Biology subjects

Lloyd, J. P. B.

Publications and source records attributed to Lloyd, J. P. B..

8 recordsLinked to original sources

bioRxiv2026

Make it so: Rapid and affordable plasmid sequencing on ONT platforms with PICARD-seq

Plasmid construction underpins molecular biology and synthetic biology, yet validation is often limited to the inserted fragment rather than the whole plasmid, and around a third of laboratory-made plasmids carry sequence errors that can affect function. Sanger sequencing scales poorly across whole plasmids, while short-read approaches cannot resolve the repeated DNA parts, such as promoters, that are common in synthetic constructs. We present PICARD-seq, a rapid nanopore-based protocol that uses off-the-shelf Tn5 rapid barcoding reagents and a MinION to sequence pools of whole plasmids in under a day, and we systematically benchmark the computational pipelines available for analysing the resulting data. Using a curated set of 25 plasmids of known sequence spanning 3.0-20.6 kbp, including various dilution series and repetitive multi-part constructs, we ran five independent replicates of each pipeline. The ONT EPI2ME Clone Validation workflow was fast (13-18 min) but stochastic, varying between replicates for both plasmids assembled and what sequence was returned; Canu outperformed the default Flye assembler, and reducing the minimum coverage parameter from 60x to 20x substantially improved assembly of large, repetitive, and dilute samples. The ensemble assembler Autocycler was slower (81-111 min) but gave the highest and most consistent rate of recovering the expected sequence. Complementary read mapping with minimap2 distinguished genuine sequence differences from assembly artefacts. Applying PICARD-seq to problematic plasmids revealed backbone concatemers, a misincorporated promoter part, and a mixed population of rearranged molecules in a repetitive construct. PICARD-seq makes routine whole-plasmid validation practical and affordable for individual laboratories.

bioinformatics↗

bioRxiv2025

CREation of an expanded plant memory gene circuit toolkit

Genetic circuits rely on modular, well-characterized genetic parts to achieve predictable cellular behaviour. Despite their widespread adoption, biological parts are complex and when used in a new molecular context can result in unexpected outcomes. Site-specific recombinases have been widely used due to their unique ability to induce precise, irreversible changes to a DNA sequence, making them ideal tools for memory logic operations. However, unexpected context-specific failures of even the most widely used recombinase, Cre, has limited the expansion and complexity of synthetic gene circuits in diverse species. Here, through a systematic analysis of Cre failure in plant gene circuits, we uncovered multiple unexpected post-recombination interactions within the transcriptional unit between the promoter region, recombinase, and their cognate recognition sites. These significantly inhibit transcriptional activity, preventing circuit functionality. Specifically, post-recombination Cre recombinase exhibits an inhibitory property by binding to lox sites, with the remaining lox site repressing transcription. By thoroughly characterizing these dynamics, we restored Cre functionality and expanded the plant-based recombinase toolkit for Cre-related recombinases, developing combinations of recombinases and cognate sites with different activation levels for constructing logic gates with stable memory functions. We further developed multiple functional split-recombinase systems for simultaneous logic using a range of dimerization domains and constructed complex logic gates, including 3-input and 6-input AND gates. Together, this exploration of component context dependency in genetic circuits, even with commonly used parts like the Cre/lox system, has implications extending beyond plant systems, and significantly expands plant memory circuit capabilities.

synthetic biology↗

bioRxiv2025

Start right to end right: authentic open reading frame selection matters

Accurate annotation of open reading frames (ORFs) is fundamental for understanding gene function and post-transcriptional regulation. A critical but often overlooked aspect of transcriptome annotation is the selection of authentic translation start sites. Many genome annotation pipelines identify the longest possible ORF in alternatively spliced transcripts, using internal methionine codons as putative start sites. However, this computational approach ignores the biological reality that ribosomes select start codons based on sequence context, not ORF length. Here, we demonstrate that this practice leads to systematic misannotation of nonsense-mediated decay (NMD) targets in the Arabidopsis thaliana Araport11 reference transcriptome. Using TranSuite software to identify authentic start codons, we reanalyzed transcriptomic data from an NMD-deficient mutant and found that correct ORF annotation more than doubles the number of identifiable NMD targets with premature termination codons followed by downstream exon junctions, from 203 to 426 transcripts. Furthermore, we show that incorrect ORF annotations can lead to erroneous protein structure predictions, potentially introducing computational artifacts into protein databases. Our findings underscore the importance of biologically informed ORF annotation for accurate assessment of post-transcriptional regulation and proteome prediction, with implications for all eukaryotic genome annotation projects.

genomics↗

bioRxiv2023

Characterization of DNA methylation reader proteins of Arabidopsis thaliana

In plants, cytosine DNA methylation (mC) is largely associated with transcriptional repression of transposable elements, but it can also be found in the body of expressed genes, referred to as gene body methylation (GbM). GbM is correlated with ubiquitously expressed genes, however its function, or absence thereof, is highly debated. The different output that mC can have raises questions as to how it is interpreted - or read - differently in these sequence and genomic contexts. To screen for potential mC binding proteins, we performed an unbiased DNA affinity pull-down assay combined with quantitative mass spectrometry using methylated DNA probes for each DNA sequence context. All mC readers known to date were found to preferentially bind to the methylated probes, along with a range of new mC binding protein candidates. Functional characterization of these mC readers, focused on the MBD and SUVH families, was undertaken by ChIP-seq mapping of genome-wide binding sites, their protein interactors, and the impact of high-order mutations on transcriptomic and epigenomic profiles. Together, this highlighted specific context preferences for these proteins, and in particular the ability of MBD2 to bind specifically to GbM. This comprehensive analysis of Arabidopsis mC readers emphasizes the complexity and interconnectivity between DNA methylation and chromatin remodelling processes in plants.

plant biology↗

bioRxiv2022

Synthetic memory circuits for programmable cell reconfiguration in plants

Plant biotechnology predominantly relies on a restricted set of genetic parts with limited capability to customize spatiotemporal and conditional expression patterns. Synthetic gene circuits have the ability to integrate multiple customizable input signals through a processing unit constructed from biological parts, to produce a predictable and programmable output. Here, we present a suite of functional recombinase-based gene circuits for use in plants. We first established a range of key gene circuit components compatible with plant cell functionality. We then used these to develop a range of operational logic gates using the identify function (activation) and negation function (repression) in Arabidopsis protoplasts and in vivo, demonstrating their utility for programmable manipulation of transcriptional activity in a complex multicellular organism. Through utilization of genetic recombination these circuits create stable long-term changes in expression and recording of past stimuli. This highly-compact programmable gene circuit platform provides new capabilities for engineering sophisticated transcriptional programs and previously unrealised traits into plants.

synthetic biology↗

bioRxiv2020

Transcriptome analysis of alternative splicing-coupled nonsense-mediated mRNA decay in human cells reveals broad regulatory potential

To explore the regulatory potential of nonsense-mediated mRNA decay (NMD) coupled with alternative splicing, we globally surveyed the transcripts targeted by this pathway via RNA-Seq analysis of HeLa cells in which NMD had been inhibited. We identified putative NMD-targeted transcripts as those with a termination codon more than 50 nucleotides upstream of an exon-exon junction (premature termination as defined by the ‘50nt rule’) and that significantly increased in abundance upon NMD inhibition. We additionally controlled for potential transcriptional up-regulation by requiring the putative NMD targets to increase in abundance substantially more than the isoforms from the same gene that do not contain a premature termination codon. This resulted in a conservative set of 2,793 transcripts derived from 2,116 genes as physiological NMD targets (9.2% of expressed transcripts and >20% of alternatively spliced genes). Our analysis identified previously inferred unproductive isoforms and numerous heretofore-uncharacterized ones. NMD-targeted transcripts were derived from genes involved in many functional categories, and are particularly enriched for RNA splicing genes as well as for those harboring ultraconserved elements. By investigating the features of all transcripts impacted by NMD, we find that the 50nt rule is a strong predictor of NMD degradation while 3’ UTR length on its own generally has only a small effect in this human cell line. Additionally, thousands more transcripts without a premature termination codon in the main coding sequence contain a uORF and display significantly increased abundance upon NMD inhibition indicating potentially widespread regulation through decay coupled with uORF translation. Our results support that alternative splicing coupled with NMD is a prevalent post-transcriptional mechanism in human cells with broad potential for biological regulation.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2020

Networks of Splice Factor Regulation by Unproductive Splicing Coupled With Nonsense Mediated mRNA Decay

BackgroundNonsense mediated mRNA decay (NMD) is an RNA surveillance pathway that degrades aberrant transcripts harboring premature termination codons. This pathway, in conjunction with alternative splicing, regulates gene expression post-transcriptionally. Nearly all serine and arginine-rich (SR) proteins and many heterogeneous nuclear ribonucleoproteins (hnRNPs) produce isoforms that can be degraded by the NMD pathway. Many splicing factors have been reported to be regulated via alternative splicing coupled to NMD. However, it is still uncharacterized that to what extent NMD contributes to the regulation of splicing factors. ResultsHere, we characterized a regulatory network of splicing factors through alternative splicing coupled to NMD. Based upon an extensive literature search, we first assembled a network that encompasses the current knowledge of splice factors repressing or activating the expression of other splicing factors through alternative splicing coupled to NMD. This regulatory network is limited, including just a handful of well-studied splicing factors. To gain a more global and less biased overview, we examined the splicing factor-mRNA interactions from public crosslinking-immunoprecipitation (CLIP)-seq data, which provides information about protein-RNA interactions. A network view of these interactions reveals extensive binding among splicing regulators. We also found that splicing factors bind more frequently to transcripts of other splicing factors than to other genes. In addition, many splicing factors are targets of NMD, and might be regulated via alternative splicing coupled to NMD, which is demonstrated by the significant overlap between the experimental network and eCLIP-network. We found that hierarchy of the splicing-factor interaction network differs from the hierarchy observed for transcription factors. ConclusionThe extensive interaction between splicing factors and transcripts of other splicing factors suggests that the potential regulation via alternative splicing coupled with NMD is widespread. The splicing factor regulation is fundamentally different from that of transcription factors.

molecular biology↗

bioRxiv2020

Polysome fractionation analysis reveals features important for human nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a translation-dependent mRNA surveillance pathway that eliminates transcripts with premature termination codons. Several studies have tried defined the features governing which transcripts are targeted to NMD. However, these approaches often rely on inhibiting core NMD factors, which often have roles in non-NMD processes within the cell. Based on reports that NMD-targeted transcripts are often bound by a single ribosome, we analyzed RNA-Seq data from a polysome fractionation experiment (TrlP-Seq) to characterize the features of NMD-targeted transcripts in human cells. This approach alleviates the need to inhibit the NMD pathway. We found that the exon junction complex (EJC) model, wherein an exon-exon junction located [≥]50 nucleotides downstream of a stop codon is predicted to elicit NMD, was a powerful predictor of transcripts with high abundance in the monosome fraction (bound by a single ribosome). This was also true for the presence of an upstream open reading frame. In contrast, as 3 UTR lengths increase, the proportion of transcripts that are most abundant in the monosome fraction does not increase. This suggests that either longer 3 UTRs do not consistently act as potent triggers of NMD or that the degradation of these transcripts is mechanistically different to other NMD-targeted transcripts. Of the ribosome-associated transcripts annotated as "non-coding", we find that a majority are bound by a single ribosome. Many of these transcripts increase in response to NMD inhibition, including the oncogenic SHNG15, suggesting many might be NMD targets. Finally, we found that retained intron transcripts without a premature termination codon are over-represented in the monosome fraction, suggesting an alternative mechanism is responsible for the low level of translation of these transcripts. In summary, our analysis finds that the EJC model is a powerful predictor of NMD-targeted transcripts, while the presence of a long 3 UTR is not.

molecular biology↗