Transcript-specific determinants of pre-mRNA splicing revealed through in vivo kinetic analyses of the 1st and 2nd chemical steps.

We generate high-precision measurements of the in vivo rates of both chemical steps of pre-mRNA splicing across the genome-wide complement of substrates in yeast by coupling metabolic labeling, multiplexed primer-extension sequencing, and kinetic modeling. We demonstrate that the rates of intron removal vary widely, splice-site sequences are primary determinants of 1st step but have little apparent impact on 2nd step rates, and the 2nd step is generally faster than the 1st step. Ribosomal protein genes (RPGs) are spliced faster than non-RPGs at each step, and RPGs share evolutionarily conserved properties that may contribute to their faster splicing. A genetic variant defective in the 1st step of the pathway reveals a genome-wide defect in the 1st step but an unexpected, transcript-specific change in the 2nd step. Our work demonstrates that extended co-transcriptional association is an important determinant of splicing rate, a conclusion at odds with recent claims of ultra-fast splicing.

The IRG1-itaconate axis protects from cholesterol-induced inflammation and atherosclerosis.

Atherosclerosis is fueled by a failure to resolve lipid-driven inflammation within the vasculature that drives plaque formation. Therapeutic approaches to reverse atherosclerotic inflammation are needed to address the rising global burden of cardiovascular disease (CVD). Recently, metabolites have gained attention for their immunomodulatory properties, including itaconate, which is generated from the tricarboxylic acid-intermediate cis-aconitate by the enzyme Immune Responsive Gene 1 (IRG1/ACOD1). Here, we tested the therapeutic potential of the IRG1-itaconate axis for human atherosclerosis. Using single-cell RNA sequencing (scRNA-seq), we found that IRG1 is up-regulated in human coronary atherosclerotic lesions compared to patient-matched healthy vasculature, and in mouse models of atherosclerosis, where it is primarily expressed by plaque monocytes, macrophages, and neutrophils. Global or hematopoietic Irg1-deficiency in mice increases atherosclerosis burden, plaque macrophage and lipid content, and expression of the proatherosclerotic cytokine interleukin (IL)-1ß. Mechanistically, absence of Irg1 increased macrophage lipid accumulation, and accelerated inflammation via increased neutrophil extracellular trap (NET) formation and NET-priming of the NLRP3-inflammasome in macrophages, resulting in increased IL-1ß release. Conversely, supplementation of the Irg1-itaconate axis using 4-octyl itaconate (4-OI) beneficially remodeled advanced plaques and reduced lesional IL-1ß levels in mice. To investigate the effects of 4-OI in humans, we leveraged an ex vivo systems-immunology approach for CVD drug discovery. Using CyTOF and scRNA-seq of peripheral blood mononuclear cells treated with plasma from CVD patients, we showed that 4-OI attenuates proinflammatory phospho-signaling and mediates anti-inflammatory rewiring of macrophage populations. Our data highlight the relevance of pursuing IRG1-itaconate axis supplementation as a therapeutic approach for atherosclerosis in humans.

Long noncoding RNA CHROMR regulates antiviral immunity in humans.

Long noncoding RNAs (lncRNAs) have emerged as critical regulators of gene expression, yet their contribution to immune regulation in humans remains poorly understood. Here, we report that the primate-specific lncRNA CHROMR is induced by influenza A virus and SARS-CoV-2 infection and coordinates the expression of interferon-stimulated genes (ISGs) that execute antiviral responses. CHROMR depletion in human macrophages reduces histone acetylation at regulatory regions of ISG loci and attenuates ISG expression in response to microbial stimuli. Mechanistically, we show that CHROMR sequesters the interferon regulatory factor (IRF)-2-dependent transcriptional corepressor IRF2BP2, thereby licensing IRF-dependent signaling and transcription of the ISG network. Consequently, CHROMR expression is essential to restrict viral infection of macrophages. Our findings identify CHROMR as a key arbitrator of antiviral innate immune signaling in humans.

ANGPTL3 deficiency impairs lipoprotein production and produces adaptive changes in hepatic lipid metabolism.

Angiopoietin-like protein 3 (ANGPTL3) is a hepatically secreted protein and therapeutic target for reducing plasma triglyceride-rich lipoproteins and low-density lipoprotein (LDL) cholesterol. Although ANGPTL3 modulates the metabolism of circulating lipoproteins, its role in triglyceride-rich lipoprotein assembly and secretion remains unknown. CRISPR-associated protein 9 (CRISPR/Cas9) was used to target ANGPTL3 in HepG2 cells (ANGPTL3-/-) whereupon we observed ∼50% reduction of apolipoprotein B100 (ApoB100) secretion, accompanied by an increase in ApoB100 early presecretory degradation via a predominantly lysosomal mechanism. Despite defective particle secretion in ANGPTL3-/- cells, targeted lipidomic analysis did not reveal neutral lipid accumulation in ANGPTL3-/- cells; rather ANGPTL3-/- cells demonstrated decreased secretion of newly synthesized triglycerides and increased fatty acid oxidation. Furthermore, RNA sequencing demonstrated significantly altered expression of key lipid metabolism genes, including targets of peroxisome proliferator-activated receptor α, consistent with decreased lipid anabolism and increased lipid catabolism. In contrast, CRISPR/Cas9 LDL receptor (LDLR) deletion in ANGPTL3-/- cells did not result in a secretion defect at baseline, but proteasomal inhibition strongly induced compensatory late presecretory degradation of ApoB100 and impaired its secretion. Additionally, these ANGPTL3-/-;LDLR-/- cells rescued the deficient LDL clearance of LDLR-/- cells. In summary, ANGPTL3 deficiency in the presence of functional LDLR leads to the production of fewer lipoprotein particles due to early presecretory defects in particle assembly that are associated with adaptive changes in intrahepatic lipid metabolism. In contrast, when LDLR is absent, ANGPTL3 deficiency is associated with late presecretory regulation of ApoB100 degradation without impaired secretion. Our findings therefore suggest an unanticipated intrahepatic role for ANGPTL3, whose function varies with LDLR status.

Detection of splice isoforms and rare intermediates using multiplexed primer extension sequencing.

Targeted RNA sequencing (RNA-seq) aims to focus coverage on areas of interest that are inadequately sampled in standard RNA-seq experiments. Here we present multiplexed primer extension sequencing (MPE-seq), an approach for targeted RNA-seq that uses complex pools of reverse-transcription primers to enable sequencing enrichment at user-selected locations across the genome. We targeted hundreds to thousands of pre-mRNA splice junctions and obtained high-precision detection of splice isoforms, including rare pre-mRNA splicing intermediates.

Multiplexed primer extension sequencing: A targeted RNA-seq method that enables high-precision quantitation of mRNA splicing isoforms and rare pre-mRNA splicing intermediates.

The study of pre-mRNA splicing has been greatly aided by the advent of RNA sequencing (RNA-seq), which enables the genome-wide detection of discrete splice isoforms. Quantification of these splice isoforms requires analysis of splicing informative sequencing reads, those that unambiguously map to a single splice isoform, including exon-intron spanning alignments corresponding to retained introns, as well as exon-exon junction spanning alignments corresponding to either canonically- or alternatively-spliced isoforms. Because most RNA-seq experiments are designed to produce sequencing alignments that uniformly cover the entirety of transcripts, only a comparatively small number of splicing informative alignments are generated for any given splice site, leading to a decreased ability to detect and/or robustly quantify many splice isoforms. To address this problem, we have recently described a method termed Multiplexed Primer Extension sequencing, or MPE-seq, which uses pools of reverse transcription primers to target sequencing to user selected loci. By targeting reverse transcription to pre-mRNA splice junctions, this approach enables a dramatic enrichment in the fraction of splicing informative alignments generated per splicing event, yielding an increase in both the precision with which splicing efficiency can be measured, and in the detection of splice isoforms including rare splicing intermediates. Here we provide a brief review of the shortcomings associated with RNA-seq that drove our development of MPE-seq, as well as a detailed protocol for implementation of MPE-seq.

SARS-CoV-2 infection triggers pro-atherogenic inflammatory responses in human coronary vessels.

Patients with coronavirus disease 2019 (COVID-19) present increased risk for ischemic cardiovascular complications up to 1 year after infection. Although the systemic inflammatory response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection likely contributes to this increased cardiovascular risk, whether SARS-CoV-2 directly infects the coronary vasculature and attendant atherosclerotic plaques remains unknown. Here we report that SARS-CoV-2 viral RNA is detectable and replicates in coronary lesions taken at autopsy from severe COVID-19 cases. SARS-CoV-2 targeted plaque macrophages and exhibited a stronger tropism for arterial lesions than adjacent perivascular fat, correlating with macrophage infiltration levels. SARS-CoV-2 entry was increased in cholesterol-loaded primary macrophages and dependent, in part, on neuropilin-1. SARS-CoV-2 induced a robust inflammatory response in cultured macrophages and human atherosclerotic vascular explants with secretion of cytokines known to trigger cardiovascular events. Our data establish that SARS-CoV-2 infects coronary vessels, inducing plaque inflammation that could trigger acute cardiovascular complications and increase the long-term cardiovascular risk.

SARS-CoV-2 infection triggers pro-atherogenic inflammatory responses in human coronary vessels.

COVID-19 patients present higher risk for myocardial infarction (MI), acute coronary syndrome, and stroke for up to 1 year after SARS-CoV-2 infection. While the systemic inflammatory response to SARS-CoV-2 infection likely contributes to this increased cardiovascular risk, whether SARS-CoV-2 directly infects the coronary vasculature and attendant atherosclerotic plaques to locally promote inflammation remains unknown. Here, we report that SARS-CoV-2 viral RNA (vRNA) is detectable and replicates in coronary atherosclerotic lesions taken at autopsy from patients with severe COVID-19. SARS-CoV-2 localizes to plaque macrophages and shows a stronger tropism for arterial lesions compared to corresponding perivascular fat, correlating with the degree of macrophage infiltration. In vitro infection of human primary macrophages highlights that SARS-CoV-2 entry is increased in cholesterol-loaded macrophages (foam cells) and is dependent, in part, on neuropilin-1 (NRP-1). Furthermore, although viral replication is abortive, SARS-CoV-2 induces a robust inflammatory response that includes interleukins IL-6 and IL-1ß, key cytokines known to trigger ischemic cardiovascular events. SARS-CoV-2 infection of human atherosclerotic vascular explants recapitulates the immune response seen in cultured macrophages, including pro-atherogenic cytokine secretion. Collectively, our data establish that SARS-CoV-2 infects macrophages in coronary atherosclerotic lesions, resulting in plaque inflammation that may promote acute CV complications and long-term risk for CV events.

Minimally invasive prostate cancer detection test using FISH probes.

PURPOSE: The ability to test for and detect prostate cancer with minimal invasiveness has the potential to reduce unnecessary prostate biopsies. This study was conducted as part of a clinical investigation for the development of an OligoFISH(®) probe panel for more accurate detection of prostate cancer. MATERIALS AND METHODS: One hundred eligible male patients undergoing transrectal ultrasound biopsies were enrolled in the study. After undergoing digital rectal examination with pressure, voided urine was collected in sufficient volume to prepare at least two slides using ThinPrep. Probe panels were tested on the slides, and 500 cells were scored when possible. From the 100 patients recruited, 85 had more than 300 cells scored and were included in the clinical performance calculations. RESULTS: Chromosomes Y, 7, 10, 20, 6, 8, 16, and 18 were polysomic in most prostate carcinoma cases. Of these eight chromosomes, chromosomes 7, 16, 18, and 20 were identified as having the highest clinical performance as a fluorescence in situ hybridization test and used to manufacture the fluorescence in situ hybridization probe panels. The OligoFISH(®) probes performed with 100% analytical specificity. When the OligoFISH(®) probes were compared with the biopsy results for each individual, the test results highly correlated with positive and negative prostate biopsy pathology findings, supporting their high specificity and accuracy. Probes for chromosomes 7, 16, 18, and 20 showed in the receiver operator characteristics analysis an area under the curve of 0.83, with an accuracy of 81% in predicting the biopsy result. CONCLUSION: This investigation demonstrates the ease of use with high specificity, high predictive value, and accuracy in identifying prostate cancer in voided urine after digital rectal examination with pressure. The test is likely to have positive impact on clinical practice and advance approaches to the detection of prostate cancer. Further evaluation is warranted.

One-year monitoring of an oligonucleotide fluorescence in situ hybridization probe panel laboratory-developed test for bladder cancer detection.

BACKGROUND: Previously, we had developed and manufactured an oligonucleotide fluorescence in situ hybridization (OligoFISH) probe panel based on the most clinically sensitive chromosomes found in a reference set of bladder carcinoma cases. The panel was clinically validated for use as a diagnostic and monitoring assay for bladder cancer, reaching 100% correlation with the results of the UroVysion test. After 1 year of using this probe panel, we present here the comparison of cytology, cystoscopy, and pathology findings to the OligoFISH probe panel results to calculate its clinical performance. MATERIALS AND METHODS: In order to calculate clinical performance, we compared the OligoFISH results to the cytology and cystoscopy/pathology findings for 147 initial diagnoses and 399 recurrence monitorings. Finally, we compared clinical performance to published values for the UroVysion test, including both low- and high-grade tumors. RESULTS: Chromosomes 3, 6, 7, and 20 were highly involved in bladder carcinoma aneuploidy. At the initial diagnosis, we obtained 90.5% (95% confidence interval [CI]: 84.5%-94.7%) accuracy, 96.8% sensitivity (95% CI: 91.0%-99.3%), 79.2% specificity (95% CI: 65.9%-87.8%), 89.2% positive predictive value (PPV; 95% CI: 81.5%-94.5%), and 93.3% negative predictive value (NPV; 95% CI: 81.7%-97.3%). When monitoring for recurrence, we obtained 85.2% accuracy (95% CI: 81.3%-88.5%), 82.0% sensitivity (95% CI: 76.0%-87.1%), 88.4% specificity (95% CI: 83.2%-92.5%), 87.7% PPV (95% CI: 82.1%-92.0%), and 83.0% NPV (95% CI: 77.3%-87.8%). When looking at low- and high-grade tumors, the test showed 100% sensitivity for high-grade tumors (95% CI: 92.5%-100%) and 87.5% sensitivity (95% CI: 68.8%-95.5%) for low-grade tumors. All the clinical parameters for the OligoFISH panel were higher than the UroVysion test's published performance. We found significantly higher clinical sensitivity and NPV at initial diagnosis and significantly higher specificity and PPV for recurrence. CONCLUSION: The OligoFISH probe panel is a fast, easy, and reproducible test for bladder cancer diagnosis and monitoring, with excellent clinical performance and utility.