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Abstract 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.

Abstract Internal modifications of mRNA have emerged as widespread and versatile regulatory mechanism to control gene expression at the post-transcriptional level. Most of these modifications are methyl groups, making S-adenosyl-L-methionine (SAM) a central metabolic hub. Here we show that metabolic labeling with a clickable metabolic precursor of SAM, propargyl-selenohomocysteine (PSH), enables detection and identification of various methylation sites. Propargylated A, C, and G nucleosides form at detectable amounts via intracellular generation of the corresponding SAM analogue. Integration into next generation sequencing enables mapping of N 6-methyladenosine (m6A) and 5-methylcytidine (m5C) sites in mRNA with single nucleotide precision (MePMe-seq). Analysis of the termination profiles can be used to distinguish m6A from 2′-O-methyladenosine (Am) and N1-methyladenosine (m1A) sites. MePMe-seq overcomes the problems of antibodies for enrichment and sequence-motifs for evaluation, which was limiting previous methodologies. Metabolic labeling via clickable SAM facilitates the joint evaluation of methylation sites in RNA and potentially DNA and proteins.

Abstract Dilated cardiomyopathy is the second most common cause for heart failure with no cure except a high-risk heart transplantation. Approximately 30% of patients harbor heritable mutations which are amenable to CRISPR-based gene therapy. However, challenges related to delivery of the editing complex and off-target concerns hamper the broad applicability of CRISPR agents in the heart. We employ a combination of the viral vector AAVMYO with superior targeting specificity of heart muscle tissue and CRISPR base editors to repair patient mutations in the cardiac splice factor Rbm20, which cause aggressive dilated cardiomyopathy. Using optimized conditions, we repair >70% of cardiomyocytes in two Rbm20 knock-in mouse models that we have generated to serve as an in vivo platform of our editing strategy. Treatment of juvenile mice restores the localization defect of RBM20 in 75% of cells and splicing of RBM20 targets including TTN. Three months after injection, cardiac dilation and ejection fraction reach wild-type levels. Single-nuclei RNA sequencing uncovers restoration of the transcriptional profile across all major cardiac cell types and whole-genome sequencing reveals no evidence for aberrant off-target editing. Our study highlights the potential of base editors combined with AAVMYO to achieve gene repair for treatment of hereditary cardiac diseases.

Abstract We present CARDIO:DE, the first freely available and distributable large German clinical corpus from the cardiovascular domain. CARDIO:DE encompasses 500 clinical routine German doctor’s letters from Heidelberg University Hospital, which were manually annotated. Our prospective study design complies well with current data protection regulations and allows us to keep the original structure of clinical documents consistent. In order to ease access to our corpus, we manually de-identified all letters. To enable various information extraction tasks the temporal information in the documents was preserved. We added two high-quality manual annotation layers to CARDIO:DE, (1) medication information and (2) CDA-compliant section classes. To the best of our knowledge, CARDIO:DE is the first freely available and distributable German clinical corpus in the cardiovascular domain. In summary, our corpus offers unique opportunities for collaborative and reproducible research on natural language processing models for German clinical texts.

Abstract Circular RNAs (circRNAs) are regulators of processes like adipogenesis. Their expression can be modulated by SNPs. We analysed links between BMI-associated SNPs and circRNAs. First, we detected an enrichment of BMI-associated SNPs on circRNA genomic loci in comparison to non-significant variants. Analysis of sex-stratified GWAS data revealed that circRNA genomic loci encompassed more genome-wide significant BMI-SNPs in females than in males. To explore whether the enrichment is restricted to BMI, we investigated nine additional GWAS studies. We showed an enrichment of trait-associated SNPs in circRNAs for four analysed phenotypes (body height, chronic kidney disease, anorexia nervosa and autism spectrum disorder). To analyse the influence of BMI-affecting SNPs on circRNA levels in vitro, we examined rs4752856 located on hsa_circ_0022025. The analysis of heterozygous individuals revealed an increased level of circRNA derived from the BMI-increasing SNP allele. We conclude that genetic variation may affect the BMI partly through circRNAs.

The noncoding RNA RN7SK regulates RNA polymerase II pausing and splicing. Here the authors deplete RN7SK in mouse and human during epidermal stem cell differentiation and reveal a novel role in orchestrating bidirectional transcription of highly expressed gene pairs.

Degradation of nonsense mediated mRNA decay (NMD) substrates is carried out by two seemingly independent pathways, SMG6-mediated endonucleolytic cleavage and/or SMG5-SMG7-induced accelerated deadenylation. Here the authors show that SMG5-SMG7 maintain NMD activity by permitting SMG6 activation.

Alternative RNA splicing varies across the brain. Its mapping at single cell resolution is unclear. Here, the authors provide a spatial and single-cell splicing atlas reporting brain region- and cell type-specific expression of different isoforms in the postnatal mouse brain.

Despite widespread transcription of LncRNA in mammalian systems, their contribution to metabolic homeostasis at the cellular and tissue level remains elusive. Here Pradas-Juni et al. describe a transcription factor–LncRNA pathway that couples hepatocyte nutrient sensing to regulation of glucose metabolism in mice.

Decreased autophagy is a hallmark of ageing, but its inter-tissue regulation is poorly understood. Here, Zhou et al. identify mir-83 in C. elegans, which is transported across tissues and suppresses autophagy, contributing to age-related decline.

Unlike transcriptional regulation of hESC identity, little is known post-transcriptionally. Here, the authors show that the RNA binding protein CSDE1 regulates core components of hESC identity, neurectoderm commitment and neurogenesis to maintain pluripotency and prevent neural differentiation.

Pluripotent stem cells are thought to require a highly active proteostatic machinery. Here, the authors show that CCT8, a subunit of the proteostatic chaperonin complex, is increased in pluripotent stem cells, and that overexpression of CCT8 in worms increases cellular proteostasis and organismal longevity.

Removal of the C. elegans germline substantially increases organismal lifespan. Here, Nakamura et al. show that the transcription factors MML-1 and MXL-2 coordinate this process in that they reduce TOR signalling and increase autophagy by regulating activity of HLH-30.