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Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic stem cell diseases characterized by cell dysplasia, ineffective hematopoiesis and risk of transformation to acute myeloid leukemia (AML). The median age of 75 years at diagnosis is associated with the presence of comorbidities, which preclude intensive therapies like allogeneic hematopoietic stem cell transplantation in most MDS patients. Risk stratification using the (Revised) International Prognostic Scoring System (IPSS/IPSS-R) is necessary to plan individualized treatment.Luspatercept (ACE-536), a specific activin receptor fusion protein, promotes late-stage erythropoiesis. Two clinical trials, PACE-MDS (phase 2) and MEDALIST (phase 3), yielded positive results in terms of improved hemoglobin levels and loss of transfusion dependence, with hardly any side effects. A phase 3 trial to compare luspatercept to ESAs (COMMANDS study) is ongoing.Luspatercept is a promising alternative to ESAs for a subset of transfusion-dependent patients with lower risk MDS, namely those with a sideroblastic phenotype who are either not suitable for or have already failed erythropoietin-based treatment. The favorable safety profile and convenient subcutaneous administration every 3 weeks are more conducive to patients' quality of life than chronic red blood cell transfusion therapy.

Ribosome profiling (Ribo-seq) has catalyzed a paradigm shift in our understanding of the translational ‘vocabulary’ of the human genome, discovering thousands of translated open reading frames (ORFs) within long non-coding RNAs and presumed untranslated regions of protein-coding genes. However, reference gene annotation projects have been circumspect in their incorporation of these ORFs due to uncertainties about their experimental reproducibility and physiological roles. Yet, it is indisputable that certain Ribo-seq ORFs make stable proteins, others mediate gene regulation, and many have medical implications. Ultimately, the absence of standardized ORF annotation has created a circular problem: while Ribo-seq ORFs remain unannotated by reference biological databases, this lack of characterisation will thwart research efforts examining their roles. Here, we outline the initial stages of a community-led effort supported by GENCODE / Ensembl, HGNC and UniProt to produce a consolidated catalog of human Ribo-seq ORFs.

Little is known about the impact of naturally occurring genetic variation on the rates with which proteins are synthesized by ribosomes. Here, we investigate how genetic influences on mRNA translational efficiency are associated with complex disease phenotypes using a panel of rat recombinant inbred lines. We identify a locus for cardiac hypertrophy that is associated with a translatome-wide and protein length-dependent shift in translational efficiency. This master regulator primarily affects the translation of very short and very long protein-coding sequences, altering the physiological stoichiometric translation rates of sarcomere proteins. Mechanistic dissection of this locus points to altered ribosome assembly, characterized by accumulation of polysome half-mers, changed ribosomal configurations and misregulation of the small nucleolar RNASNORA48. We postulate that this locus enhances a pre-existing negative correlation between protein length and translation initiation in diseased hearts. Our work shows that a single genomic locus can trigger a complex, translation-driven molecular mechanism that contributes to phenotypic variability between individuals.Graphical AbstractHighlightsGenetic variability impacts protein synthesis rates in a rat model for cardiac hypertrophyAtranslocus affects stoichiometric translation rates of cardiac sarcomeric proteinsThis master regulator locus induces a global, protein length-dependent shift in translationDysregulated ribosome assembly induces half-mer formation and affects translation initiation rate

Long noncoding RNAs (lncRNAs) are a heterogenous group of RNAs, which can encode small proteins. The extent to which developmentally regulated lncRNAs are translated and whether the produced microproteins are relevant for human development is unknown. Using a human embryonic stem cell (hESC)-based pancreatic differentiation system, we show that many lncRNAs in direct vicinity of lineage-determining transcription factors (TFs) are dynamically regulated, predominantly cytosolic, and highly translated. We genetically ablated ten such lncRNAs, most of them translated, and found that nine are dispensable for pancreatic endocrine cell development. However, deletion of LINC00261 diminishes insulin+ cells, in a manner independent of the nearby TF FOXA2. One-by-one disruption of each of LINC00261's open reading frames suggests that the RNA, rather than the produced microproteins, is required for endocrine development. Our work highlights extensive translation of lncRNAs during hESC pancreatic differentiation and provides a blueprint for dissection of their coding and noncoding roles.

Long noncoding RNAs (lncRNAs) are a heterogenous group of RNAs, which can encode small proteins. The extent to which developmentally regulated lncRNAs are translated and whether the produced microproteins are relevant for human development is unknown. Here, we show that many lncRNAs in direct vicinity of lineage-determining transcription factors (TFs) are dynamically regulated, predominantly cytosolic, and highly translated during pancreas development. We genetically ablated ten such lncRNAs, most of them translated, and found that nine are dispensable for endocrine cell differentiation. However, deletion of LINC00261 diminishes generation of insulin+ endocrine cells, in a manner independent of the nearby TF FOXA2. Systematic deletion of each of LINC00261’s seven poorly conserved microproteins shows that the RNA, rather than the microproteins, is required for endocrine development. Our work highlights extensive translation of lncRNAs into recently evolved microproteins during human pancreas development and provides a blueprint for dissection of their coding and noncoding roles.Graphical AbstractHighlightsExtensive lncRNA translation and microprotein production during human pancreas developmentA small-scale loss-of-function screen shows most translated lncRNAs are dispensableLINC00261 is highly translated and regulates endocrine cell differentiationDeleting LINC00261’s evolutionary young microproteins reveals no essential roles

Cardiac microproteins are an emerging class of important regulators of cellular homeostasis, often encoded by presumed long noncoding RNAs. With only few described so far, their prevalence and biological significance largely remains unclear. To create an atlas of cardiac-expressed microproteins, we performed ribosome profiling on 80 human heart samples. This revealed the translation of hundreds of previously undetected microproteins, which we validated in vivo by targeted mass spectrometry. A single heart-specific microprotein caught our attention as it showed strong expression coregulation with components of mitochondrial OXPHOS pathway. We next deleted this microprotein's lncRNA host gene in hiPSCs using CRISPR/Cas9, and, upon differentiation into 30-d old cardiomyocytes, find a strong expression upregulation of genes involved in oxidative phosphorylation and calcium handling. To investigate the impact of the microprotein KO on cardiomyocyte mechanical function, we assessed sarcomere dynamics using an endogenous TTN-GFP tag, enabling live cell imaging analysis with the SarcTrack algorithm. Preliminary analysis sowes a putative increase in sarcomere contraction and relaxation times, suggesting a repressive regulatory function of the eliminated microprotein encoding lncRNA.

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.

BackgroundSeveral long noncoding RNAs (lncRNAs) have been shown to function as central components of molecular machines that play fundamental roles in biology. While the number of annotated lncRNAs in mammalian genomes has greatly expanded, their functions remain largely uncharacterized. This is compounded by the fact that identifying lncRNA loci that have robust and reproducible phenotypes when mutated has been a challenge.ResultsWe previously generated a cohort of 20 lncRNA loci knockout mice. Here, we extend our initial study and provide a more detailed analysis of the highly conserved lncRNA locus, Taurine Upregulated Gene 1 (Tug1). We report that Tug1 knockout male mice are sterile with complete penetrance due to a low sperm count and abnormal sperm morphology. Having identified a lncRNA loci with a robust phenotype, we wanted to determine which, if any, potential elements contained in the Tug1 genomic region (DNA, RNA, protein, or the act of transcription) have activity. Using engineered mouse models and cell-based assays, we provide evidence that the Tug1 locus harbors three distinct regulatory activities – two noncoding and one coding: (i) a cis DNA repressor that regulates many neighboring genes, (ii) a lncRNA that can regulate genes by a trans-based function, and finally (iii) Tug1 encodes an evolutionary conserved peptide that when overexpressed impacts mitochondrial membrane potential.ConclusionsOur results reveal an essential role for the Tug1 locus in male fertility and uncover three distinct regulatory activities in the Tug1 locus, thus highlighting the complexity present at lncRNA loci.