Biology subjects

Holley, C. L.

Publications and source records attributed to Holley, C. L..

11 recordsLinked to original sources

bioRxiv2026

CpxR and HicB exert independent regulatory action on the gonococcal hicAB-encoded toxin-antitoxin system

The continued emergence of Neisseria gonorrhoeae (Ng) isolates resistant to front-line antibiotics has focused efforts on understanding how alternative therapies, such as the expanded use of gentamicin (Gen), might counteract this global public health problem. Focusing on Gen as a viable alternative antibiotic for the treatment of gonorrheal infections, we previously used RNA-seq to determine if sub-lethal levels of Gen might impact gonococci on a transcriptional level and showed that expression of the putative HicA-HicB toxin-antitoxin (TA) system was increased in response to sub-lethal Gen. Importantly, loss of this TA system resulted in reduction of Ng biofilm formation in a strain specific manner. Focusing on this strain specificity, we found that the CpxR/CpxA two-component system (TCS) influences expression of the hicAB operon independently of HicB autoregulation. We now report that CpxR selectively binds to the hicAB operon to enhance expression of hicAB but does not interfere with binding of HicB to the promoter region. Furthermore, we show that single base pair differences in the intergenic region between hicA and hicB impact regulation by CpxR. Hence, the regulation of the HicAB TA in gonococcal strains is a highly coordinated response that can involve autoregulation by HicB and the CpxRA TCS. We propose that this dual regulatory scheme maximizes the ability of Ng to respond to Gen and hostile environmental conditions.

microbiology↗

bioRxiv2025

Genome-wide profiling of RNA 2'-O-methylation in neurons and identification of orphan snoRNA targets

We have compared genome-wide patterns of RNA 2-O-methylation (Nm) between two isogenic pairs of neurons. Each pair includes one line harboring a small deletion of orphan box C/D snoRNAs (SNORD116s) from the paternal chr15q11-q13 region. One isogenic pair also differs in expression of SNORD113/114 snoRNAs from chr14q32.2. Wild-type and modified cells were differentiated into cortical neurons, and genome-wide patterns of Nm identified. Neurons display a distinctive signature of rRNA modification compared to undifferentiated stem cells. We further identified thousands of shared Nm sites in mRNAs, lncRNAs and small RNAs. Most sites do not exhibit canonical complementarity to snoRNAs, but a number exhibit strong complementarity to U3 snoRNA, not previously shown to direct Nm. Evidence from cross-linking and sequencing of hybrids (CLASH) suggests that U3 is proximally associated with a subset of 2-O-methylation events. Finally, we identify a number of apparent canonical targets of SNORD113, SNORD114 and SNORD116 snoRNAs. These data present a comprehensive characterization of the Nm landscape in neurons and, for the first time, allow the assignment of Nm sites targeted by specific orphan snoRNAs associated with neurodevelopmental and other disorders.

molecular biology↗

bioRxiv2025

snoCLASH Reveals Extensive snoRNA-mRNA Interaction Networks

Small nucleolar RNAs (snoRNAs) are classically defined as guides for modification of ribosomal RNA (rRNA) and small nuclear RNA (snRNA), yet increasing evidence suggests that box C/D snoRNAs also interact with non-canonical transcripts. Systematic discovery of such interactions has been hindered by overwhelming rRNA abundance and technical limitations in RNA-RNA capture. Here, we present snoCLASH, an optimized snoRNA:RNA binding protein (RBP)-based crosslinking, ligation, and sequencing framework that integrates phenol-toluol extraction, polyA enrichment, nuclear fractionation, rRNA depletion, and dual-reference chimeric read analysis to enable transcriptome-scale identification of snoRNA-non-rRNA interactions. Applying this approach reveals thousands of snoRNA-associated mRNA regions spanning coding and regulatory elements and enriched for RBPs linked to epitranscriptomic regulation. Using this framework, we identify high-confidence snoRNA-mRNA interactions and functionally validate OGA mRNA as a snoCLASH-discovered target undergoes snoRNA-dependent 2-O-methylation with downstream effects on protein expression. Together, this work establishes snoCLASH as a platform for discovering and validating non-canonical snoRNA targets beyond rRNA. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=56 SRC="FIGDIR/small/693487v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@13d9a4corg.highwire.dtl.DTLVardef@15d12fdorg.highwire.dtl.DTLVardef@189ac06org.highwire.dtl.DTLVardef@17ceeea_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Rpl13a snoRNAs Downregulate Smooth Muscle Cell COX4I2 and Promote Neointimal Hyperplasia

BACKGROUNDReactive oxygen species (ROS) augment the activation of vascular smooth muscle cells (SMCs) and promote neointimal hyperplasia evoked by arterial injury or atherogenesis. We have previously shown that small nucleolar RNAs (snoRNAs) from the Rpl13a locus are key regulators of cellular ROS levels. METHODSUsing mice deficient in the Rpl13a snoRNAs, we tested whether these snoRNAs regulate SMC activation in vitro and in vivo. Carotid endothelial denudation was used to provoke neointimal hyperplasia in wild-type (WT) and snoRNA knockout (snoKO) mice, which lack all four intronically-encoded Rpl13a snoRNAs. Primary SMCs from WT and snoKO mice were used for in vitro functional and proteomic analyses. HEK293T cells with specific snoRNA deletions were used to test for snoRNA-guided 2-O-methylation of mRNA. RESULTSArterial ROS levels, inflammation, and carotid artery neointimal hyperplasia were reduced in snoKO compared with WT mice. In vitro, snoKO SMCs demonstrated lower ROS levels and less migration, proliferation, and inflammatory signaling than WT SMCs. Reduced ROS levels in snoKO SMCs and aortas correlated with upregulation of the mitochondrial protein COX4I2, which is associated with reduced mitochondrial ROS under normoxic conditions. Deleting the snoRNA U32A in human HEK293T cells decreased 2-O-methylation of COX4I2 mRNA and upregulated COX4I2 protein without changing COX4I2 mRNA levels. Silencing Cox4i2 in snoKO SMCs upregulated SMC ROS to WT levels. CONCLUSIONSRpl13a snoRNAs are important drivers of SMC activation and neointimal hyperplasia. Rpl13a snoRNAs augment SMC ROS levels, at least in part, by post-transcriptional downregulation of COX4I2 expression.

molecular biology↗

bioRxiv2025

Gentamicin induction of the gonococcal hicAB toxin-antitoxin encoding system and 2 impact on gene expression influencing biofilm formation and in vivo fitness in a strain specific manner

The continued emergence of Neisseria gonorrhoeae (Ng) isolates resistant to first-line antibiotics has focused efforts on understanding how alternative therapies such as expanded use of gentamicin (Gen) might counteract this global public health problem. Focusing on Gen as a viable alternative antibiotic for treatment of gonorrheal infections, we used RNA-Seq to determine if sub-lethal levels of Gen might impact gonococci on a transcriptional level. We found that sub-lethal Gen levels altered expression of 23 genes in Ng strain FA19. Many of the differentially regulated genes were associated with known stress responses elaborated by Ng under different harmful conditions. We found that the transcripts of the hicAB operon, which encodes a putative HicA-HicB toxin-antitoxin system that is encoded by tandem genes with the prophage Ngo {varphi}3, were increased in response to Gen. While loss of hicAB did not impact gonococcal susceptibility to a variety of antimicrobial agents or harmful environmental conditions it did reduce biofilm formation in Ng strains F62, FA1090, WHO X and CDC200 but not that of strain FA19. Further, in strain F62, but not FA19, loss of hicAB reduced the in vivo fitness of Ng during experimental lower genital tract infection of female mice. Further, we found that expression of hicAB can influence levels of the norB transcript, which encodes the nitrate reductase shown previously to be upregulated in gonococcal biofilms. We propose that sub-lethal Gen has the capacity to influence gonococcal pathogenesis through the action of the HicAB toxin-antitoxin system. ImportanceDuring antibiotic treatment bacteria can be exposed to sub-lethal levels that could serve as a stress signal resulting in changes in gene expression. The continued emergence of multi-drug resistant strains of Ng has rekindled interest in expanded use of gentamicin (Gen) for treatment of gonorrheal infections. We report that sub-lethal levels of Gen can influence levels of Ng transcripts including that of the gonococcal hicAB-encoded toxin-antitoxin (TA) locus, which is embedded within an integrated prophage, While loss of this TA locus did not impact Ng susceptibility to Gen it reduced the biofilm forming ability of 4/5 Ng strains. Further, in an examined strain in this group we found that Ng fitness during experimental infection was negatively impacted. We propose that that levels of the hicA-hicB transcripts can be increased by sub-lethal levels of an antibiotic used in treatment of gonorrhea and that this could influence pathogenicity.

microbiology↗

bioRxiv2024

Generating snoRNA-guided Programmable 2'O-methylation

Small nucleolar RNAs (snoRNAs) are critical in guiding post-transcriptional modifications like 2-O-methylation (Nm), which play crucial roles in downstream processes such as splicing and translation. This study tests a novel method for Nm validation, addressing a significant gap in modern Nm research, and offers insight into the intricacies of snoRNA-guided Nm. While mapping of Nm modifications has seen significant improvement within the past decade, no major techniques have been able to validate these potential sites. Additionally, many mapping techniques lack consensus among proposed Nm sites, especially on mRNAs. Without a proper validation technique, Nm research lags compared to its peer post-transcriptional modifications. The RNase H-based Nm-VAQ assay used here quantifies 2-O-methylation at single nucleotide resolution across various RNA species including rRNA, snRNA, and mRNA. Its optimization for mRNA allows for an unprecedented way to study the effects of Nm modifications in low abundance transcripts. Utilizing this, the study also explores the potential of creating synthetic snoRNAs to guide Nm modifications. Exogenous snoRNAs are shown to rescue Nm in genetic knockout models and can be mutated to guide Nm at any location along the target RNA transcript. Preliminary work indicates that synthetic snoRNAs demonstrate the ability to modify luciferase, impacting translation efficiency. Targeting an exon increases mRNA abundance but decreases protein expression, consistent with previous findings on Pxdn mRNA. These findings set the scene for novel understanding of the relationship between snoRNA abundance, 2-O-methylation efficiency, and Nms impact on gene expression.

molecular biology↗

bioRxiv2022

ACE2 is necessary for SARS-CoV-2 infection and sensing by macrophages but not sufficient for productive viral replication

Macrophages are key cellular contributors to COVID-19 pathogenesis. Whether SARS-CoV-2 can enter macrophages, replicate and release new viral progeny remains controversial. Similarly, whether macrophages need to sense replicating virus to drive cytokine release is also unclear. Macrophages are heterogeneous cells poised to respond to their local microenvironment, and accordingly, the SARS-CoV-2 entry receptor ACE2 is only present on a subset of macrophages at sites of human infection. Here, we use in vitro approaches to investigate how SARS-CoV-2 interacts with ACE2-negative and ACE2-positive human macrophages and determine how these macrophage populations sense and respond to SARS-CoV-2. We show that SARS-CoV-2 does not replicate within ACE2-negative human macrophages and does not induce pro-inflammatory cytokine expression. By contrast, ACE2 expression in human macrophages permits SARS-CoV-2 entry, replication, and virion release. ACE2-expressing macrophages sense replicating virus to trigger pro-inflammatory and anti-viral programs that limit virus release. These combined findings resolve several controversies regarding macrophage-SARS-CoV-2 interactions and identify a signaling circuit by which macrophages sense SARS-CoV-2 cell entry and respond by restricting viral replication. One sentence summaryLack of macrophage ACE2 expression precludes SARS-CoV-2 entry and sensing, while ACE2-expressing macrophages sense intramacrophage SARS-CoV-2 replication to induce rapid anti-viral responses that limit new virion release.

immunology↗

bioRxiv2021

Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique

Pseudouridine ({Psi}) is the most common non-canonical ribonucleoside present on mammalian non-coding RNAs (ncRNAs), including rRNAs, tRNAs and snRNAs, where it contributes [~]7% of the total uridine level. However, {Psi} constitutes only [~]0.1% of the uridines present on mRNAs and its effect on mRNA function remains unclear. {Psi} residues have been shown to inhibit the detection of exogenous RNA transcripts by host innate immune factors, thus raising the possibility that viruses might have subverted the addition of {Psi} residues to mRNAs by host pseudouridine synthase (PUS) enzymes as a way to inhibit antiviral responses in infected cells. Here, we describe and validate a novel antibody-based {Psi} mapping technique called photo-crosslinking assisted {Psi} sequencing (PA-{Psi}-seq) and use it to map {Psi} residues on not only multiple cellular RNAs but also on the mRNAs and genomic RNA encoded by HIV-1. We describe several 293T-derived cell lines in which human PUS enzymes previously reported to add {Psi} residues to human mRNAs, specifically PUS1, PUS7 and TRUB1/PUS4, were inactivated by gene editing. Surprisingly, while this allowed us to assign several sites of {Psi} addition on cellular mRNAs to each of these three PUS enzymes, the {Psi} sites present on HIV-1 transcripts remained unaffected. Moreover, loss of PUS1, PUS7 or TRUB1 function did not significantly reduce the level of {Psi} residues detected on total human mRNA below the [~]0.1% level seen in wild type cells, thus implying that the PUS enzyme(s) that adds the bulk of {Psi} residues to human mRNAs remains to be defined.

molecular biology↗

bioRxiv2020

Snord67 promotes lymph node metastasis and regulates U6-mediated alternative splicing in breast cancer

Small nucleolar RNAs (snoRNAs) have long been considered "housekeeping genes", important for ribosomal biogenesis and protein synthesis. However, there is increasing evidence that this largely ignored class of non-coding RNAs (ncRNAs) also have wide-ranging, non-canonical functions in diseases, including cancer. SnoRNAs have been shown to have both oncogenic and tumor suppressor roles, yet whether snoRNAs regulate metastasis is unknown. Here we show that expression of certain snoRNAs are enriched in lymph node (LN) metastases in a micro-surgical, immune-competent mouse model of breast cancer. We identify the snoRNA Snord67 as a key regulator of LN metastasis. Knockout of Snord67 resulted in significantly decreased LN tumor growth and subsequent development of distant metastases. This was associated with loss of targeted 2-O-methylation on the small nuclear RNA U6, a component of the spliceosome. RNA sequencing revealed distinct alternative splicing patterns in Snord67 knockout cells. Using rapid autopsy breast cancer cases, we found that matched human primary tumor and LN metastases revealed similar alternatively spliced genes, including several that are known to contribute to cancer. These results demonstrate that Snord67 is critical for growth of LN metastases and subsequent spread to distant metastases, and suggest that snoRNA-guided modifications of the spliceosome represent a previously unappreciated, yet targetable pathway in cancer.

cancer biology↗

bioRxiv2019

Altered m6A modification of specific cellular transcripts affects Flaviviridae infection

The RNA modification N6-methyladenosine (m6A) can modulate mRNA fate and thus affect many biological processes. We analyzed m6A modification across the transcriptome following infection by dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), and hepatitis C virus (HCV). We found that infection by these viruses in the Flaviviridae family alters m6A modification of specific cellular transcripts, including RIOK3 and CIRBP. During viral infection, the addition of m6A to RIOK3 promotes its translation, while loss of m6A in CIRBP promotes alternative splicing. Importantly, we found that activation of innate immune sensing or the endoplasmic reticulum (ER) stress response by viral infection contributes to the changes in m6A modification in RIOK3 and CIRBP, respectively. Further, several transcripts with infection-altered m6A profiles, including RIOK3 and CIRBP, encode proteins that influence DENV, ZIKV, and HCV infection. Overall, this work reveals that cellular signaling pathways activated during viral infection lead to alterations in m6A modification of host mRNAs to regulate infection.

molecular biology↗

bioRxiv2019

Extensive epitranscriptomic methylation of A and C residues on murine leukemia virus transcripts enhances viral gene expression

While it has been known for several years that viral RNAs are subject to the addition of several distinct covalent modifications to individual nucleotides, collectively referred to as epitranscriptomic modifications, the effect of these editing events on viral gene expression has been controversial. Here, we report the purification of murine leukemia virus (MLV) genomic RNA to homogeneity and show that this viral RNA contains levels of N6-methyladenosine (m6A), 5-methylcytosine (m5C) and 2O-methylated (Nm) ribonucleotides that are an order of magnitude higher than detected on bulk cellular mRNAs. Mapping of m6A and m5C residues on MLV transcripts identified multiple discrete editing sites and allowed the construction of MLV variants bearing silent mutations that removed a subset of these sites. Analysis of the replication potential of these mutants revealed a modest but significant attenuation in viral replication in 3T3 cells in culture. Consistent with a positive role for m6A and m5C in viral replication, we also demonstrate that overexpression of the key m6A reader protein YTHDF2 enhances MLV replication, while downregulation of the m5C writer NSUN2 inhibits MLV replication.\n\nImportanceThe data presented in this manuscript demonstrate that MLV RNAs bear an exceptionally high level of the epitranscriptomic modifications m6A, m5C and Nm, thus suggesting that these each facilitate some aspect of the viral replication cycle. Consistent with this hypothesis, we demonstrate that mutational removal of a subset of these m6A or m5C modifications from MLV transcripts inhibits MLV replication in cis and a similar result was also observed upon manipulation of the level of expression of key cellular epitranscriptomic cofactors in trans. Together, these results argue that the addition of several different epitranscriptomic modifications to viral transcripts stimulates viral gene expression and suggest that MLV has therefore evolved to maximize the level of these modifications that are added to viral RNAs.

microbiology↗