Biology subjects

Dunning Hotopp, J. C.

Publications and source records attributed to Dunning Hotopp, J. C..

9 recordsLinked to original sources

bioRxiv2026

Dynamic regulation of long non-coding RNAs across asexual andgametocyte development in Plasmodium falciparum

Long non-coding RNAs (lncRNAs) are critical regulators of gene expression in eukaryotes. Short reads from Illumina sequencing, reverse transcriptase artefacts, and incomplete second-strand degradation in strand-specific cDNA libraries hamper genome-wide identification of lncRNAs, especially in gene-dense genomes such as Plasmodium. Here, we integrated long-read Oxford Nanopore Technology direct RNA sequencing, ribosome profiling, and single-cell transcriptomics to generate a robust and stage-specific characterization of P. falciparum lncRNAs. We generated comprehensive annotations of lncRNAs expressed in both asexual and sexual blood stages and confirmed their non-coding nature using ribosome profiling. Most lncRNAs showed pronounced stage-specific expression and appeared to be particularly abundant in mature gametocytes. Single-cell RNA sequencing revealed differential expression of many lncRNAs in female and male gametocytes, suggesting important roles in gametocytogenesis and transmission. Many lncRNAs are located antisense to protein-coding genes and are co-expressed with their sense mRNA, possibly from putative bidirectional promoters, while others overlap mRNA coding sequences or 3 untranslated regions and showed negatively correlated expression patterns. Overall, our study shows the prevalence of P. falciparum lncRNAs and highlights their possible roles in controlling the regulation of gene expression, particularly during gametocytogenesis.

genomics↗

bioRxiv2025

The UTRs of Leishmania donovani vary in length and are enriched in potential regulatory structures

Leishmania spp. regulate gene expression largely post-transcriptionally, yet untranslated regions (UTRs) remain poorly delineated. We generated high-quality genome and transcriptome datasets for Leishmania donovani strain 1S2D (Ld1S) by combining PacBio HiFi de novo assembly with Oxford Nanopore direct RNA sequencing of promastigotes and axenic amastigotes. The genome assembly consists of 65 scaffolds totaling [~]33.3 Mb. Structural comparisons to LdBPK282A1 revealed numerous rearrangements, including some reshufling genes among polycistronic transcription units and validated by polycistronic reads from RNA sequencing. Promastigote and amastigote RNA sequencing produced 469,010 and 46,729 monocistronic reads containing a spliced-leader and a polyA tail sequences, defining 8,479 transcripts and supporting 7,415 of the 7,969 annotated protein coding genes, as well as 604 putative long non-coding RNAs. We annotated UTRs for 4,921 genes and observed that putative RNA G-quadruplexes were markedly enriched in UTRs. We also noted that 31.9% and 11.5% were expressed into multiple isoforms in promastigotes and amastigotes, respectively. Collectively, these data provide a comprehensive annotation of L. donovani genes and their UTRs and reveal widespread and stage-specific UTR length polymorphisms, and, overall, points to an important role of 3 UTR in post-transcriptional regulation in L. donovani. Author SummaryLeishmania donovani parasites cause visceral leishmaniasis, a deadly disease affecting hundreds of thousands of people worldwide. Unlike most eukaryotes, these parasites do not regulate their genes mainly at the level of transcription. Instead, control happens after the genes are transcribed, and much of this regulation depends on regions of RNA that are not translated into protein, called untranslated regions (UTRs). However, UTRs in Leishmania have remained poorly characterized. Here we generated high-quality genome and transcriptome resources for a clinical strain of L. donovani. By combining state-of-the-art long-read sequencing technologies, we precisely mapped thousands of UTRs and discovered that many genes produce transcripts with variable UTR lengths that differ between parasite life stages. We also found that UTRs are enriched in RNA structures called G-quadruplexes, which are known to influence gene regulation. These findings provide the most comprehensive view to date of UTRs in Leishmania and highlight their central role in controlling how genes are expressed during the parasites life cycle. Our work lays a foundation for future studies aimed at understanding parasite biology and identifying new targets for intervention.

genomics↗

bioRxiv2025

Degradation of ribosomal RNA during Plasmodium falciparum gametocytogenesis

The life cycle of Plasmodium falciparum is characterized by complex regulatory changes that allow adaptation of the parasites to different environmental conditions, which are especially pronounced during transmission between the mammalian host and the insect vector. Previous studies have shown that P. falciparum uses three types of ribosomal RNAs (rRNA A-, S1- and S2-types) at different stages of its life cycle. We used Oxford Nanopore Technologies (ONT) direct RNA sequencing to investigate the dynamics of rRNA usage throughout the parasites intraerythrocytic development, as well as in salivary gland sporozoites. Our study revealed a preponderance of A-type rRNAs during the intraerythrocytic cycle and gametocytogenesis, while S-type rRNAs slowly increase in abundance in mosquito stages starting three days post infection. Salivary gland sporozoites showed an even proportion of all rRNA types. By examining the length distributions of rRNA molecules, we detected an extensive and specific degradation of rRNAs during gametocytogenesis, starting in stage II gametocytes and continuing until the final stages of gametocyte development. We hypothesize that rRNA degradation may be linked to the global translational repression and metabolic quiescence described in stage V gametocytes, similar to mechanisms observed in bacterial and eukaryotic stress responses.

microbiology↗

bioRxiv2023

Common Analysis of Direct RNA SequencinG CUrrently Leads to Misidentification of 5-Methylcytosine Modifications at GCU Motifs

RNA modifications, such as methylation, can be detected with Oxford Nanopore Technologies direct RNA sequencing. One commonly used tool for detecting 5-methylcytosine (m5C) modifications is Tombo, which uses an "Alternative Model" to detect putative modifications from a single sample. We examined direct RNA sequencing data from diverse taxa including virus, bacteria, fungi, and animals. The algorithm consistently identified a 5-methylcytosine at the central position of a GCU motif. However, it also identified a 5-methylcytosine in the same motif in fully unmodified in vitro transcribed RNA, suggesting that this a frequent false prediction. In the absence of further validation, several published predictions of 5-methylcytosine in human coronavirus and human cerebral organoid RNA in a GCU context should be reconsidered. IMPORTANCEThe detection of chemical modifications to RNA is a rapidly expanding field within epigenetics. Nanopore sequencing technology provides an attractive means of detecting these modifications directly on the RNA, but accurate modification predictions are dependent upon the software developed to interpret the sequencing results. One of these tools, Tombo, allows users to detect modifications using sequencing results from a single RNA sample. However, we find that this method falsely predicts modifications in a specific sequence context across a variety of RNA samples, including RNA that lacks modifications. Results from previous publications include predictions in human coronaviruses with this sequence context and should be reconsidered. Our results highlight the importance of using RNA modification detection tools with caution in the absence of a control RNA sample for comparison.

genomics↗

bioRxiv2021

Mucoricin is a Ricin-Like Toxin that is Critical for the Pathogenesis of Mucormycosis

Fungi of the order Mucorales cause mucormycosis, a lethal infection with an incompletely understood pathogenesis. We now demonstrate that Mucorales fungi produce a toxin that plays a central role in virulence. Polyclonal antibodies against this toxin inhibit its ability to damage human cells in vitro, and prevent hypovolemic shock, organ necrosis, and death in mice with mucormycosis. RNAi inhibition of the toxin in Rhizopus delemar, compromises the ability of the fungus to damage host cells and attenuates virulence in mice. This 17 kDa toxin has structural and functional features of the plant toxin, ricin, including the ability to inhibit protein synthesis by its N-glycosylase activity, the existence of a motif that mediates vascular leak, and a lectin sequence. Antibodies against the toxin inhibit R. delemar- or toxin-mediated vascular permeability in vitro and cross-react with ricin. A monoclonal anti-ricin B chain antibody binds to the toxin and also inhibits its ability to cause vascular permeability. Therefore, we propose the name "mucoricin" for this toxin. Not only is mucoricin important in the pathogenesis of mucormycosis but our data suggest that a ricin- like toxin is produced by organisms beyond the plant and bacterial kingdoms. Importantly, mucoricin should be a promising therapeutic target.

microbiology↗

bioRxiv2020

Comparison of long read sequencing technologies in resolving bacteria and fly genomes

BackgroundThe newest generation of DNA sequencing technology is highlighted by the ability to sequence reads hundreds of kilobases in length, and the increased availability of long read data has democratized the genome sequencing and assembly process. PacBio and Oxford Nanopore Technologies (ONT) have pioneered competitive long read platforms, with more recent work focused on improving sequencing throughput and per-base accuracy. Released in 2019, the PacBio Sequel II platform advertises substantial enhancements over previous PacBio systems. ResultsWe used whole-genome sequencing data produced by two PacBio platforms (Sequel II and RS II) and two ONT protocols (Rapid Sequencing and Ligation Sequencing) to compare assemblies of the bacteria Escherichia coli and the fruit fly Drosophila ananassae. Sequel II assemblies had higher contiguity and consensus accuracy relative to other methods, even after accounting for differences in sequencing throughput. ONT RAPID libraries had the fewest chimeric reads in addition to superior quantification of E. coli plasmids versus ligation-based libraries. The quality of assemblies can be enhanced by adopting hybrid approaches using Illumina libraries for bacterial genome assemblies or combined ONT and Sequel II libraries for eukaryotic genome assemblies. Genome-wide DNA methylation could be detected using both technologies, however ONT libraries enabled the identification of a broader range of known E. coli methyltransferase recognition motifs in addition to undocumented D. ananassae motifs. ConclusionsThe ideal choice of long read technology may depend on several factors including the question or hypothesis under examination. No single technology outperformed others in all metrics examined.

genomics↗

bioRxiv2018

Cost Effective, Experimentally Robust Differential Expression Analysis for Human/Mammalian, Pathogen, and Dual-Species Transcriptomics

As sequencing read length has increased, researchers have quickly adopted longer reads for their experiments. Here, we examine host-pathogen interaction studies to assess if using longer reads is warranted. Six diverse datasets encountered in studies of host-pathogen interactions were used to assess what genomic attributes might affect the outcome of differential gene expression analysis including: gene density, operons, gene length, number of introns/exons, and intron length. Principal components analysis, hierarchical clustering with bootstrap support, and regression analyses of pairwise comparisons were undertaken on the same reads, looking at all combinations of paired and unpaired reads trimmed to 36,54,72, and 101-bp. For E coli, 36-bp single end reads performed as well as any other read length and as well as paired end reads. For all other comparisons, 54-bp and 72-bp reads were typically equivalent and different from 36-bp and 101-bp reads. Read pairing improved the outcome in several, but not all, comparisons in no discernable pattern, such that using paired reads is recommended in most scenarios. No specific genome attribute appeared to influence the data. However, experiments with an a priori expected greater biological complexity had more variable results with all read lengths relative to those with decreased complexity. When combined with cost, 54-bp paired end reads provided the most robust, internally reproducible results across all comparisons. However, using 36-bp single end reads may be desirable for bacterial samples, although possibly only if the transcriptional response is expected a priori to be robust.\n\nDATA SUMMARYO_LIThe human only CSHL Encode data set (1) was downloaded from ftp://hgdownload.cse.ucsc.edu/goldenPath/hgl9/encodeDCC/wgEncodeCshlLongRnaSeq/.\nC_LIO_LIThe data from mice vaginas infected with Candida albicans (2) was downloaded from the SRA (url - https://trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP057050).\nC_LIO_LIThe data from Aspergillus fumigatus cells in contact with human cells was downloaded from the SRA (url - https://www.ncbi.nlm.nih.gov/bioproject/399754).\nC_LIO_LIThe data from a strand-specific library from a study comparing C. albicans cells in contact with human cells with those in media (3) was downloaded from the SRA (url - https://trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP011085).\nC_LIO_LIThe data from C. albicans in culture media (3) was downloaded from the SRA (url - https://trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP011085).\nC_LIO_LIThe data from Escherichia coli grown in different media (4) was downloaded from the SRA (url - https://trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP056578).\nC_LI\n\nI/We confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. {boxtimes}\n\nIMPACT STATEMENTAs sequencing technologies improve, sequencing costs decrease and read lengths increase. We examine host-pathogen interaction studies to assess if using these longer reads is warranted given their increased cost relative to using the same number of shorter reads. To this end we compared the use of various read lengths and read pairing for six diverse host-pathogen datasets with varying genomic attributes including: gene density, operons, gene length, number of introns/exons, and intron length. We find that in the bacterial sample, 36-bp single end reads performed as well as any other read length and as well as paired end reads. When combined with cost, 54-bp paired end reads provided the most robust, internally reproducible results for all other comparisons. Read pairing improved the outcome in several, but not all, comparisons in no discernable pattern, such that using paired reads is recommended in most scenarios. No specific genome attribute appeared to influence the data.

genomics↗

bioRxiv2018

Using Core Genome Alignments to Assign Bacterial Species

With the exponential increase in the number of bacterial taxa with genome sequence data, a new standardized method is needed to assign bacterial species designations using genomic data that is consistent with the classically-obtained taxonomy. This is particularly acute for unculturable obligate intracellular bacteria like those in the Rickettsiales, where classical methods like DNA-DNA hybridization cannot be used to define species. Within the Rickettsiales, species designations have been applied inconsistently, often obfuscating the relationship between organisms and the context for experimental results. In this study, we generated core genome alignments for a wide range of genera with classically defined species, including Arcobacter, Caulobacter, Erwinia, Neisseria, Polaribacter, Ralstonia, Thermus, as well as genera within the Rickettsiales including Rickettsia, Orientia, Ehrlichia, Neoehrlichia, Anaplasma, eorickettsia, and Wolbachia. A core genome alignment sequence identity (CGASI) threshold of 96.8% was found to maximize the prediction of classically-defined species. Using the CGASI cutoff, the Wolbachia genus can be delineated into species that differ from the currently used supergroup designations, while the Rickettsia genus is delineated into nine species, as opposed to the current 27 species. Additionally, we find that core genome alignments cannot be constructed between genomes belonging to different genera, establishing a bacterial genus cutoff that suggests the need to create new genera from the Anaplasma and Neorickettsia. By using core genome alignments to assign taxonomic designations, we aim to provide a high-resolution, robust method for bacterial nomenclature that is aligned with classically-obtained results.

microbiology↗

bioRxiv2017

Grafting or Pruning in the Animal Tree: Lateral Gene Transfer and Gene Loss?

Lateral gene transfer (LGT) into multicellular eukaryotes with differentiated tissues, particularly gonads, continues to be met with skepticism by many prominent evolutionary and genomic biologists. A detailed examination of 26 animal genomes identifed putative LGTs in invertebrate and vertebrate genomes, concluding that there are fewer predicted LGTs in vertebrates/chordates than invertebrates, but there is still evidence of LGT into chordates, including humans. More recently, a reanalysis a subset of these putative LGTs into vertebrates concluded that there is not horizontal gene transfer in the human genome. One of the genes in dispute is an N-acyl-aromatic-L-amino acid amidohydrolase (ENSG00000132744), which encodes ACY3, which was initially identified as a putative bacteria-chordate LGT but was later debunked has a significant BLAST match to a more recently deposited genome of Saccoglossus kowalevskii, a flatworm, Metazoan, and hemichordate. Using BLAST searches, HMM searches, and phylogenetics to better understand the evidence for lateral gene transfer, gene loss, and rate variation in ACY3/ASPA homologues, the most parsimonious explanation for the distribution of ACY3/ASPA genes in eukaryotes likely involves both gene loss and lateral gene transfer, albeit lateral gene transfer that occurred hundreds of millions of years ago prior to the divergence of gnathostomes and even longer and prior to the divergence of bilateria. Given the many known, well-characterized, and adaptive lateral gene transfers from bacteria to insects and nematodes, lateral gene transfers at these time scales in the ancestors of humans is expected.

genomics↗