Biology subjects

Simpson, G. G.

Publications and source records attributed to Simpson, G. G..

10 recordsLinked to original sources

bioRxiv2025

Disruption of the mRNA m6A writer complex triggers autoimmunity in Arabidopsis

Distinguishing self from non-self is crucial to direct immune responses against pathogens. Unmodified RNAs stimulate human innate immunity, but RNA modifications suppress this response. mRNA m6A modification is essential for Arabidopsis thaliana viability. However, the molecular basis of the impact of mRNA m6A depletion is poorly understood. Here, we show that disruption of the Arabidopsis mRNA m6A writer complex triggers autoimmunity. Most gene expression changes in m6A writer complex vir-1 mutants grown at 17{degrees}C are explained by defence gene activation and are suppressed at 27{degrees}C, consistent with the frequent temperature sensitivity of Arabidopsis immunity. Accordingly, we found enhanced pathogen resistance and increased premature cell death in vir-1 mutants at 17{degrees}C but not 27{degrees}C. Global temperature-sensitive mRNA poly(A) tail length changes accompany these phenotypes. Our results demonstrate that autoimmunity is a major phenotype of mRNA m6A writer complex mutants, with important implications for interpreting the role of this modification. Furthermore, we open the broader question of whether unmodified RNA triggers immune signalling in plants.

plant biology↗

bioRxiv2023

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.

molecular biology↗

bioRxiv2023

The evolution of splice site sequence preference is linked to the U6 snRNA m6A methyltransferase METTL16

Eukaryotic genes are interrupted by introns that are removed from transcribed RNAs by splicing. The extent of alternative splicing is the best genomic predictor of developmental complexity, yet it is unclear what mediates change in patterns of splicing complexity between species. Here we show that variation in 5 splice site sequence preferences correlate with the presence of the U6 snRNA methyltransferase METTL16 and the splicing factor SNRNP27K. We used inter-species association mapping with Saccharomycotina species to correlate splicing phenotypes with the presence or absence of splicing factors. 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 +4U. 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 conclude that variation in concerted processes of 5 splice site selection by U6 snRNA is crucial to evolutionary change in splicing complexity.

bioinformatics↗

bioRxiv2022

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, FIO1, 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 and reinforces splicing fidelity at elevated temperature. 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.

molecular biology↗

bioRxiv2021

Yanocomp: robust prediction of m6A modifications in individual nanopore direct RNA reads.

Yanocomp is a tool for predicting the positions and stoichiometries of RNA modifications in Nanopore direct RNA sequencing data. It uses general mixture models to identify differentially modified sites between two conditions, with good support for replicates. Yanocomp models across adjacent kmers and uses a uniform component to account for outliers, improving the accuracy of single molecule predictions. Consequently, Yanocomp can be used to measure modification stoichiometry, and correlate modifications with other RNA processing events. AvailabilityYanocomp is available under an MIT license at www.github.com/bartongroup/yanocomp.

bioinformatics↗

bioRxiv2021

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 greater yam combined with a dense genetic map derived from African breeding populations. The genome sequence reveals an ancient lineage-specific genome duplication, followed by extensive genome-wide reorganization. Using our new genomic tools we find quantitative trait loci for susceptibility 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.

genomics↗

bioRxiv2020

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 receptor (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.

molecular biology↗

bioRxiv2020

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.

bioinformatics↗

bioRxiv2019

Nanopore direct RNA sequencing maps an Arabidopsis N6 methyladenosine epitranscriptome

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.

molecular biology↗

bioRxiv2016

How well do RNA-Seq differential gene expression tools perform in higher eukaryotes?

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 distribution of gene expression. A recent highly replicated study revealed that RNA-seq gene expression measurements in yeast are best represented as being drawn from an underlying negative binomial distribution. In this paper, the statistical properties of gene expression in the higher eukaryote Arabidopsis thaliana are shown to be essentially identical to those from yeast despite the large increase in the size and complexity of the transcriptome: Gene expression measurements from this model plant species are consistent with being drawn from an underlying negative binomial or log-normal distribution and the false positive rate performance of nine widely used DGE tools is not strongly affected by the additional size and complexity of the A. thaliana transcriptome. For RNA-seq data, we therefore recommend the use of DGE tools that are based on the negative binomial distribution.

genomics↗