Biology subjects

Yates, P. A.

Publications and source records attributed to Yates, P. A..

8 recordsLinked to original sources

bioRxiv2024

A high-throughput protein tagging toolkit that retains endogenous UTRs for studying gene regulation in Kinetoplastids.

Kinetoplastid parasites cause diseases that threaten human and animal health. To survive transitions between vertebrate hosts and insect vectors, these parasites rely on precise regulation of gene expression to adapt to environmental changes. Since gene regulation in Kinetoplastids is primarily post-transcriptional, developing efficient genetic tools for modifying genes at their endogenous loci while preserving regulatory mRNA elements is crucial for studying their complex biology. We present a CRISPR/Cas9-based tagging system that preserves untranslated regulatory elements and uses a viral 2A peptide from Thosea asigna to generate two separate proteins from a single transcript: a drug-selectable marker and a tagged protein of interest. This dual-function design maintains native control elements, allowing discrimination between regulation of transcript abundance, translational efficiency, and post-translational events. We validate the system by tagging six Trypanosoma brucei proteins and demonstrate: (i) high-efficiency positive selection and separation of drug-selectable marker and target protein, (ii) preservation of regulatory responses to environmental cues like heat shock and iron availability, and (iii) maintenance of stage-specific regulation during developmental transitions. This versatile toolkit is applicable to all kinetoplastids amenable to CRISPR/Cas9 editing, providing a powerful reverse genetic tool for studying post-transcriptional regulation and protein function in organisms where post-transcriptional control is dominant.

molecular biology↗

bioRxiv2022

ASAR RNAs control replication through interactions with dozens of RNA binding proteins

ASARs are a family of very-long noncoding RNAs that control replication timing on individual human autosomes, and are essential for chromosome stability. The eight known ASAR lncRNAs remain closely associated with their parent chromosomes. Analysis of RNA-protein interaction data (from ENCODE) revealed numerous RBPs with significant interactions with multiple ASAR lncRNAs, with several hnRNPs as abundant interactors. An [~]7kb domain within the ASAR6-141 lncRNA shows a striking density of RBP interaction sites. Genetic deletion and ectopic integration assays indicate that this [~]7kb RNA binding protein domain contains functional sequences for controlling replication timing of entire chromosomes in cis. shRNA-mediated depletion of 10 different RNA binding proteins, including HNRNPA1, HNRNPC, HNRNPL, HNRNPM, HNRNPU, or HNRNPUL1, results in dissociation of ASAR lncRNAs from their chromosome territories, and disrupts the synchronous replication that occurs on all autosome pairs, recapitulating the effect of individual ASAR knockouts on a genome-wide scale. Our results further demonstrate the role that ASARs play during the temporal order of genome-wide replication, and we propose that ASARs function as essential RNA scaffolds for the assembly of hnRNP complexes that help maintain the structural integrity of each mammalian chromosome.

genetics↗

bioRxiv2022

Epigenetic Control of Hundreds of Chromosome-Associated lncRNA Genes Essential for Replication and Stability

ASARs are long noncoding RNA genes that control replication timing of entire human chromosomes in cis. The three known ASAR genes are located on human chromosomes 6 and 15, and are essential for chromosome integrity. To identify ASARs on all human chromosomes we utilized a set of distinctive ASAR characteristics that allowed for the identification of hundreds of autosomal loci with epigenetically controlled, allele-restricted behavior in expression and replication timing of coding and noncoding genes, and is distinct from genomic imprinting. Disruption of noncoding RNA genes at five of five tested loci resulted in chromosome-wide delayed replication and chromosomal instability, validating their ASAR activity. In addition to the three known essential cis-acting chromosomal loci, origins, centromeres, and telomeres, we propose that all mammalian chromosomes also contain "Inactivation/Stability Centers" that display allele-restricted epigenetic regulation of protein coding and noncoding ASAR genes that are essential for replication and stability of each chromosome.

genetics↗

bioRxiv2021

Novel aspects of iron homeostasis in pathogenic bloodstream form Trypanosoma brucei

Iron is an essential regulatory signal for virulence factors in many pathogens. Mammals and bloodstream form (BSF) Trypanosoma brucei obtain iron by receptor-mediated endocytosis of transferrin bound to receptors (TfR) but the mechanisms by which T. brucei subsequently handles iron remains enigmatic. Here, we analyse the transcriptome of T. brucei cultured in iron-rich and iron-poor conditions. We show that adaptation to iron-deprivation induces upregulation of TfR, a cohort of parasite-specific genes (ESAG3, PAGS), genes involved in glucose uptake and glycolysis (THT1 and hexokinase), endocytosis (Phosphatidic Acid Phosphatase, PAP2), and most notably a divergent RNA binding protein RBP5, indicative of a non-canonical mechanism for regulating intracellular iron levels. We show that cells depleted of TfR by RNA silencing import free iron as a compensatory survival strategy. The TfR and RBP5 iron response are reversible by genetic complementation, the response kinetics are similar, but the regulatory mechanisms are distinct. Increased TfR protein is due to increased mRNA. Increased RBP5 expression, however, occurs by a post-transcriptional feedback mechanism whereby RBP5 interacts with its own, and with PAP2 mRNAs. Further observations suggest that increased RBP5 expression in iron-deprived cells has a maximum threshold as ectopic overexpression above this threshold disrupts normal cell cycle progression resulting in an accumulation of anucleate cells and cells in G2/M phase. This phenotype is not observed with overexpression of RPB5 containing a point mutation (F61A) in its single RNA Recognition Motif. Our experiments shed new light on how T. brucei BSFs reorganise their transcriptome to deal with iron stress revealing the first iron responsive RNA binding protein that is co-regulated with TfR, is important for cell viability and iron homeostasis; two essential processes for successful proliferation. Author SummaryAfrican trypanosomes are single-celled extracellular parasites of humans and animals relying on essential host nutrients for survival. They satisfy their iron needs by capturing host transferrin-bound iron using a surface-localised transferrin receptor (TfR) that is structurally distinct from its host counterpart. Little is known about the trypanosome response to fluctuations in host iron availability, with the exception of modulated TfR expression. We show that unlike other eukaryotes, at the transcriptome level, trypanosomes do not regulate iron-dependent enzymes as a mechanism to cope with iron deprivation. Instead, we identify a group of novel iron responsive trypanosome-specific genes, particularly an RNA Binding Protein RBP5 that is responsive to iron levels, albeit mediated by a distinct mechanism from TfR. We show that although RBP5 expression is elevated at the mRNA and protein levels, increased abundance above a maximum threshold is toxic. The trypanosome TfR has been suggested as a therapeutic target, but whether it is essential for optimal host colonisation is unclear. Our data demonstrate that trypanosomes efficiently import free iron from their environment independent of TfR suggesting that alternative iron uptake pathways exist, and that any therapeutic interventions targeting TfR must be evaluated with caution.

microbiology↗

bioRxiv2020

Distinct cis-acting elements govern purine-responsive regulation of the Leishmania donovani nucleoside transporters, LdNT1 and LdNT2

Purine salvage from the host is an obligatory process for all protozoan parasites. In Leishmania donovani, this is accomplished by four membrane nucleoside and nucleobase transporters, or LdNTs. Previously, we demonstrated that purine starvation invokes a robust stress response in Leishmania and characterized the proteomic changes involved. However, because Leishmania do not control the transcription of individual genes, the underlying mechanisms responsible for these changes were ill-defined. LdNT1 and LdNT2 are among the most rapidly and significantly upregulated genes in purine-starved L. donovani parasites. Thus, to better understand post-transcriptional mechanisms of purine-responsive gene expression, we have examined regulation of these genes in molecular detail. Here we report that LdNT1 and LdNT2 are controlled by distinct cis-acting elements. In the case of LdNT2, mRNA abundance and translational enhancement under purine stress depend on a 76 nt-long polypyrimidine tract encoded in the LdNT2 mRNA 3-UTR. Transcripts containing the LdNT2 polypyrimidine tract were found to localize to discrete cytoplasmic foci in purine-replete cells, suggesting that the LdNT2 message may be stored in RNA granules at steady-state. In the case of LdNT1, we found that purine-responsiveness is conferred by a 48 nt-long polypyrimidine tract and additional upstream element, termed UE1. Both features are independently required for regulation, with the polypyrimidine tract and UE1 controlling mRNA abundance and translation, respectively. Together, these results highlight a remarkable degree of complexity in the regulation of the Leishmania purine stress response and set the stage for future investigations to identify the larger network of RNA-protein and protein-protein interactions involved.

genetics↗

bioRxiv2020

Purine-responsive expression of the Leishmania donovani NT3 purine nucleobase transporter is mediated by a conserved RNA stem-loop

The ability to modulate gene expression in response to changes in the host environment is essential for survival of the kinetoplastid parasite Leishmania. Unlike most eukaryotes, gene expression in kinetoplastids is predominately regulated post-transcriptionally. Consequently, RNA-binding proteins (RBPs) and mRNA-encoded sequence elements serve as primary determinants of gene regulation in these organisms; however, few have been ascribed roles in specific stress-response pathways. Leishmania lack the capacity for de novo purine synthesis and must scavenge these essential nutrients from the host. Leishmania have evolved a robust stress response to withstand sustained periods of purine scarcity during their lifecycle. The purine nucleobase transporter, LdNT3, is among the most substantially upregulated proteins in purine-starved L. donovani. Here we report that the post-translational stability of the LdNT3 protein is unchanged in response to purine starvation. Instead, LdNT3 upregulation is primarily mediated by a 33 nucleotide (nt) sequence in the LdNT3 mRNA 3-untranslated region that is predicted to adopt a stem-loop structure. While this sequence is highly conserved within the mRNAs of orthologous transporters in multiple kinetoplastid species, putative stem-loops from L. donovani and Trypanosoma brucei nucleobase transporter mRNAs are not functionally interchangeable for purine-responsive regulation. Through mutational analysis of the element, we demonstrate that species specificity is attributable to just three variant bases within the predicted loop. Finally, we provide evidence that the abundance of the trans-acting factor that binds the LdNT3 stem-loop in vivo is substantially higher than required for regulation of LdNT3 alone, implying a potential role in regulating other purine-responsive genes.

molecular biology↗

bioRxiv2019

REAVER: Improved Analysis of High-resolution Vascular Network Images Revealed Through Round-robin Rankings of Accuracy and Precision

Alterations in vascular networks, including angiogenesis and capillary regression, play key roles in disease, wound healing, and development. Imaging of microvascular networks can reveal their spatial structures, but effective study of network architecture requires methods to accurately quantify them using a variety of metrics. We present REAVER (Rapid Editable Analysis of Vessel Elements Routine), a freely available open source tool that researchers can use to analyze and quantify high resolution fluorescent images of blood vessel networks, and assess its performance compared to alternative state-of-the-art image analysis software programs. Top performing programs for each metric are identified by assigning a rank based on statistical multiple comparisons of accuracy and precision, modeled as matches in a round-robin style tournament. This comparison method yields a clearly defined and consistent standard for characterizing program performance, avoiding the use of non-standard ad hoc interpretations of multiple comparisons between programs. Using this comparison method and a dataset of manually analyzed images as a ground-truth, we show that REAVER was the top ranked program for both accuracy and precision for all metrics quantified, including vessel length density, vessel area fraction, mean vessel diameter, and branchpoint count. REAVER can be used to quantify differences in blood vessel architectures between study groups, which makes it particularly useful in experiments designed to evaluate the effects of different external perturbations (e.g. drugs or disease states).

bioinformatics↗

bioRxiv2019

Pericyte Bridges in Homeostasis and Hyperglycemia: Reconsidering Pericyte Dropout and Microvascular Structures

Diabetic retinopathy threatens the vision of a third of diabetic patients. Progression of the disease is attributed to the dropout of pericytes, a cell type that enwraps and stabilizes the microvasculature. In tandem with this presumptive pericyte dropout, there is enriched formation of structures assumed to be remnants of collapsed or regressed vessels, previously classified as acellular capillaries, string vessels, and basement membrane bridges. Instead of endothelial cells, we show that pericytes colocalize with basement membrane bridges, and both bridging structures are enriched by cell-specific knockout of KLF4 and reversibly enriched with elevation of Ang-2, PDGF-BB, and blood sugar. Our data suggests that pericyte counts from retinal digests have misclassified pericyte bridges as endothelial structures and have exaggerated the role of pericyte loss in DR progression. In vivo imaging of corneal limbal vessels demonstrates pericyte migration off-vessel, implicating pericyte movement in formation of pericyte bridges and pathogenesis of diabetic retinopathy.

cell biology↗