Biology subjects

Gregory, B. D.

Publications and source records attributed to Gregory, B. D..

9 recordsLinked to original sources

bioRxiv2026

Leaves in Transition: Single nuclei RNA sequencing provides insights into sorghums juvenile and adult phases

The transition from juvenile to adult phase (JA) is a key developmental process in plants, driven by conserved pathways that shape growth and stress responses. In monocots such as sorghum, this transition influences traits linked to environmental resilience, yet the regulation of micro-anatomical features such as trichomes and bulliform cells remains poorly understood. Here, we use single-nucleus RNA sequencing (snRNA-seq) to generate high-resolution gene expression maps of juvenile and adult sorghum leaves. Contrary to the traditional developmental model, we find that trichomes are present in both juvenile and adult leaves, with stage-dependent differences in gene expression. We also observe that bulliform cells, typically considered adult-specific, are present in juvenile leaves. At the transcriptomic level, cell populations cluster more strongly by developmental stage than by cell type, with juvenile cells grouping with other juvenile cells and adult cells with adult counterparts, indicating a dominant effect of developmental state on gene expression. In addition, we identify enrichment of dhurrin biosynthetic genes in trichome-associated cells as well as elevated expression of aquaporin genes in these cells, pointing to a potential role for trichomes in coordinating defense and water-related processes. Together, these findings refine current models of sorghum development and suggest that key epidermal cells and their associated functions may be established earlier than previously known. Collectively, this study provides a single nucleus resolved framework for understanding how developmental stage shapes gene expression in sorghum leaves.

plant biology↗

bioRxiv2025

Arabidopsis DXO1 is an evolutionarily diverged homolog that impacts ribosome loading and mRNA surveillance

The Rai1/Dxo1/DXO protein family are involved in eukaryotic 5'-end RNA quality control by hydrolyzing non-canonical mRNA cap structures, such as the NAD+ 5'-cap. The plant DXO1 ortholog shows distinct biochemical properties, compared to its fungal and mammalian counterparts, and only minor phenotypic traits can be attributed to its NAD+-decapping (deNADding) activity. On the other hand, the chloroplast and growth defects observed in dxo1 null mutants appear linked to the presence of a serine-rich domain at its N-terminus. Here, we report that plant DXO1 is a distinct ortholog that likely diverged earlier than its fungal and mammalian counterparts. We attribute this to the presence of a serine-rich N-terminal domain, which is found almost exclusively in plants and likely arose during the emergence of angiosperms. Degradome profiling revealed that Arabidopsis DXO1 contributes to mRNA surveillance, via examination of exon junction complex and ribosome footprints, and this activity is reliant on the presence of the serine-rich N-terminal domain in Arabidopsis. Furthermore, polysome profiling revealed that plants lacking DXO1 have severe ribosome loading defects, which may explain the loss of mRNA surveillance and alterations in pre-rRNA processing. Taken together, our data leads us to propose a model whereby Arabidopsis DXO1 is involved in 5'-end RNA quality control, which is critical for ribosome loading that impacts translational fidelity and translation-dependent mRNA surveillance.

plant biology↗

bioRxiv2025

HAMRLNC: A Comprehensive Pipeline for High-throughput Analysis of Modified Ribonucleotides and Long Non-Coding Ribonucleic Acids

As sequencing technologies advance and costs decline, there has been a surge in the application of RNA sequencing (RNA-seq) in understanding biological processes. In addition to the typical uses of RNA-seq for transcriptomics, gene annotation, novel gene discovery, and network analysis, these data can enable a deeper understanding of cellular processes through the identification of RNA modifications (epitranscriptome) and long non-coding RNAs (lncRNAs). To expedite discovery, we developed a portable, centralized computational pipeline for High-throughput Annotation of Modified Ribonucleotides and Long Non-Coding ribonucleic acids (HAMRLNC). HAMRLNC differs from existing methods by incorporating three workflows for quantifying transcript abundance, inferring RNA modifications, and lncRNA annotation using the same RNA- seq pre-processing and mapping steps. This facilitates reproducibility across multiple analyses and allows researchers to perform post-hoc analyses of archived sequencing data. In addition, we include novel analysis features to enable downstream visualization of annotated modified RNAs. HAMRLNC generates over a dozen well-defined and labeled figures as output, including gene ontology heatmaps, modification enrichment landscape, and modification clustering statistics. Availability and ImplementationHAMRLNC is an open-source software, and the source code is available at https://github.com/bdgregory/HAMRLNC. The pipeline can be installed and used through a docker container (https://hub.docker.com/r/chosenobih/HAMRLNC/tags). HAMRLNC is also available as an app in the CyVerse Discovery Environment https://de.cyverse.org/. Supplementary informationSupplementary data are available at BioRxiv online.

bioinformatics↗

bioRxiv2025

A field-focused systems approach reveals mRNA covalent modifications linked to sorghum growth and development under drought stress

RNA Covalent Modifications (RCMs) are post-transcriptional chemical alterations that influence RNA stability and translation efficiency, thus play critical roles in eukaryotic growth and development. However, their role in regulating plant performance under abiotic stress remain largely unexplored. Here, we integrated multi-omics data in six Sorghum bicolor accessions under water-limiting conditions in the field to explore the relationship between RCMs and drought response. Within a stress and photosynthesis-associated gene co-expression module, we identified SbDUS2, a member of family of enzymes, conserved across eukaryotes, which catalyzes the reduction of uracil to dihydrouridine (DHU) on RNA molecules. DHU-modified transcripts in this module were enriched for photosynthetic functions and showed strong correlation with photosynthetic traits. To elucidate the function of this RCM, we characterized loss of function dus2 mutants in the genetic model, Arabidopsis thaliana. Under control conditions, these DHU-deficient mutants exhibited impaired germination and delayed development. Furthermore, when exposed to heat or water-limiting conditions, these mutants showed significantly reduced net CO2 assimilation and survival. Using multiple transcriptome-wide RNA stability assays, we demonstrated that transcripts associated with lower DHU level in a dus2 background generally exhibited increased stability compared to Col-0 controls. Particularly, lack of DUS2 led to the hyperstability of photosynthesis-related transcripts, impeding their turnover and likely preventing proper photosynthetic acclimation during stress. We propose a model based on these data where DHU acts as a critical post-transcriptional regulator marking mRNAs for rapid turnover under stress, highlighting an overlooked regulatory layer contributing to plant resilience.

plant biology↗

bioRxiv2023

Expansion of human hematopoietic stem cells by inhibiting translation

Hematopoietic stem cell (HSC) transplantation using umbilical cord blood (UCB) is a potentially life-saving treatment for leukemia and bone marrow failure but is limited by the low number of HSCs in UCB. The loss of HSCs after ex vivo manipulation is also a major obstacle to gene editing for inherited blood disorders. HSCs require a low rate of translation to maintain their capacity for self-renewal, but hematopoietic cytokines used to expand HSCs stimulate protein synthesis and impair long-term self-renewal. We previously described cytokine-free conditions that maintain but do not expand human and mouse HSCs ex vivo. Here we performed a high throughput screen and identified translation inhibitors that allow ex vivo expansion of human HSCs while minimizing cytokine exposure. Transplantation assays show a [~]5-fold expansion of long-term HSCs from UCB after one week of culture in low cytokine conditions. Single cell transcriptomic analysis demonstrates maintenance of HSCs expressing mediators of the unfolded protein stress response, further supporting the importance of regulated proteostasis in HSC maintenance and expansion. These culture conditions maintain adult HSCs after CRISPR/Cas9 editing of the BCL11A+58 enhancer, overcoming a major obstacle to ex vivo gene correction for human hemoglobinopathies. SignificanceO_LIWe report ex vivo expansion of human hematopoietic stem cells from umbilical cord blood using low cytokines and small molecule inhibitors of GSK-3, mTORC1, and the translation initiation factor eIF4E. C_LIO_LIOur method maintains human HSCs ex vivo after gene editing with conventional CRISPR/Cas9, lipid nanoparticle delivery, and base editing approaches, overcoming a major obstacle to gene editing for inherited blood disorders. C_LI

cell biology↗

bioRxiv2021

A conserved long intergenic non-coding RNA containing snoRNA sequences, lncCOBRA1, affects Arabidopsis germination and development

Long non-coding RNAs (lncRNAs) are an increasingly studied group of non-protein-coding transcripts with a wide variety of molecular functions gaining attention for their roles in numerous biological processes. Nearly 6,000 lncRNAs have been identified in Arabidopsis thaliana but many have yet to be studied. Here, we examine a class of previously uncharacterized lncRNAs termed CONSERVED IN BRASSICA RAPA (lncCOBRA) transcripts that were previously identified for their high level of sequence conservation in the related crop species Brassica rapa, their nuclear-localization and protein-bound nature. In particular, we focus on lncCOBRA1 and demonstrate that its abundance is highly tissue and developmental specific, with particularly high levels early in germination. lncCOBRA1 contains two snoRNAs domains within it, making it the first sno-lincRNA example in a non-mammalian system, though we find that it is processed differently than its mammalian counterparts. We further show that plants lacking lncCOBRA1 display patterns of delayed gemination and are overall smaller than wild-type plants. Lastly, we identify the proteins that interact with lncCOBRA1 and examine the protein-protein interaction network of lncCOBRA1-interacting proteins.

plant biology↗

bioRxiv2019

HIPPIE2: a method for fine-scale identification of physically interacting chromatin regions

Most regulatory chromatin interactions are mediated by various transcription factors (TFs) and involve physically-interacting elements such as enhancers, insulators, or promoters. To map these elements and interactions, we developed HIPPIE2 which analyzes raw reads from high-throughput chromosome conformation (Hi-C) experiments to identify fine-scale physically-interacting regions (PIRs). Unlike standard genome binning approaches (e.g., 10K-1Mbp bins), HIPPIE2 dynamically calls physical locations of PIRs with better precision and higher resolution based on the pattern of restriction events and relative locations of interacting sites inferred from the sequencing readout.\n\nWe applied HIPPIE2 to in situ Hi-C datasets across 6 human cell lines (GM12878, IMR90, K562, HMEC, HUVEC, NHEK) with matched ENCODE and Roadmap functional genomic data. HIPPIE2 detected 1,042,738 distinct PIRs across cell lines, with high resolution (average PIR length of 1,006bps) and high reproducibility (92.3% in GM12878 replicates). 32.8% of PIRs were shared among cell lines. PIRs are enriched for epigenetic marks (H3K27ac, H3K4me1) and open chromatin, suggesting active regulatory roles. HIPPIE2 identified 2.8M significant intrachromosomal PIR-PIR interactions, 27.2% of which were enriched for TF binding sites. 50,608 interactions were enhancer-promoter interactions and were enriched for 33 TFs (31 in enhancers/29 in promoters), several of which are known to mediate DNA looping/long-distance regulation. 29 TFs were enriched in >1 cell line and 4 were cell line-specific. These findings demonstrate that the dynamic approach used in HIPPIE2 (https://bitbucket.com/wanglab-upenn/HIPPIE2) characterizes PIR-PIR interactions with high resolution and reproducibility.

bioinformatics↗

bioRxiv2018

Isolation and sequencing of AGO-bound RNAs reveals characteristics of mammalian stem-loop processing in vivo

MicroRNA precursors (pre-miRNAs) are short hairpin RNAs that are rapidly processed into mature microRNAs (miRNAs) in the cytoplasm. Due to their low abundance in cells, sequencing-based studies of pre-miRNAs have been limited. We successfully enriched for and deep sequenced pre-miRNAs in human cells by capturing these RNAs during their interaction with Argonaute (AGO) proteins. Using this approach, we detected > 350 pre-miRNAs in human cells and > 250 pre-miRNAs in a reanalysis of a similar study in mouse cells. We uncovered widespread trimming and non-templated additions to the 3 ends of pre- and mature miRNAs. Additionally, we created an index for microRNA precursor processing efficiency. This analysis revealed a subset of pre-miRNAs that produce low levels of mature miRNAs despite abundant precursors, including an annotated miRNA in the 5 UTR of the DiGeorge syndrome critical region 8 mRNA transcript. This led us to search for AGO-associated stem-loops originating from other mRNA species, which identified hundreds of putative pre-miRNAs derived from human and mouse mRNAs. In summary, we provide a wealth of information on mammalian pre-miRNAs, and identify novel microRNA and microRNA-like elements localized in mRNAs.

molecular biology↗

bioRxiv2017

CoGe LoadExp+: A web-based suite that integrates next-gen sequencing data analysis workflows and visualization

To make genomic and epigenomic analyses more widely available to the biological research community, we have created LoadExp+, a suite of bioinformatics workflows integrated with the web-based comparative genomics platform, CoGe. LoadExp+ allows users to perform transcriptomic (RNA-seq), epigenomic (bisulfite-seq), chromatin-binding (ChIP-seq), variant identification (SNPs), and population genetics analyses against any genome in CoGe, including genomes integrated by users themselves. Through LoadExp+s integration with CoGes existing features, all analyses are available for visualization and additional downstream processing, and are available for export to CyVerses data management and analysis platforms. LoadExp+ provides easy-to-use functionality to manage genomics and epigenomics data throughout its entire lifecycle and facilitates greater accessibility of genomics analyses to researchers of all skill levels. LoadExp+ can be accessed at https://genomevolution.org.

bioinformatics↗