Detecting m6A at single-molecular resolution via direct RNA sequencing and realistic training data.

Direct RNA sequencing offers the possibility to simultaneously identify canonical bases and epi-transcriptomic modifications in each single RNA molecule. Thus far, the development of computational methods has been hampered by the lack of biologically realistic training data that carries modification labels at molecular resolution. Here, we report on the synthesis of such samples and the development of a bespoke algorithm, mAFiA (m6A Finding Algorithm), that accurately detects single m6A nucleotides in both synthetic RNAs and natural mRNA on single read level. Our approach uncovers distinct modification patterns in single molecules that would appear identical at the ensemble level. Compared to existing methods, mAFiA also demonstrates improved accuracy in measuring site-level m6A stoichiometry in biological samples.

Spine Stereotactic Body Radiation Therapy Without Immobilization: Detailed Analysis of Intrafraction Motion Using High-Frequency kV Imaging During Irradiation.

PURPOSE: Spine stereotactic body radiation therapy (SBRT) requires high positioning accuracy and a stable patient to maximize target coverage and reduce excessive irradiation to organs at risk. Positional verification during spine SBRT delivery helps to ensure accurate positioning for all patients. We report our experience with noninvasive 3-dimensional target position monitoring during volumetric modulated arc therapy of spine metastases in nonimmobilized patients positioned using only a thin mattress and simple arm and knee supports. METHODS AND MATERIALS: Fluoroscopic planar kV images were acquired at 7 frames/s using the on-board imaging system during volumetric modulated arc therapy spine SBRT. Template matching and triangulation were used to track the target in vertical, longitudinal, and lateral directions. If the tracking trace deviated >1 mm from the planned position in ≥1 direction, treatment was manually interrupted and 6-dimensional cone beam computed tomography (CBCT)-based couch correction was performed. Tracking data were used to retrospectively analyze the target position. Positional data, agreement with CBCT, correlation between position of the couch and direction of any positional correction, and treatment times were analyzed. RESULTS: In total, 175 fractions were analyzed. Delivery was interrupted 83 times in 66 fractions for a deviation >1 mm. In 97% of cases the difference between tracking data and subsequent clinical shift performed after the CBCT match was ≤0.5 mm. Lateral/longitudinal shift performed after intervention correlated with the couch roll/pitch at the start of treatment (correlation coefficient, -0.63/0.53). Mean (SD; range) time between start of first imaging and end of the last arc was 15.2 minutes (5.1; 7.6-36.3). CONCLUSIONS: Spine tracking during irradiation can be used to prompt an intervention CBCT scan and repositioning so that a spine SBRT target deviates by ≤1 mm from the planned position, even in nonimmobilized patients. kV tracking and CBCT are in good agreement. The data support verification CBCT after all 6 degrees-of-freedom positional corrections in nonimmobilized spine SBRT patients.

Insight into the mechanism of AML del(9q) progression: hnRNP K targets the myeloid master regulators CEBPA (C/EBPα) and SPI1 (PU.1).

Deletions on the long arm of chromosome 9 (del(9q)) are recurrent abnormalities in about 2 % of acute myeloid leukemia cases, which usually involve HNRNPK and are frequently associated with other known aberrations. Based on an Hnrnpk haploinsufficient mouse model, a recent study demonstrated a function of hnRNP K in pathogenesis of myeloid malignancies via the regulation of cellular proliferation and myeloid differentiation programs. Here, we provide evidence that reduced hnRNP K expression results in the dysregulated expression of C/EBPα and additional transcription factors. CyTOF analysis revealed monocytic skewing with increased levels of mature myeloid cells. To explore the role of hnRNP K during normal and pathological myeloid differentiation in humans, we characterized hnRNP K-interacting RNAs in human AML cell lines. Notably, RNA-sequencing revealed several mRNAs encoding key transcription factors involved in the regulation of myeloid differentiation as targets of hnRNP K. We showed that specific sequence motifs confer the interaction of SPI1 and CEBPA 5' and 3'UTRs with hnRNP K. The siRNA mediated reduction of hnRNP K in human AML cells resulted in an increase of PU.1 and C/EBPα that is most pronounced for the p30 isoform. The combinatorial treatment with the inducer of myeloid differentiation valproic acid resulted in increased C/EBPα expression and myeloid differentiation. Together, our results indicate that hnRNP K post-transcriptionally regulates the expression of myeloid master transcription factors. These novel findings can inaugurate novel options for targeted treatment of AML del(9q) by modulation of hnRNP K function.

Detection of queuosine and queuosine precursors in tRNAs by direct RNA sequencing.

Queuosine (Q) is a complex tRNA modification found in bacteria and eukaryotes at position 34 of four tRNAs with a GUN anticodon, and it regulates the translational efficiency and fidelity of the respective codons that differ at the Wobble position. In bacteria, the biosynthesis of Q involves two precursors, preQ0 and preQ1, whereas eukaryotes directly obtain Q from bacterial sources. The study of queuosine has been challenging due to the limited availability of high-throughput methods for its detection and analysis. Here, we have employed direct RNA sequencing using nanopore technology to detect the modification of tRNAs with Q and Q precursors. These modifications were detected with high accuracy on synthetic tRNAs as well as on tRNAs extracted from Schizosaccharomyces pombe and Escherichia coli by comparing unmodified to modified tRNAs using the tool JACUSA2. Furthermore, we present an improved protocol for the alignment of raw sequence reads that gives high specificity and recall for tRNAs ex cellulo that, by nature, carry multiple modifications. Altogether, our results show that 7-deazaguanine-derivatives such as queuosine are readily detectable using direct RNA sequencing. This advancement opens up new possibilities for investigating these modifications in native tRNAs, furthering our understanding of their biological function.

Adaptive sampling for nanopore direct RNA-sequencing.

Nanopore long-read sequencing enables real-time monitoring and controlling of individual nanopores. This allows us to enrich or deplete specific sequences in DNA sequencing in a process called "adaptive sampling." So far, adaptive sampling (AS) was not applicable to the direct sequencing of RNA. Here, we show that AS is feasible and useful for direct RNA sequencing (DRS), which has its specific technical and biological challenges. Using a well-controlled in vitro transcript-based model system, we identify essential characteristics and parameter settings for AS in DRS, as the superior performance of depletion over enrichment. Here, the efficiency of depletion is close to the theoretical maximum. Additionally, we demonstrate that AS efficiently depletes specific transcripts in transcriptome-wide sequencing applications. Specifically, we applied our AS approach to poly(A)-enriched RNA samples from human-induced pluripotent stem cell-derived cardiomyocytes and mouse whole heart tissue and show efficient 2.5- to 2.8-fold depletion of highly abundant mitochondrial-encoded transcripts. Finally, we characterize depletion and enrichment performance for complex transcriptome subsets, that is, at the level of the entire Chromosome 11, proving the general applicability of direct RNA AS. Our analyses provide evidence that AS is especially useful to enable the detection of lowly expressed transcripts and reduce the sequencing of highly abundant disturbing transcripts.

Comprehensive identification of diverse ribosomal RNA modifications by targeted nanopore direct RNA sequencing and JACUSA2.

Ribosomal RNAs are decorated by numerous post-transcriptional modifications whose exact roles in ribosome biogenesis, function, and human pathophysiology remain largely unknown. Here, we report a targeted direct rRNA sequencing approach involving a substrate selection step and demonstrate its suitability to identify differential modification sites in combination with the JACUSA2 software. We compared JACUSA2 to other tools designed for RNA modification detection and show that JACUSA2 outperforms other software with regard to detection of base modifications such as methylation, acetylation and aminocarboxypropylation. To illustrate its widespread usability, we applied our method to a collection of CRISPR-Cas9 engineered colon carcinoma cells lacking specific enzymatic activities responsible for particular rRNA modifications and systematically compared them to isogenic wild-type RNAs. Besides the numerous 2'-O methylated riboses and pseudouridylated residues, our approach was suitable to reliably identify differential base methylation and acetylation events. Importantly, our method does not require any prior knowledge of modification sites or the need to train complex models. We further report for the first time detection of human rRNA modifications by direct RNA-sequencing on Flongle flow cells, the smallest-scale nanopore flow cell available to date. The use of these smaller flow cells reduces RNA input requirements, making our workflow suitable for the analysis of samples with limited availability and clinical work.

Characterizing alternative splicing effects on protein interaction networks with LINDA.

MOTIVATION: Alternative RNA splicing plays a crucial role in defining protein function. However, despite its relevance, there is a lack of tools that characterize effects of splicing on protein interaction networks in a mechanistic manner (i.e. presence or absence of protein-protein interactions due to RNA splicing). To fill this gap, we present Linear Integer programming for Network reconstruction using transcriptomics and Differential splicing data Analysis (LINDA) as a method that integrates resources of protein-protein and domain-domain interactions, transcription factor targets, and differential splicing/transcript analysis to infer splicing-dependent effects on cellular pathways and regulatory networks. RESULTS: We have applied LINDA to a panel of 54 shRNA depletion experiments in HepG2 and K562 cells from the ENCORE initiative. Through computational benchmarking, we could show that the integration of splicing effects with LINDA can identify pathway mechanisms contributing to known bioprocesses better than other state of the art methods, which do not account for splicing. Additionally, we have experimentally validated some of the predicted splicing effects that the depletion of HNRNPK in K562 cells has on signalling.

Mapping of RNA Modifications by Direct Nanopore Sequencing and JACUSA2.

RNA modifications exist in all kingdom of life. Several different types of base or ribose modifications are now summarized under the term "epitranscriptome." With the advent of high-throughput sequencing technologies, much progress has been made in understanding RNA modification biology and how these modifications can influence many aspects of RNA life. The most widespread internal modification on mRNA is m6A, which has been implicated in physiological processes as well as disease pathogenesis. Here, we provide a workflow for the mapping of m6A sites using Nanopore direct RNA sequencing data. Our strategy employs pairwise comparison of basecalling error profiles with JACUSA2. We outline a general strategy for RNA modification detection on mRNA and describe two specific use cases on m6A detection in detail. Use case 1: a sample of interest with modifications (e.g., "wild-type" sample) is compared to a sample lacking a specific modification type (e.g., "knockout" sample, here METTL3-KO) or Use case 2: a sample of interest with modifications is compared to a sample lacking all modifications (e.g., in vitro transcribed cDNA). We provide a detailed protocol on experimental and computational aspects. Extensive online material provides a snakemake pipeline to identify m6A positions in mRNA and to validate the results against a miCLIP-derived m6A reference set. The general strategy is flexible and can be easily adapted by users in different application scenarios.

Full-Length Spatial Transcriptomics Reveals the Unexplored Isoform Diversity of the Myocardium Post-MI.

We introduce Single-cell Nanopore Spatial Transcriptomics (scNaST), a software suite to facilitate the analysis of spatial gene expression from second- and third-generation sequencing, allowing to generate a full-length near-single-cell transcriptional landscape of the tissue microenvironment. Taking advantage of the Visium Spatial platform, we adapted a strategy recently developed to assign barcodes to long-read single-cell sequencing data for spatial capture technology. Here, we demonstrate our workflow using four short axis sections of the mouse heart following myocardial infarction. We constructed a de novo transcriptome using long-read data, and successfully assigned 19,794 transcript isoforms in total, including clinically-relevant, but yet uncharacterized modes of transcription, such as intron retention or antisense overlapping transcription. We showed a higher transcriptome complexity in the healthy regions, and identified intron retention as a mode of transcription associated with the infarct area. Our data revealed a clear regional isoform switching among differentially used transcripts for genes involved in cardiac muscle contraction and tissue morphogenesis. Molecular signatures involved in cardiac remodeling integrated with morphological context may support the development of new therapeutics towards the treatment of heart failure and the reduction of cardiac complications.

Targeted Analysis of circRNA Expression in Patient Samples by Lexo-circSeq.

Recently, circular RNAs (circRNAs) have been extensively studied in animals and plants. circRNAs are generated by backsplicing from the same linear transcripts that are canonically spliced to produce, for example, mature mRNAs. circRNAs exhibit tissue-specific expression and are potentially involved in many diseases, among them cardiovascular diseases. The comprehensive analysis of circRNA expression patterns across larger patient cohorts requires a streamlined and cost-effective workflow designed to meet small input requirements. In this article, we present Lexo-circSeq, a targeted RNA sequencing approach that can profile up to 110 circRNAs and their corresponding linear transcripts in one experiment. We established Lexo-circSeq employing total human heart RNA and show that our protocol can detect depletion of a specific circRNA in hiPSC-derived cardiomyocytes. Finally, Lexo-circSeq was applied to biopsies from patients diagnosed with dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), respectively. Interestingly, our results indicate that circular-to-linear-ratios for circSLC8A1 and circRBM33 are deregulated in cardiomyopathy.

RNA modification mapping with JACUSA2.

Several high-throughput antibody-free methods for RNA modification detection from sequencing data have been developed. We present JACUSA2 as a versatile software solution and comprehensive analysis framework for RNA modification detection assays that are based on either the Illumina or Nanopore platform. Importantly, JACUSA2 can integrate information from multiple experiments, such as replicates and different conditions, and different library types, such as first- or second-strand cDNA libraries. We demonstrate its utility, showing analysis workflows for N6-methyladenosine (m6A) and pseudouridine (Ψ) detection on Illumina and Nanopore sequencing data sets. Our software and its R helper package are available as open source solutions.

Seroprevalence of anti-Leptospira antibodies in equines and associated workers-Isolation of Leptospira interrogans serogroup Canicola from equine urine.

To investigate seroprevalence of anti-Leptospira antibodies in equines and associated workers in Uruguay, 891 equine and 150 human sera were drawn; 212 equine urine samples were also taken for culture. Environmental conditions and equine raising or managing practices were recorded in all 72 visited establishments; epidemiological information was obtained from each worker. Microscopic agglutination technique (MAT) was performed with 10 Leptospira strains for equines and 18 for human sera, that were also studied with IgM indirect immunofluorescence (IgM-IIF). Equine titres ≥100 were considered positive, and human sera titres ≥200 suggested probable recent or past infection. Urines were cultured in Ellinghausen-McCullough-Johnson-Harris (EMJH) media; local identification of one obtained isolate with lipL32 PCR, Multiple Locus Variable number tandem repeat Analysis and partial rrs gene sequencing, were completed at Institut Pasteur, Paris. Estimated reactivity was 61.3% for equines, which was higher than the studied bovine national levels (21%) and mainly observed with Icterohaemorrhagiae serogroup (40.3%), Sejroe, Canicola, Pomona or Ballum. Aged animals from slaughterhouses and cattle farms were the most frequently positive. Multiple regression analysis confirmed a significant association between seropositivity and equine age. Only one positive culture could be fully studied, and confirmed to be Leptospira interrogans serogroup Canicola; it was added to the MAT antigen panel and revealed fairly frequent reaction with equine and human sera. Three workers (2%) showed titres = 200 with Icterohaemorrhagiae or Canicola serogroups, without recent clinical manifestations. Their attended equines reacted with the same serogroups, suggesting common source infections or infection transmitted by equines. Three other humans yielded titres = 100, and none of the 150 showed an IgM-IIF-positive result. Equines seem not to be an important origin of regional human leptospirosis, except perhaps during acute animal infection. More culture work is required to study intensity and lapses of leptospiruria, as well as to further identify circulating strains.

Improved nanopore direct RNA sequencing of cardiac myocyte samples by selective mt-RNA depletion.

RNA sequencing is a powerful tool to analyze gene expression transcriptome wide. However, RNA sequencing in general and especially the recently developed methods of long read RNA sequencing are still low-throughput and cost-intensive. Here, one important design choice is to concentrate the sequencing capacity on specific parts of the transcriptome. Especially, abundant transcripts as ribosomal RNAs may dominate the available sequencing space, if not removed prior to sequencing. Several methods exist to reduce ribosomal RNA read numbers: either based on enrichment of the relevant fraction (polyA+ RNA) or depletion, respectively degradation of ribosomal RNAs. Furthermore, commercial kits are available to deplete globin transcripts from blood samples. However, so far, no solution exists to deal with other tissue-specific highly abundant transcripts. This is especially of interest in the heart and other muscle derived samples, where reads originating from mitochondrial RNAs make up to 30% of reads in polyA+ selected libraries and around 70% in single cell sequencing experiments. We present a simple method to diminish sequencing of mitochondrial RNAs in Oxford Nanopore direct RNA sequencing libraries by RNase H based clipping of the polyA tail. We show that mt-clipping enables enhanced detection of cytoplasmic mRNAs, among them genes involved in heart development and pathogenesis. Mt-clipping may be applied as well to other sequencing protocols that are based on oligo(dT) priming and can be easily adapted to other tissue-specific high-abundant transcripts.

Relationship between Treatment Plan Dosimetry, Toxicity, and Survival following Intensity-Modulated Radiotherapy, with or without Chemotherapy, for Stage III Inoperable Non-Small Cell Lung Cancer.

Concurrent chemoradiotherapy (cCRT) is the preferred treatment for stage III NSCLC because surgery containing multimodality treatment is often not appropriate. Alternatives, often for less fit patients, include sequential CRT and RT alone. Many reports describing the relationship between overall survival (OS), toxicity, and dosimetry are based on clinical trials, with strict criteria for patient selection. We performed an institutional analysis to study the relationship between dosimetric parameters, toxicity, and OS in inoperable patients with stage III NSCLC treated with (hybrid) IMRT/VMAT-based techniques in routine clinical practice. Eligible patients had undergone treatment with radical intent using cCRT, sCRT, or RT alone, planned to a total dose ≥ 50 Gy delivered in ≥15 fractions. All analyses were performed for two patient groups, (1) cCRT (n = 64) and (2) sCRT/RT (n = 65). The toxicity rate differences between the two groups were not significant, and OS was 29 and 17 months, respectively. For sCRT/RT, no dosimetric factors were associated with OS, whereas for cCRT, PTV-volume, esophagus V50 Gy, and contralateral lung V5 Gy were associated. cCRT OS was significantly lower in patients with esophagitis ≥ G2. The overall rate of ≥G3 pneumonitis was low (3%), and the rate of high-grade esophagitis the OS in this real-world patient population was comparable to those reported in clinical trials. Based on this hypothesis-generating data, more aggressive esophageal sparing merits consideration. Institutional auditing and benchmarking of the planning strategy, dosimetry, and outcome have an important role to play in the continuous quality improvement process.

A comparison of metabolic labeling and statistical methods to infer genome-wide dynamics of RNA turnover.

Metabolic labeling of newly transcribed RNAs coupled with RNA-seq is being increasingly used for genome-wide analysis of RNA dynamics. Methods including standard biochemical enrichment and recent nucleotide conversion protocols each require special experimental and computational treatment. Despite their immediate relevance, these technologies have not yet been assessed and benchmarked, and no data are currently available to advance reproducible research and the development of better inference tools. Here, we present a systematic evaluation and comparison of four RNA labeling protocols: 4sU-tagging biochemical enrichment, including spike-in RNA controls, SLAM-seq, TimeLapse-seq and TUC-seq. All protocols are evaluated based on practical considerations, conversion efficiency and wet lab requirements to handle hazardous substances. We also compute decay rate estimates and confidence intervals for each protocol using two alternative statistical frameworks, pulseR and GRAND-SLAM, for over 11 600 human genes and evaluate the underlying computational workflows for their robustness and ease of use. Overall, we demonstrate a high inter-method reliability across eight use case scenarios. Our results and data will facilitate reproducible research and serve as a resource contributing to a fuller understanding of RNA biology.

P23 Acts as Functional RBP in the Macrophage Inflammation Response.

Macrophages exert the primary cellular immune response. Pathogen components like bacterial lipopolysaccharides (LPS) stimulate macrophage migration, phagocytotic activity and cytokine expression. Previously, we identified the poly(A)+ RNA interactome of RAW 264.7 macrophages. Of the 402 RNA-binding proteins (RBPs), 32 were classified as unique in macrophages, including nineteen not reported to interact with nucleic acids before. Remarkably, P23 a HSP90 co-chaperone, also known as cytosolic prostaglandin E2 synthase (PTGES3), exhibited differential poly(A)+ RNA binding in untreated and LPS-induced macrophages. To identify mRNAs bound by P23 and to elucidate potential regulatory RBP functions in macrophages, we immunoprecipitated P23 from cytoplasmic extracts of cross-linked untreated and LPS-induced cells. RNAseq revealed that enrichment of 44 mRNAs was reduced in response to LPS. Kif15 mRNA, which encodes kinesin family member 15 (KIF15), a motor protein implicated in cytoskeletal reorganization and cell mobility was selected for further analysis. Noteworthy, phagocytic activity of LPS-induced macrophages was enhanced by P23 depletion. Specifically, in untreated RAW 264.7 macrophages, decreased P23 results in Kif15 mRNA destabilization, diminished KIF15 expression and accelerated macrophage migration. We show that the unexpected RBP function of P23 contributes to the regulation of macrophage phagocytotic activity and migration.

Methylated HNRNPK acts on RPS19 to regulate ALOX15 synthesis in erythropoiesis.

Post-transcriptional control is essential to safeguard structural and metabolic changes in enucleated reticulocytes during their terminal maturation to functional erythrocytes. The timely synthesis of arachidonate 15-lipoxygenase (ALOX15), which initiates mitochondria degradation at the final stage of reticulocyte maturation is regulated by the multifunctional protein HNRNPK. It constitutes a silencing complex at the ALOX15 mRNA 3' untranslated region that inhibits translation initiation at the AUG by impeding the joining of ribosomal 60S subunits to 40S subunits. To elucidate how HNRNPK interferes with 80S ribosome assembly, three independent screens were applied. They consistently demonstrated a differential interaction of HNRNPK with RPS19, which is localized at the head of the 40S subunit and extends into its functional center. During induced erythroid maturation of K562 cells, decreasing arginine dimethylation of HNRNPK is linked to a reduced interaction with RPS19 in vitro and in vivo. Dimethylation of residues R256, R258 and R268 in HNRNPK affects its interaction with RPS19. In noninduced K562 cells, RPS19 depletion results in the induction of ALOX15 synthesis and mitochondria degradation. Interestingly, residue W52 in RPS19, which is frequently mutated in Diamond-Blackfan Anemia (DBA), participates in specific HNRNPK binding and is an integral part of a putative aromatic cage.

On the optimal design of metabolic RNA labeling experiments.

Massively parallel RNA sequencing (RNA-seq) in combination with metabolic labeling has become the de facto standard approach to study alterations in RNA transcription, processing or decay. Regardless of advances in the experimental protocols and techniques, every experimentalist needs to specify the key aspects of experimental design: For example, which protocol should be used (biochemical separation vs. nucleotide conversion) and what is the optimal labeling time? In this work, we provide approximate answers to these questions using the asymptotic theory of optimal design. Specifically, we investigate, how the variance of degradation rate estimates depends on the time and derive the optimal time for any given degradation rate. Subsequently, we show that an increase in sample numbers should be preferred over an increase in sequencing depth. Lastly, we provide some guidance on use cases when laborious biochemical separation outcompetes recent nucleotide conversion based methods (such as SLAMseq) and show, how inefficient conversion influences the precision of estimates. Code and documentation can be found at https://github.com/dieterich-lab/DesignMetabolicRNAlabeling.

Characterization of acute myeloid leukemia with del(9q) - Impact of the genes in the minimally deleted region.

Acute myeloid leukemia is an aggressive disease that arises from clonal expansion of malignant hematopoietic precursor cells of the bone marrow. Deletions on the long arm of chromosome 9 (del(9q)) are observed in 2% of acute myeloid leukemia patients. Our deletion analysis in a cohort of 31 del(9q) acute myeloid leukemia patients further supports the importance of a minimally deleted region composed of seven genes potentially involved in leukemogenesis: GKAP1, KIF27, C9ORF64, HNRNPK, RMI1, SLC28A3 and NTRK2. Importantly, among them HNRNPK, encoding heterogeneous nuclear ribonucleoprotein K is proposed to function in leukemogenesis. We show that expression of HNRNPK and the other genes of the minimally deleted region is significantly reduced in patients with del(9q) compared with normal karyotype acute myeloid leukemia. Also, two mRNAs interacting with heterogeneous nuclear ribonucleoprotein K, namely CDKN1A and CEBPA are significantly downregulated. While the deletion size is not correlated with outcome, associated genetic aberrations are important. Patients with an additional t(8;21) show a good prognosis. RUNX1-RUNX1T1, which emerges from the t(8;21) leads to transcriptional down-regulation of CEBPA. Acute myeloid leukemia patients with mutations in CEBPA have a good prognosis as well. Interestingly, in del(9q) patients with CEBPA mutation mRNA levels of HNRNPK and the other genes located in the minimally deleted region is restored to normal karyotype level. Our data indicate that a link between CEBPA and the genes of the minimally deleted region, among them HNRNPK contributes to leukemogenesis in acute myeloid leukemia with del(9q).