Cytosolic RNA binding of the mitochondrial TCA cycle enzyme malate dehydrogenase.

Several enzymes of intermediary metabolism have been identified to bind RNA in cells, with potential consequences for the bound RNAs and/or the enzyme. In this study, we investigate the RNA-binding activity of the mitochondrial enzyme malate dehydrogenase 2 (MDH2), which functions in the tricarboxylic acid (TCA) cycle and the malate-aspartate shuttle. We confirmed RNA binding of MDH2 using orthogonal biochemical assays and performed enhanced cross-linking and immunoprecipitation (eCLIP) to identify the cellular RNAs associated with endogenous MDH2.

Systematic analysis of RNA-binding proteins identifies targetable therapeutic vulnerabilities in osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumor with a strong tendency to metastasize, limiting the prognosis of affected patients. Genomic, epigenomic and transcriptomic analyses have demonstrated the exquisite molecular complexity of this tumor, but have not sufficiently defined the underlying mechanisms or identified promising therapeutic targets. To systematically explore RNA-protein interactions relevant to OS, we define the RNA interactomes together with the full proteome and the transcriptome of cells from five malignant bone tumors (four osteosarcomata and one malignant giant cell tumor of the bone) and from normal mesenchymal stem cells and osteoblasts.

Translational control of Ybx1 expression regulates cardiac function in response to pressure overload in vivo.

RNA-protein interactions are central to cardiac function, but how activity of individual RNA-binding protein is regulated through signaling cascades in cardiomyocytes during heart failure development is largely unknown. The mechanistic target of rapamycin kinase is a central signaling hub that controls mRNA translation in cardiomyocytes; however, a direct link between mTOR signaling and RNA-binding proteins in the heart has not been established. Integrative transcriptome and translatome analysis revealed mTOR dependent translational upregulation of the RNA binding protein Ybx1 during early pathological remodeling independent of mRNA levels.

htseq-clip: a toolset for the preprocessing of eCLIP/iCLIP datasets.

Summary: Transcriptome-wide detection of binding sites of RNA-binding proteins is achieved using Individual-nucleotide crosslinking and immunoprecipitation (iCLIP) and its derivative enhanced CLIP (eCLIP) sequencing methods. Here, we introduce htseq-clip, a python package developed for preprocessing, extracting and summarizing crosslink site counts from i/eCLIP experimental data. The package delivers crosslink site count matrices along with other metrics, which can be directly used for filtering and downstream analyses such as the identification of differential binding sites.

Riboregulation of Enolase 1 activity controls glycolysis and embryonic stem cell differentiation.

Differentiating stem cells must coordinate their metabolism and fate trajectories. Here, we report that the catalytic activity of the glycolytic enzyme Enolase 1 (ENO1) is directly regulated by RNAs leading to metabolic rewiring in mouse embryonic stem cells (mESCs). We identify RNA ligands that specifically inhibit ENO1's enzymatic activity in vitro and diminish glycolysis in cultured human cells and mESCs.

A Pipeline for Analyzing eCLIP and iCLIP Data with Htseq-clip and DEWSeq.

Individual-nucleotide crosslinking and immunoprecipitation (iCLIP) sequencing and its derivative enhanced CLIP (eCLIP) sequencing are methods for the transcriptome-wide detection of binding sites of RNA-binding proteins (RBPs). This chapter provides a stepwise tutorial for analyzing iCLIP and eCLIP data with replicates and size-matched input (SMI) controls after read alignment using our open-source tools htseq-clip and DEWSeq. This includes the preparation of gene annotation, extraction, and preprocessing of truncation sites and the detection of significantly enriched binding sites using a sliding window based approach suitable for different binding modes of RBPs.

Identification of dynamic RNA-binding proteins uncovers a Cpeb4-controlled regulatory cascade during pathological cell growth of cardiomyocytes.

RNA-binding proteins (RBPs) control critical aspects of cardiomyocyte function, but the repertoire of active RBPs in cardiomyocytes during the growth response is largely unknown. We define RBPs in healthy and diseased cardiomyocytes at a system-wide level by RNA interactome capture. This identifies 67 cardiomyocyte-specific RBPs, including several contractile proteins.

CREB-binding protein (CBP) gene family regulates planarian survival and stem cell differentiation.

In developmental biology, the regulation of stem cell plasticity and differentiation remains an open question. CBP(CREB-binding protein)/p300 is a conserved gene family that functions as a transcriptional co-activator and plays important roles in a wide range of cellular processes, including cell death, the DNA damage response, and tumorigenesis. The acetyl transferase activity of CBPs is particularly important, as histone and non-histone acetylation results in changes in chromatin architecture and protein activity that affect gene expression.

Abrogation of Gap Junctional Communication in ES Cells Results in a Disruption of Primitive Endoderm Formation in Embryoid Bodies.

Gap junctional intercellular communication (GJIC) has been suggested to be involved in early embryonic development but the actual functional role remained elusive. Connexin (Cx) 43 and Cx45 are co-expressed in embryonic stem (ES) cells, form gap junctions and are considered to exhibit adhesive function and/or to contribute to the establishment of defined communication compartments. Here, we describe the generation of Cx43/Cx45-double deficient mouse ES cells to achieve almost complete breakdown of GJIC.

Excision of viral reprogramming cassettes by Cre protein transduction enables rapid, robust and efficient derivation of transgene-free human induced pluripotent stem cells.

Integrating viruses represent robust tools for cellular reprogramming; however, the presence of viral transgenes in induced pluripotent stem cells (iPSCs) is deleterious because it holds the risk of insertional mutagenesis leading to malignant transformation. Here, we combine the robustness of lentiviral reprogramming with the efficacy of Cre recombinase protein transduction to derive iPSCs devoid of transgenes. By genome-wide analysis and targeted differentiation towards the cardiomyocyte lineage, we show that transgene-free iPSCs are superior to iPSCs before Cre transduction.