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Abstract RNA editing is a highly conserved process. Adenosine deaminase acting on RNA (ADAR) mediated deamination of adenosine (A-to-I editing) is associated with human disease and immune checkpoint control. Functional implications of A-to-I editing are currently of broad interest to academic and industrial research as underscored by the fast-growing number of clinical studies applying base editors as therapeutic tools. Analyzing the dynamics of A-to-I editing, in a biological or therapeutic context, requires the sensitive detection of differential A-to-I editing, a currently unmet need. We introduce the local differential editing index (LoDEI) to detect differential A-to-I editing in RNA-seq datasets using a sliding-window approach coupled with an empirical q value calculation that detects more A-to-I editing sites at the same false-discovery rate compared to existing methods. LoDEI is validated on known and novel datasets revealing that the oncogene MYCN increases and that a specific small non-coding RNA reduces A-to-I editing.

Abstract Improved risk stratification of patients suspected of prostate cancer prior to biopsy continues to be an unmet clinical need. ExoDx Prostate (IntelliScore) “EPI” is a non-invasive urine test utilizing RNA from exosomes to provide a risk score that correlates with the likelihood of finding high grade prostate cancer at biopsy. Here, we present the results from a prospective clinical validation study of EPI-CE, a CE-marked in-vitro diagnostic (IVD) assay, specifically developed for use in European clinical laboratories. The study (NCT04720599) enrolled patients with ≥ 50 years, PSA 2–10 ng/mL, prior to MRI, who were scheduled for initial biopsy. First catch urine samples were collected from participants without prior digital rectal examination or prostate massage. Exosomal RNA was isolated and expression levels of three biomarkers ERG, PCA3 and SPDEF were analyzed according to the EPI-CE Instructions For Use. In the study cohort of N = 109 patients, EPI-CE was validated to have a Negative Predictive Value of 89%, a Sensitivity of 92% and a superior performance to two commonly used multiparametric risk calculators (PCPT and ERSPC) in both Receiver Operating Characteristics with a higher Area Under the Curve for EPI-CE 0.67 (95% CI 0.56–0.77) versus PCPT 0.59 (95% CI 0.47–0.71) and ERSPC 0.60 (95% CI 0.49–0.72) and higher Net Benefits analysis across a wide range of risk acceptance levels. This is the first clinical study reporting on the performance of EPI-CE. We demonstrate that EPI-CE provides information beyond standard clinical parameters and provides a better risk assessment prior to MRI, of patients suspected of prostate cancer, than the commonly used multiparametric risk calculators.

Abstract Background Initial prostate biopsy often fails to identify prostate cancer resulting in patient anxiety, especially when clinical features such as prostate specific antigen (PSA) remain elevated, leading to the need for repeat biopsies. Prostate biomarker tests, such as the ExoDx™ Prostate (IntelliScore), or EPI test, have been shown to provide individualized risk assessment of clinically significant prostate cancer at initial biopsy; however, the performance in the repeat biopsy setting is not well established. Methods As part of a previous prospective clinical validation study evaluating the performance of the EPI test, we collected first-catch, non-DRE urine samples across 22 sites from men with at least one prior negative biopsy scheduled to undergo a repeat prostate biopsy to rule out prostate cancer. All men were 50 years or older with a PSA 2–10 ng/mL. Exosomal mRNA was extracted and expression of three genomic markers, PCA3, ERG and SPDEF was measured. The resulting EPI score was correlated with biopsy results. Results 229 men with a prior negative biopsy underwent repeat biopsies. ExoDx Prostate demonstrated good performance ruling out high-grade (Grade group 2, GG2, or higher) prostate cancer (HGPCa) using the previously validated 15.6 cut point in the initial biopsy setting. The EPI test yielded an NPV of 92% independent of other clinical features and would have avoided 26% of unnecessary biopsies while missing only five patients with HGPCa (2.1%). Furthermore, the EPI test provided additional information at a cut-point of 20 and 29.6 with an NPV of 94%, potentially delaying 35 and 61% of unnecessary biopsies, respectively. AUC curves and Net Health Benefit Analyses demonstrated superior performance of ExoDx Prostate over PSA and clinical only risk calculators, i.e. ERSPC. Conclusions The EPI test provided good performance using the 15.6 cut-point for ruling out HGPCa / GG2 or higher in men undergoing a repeat prostate biopsy with a PSA of 2–10 ng/ml. Furthermore, the test utilizes gene expression data independent of clinical features to predict the likelihood of HGPCa / GG2 on a subsequent needle biopsy.

For transcription through chromatin, RNA polymerase (Pol) II associates with elongation factors (EFs). Here we show that many EFs crosslink to RNA emerging from transcribing Pol II in the yeast Saccharomyces cerevisiae. Most EFs crosslink preferentially to mRNAs, rather than unstable non-coding RNAs. RNA contributes to chromatin association of many EFs, including the Pol II serine 2 kinases Ctk1 and Bur1 and the histone H3 methyltransferases Set1 and Set2. The Ctk1 kinase complex binds RNA in vitro, consistent with direct EF-RNA interaction. Set1 recruitment to genes in vivo depends on its RNA recognition motifs (RRMs). These results strongly suggest that nascent RNA contributes to EF recruitment to transcribing Pol II. We propose that EF-RNA interactions facilitate assembly of the elongation complex on transcribed genes when RNA emerges from Pol II, and that loss of EF-RNA interactions upon RNA cleavage at the polyadenylation site triggers disassembly of the elongation complex.

Abstract Background The definite diagnosis of prion diseases such as Creutzfeldt-Jakob disease (CJD) in humans or bovine spongiform encephalopathy (BSE) in cattle currently relies on the post mortem detection of the pathological form of the prion protein (PrPSc) in brain tissue. Infectivity studies indicate that PrPSc may also be present in body fluids, even at presymptomatic stages of the disease, albeit at concentrations well below the detection limits of currently available analytical methods. Results We developed a highly sensitive method for detecting prion protein aggregates that takes advantage of kinetic differences between seeded and unseeded polymerization of prion protein monomers. Detection of the aggregates was carried out by flow cytometry. In the presence of prion seeds, the association of labelled recombinant PrP monomers in plasma and serum proceeds much more efficiently than in the absence of seeds. In a diagnostic model system, synthetic PrP aggregates were detected down to a concentration of approximately 10-8 nM [0.24 fg/ml]. A specific signal was detected in six out of six available serum samples from BSE-positive cattle. Conclusion We have developed a method based on seed-dependent PrP fibril formation that shows promising results in differentiating a small number of BSE-positive serum samples from healthy controls. This method may provide the basis for an ante mortem diagnostic test for prion diseases.

Background: Discriminating indolent from clinically significant prostate cancer (PCa) in the initial biopsy setting remains an important issue. Prospectively evaluated diagnostic assays are necessary to ensure efficacy and clinical adoption., Objective: Performance and utility assessment of ExoDx Prostate (IntelliScore) (EPI) urine exosome gene expression assay versus standard clinical parameters for discriminating Grade Group (GG) ≥2 PCa from GG1 PCa and benign disease on initial biopsy., Design, Setting, and Participants: A two-phase adaptive clinical utility study (NCT03031418) comparing EPI results with biopsy outcomes in men, with age ≥50 yr and prostate-specific antigen (PSA) 2-10ng/ml, scheduled for initial prostate biopsy. After EPI performance assessment during phase I, a clinical implementation document (ie, CarePath) was developed for utilizing the EPI test in phase II, where the biopsy decision is uncertain., Outcome Measurements and Statistical Analysis: Performance evaluation of the EPI test in patients enrolled in phase I and publication of a consensus CarePath for phase II., Results and Limitations: In a total of 503 patients, with median age of 64 yr, median PSA 5.4ng/ml, 14% African American, 70% Caucasian, 53% positive biopsy rate (22% GG1, 17% GG2, and 15% ≥ GG3), EPI was superior to an optimized model of standard clinical parameters with an area under the curve (AUC) 0.70 versus 0.62, respectively, comparable with previously published results (n=519 patients, EPI AUC 0.71). Validated cut-point 15.6 would avoid 26% of unnecessary prostate biopsies and 20% of total biopsies, with negative predictive value (NPV) 89% and missing 7% of ≥GG2 PCa. Alternative cut-point 20 would avoid 40% of unnecessary biopsies and 31% of total biopsies, with NPV 89% and missing 11% of ≥GG2 PCa. The clinical investigators reached consensus recommending use of the 15.6 cut-point for phase II. Outcome of the decision impact cohort in phase II will be reported separately., Conclusions: EPI is a noninvasive, easy-to-use, gene expression urine assay, which has now been successfully validated in over 1000 patients across two prospective validation trials to stratify risk of ≥GG2 from GG1 cancer and benign disease. The test improves identification of patients with higher grade disease and would reduce the total number of unnecessary biopsies., Patient Summary: It is challenging to predict which men are likely to have high-grade prostate cancer (PCa) at initial biopsy with prostate-specific antigen 2-10ng/ml. This study further demonstrates that the ExoDx Prostate (IntelliScore) test can predict ≥GG2 PCa at initial biopsy and defer unnecessary biopsies better than existing risk calculator's and standard clinical data., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

For transcription through chromatin, RNA polymerase (Pol) II associates with elongation factors (EFs). Here we show that many EFs crosslink to RNA emerging from transcribing Pol II in the yeast Saccharomyces cerevisiae . Most EFs crosslink preferentially to mRNAs, rather than unstable non-coding RNAs. RNA contributes to chromatin association of many EFs, including the Pol II serine 2 kinases Ctk1 and Bur1 and the histone H3 methyltransferases Set1 and Set2. The Ctk1 kinase complex binds RNA in vitro, consistent with direct EF-RNA interaction. Set1 recruitment to genes in vivo depends on its RNA recognition motifs (RRMs). These results strongly suggest that nascent RNA contributes to EF recruitment to transcribing Pol II. We propose that EF-RNA interactions facilitate assembly of the elongation complex on transcribed genes when RNA emerges from Pol II, and that loss of EF-RNA interactions upon RNA cleavage at the polyadenylation site triggers disassembly of the elongation complex.

At the end of protein-coding genes, RNA polymerase (Pol) II undergoes a concerted transition that involves 3'-processing of the pre-mRNA and transcription termination. Here, we present a genome-wide analysis of the 3'-transition in budding yeast. We find that the 3'-transition globally requires the Pol II elongation factor Spt5 and factors involved in the recognition of the polyadenylation (pA) site and in endonucleolytic RNA cleavage. Pol II release from DNA occurs in a narrow termination window downstream of the pA site and requires the "torpedo" exonuclease Rat1 (XRN2 in human). The Rat1-interacting factor Rai1 contributes to RNA degradation downstream of the pA site. Defects in the 3'-transition can result in increased transcription at downstream genes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Biogenesis of eukaryotic messenger ribonucleoprotein complexes (mRNPs) involves the synthesis, splicing, and 3' processing of pre-mRNA, and the assembly of mature mRNPs for nuclear export. We mapped 23 mRNP biogenesis factors onto the yeast transcriptome, providing 10(4)-10(6) high-confidence RNA interaction sites per factor. The data reveal how mRNP biogenesis factors recognize pre-mRNA elements in vivo. They define conserved interactions between splicing factors and pre-mRNA introns, including the recognition of intron-exon junctions and the branchpoint. They also identify a unified arrangement of 3' processing factors at pre-mRNA polyadenylation (pA) sites in yeast and human, which results from an A-U sequence bias at pA sites. Global data analysis indicates that 3' processing factors have roles in splicing and RNA surveillance, and that they couple mRNP biogenesis events to restrict nuclear export to mature mRNPs., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Pervasive transcription of eukaryotic genomes stems to a large extent from bidirectional promoters that synthesize mRNA and divergent noncoding RNA (ncRNA). Here, we show that ncRNA transcription in the yeast S. cerevisiae is globally restricted by early termination that relies on the essential RNA-binding factor Nrd1. Depletion of Nrd1 from the nucleus results in 1,526 Nrd1-unterminated transcripts (NUTs) that originate from nucleosome-depleted regions (NDRs) and can deregulate mRNA synthesis by antisense repression and transcription interference. Transcriptome-wide Nrd1-binding maps reveal divergent NUTs at most promoters and antisense NUTs in most 3' regions of genes. Nrd1 and its partner Nab3 preferentially bind RNA motifs that are depleted in mRNAs and enriched in ncRNAs and some mRNAs whose synthesis is controlled by transcription attenuation. These results define a global mechanism for transcriptome surveillance that selectively terminates ncRNA synthesis to provide promoter directionality and to suppress antisense transcription., (Copyright © 2013 Elsevier Inc. All rights reserved.)