Biology subjects

Rennie, S.

Publications and source records attributed to Rennie, S..

13 recordsLinked to original sources

bioRxiv2026

Systematic benchmark of demultiplexing workflows for direct RNA sequencing

Direct RNA nanopore sequencing offers the capacity to directly profile native RNAs, enabling detection of diverse epitranscriptomic modifications based on current changes as the RNA passes through a pore. Innovations in chemistry, hardware and bioinformatic tools have increased sequencing capacity and accuracy, creating demand for sequencing multiple samples within individual runs. Two recent solutions enable barcoding and demultiplexing of samples, but independent comparisons of their performance is limited. Here we benchmark WarpDemuX and SeqTagger using orthogonal ground truths derived from species identity and matched IVT spike-ins across four RNA004 flow cells. Both achieve high demultiplexing precision but differ in their trade-offs in precision and retention. SeqTagger provided strong performance at its recommended settings, whilst WarpDemuX performance varied by model, barcode and read quality. IVT-derived score cutoffs allowed dataset-specific calibration and increased WarpDeMux assignment yield, although precision achieved on IVTs was not consistently reproduced in species reads. These results support matched IVTs as internal controls for guiding choice of demultiplexing settings, while demonstrating the need to validate demultiplexing precision in biological RNA populations.

genomics↗

bioRxiv2025

PUMILIOs and m6A-ECT2/ECT3 share mRNA targets and exert opposing control over organogenesis

PUMILIO/Fem-3 Binding Factor (PUF) proteins are conserved eukaryotic mRNA-binding proteins. Higher plants encode an expanded family of PUF proteins with 26 members in Arabidopsis thaliana of which 6 (PUM1-PUM6) have a domain organization equivalent to metazoan PUF proteins implicated in mRNA regulation. Here, we show that the PUF proteins PUM2 and PUM6 are expressed in mitotically active cells of root and shoot meristems, while PUM5 is mostly expressed in the differentiation zone of root meristems exiting from active division. Increased PUM2 dosage in proliferating cells causes growth repression and developmental delay reminiscent of mutants with defects in cytoplasmic YTHDF proteins that bind to N6-methyladenosine (m6A) in mRNA. Remarkably, even weak defects in the m6A-YTHDF system enhance the effect of increased PUM2 dosage. In line with this genetic interaction, mRNA targets overlap significantly between PUFs and the major YTHDF proteins ECT2 and ECT3. Furthermore, the enrichment of the core PUF recognition motif URUAY around m6A sites is largely explained by PUF targeting, not by direct URUAY methylation as previously suggested. Our results indicate that the balance between the reciprocal activities of the deeply conserved cytoplasmic RNA binding protein families PUF and YTHDF is an important element of growth control in plants.

plant biology↗

bioRxiv2025

An epitranscriptomic map of RNA modifications on integrated HPV18 expressed from HeLa cells

Human papillomavirus (HPV)-driven cancers remain a major global health burden, and understanding how post-transcriptional regulation shapes viral gene expression may inform new therapeutic strategies. Here, we reanalysed independently generated public HeLa datasets to identify candidate RNA modification sites on integrated HPV18 transcripts expressed from the HeLa genome. Using Oxford Nanopore direct RNA sequencing of native RNAs and in vitro transcribed controls, together with GLORI, eTAM-seq and staged 4sU-GLORI datasets, we identified a set of candidate m6A sites on HPV18 early transcripts. m6A levels at a subset of these sites were reduced following perturbation of the m6A pathway, most clearly after METTL3 inhibition, WTAP knockdown or FTO overexpression. We further show that the E6*I-proximal m6A site at position 224 is enriched on unspliced transcripts in direct RNA sequencing, eTAM-seq and staged 4sU-GLORI data. In contrast, we find no convincing evidence for m5C or pseudouridine within the HPV18 regions covered by these datasets. Together, our analyses provide a candidate map of RNA modifications on HeLa-expressed HPV18 early transcripts.

genomics↗

bioRxiv2025

Genome-wide rules of transcription factor cooperativity revealed through in silico binding site ablation

Transcription factor (TF) cooperativity plays a critical role in gene regulation. However, the underlying genomic rules remain unclear, calling for scalable methods to characterize the TF binding site (motif) syntax of regulatory elements. Here, we introduce DeepCompARE, a lightweight model paired with an in silico ablation (ISA) framework for genome-wide analysis of regulatory sequences. Our framework enables precise interpretation of the motif syntax governing chromatin accessibility, enhancer activity, and promoter function. We find that most TF motifs are pairwise independent, indicating a default additive behavior of TFs, and define a cooperativity score to quantify deviations from this baseline. This reveals synergy and redundancy as opposite effects along the same cooperative spectrum. TF redundancy is linked to promoter activity and broad expression, whereas TF synergy is associated with enhancer activity, physical interactions, and cell-type specificity. Our framework provides a quantitative model for TF cooperativity, offering new insights into gene regulatory logic.

bioinformatics↗

bioRxiv2024

ALBA proteins facilitate cytoplasmic YTHDF-mediated reading of m6A in plants

N6-methyladenosine (m6A) exerts many of its regulatory effects on eukaryotic mRNAs by recruiting cytoplasmic YT521-B homology domain family (YTHDF) proteins. Here, we show that in Arabidopsis, the interaction between m6A and the major YTHDF protein ECT2 also involves the mRNA-binding ALBA protein family. ALBA and YTHDF proteins physically associate via a deeply conserved short linear motif in the intrinsically disordered region of YTHDF proteins, their mRNA target sets overlap, and ALBA4 binding sites are juxtaposed to m6A sites. These binding sites correspond to pyrimidine-rich elements previously found to be important for m6A binding of ECT2. Accordingly, both biological functions of ECT2 and its binding to m6A targets in vivo require ALBA association. Our results introduce the YTHDF-ALBA complex as the functional cytoplasmic m6A-reader in plants and define a molecular foundation for the concept of facilitated m6A reading that increases the potential for combinatorial control of biological m6A effects.

molecular biology↗

bioRxiv2022

Plant YTHDF proteins are direct effectors of antiviral immunity against an m6A-containing RNA virus

In virus-host interactions, nucleic acid-directed first lines of defense that allow viral clearance without compromising growth are of paramount importance. Plants use the RNA interference pathway as such a basal antiviral immune system, but additional RNA-based mechanisms of defense also exist. The infectivity of the plant positive strand RNA virus alfalfa mosaic virus (AMV) relies on demethylation of viral RNA by recruitment of the cellular N6-methyladenosine (m6A) demethylase ALKBH9B, but how demethylation of viral RNA promotes AMV replication remains unknown. Here, we show that inactivation of the cytoplasmic YT521-B homology domain (YTH)-containing m6A-binding proteins, ECT2, ECT3, and ECT5 is sufficient to restore AMV infectivity in partially resistant alkbh9b mutants. We also show that the antiviral function of ECT2 is distinct from its previously demonstrated function in promotion of proliferation of primordial cells, because an ECT2 mutant carrying a small deletion in its intrinsically disordered region is partially compromised for antiviral defense, but not for developmental functions. These results indicate that the m6A-YTH axis constitutes a novel branch of basal antiviral immunity in plants.

plant biology↗

bioRxiv2022

Transcription factor expression is the main determinant of variability in gene co-activity

Many genes are co-expressed and form genomic domains of coordinated gene activity. However, the regulatory determinants of domain co-activity remain unclear. Here, we leverage human individual variation in gene expression to characterize the co-regulatory processes underlying domain co-activity and systematically quantify their effect sizes. We employ transcriptional decomposition to extract from RNA expression data an expression component related to co-activity revealed by genomic positioning. This strategy reveals close to 1,500 co-activity domains, covering most expressed genes, of which the large majority are invariable across individuals. Focusing specifically on domains with high variability in co-activity reveals that contained genes have a higher sharing of eQTLs, a higher variability in enhancer interactions, and an enrichment of binding by variably expressed transcription factors compared to genes within non-variable domains. Through careful quantification of the relative contributions of regulatory processes underlying co-activity, we find transcription factor expression levels to be the main determinant of gene co-activity. Our results indicate that distal trans effects contribute more than local genetic variation to individual variation in co-activity domains.

bioinformatics↗

bioRxiv2021

Promoter sequence and architecture determine expression variability and confer robustness to genetic variants

Genetic and environmental exposures cause variability in gene expression. Although most genes are affected in a population, their effect sizes vary greatly, indicating the existence of regulatory mechanisms that could amplify or attenuate expression variability. Here, we investigate the relationship between the sequence and transcription start site architectures of promoters and their expression variability across human individuals. We find that expression variability can be largely explained by a promoters DNA sequence and its binding sites for specific transcription factors. We show that promoter expression variability reflects the biological process of a gene, demonstrating a selective trade-off between stability for metabolic genes and plasticity for responsive genes and those involved in signaling. Promoters with a rigid transcription start site architecture are more prone to have variable expression and to be associated with genetic variants with large effect sizes, while a flexible usage of transcription start sites within a promoter attenuates expression variability and limits genotypic effects. Our work provides insights into the variable nature of responsive genes and reveals a novel mechanism for supplying transcriptional and mutational robustness to essential genes through multiple transcription start site regions within a promoter.

genomics↗

bioRxiv2021

hyperTRIBER: a flexible R package for the analysis of differential RNA editing

AO_SCPLOWBSTRACTC_SCPLOWRNA editing by ADAR (adenosine deaminase acting on RNA) is gaining an increased interest in the field of post-transcriptional regulation. Fused to an RNA-binding protein (RBP) of interest, the catalytic activity of ADAR results in A-to-I RNA edits, whose identification will determine RBP-bound RNA transcripts. However, the computational tools available for their identification and differential RNA editing statistical analysis are limited or too specialised for general-purpose usage. Here we present hyperTRIBER, a flexible suite of tools, wrapped into a convenient R package, for the detection of differential RNA editing. hyperTRIBER is applicable to complex scenarios and experimental designs, and provides a robust statistical framework allowing for the control for coverage of reads at a given base, the total expression level and other co-variates. We demonstrate the capabilities of our approach on HyperTRIBE RNA-seq data for the detection of bound RNAs by the N6-methyladenosine (m6A) reader protein ECT2 in Arabidopsis roots. We show that hyperTRIBER finds edits with a high statistical power, even where editing proportions and RNA transcript expression levels are low, together demonstrating its usability and versatility for analysing differential RNA editing.

bioinformatics↗

bioRxiv2021

The Arabidopsis m6A-binding proteins ECT2 and ECT3 bind largely overlapping mRNA target sets and influence target mRNA abundance, not alternative polyadenylation

Gene regulation via N6-methyladenosine (m6A) in mRNA involves RNA-binding proteins that recognize m6A via a YT521-B homology (YTH) domain. The plant YTH domain proteins ECT2 and ECT3 act genetically redundantly in stimulating cell proliferation during organogenesis, but several fundamental questions regarding their mode of action remain unclear. Here, we use HyperTRIBE (targets of RNA-binding proteins identified by editing) to show that most ECT2 and ECT3 targets overlap, with only few examples of preferential targeting by either of the two proteins. HyperTRIBE in different mutant backgrounds also provides direct views of redundant and specific target interactions of the two proteins. We also show that contrary to conclusions of previous reports, ECT2 does not accumulate in the nucleus. Accordingly, inactivation of ECT2, ECT3 and their surrogate ECT4 does not change patterns of polyadenylation site choice in ECT2/3 target mRNAs, but does lead to lower steady state accumulation of target mRNAs. In addition, mRNA and microRNA expression profiles show indications of stress response activation in ect2/ect3/ect4 mutants, likely via indirect effects. Thus, previous suggestions of control of alternative polyadenylation by ECT2 are not supported by evidence, and ECT2 and ECT3 act largely redundantly to regulate target mRNA, including its abundance, in the cytoplasm.

molecular biology↗

bioRxiv2021

Principles of mRNA targeting and regulation via the Arabidopsis m6A-binding proteins ECT2 and ECT3

Specific recognition of N6-methyladenosine (m6A) in mRNA by RNA-binding proteins containing a YT521-B homology (YTH) domain is important in eukaryotic gene regulation. The Arabidopsis YTH-domain protein ECT2 is thought to bind to mRNA at URU(m6A)Y sites, yet RR(m6A)CH is the canonical m6A consensus site in all eukaryotes and ECT2 functions require m6A binding activity. Here, we apply iCLIP (individual-nucleotide resolution cross-linking and immunoprecipitation) and HyperTRIBE (targets of RNA-binding proteins identified by editing) to define high-quality target sets of ECT2, and analyze the patterns of enriched sequence motifs around ECT2 crosslink sites. Our analyses show that ECT2 does in fact bind to RR(m6A)CH. Pyrimidine-rich motifs are enriched around, but not at m6A-sites, reflecting a preference for N6-adenosine methylation of RRACH/GGAU islands in pyrimidine-rich regions. Such motifs, particularly oligo-U and UNUNU upstream of m6A sites, are also implicated in ECT2 binding via its intrinsically disordered region (IDR). Finally, URUAY-type motifs are enriched at ECT2 crosslink sites, but their distinct properties suggest function as sites of competition between binding of ECT2 and as yet unidentified RNA-binding proteins. Our study provides coherence between genetic and molecular studies of m6A-YTH function in plants, and reveals new insight into the mode of RNA recognition by YTH-domain-containing proteins.

molecular biology↗

bioRxiv2017

Transcription start site analysis reveals widespread divergent transcription in D. melanogaster and core promoter encoded enhancer activities

Mammalian gene promoters and enhancers share many properties. They are composed of a unified promoter architecture of divergent transcripton initiation and gene promoters may exhibit enhancer function. However, it is currently unclear how expression strength of a regulatory element relates to its enhancer strength and if the unifying architecture is conserved across Metazoa. Here we investigate the transcription initiation landscape and its associated RNA decay in D. melanogaster. Surprisingly, we find that the majority of active gene-distal enhancers and a considerable fraction of gene promoters are divergently transcribed. We observe quantitative relationships between enhancer potential, expression level and core promoter strength, providing an explanation for indirectly related histone modifications that are reflecting expression levels. Lowly abundant unstable RNAs initiated from weak core promoters are key characteristics of gene-distal developmental enhancers, while the housekeeping enhancer strengths of gene promoters reflect their expression strengths. The different layers of regulation mediated by gene-distal enhancers and gene promoters are also reflected in chromatin interaction data. Our results suggest a unified promoter architecture of many D. melanogaster regulatory elements, that is universal across Metazoa, whose regulatory functions seem to be related to their core promoter elements.

genomics↗

bioRxiv2017

Transcriptional decomposition reveals active chromatin architectures and cell specific regulatory interactions

Transcriptional regulation is tightly coupled with chromosomal positioning and three-dimensional chromatin architecture. However, it is unclear what proportion of transcriptional activity is reflecting such organisation, how much can be informed by RNA expression alone, and how this impacts disease. Here, we develop a transcriptional decomposition approach separating the proportion of expression associated with genome organisation from independent effects not directly related to genomic positioning.\n\nWe show that positionally attributable expression accounts for a considerable proportion of total levels and is highly informative of topological associating domain activities and organisation, revealing boundaries and chromatin compartments. Furthermore, expression data alone accurately predicts individual enhancer-promoter interactions, drawing features from expression strength, stabilities, insulation and distance. We further characterise commonalities and differences across predictions in 76 human cell types, observing extensive sharing of domains, yet highly cell-type specific enhancer-promoter interactions and strong enrichments in relevant trait-associated variants. Our work demonstrates a close relationship between transcription and chromatin architecture, presenting a novel strategy and an unprecedented resource for investigating regulatory organisations and interpretations of disease associated genetic variants across cell types.

genomics↗