Intervention with metabolites emulating endogenous cell transitions accelerates muscle regeneration in young and aged mice.

Tissue regeneration following an injury requires dynamic cell-state transitions that allow for establishing the cell identities required for the restoration of tissue homeostasis and function. Here, we present a biochemical intervention that induces an intermediate cell state mirroring a transition identified during normal differentiation of myoblasts and other multipotent and pluripotent cells to mature cells. When applied in somatic differentiated cells, the intervention, composed of one-carbon metabolites, reduces some dedifferentiation markers without losing the lineage identity, thus inducing limited reprogramming into a more flexible cell state. Moreover, the intervention enabled accelerated repair after muscle injury in young and aged mice. Overall, our study uncovers a conserved biochemical transitional phase that enhances cellular plasticity in vivo and hints at potential and scalable biochemical interventions of use in regenerative medicine and rejuvenation interventions that may be more tractable than genetic ones.

Overcoming muscle stem cell aging.

Reduced muscle strength and mass is one of the hallmarks of physiological aging in humans and can result in severe impairment of the quality of life. In part this is caused by a functional loss of the highly specialized muscle stem cells (MuSCs), which in healthy conditions provide maintenance, growth, and regeneration. Recent progress in understanding of the stem cell niche and results from single cell technologies reveal exciting insights at unprecedented detail into MuSCs and muscle biology during aging. Here, we review this field and discuss the implications of current findings with a focus on cellular reprogramming approaches as a novel therapeutic avenue for age-related muscle decline.

In vivo partial cellular reprogramming enhances liver plasticity and regeneration.

Mammals have limited regenerative capacity, whereas some vertebrates, like fish and salamanders, are able to regenerate their organs efficiently. The regeneration in these species depends on cell dedifferentiation followed by proliferation. We generate a mouse model that enables the inducible expression of the four Yamanaka factors (Oct-3/4, Sox2, Klf4, and c-Myc, or 4F) specifically in hepatocytes. Transient in vivo 4F expression induces partial reprogramming of adult hepatocytes to a progenitor state and concomitantly increases cell proliferation. This is indicated by reduced expression of differentiated hepatic-lineage markers, an increase in markers of proliferation and chromatin modifiers, global changes in DNA accessibility, and an acquisition of liver stem and progenitor cell markers. Functionally, short-term expression of 4F enhances liver regenerative capacity through topoisomerase2-mediated partial reprogramming. Our results reveal that liver-specific 4F expression in vivo induces cellular plasticity and counteracts liver failure, suggesting that partial reprogramming may represent an avenue for enhancing tissue regeneration.

Unlocking Tissue Regenerative Potential by Epigenetic Reprogramming.

The regeneration potential of axons projecting from retinal ganglion cells (RGCs) is lost shortly after birth. In Nature, Lu et al. (2020) demonstrate that epigenetic reprogramming of RGCs by overexpression of Oct4, Sox2, and Klf4 leads to axon regeneration and restoration of vision in a glaucoma model and aged mice.

Expanding the Toolbox and Targets for Gene Editing.

Genome editing holds great promise for treating a range of human genetic diseases. While emerging clustered regularly interspaced short-palindromic repeats (CRISPR) technologies allow editing of the nuclear genome, it is still not possible to precisely manipulate mitochondrial DNA (mtDNA). Here, we summarize past developments and recent advances in nuclear and mitochondrial genome editing.

Single-cell omics in ageing: a young and growing field.

Organismal ageing results from interlinked molecular changes in multiple organs over time. The study of ageing at the molecular level is complicated by varying decay characteristics and kinetics-both between and within organs-driven by intrinsic and extracellular factors. Emerging single-cell omics methods allow for molecular and spatial profiling of cells, and probing of regulatory states and cell-fate determination, thus providing promising tools for unravelling the heterogeneous process of ageing and making it amenable to intervention. These new strategies are enabled by advances in genomic, epigenomic and transcriptomic technologies. Combined with methods for proteome and metabolome analysis, single-cell techniques provide multidimensional, integrated data with unprecedented detail and throughput. Here, we provide an overview of the current state, and perspectives on the future, of this emerging field. We discuss how single-cell approaches can advance understanding of mechanisms underlying organismal ageing and aid in the development of interventions for ageing and ageing-associated diseases.

The Translational Landscape of the Human Heart.

Gene expression in human tissue has primarily been studied on the transcriptional level, largely neglecting translational regulation. Here, we analyze the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation. We show extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner. Translation downstream of predicted disease-causing protein-truncating variants appears to be frequent, suggesting inefficient translation termination. We identify hundreds of previously undetected microproteins, expressed from lncRNAs and circRNAs, for which we validate the protein products in vivo. The translation of microproteins is not restricted to the heart and prominent in the translatomes of human kidney and liver. We associate these microproteins with diverse cellular processes and compartments and find that many locate to the mitochondria. Importantly, dozens of microproteins are translated from lncRNAs with well-characterized noncoding functions, indicating previously unrecognized biology.

Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function.

Hundreds of circular RNAs (circRNAs) are highly abundant in the mammalian brain, often with conserved expression. Here we show that the circRNA Cdr1as is massively bound by the microRNAs (miRNAs) miR-7 and miR-671 in human and mouse brains. When the Cdr1as locus was removed from the mouse genome, knockout animals displayed impaired sensorimotor gating-a deficit in the ability to filter out unnecessary information-which is associated with neuropsychiatric disorders. Electrophysiological recordings revealed dysfunctional synaptic transmission. Expression of miR-7 and miR-671 was specifically and posttranscriptionally misregulated in all brain regions analyzed. Expression of immediate early genes such as Fos, a direct miR-7 target, was enhanced in Cdr1as-deficient brains, providing a possible molecular link to the behavioral phenotype. Our data indicate an in vivo loss-of-function circRNA phenotype and suggest that interactions between Cdr1as and miRNAs are important for normal brain function.

A map of human circular RNAs in clinically relevant tissues.

Cellular circular RNAs (circRNAs) are generated by head-to-tail splicing and are present in all multicellular organisms studied so far. Recently, circRNAs have emerged as a large class of RNA which can function as post-transcriptional regulators. It has also been shown that many circRNAs are tissue- and stage-specifically expressed. Moreover, the unusual stability and expression specificity make circRNAs important candidates for clinical biomarker research. Here, we present a circRNA expression resource of 20 human tissues highly relevant to disease-related research: vascular smooth muscle cells (VSMCs), human umbilical vein cells (HUVECs), artery endothelial cells (HUAECs), atrium, vena cava, neutrophils, platelets, cerebral cortex, placenta, and samples from mesenchymal stem cell differentiation. In eight different samples from a single donor, we found highly tissue-specific circRNA expression. Circular-to-linear RNA ratios revealed that many circRNAs were expressed higher than their linear host transcripts. Among the 71 validated circRNAs, we noticed potential biomarkers. In adenosine deaminase-deficient, severe combined immunodeficiency (ADA-SCID) patients and in Wiskott-Aldrich-Syndrome (WAS) patients' samples, we found evidence for differential circRNA expression of genes that are involved in the molecular pathogenesis of both phenotypes. Our findings underscore the need to assess circRNAs in mechanisms of human disease. KEY MESSAGES: circRNA resource catalog of 20 clinically relevant tissues. circRNA expression is highly tissue-specific. circRNA transcripts are often more abundant than their linear host RNAs. circRNAs can be differentially expressed in disease-associated genes.

Identification and Characterization of Circular RNAs As a New Class of Putative Biomarkers in Human Blood.

Covalently closed circular RNA molecules (circRNAs) have recently emerged as a class of RNA isoforms with widespread and tissue specific expression across animals, oftentimes independent of the corresponding linear mRNAs. circRNAs are remarkably stable and sometimes highly expressed molecules. Here, we sequenced RNA in human peripheral whole blood to determine the potential of circRNAs as biomarkers in an easily accessible body fluid. We report the reproducible detection of thousands of circRNAs. Importantly, we observed that hundreds of circRNAs are much higher expressed than corresponding linear mRNAs. Thus, circRNA expression in human blood reveals and quantifies the activity of hundreds of coding genes not accessible by classical mRNA specific assays. Our findings suggest that circRNAs could be used as biomarker molecules in standard clinical blood samples.

Analysis of intron sequences reveals hallmarks of circular RNA biogenesis in animals.

Circular RNAs (circRNAs) are a large class of animal RNAs. To investigate possible circRNA functions, it is important to understand circRNA biogenesis. Besides human ALU repeats, sequence features that promote exon circularization are largely unknown. We experimentally identified circRNAs in C. elegans. Reverse complementary sequences between introns bracketing circRNAs were significantly enriched in comparison to linear controls. By scoring the presence of reverse complementary sequences in human introns, we predicted and experimentally validated circRNAs. We show that introns bracketing circRNAs are highly enriched in RNA editing or hyperediting events. Knockdown of the double-strand RNA-editing enzyme ADAR1 significantly and specifically upregulated circRNA expression. Together, our data support a model of animal circRNA biogenesis in which competing RNA-RNA interactions of introns form larger structures that promote circularization of embedded exons, whereas ADAR1 antagonizes circRNA expression by melting stems within these interactions.

circRNA biogenesis competes with pre-mRNA splicing.

Circular RNAs (circRNAs) are widely expressed noncoding RNAs. However, their biogenesis and possible functions are poorly understood. Here, by studying circRNAs that we identified in neuronal tissues, we provide evidence that animal circRNAs are generated cotranscriptionally and that their production rate is mainly determined by intronic sequences. We demonstrate that circularization and splicing compete against each other. These mechanisms are tissue specific and conserved in animals. Interestingly, we observed that the second exon of the splicing factor muscleblind (MBL/MBNL1) is circularized in flies and humans. This circRNA (circMbl) and its flanking introns contain conserved muscleblind binding sites, which are strongly and specifically bound by MBL. Modulation of MBL levels strongly affects circMbl biosynthesis, and this effect is dependent on the MBL binding sites. Together, our data suggest that circRNAs can function in gene regulation by competing with linear splicing. Furthermore, we identified muscleblind as a factor involved in circRNA biogenesis.

Circular RNAs are a large class of animal RNAs with regulatory potency.

Circular RNAs (circRNAs) in animals are an enigmatic class of RNA with unknown function. To explore circRNAs systematically, we sequenced and computationally analysed human, mouse and nematode RNA. We detected thousands of well-expressed, stable circRNAs, often showing tissue/developmental-stage-specific expression. Sequence analysis indicated important regulatory functions for circRNAs. We found that a human circRNA, antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as), is densely bound by microRNA (miRNA) effector complexes and harbours 63 conserved binding sites for the ancient miRNA miR-7. Further analyses indicated that CDR1as functions to bind miR-7 in neuronal tissues. Human CDR1as expression in zebrafish impaired midbrain development, similar to knocking down miR-7, suggesting that CDR1as is a miRNA antagonist with a miRNA-binding capacity ten times higher than any other known transcript. Together, our data provide evidence that circRNAs form a large class of post-transcriptional regulators. Numerous circRNAs form by head-to-tail splicing of exons, suggesting previously unrecognized regulatory potential of coding sequences.

The SNF2-like helicase HELLS mediates E2F3-dependent transcription and cellular transformation.

The activating E2F-transcription factors are best known for their dependence on the Retinoblastoma protein and their role in cellular proliferation. E2F3 is uniquely amplified in specific human tumours where its expression is inversely correlated with the survival of patients. Here, E2F3B interaction partners were identified by mass spectrometric analysis. We show that the SNF2-like helicase HELLS interacts with E2F3A in vivo and cooperates with its oncogenic functions. Depletion of HELLS severely perturbs the induction of E2F-target genes, hinders cell-cycle re-entry and growth. Using chromatin immmunoprecipitation coupled to sequencing, we identified genome-wide targets of HELLS and E2F3A/B. HELLS binds promoters of active genes, including the trithorax-related MLL1, and co-regulates E2F3-dependent genes. Strikingly, just as E2F3, HELLS is overexpressed in human tumours including prostate cancer, indicating that either factor may contribute to the malignant progression of tumours. Our work reveals that HELLS is important for E2F3 in tumour cell proliferation.