drawn from an underlying negative binomial distribution than either a log-normal distribution or a normal distribution, and that the size and complexity of the A.thaliana transcriptome does not influence the false positive rate performance of nine widely used DGE tools tested here. We therefore recommend the use of DGE tools that are based on the negative binomial distribution. AVAILABILITY AND IMPLEMENTATION: The raw data for the 17 WT Arabidopsis thaliana datasets is available from the European Nucleotide Archive (E-MTAB-5446). The processed and aligned data can be visualized in context using IGB (Freese et al., 2016), or downloaded directly, using our publicly available IGB quickload server at https://compbio.lifesci.dundee.ac.uk/arabidopsisQuickload/public_quickload/ under 'RNAseq>Froussios2019'. All scripts and commands are available from github at https://github.com/bartongroup/KF_arabidopsis-GRNA. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use?

RNA-seq is now the technology of choice for genome-wide differential gene expression experiments, but it is not clear how many biological replicates are needed to ensure valid biological interpretation of the results or which statistical tools are best for analyzing the data. An RNA-seq experiment with 48 biological replicates in each of two conditions was performed to answer these questions and provide guidelines for experimental design. With three biological replicates, nine of the 11 tools evaluated found only 20%-40% of the significantly differentially expressed (SDE) genes identified with the full set of 42 clean replicates. This rises to >85% for the subset of SDE genes changing in expression by more than fourfold. To achieve >85% for all SDE genes regardless of fold change requires more than 20 biological replicates. The same nine tools successfully control their false discovery rate at ≲5% for all numbers of replicates, while the remaining two tools fail to control their FDR adequately, particularly for low numbers of replicates. For future RNA-seq experiments, these results suggest that at least six biological replicates should be used, rising to at least 12 when it is important to identify SDE genes for all fold changes. If fewer than 12 replicates are used, a superior combination of true positive and false positive performances makes edgeR and DESeq2 the leading tools. For higher replicate numbers, minimizing false positives is more important and DESeq marginally outperforms the other tools.

Transcription termination and chimeric RNA formation controlled by Arabidopsis thaliana FPA.

Alternative cleavage and polyadenylation influence the coding and regulatory potential of mRNAs and where transcription termination occurs. Although widespread, few regulators of this process are known. The Arabidopsis thaliana protein FPA is a rare example of a trans-acting regulator of poly(A) site choice. Analysing fpa mutants therefore provides an opportunity to reveal generic consequences of disrupting this process. We used direct RNA sequencing to quantify shifts in RNA 3' formation in fpa mutants. Here we show that specific chimeric RNAs formed between the exons of otherwise separate genes are a striking consequence of loss of FPA function. We define intergenic read-through transcripts resulting from defective RNA 3' end formation in fpa mutants and detail cryptic splicing and antisense transcription associated with these read-through RNAs. We identify alternative polyadenylation within introns that is sensitive to FPA and show FPA-dependent shifts in IBM1 poly(A) site selection that differ from those recently defined in mutants defective in intragenic heterochromatin and DNA methylation. Finally, we show that defective termination at specific loci in fpa mutants is shared with dicer-like 1 (dcl1) or dcl4 mutants, leading us to develop alternative explanations for some silencing roles of these proteins. We relate our findings to the impact that altered patterns of 3' end formation can have on gene and genome organisation.

Message ends: RNA 3' processing and flowering time control.

Plants control the time at which they flower in order to ensure reproductive success. This control is underpinned by precision in gene regulation acting through genetically separable pathways. The genetic dissection of this process in the model plant Arabidopsis thaliana has led to the recurrent identification of plant-specific and highly conserved RNA 3' end processing factors required to control flowering by specifically controlling transcription of mRNA encoding the floral repressor FLOWERING LOCUS C (FLC). Here, we review the features of these RNA-processing and RNA-associated proteins, and the complex architecture of coding and non-coding RNA transcription at the FLC locus. We discuss alternative concepts that might explain how these RNA-processing events regulate FLC transcription and hence control flowering time.

Noncanonical translation initiation of the Arabidopsis flowering time and alternative polyadenylation regulator FCA.

The RNA binding protein FCA regulates the floral transition and is required for silencing RNAs corresponding to specific noncoding sequences in the Arabidopsis thaliana genome. Through interaction with the canonical RNA 3' processing machinery, FCA affects alternative polyadenylation of many transcripts, including antisense RNAs at the locus encoding the floral repressor FLC. This potential for widespread alteration of gene regulation clearly needs to be tightly regulated, and we have previously shown that FCA expression is autoregulated through poly(A) site choice. Here, we show distinct layers of FCA regulation that involve sequences within the 5' region that regulate noncanonical translation initiation and alter the expression profile. FCA translation in vivo occurs exclusively at a noncanonical CUG codon upstream of the first in-frame AUG. We fully define the upstream flanking sequences essential for its selection, revealing features that distinguish this from other non-AUG start site mechanisms. Bioinformatic analysis identified 10 additional Arabidopsis genes that likely initiate translation at a CUG codon. Our findings reveal further unexpected complexity in the regulation of FCA expression with implications for its roles in regulating flowering time and gene expression and more generally show plant mRNA exceptions to AUG translation initiation.

Inter-species association mapping links splice site evolution to METTL16 and SNRNP27K.

Eukaryotic genes are interrupted by introns that are removed from transcribed RNAs by splicing. Patterns of splicing complexity differ between species, but it is unclear how these differences arise. We used inter-species association mapping with Saccharomycotina species to correlate splicing signal phenotypes with the presence or absence of splicing factors. Here, we show that variation in 5' splice site sequence preferences correlate with the presence of the U6 snRNA N6-methyladenosine methyltransferase METTL16 and the splicing factor SNRNP27K. The greatest variation in 5' splice site sequence occurred at the +4 position and involved a preference switch between adenosine and uridine. Loss of METTL16 and SNRNP27K orthologs, or a single SNRNP27K methionine residue, was associated with a preference for +4 U. These findings are consistent with splicing analyses of mutants defective in either METTL16 or SNRNP27K orthologs and models derived from spliceosome structures, demonstrating that inter-species association mapping is a powerful orthogonal approach to molecular studies. We identified variation between species in the occurrence of two major classes of 5' splice sites, defined by distinct interaction potentials with U5 and U6 snRNAs, that correlates with intron number. We conclude that variation in concerted processes of 5' splice site selection by U6 snRNA is associated with evolutionary changes in splicing signal phenotypes.

U6 snRNA m6A modification is required for accurate and efficient cis- and trans-splicing of C. elegans mRNAs.

pre-mRNA splicing is a critical feature of eukaryotic gene expression. Many eukaryotes use cis-splicing to remove intronic sequences from pre-mRNAs. In addition to cis-splicing, many organisms use trans-splicing to replace the 5' ends of mRNAs with a non-coding spliced-leader RNA. Both cis- and trans-splicing rely on accurately recognising splice site sequences by spliceosomal U snRNAs and associated proteins. Spliceosomal snRNAs carry multiple RNA modifications with the potential to affect different stages of pre-mRNA splicing. Here, we show that m6A modification of U6 snRNA A43 by the RNA methyltransferase METT-10 is required for accurate and efficient cis- and trans-splicing of C. elegans pre-mRNAs. The absence of U6 snRNA m6A modification primarily leads to alternative splicing at 5' splice sites. Furthermore, weaker 5' splice site recognition by the unmodified U6 snRNA A43 affects splicing at 3' splice sites. U6 snRNA m6A43 and the splicing factor SNRNP27K function to recognise an overlapping set of 5' splice sites with an adenosine at +4 position. Finally, we show that U6 snRNA m6A43 is required for efficient SL trans-splicing at weak 3' trans-splice sites. We conclude that the U6 snRNA m6A modification is important for accurate and efficient cis- and trans-splicing in C. elegans.

Two zinc finger proteins with functions in m6A writing interact with HAKAI.

The methyltransferase complex (m6A writer), which catalyzes the deposition of N6-methyladenosine (m6A) in mRNAs, is highly conserved across most eukaryotic organisms, but its components and interactions between them are still far from fully understood. Here, using in vivo interaction proteomics, two HAKAI-interacting zinc finger proteins, HIZ1 and HIZ2, are discovered as components of the Arabidopsis m6A writer complex. HAKAI is required for the interaction between HIZ1 and MTA (mRNA adenosine methylase A). Whilst HIZ1 knockout plants have normal levels of m6A, plants in which it is overexpressed show reduced methylation and decreased lateral root formation. Mutant plants lacking HIZ2 are viable but have an 85% reduction in m6A abundance and show severe developmental defects. Our findings suggest that HIZ2 is likely the plant equivalent of ZC3H13 (Flacc) of the metazoan m6A-METTL Associated Complex.

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.

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.

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.

Arabidopsis RNA immunoprecipitation.

RNA-binding proteins are key regulators of plant gene expression. Consistent with this, the Arabidopsis genome encodes many RNA-binding proteins that are genetically required for normal development and for responding to environmental changes. However, the direct RNA targets and RNA processing events that these RNA-binding proteins control are poorly understood. In order to facilitate the functional characterization of RNA-binding proteins, we have applied the RNA immunoprecipitation assay to Arabidopsis. Working with the U2B''-U2 snRNA interaction as a model experimental system, we show that treatment of intact plants with formaldehyde allows immunocapture of U2 snRNA using antibodies that recognize U2B'' fused to the generic GFP tag. When coupled with recent developments in whole-genome tiling arrays and high-throughput next-generation sequencing, this combination of procedures and technology has the potential to allow systematic functional analysis of plant RNA-binding proteins.

Alternative polyadenylation of antisense RNAs and flowering time control.

Flowering time is controlled by precision in gene regulation mediated by different pathways. Two Arabidopsis thaliana components of the autonomous flowering pathway, FCA and FPA, function as genetically independent trans-acting regulators of alternative cleavage and polyadenylation. FCA and FPA directly associate with chromatin at the locus encoding the floral repressor FLC, but appear to control FLC transcription by mediating alternative polyadenylation of embedded non-coding antisense RNAs. These findings prompt the re-examination of how other factors control FLC expression, as it is formally possible that they function primarily to control alternative processing of antisense RNAs. As co-expressed sense and antisense gene pairs are widespread in eukaryotes, alternative processing of antisense RNAs may represent a significant form of gene regulation.

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.

The autonomous pathway: epigenetic and post-transcriptional gene regulation in the control of Arabidopsis flowering time.

Mechanisms that mediate the control of flowering time have been accessed through a molecular genetic approach in Arabidopsis. Flowering is regulated by different pathways and, in the past year, all of the known components of the so-called autonomous pathway have been identified. The autonomous pathway comprises a combination of factors involved in RNA processing and epigenetic regulation that downregulate the floral repressor, FLOWERING LOCUS C (FLC). However, components of the autonomous pathway are more widely conserved in plant species other than Arabidopsis than is FLC. Therefore, the broadest lessons we learn from dissecting the function of the autonomous pathway may be in revealing how precision in regulated gene expression is delivered.

Regulated RNA processing in the control of Arabidopsis flowering.

Flowering time is controlled in order to ensure reproductive success. Molecular genetic analyses in Arabidopsis thaliana have identified many genes regulating this developmental switch. One group of factors which promote flowering do so by down-regulating the expression of the MADS-box floral repressor, FLC. RNA processing appears to play an important role in this regulation as genes within this group encode RNA binding proteins (FCA, FPA and FLK) and an mRNA 3' end processing factor (FY). FCA promotes flowering and negatively autoregulates its own expression post-transcriptionally through a mechanism that involves alternative polyadenylation. FCA physically interacts with FY and this interaction is required for the function FY performs in flowering control and in FCA autoregulation. Potential similarities are emerging in the molecular mechanisms controlling FLC expression and those controlling the floral homeotic gene, AGAMOUS. In addition, microRNAs have been shown to regulate plant developmental processes including the timing to flower. Together, these new data indicate that post-transcriptional regulation of gene expression plays an important role in regulating the floral transition.

Crystal Structure of the SPOC Domain of the Arabidopsis Flowering Regulator FPA.

The Arabidopsis protein FPA controls flowering time by regulating the alternative 3'-end processing of the FLOWERING LOCUS (FLC) antisense RNA. FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain. The SPOC domain is highly conserved among FPA homologs in plants, but the conservation with the domain in other SPEN proteins is much lower. We have determined the crystal structure of Arabidopsis thaliana FPA SPOC domain at 2.7 Å resolution. The overall structure is similar to that of the SPOC domain in human SMRT/HDAC1 Associated Repressor Protein (SHARP), although there are also substantial conformational differences between them. Structural and sequence analyses identify a surface patch that is conserved among plant FPA homologs. Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3'-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain.