Biology subjects

Schwarzl, T.

Publications and source records attributed to Schwarzl, T..

9 recordsLinked to original sources

bioRxiv2024

RNA promotes mitochondrial import of F1-ATP synthase subunit alpha (ATP5A1)

Most mitochondrial proteins are encoded by the nuclear genome, translated as precursor proteins in the cytosol and matured during directed import into the mitochondria 1. For many mitochondrial proteins this process is carefully regulated to meet demand and to avoid mitochondrial stress 2,3,4. Recently, mitochondrial F1-ATP synthase subunits have been found to interact with RNA across various eukaryotes. This includes genome wide RNA-interactome studies from yeast 5-7, fruit flies 8,9, plants 10-12, mice 13-17 and humans 18-24. To shed light on this unexpected observation, we determined the interacting cellular RNAs of ATP5A1 and the subcellular sites of interaction. Using RNA binding-deficient mutants of ATP5A1 and functional assays, we show that specific cytosolic RNAs bind ATP5A1 precursor proteins at the outer surface of mitochondria and promote their mitochondrial import both in vitro and in cellulo. These findings add an unexpected twist to understanding mitochondrial protein import and expand the growing list of riboregulated cellular processes.

cell biology↗

bioRxiv2023

Cytosolic RNA binding of the mitochondrial TCA cycle enzyme malate dehydrogenase (MDH2)

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 in cellulo RNA-binding of MDH2 using orthogonal biochemical assays and performed enhanced crosslinking and immunoprecipitation (eCLIP) to identify the cellular RNAs associated with endogenous MDH2. Surprisingly, MDH2 preferentially binds cytosolic over mitochondrial RNAs, although the latter are abundant in the milieu of the mature protein. Subcellular fractionation followed by RNA-binding assays revealed that MDH2-RNA interactions occur predominantly outside of mitochondria. We also found that a cytosolically-retained N-terminal deletion mutant of MDH2 is competent to bind RNA, indicating that mitochondrial targeting is dispensable for MDH2-RNA interactions. MDH2 RNA binding increased when cellular NAD+ levels (MDH2s co-factor) was pharmacologically diminished, suggesting that the metabolic state of cells affects RNA binding. Taken together, our data implicate an as yet unidentified function of MDH2 binding RNA in the cytosol.

cell biology↗

bioRxiv2022

Improved discovery of RNA-binding protein binding sites in eCLIP data using DEWSeq

Enhanced crosslinking and immunoprecipitation (eCLIP) sequencing is a powerful method for transcriptome-wide detection of binding sites of RNA-binding proteins (RBPs). However, identified crosslink sites can profoundly deviate from experimentally established functional elements of even well-studied RBPs. Current peak-calling strategies result in low replication and high false-positive rates. Here, we present the R/Bioconductor package DEWSeq that makes full use of replicate information and size-matched input controls. We benchmarked DEWSeq on 107 RBPs for which both eCLIP data and RNA sequence motifs are available and were able to more than double the number of motif-containing binding regions relative to standard eCLIP processing (2.3-fold median). The improvement not only relates to the number of binding sites (e.g., 3.1-fold of known motifs for RBFOX2), but also their subcellular localisation (e.g., 1.9-fold of mitochondrial genes for FASTKD2) and structural targets (e.g., 2.2-fold increase of stem-loop regions for SLBP). DEWSeq therefore shows promise as an improved processing method for eCLIP protein-RNA interaction data.

molecular biology↗

bioRxiv2022

Small non-coding RNA Interactome Capture reveals pervasive, carbon source-dependent tRNA engagement of yeast glycolytic enzymes

Small non-coding RNAs fulfill key functions in cellular and organismal biology, typically working in concert with RNA-binding proteins (RBPs). While proteome-wide methodologies have enormously expanded the repertoire of known RBPs, these methods do not distinguish RBPs binding to small non-coding RNAs from the rest. To specifically identify this relevant subclass of RBPs, we developed small non-coding RNA interactome capture (snRIC2C) based on the differential RNA-binding capacity of silica matrices (2C). We define the S. cerevisiae proteome of nearly 300 proteins that specifically binds to RNAs smaller than 200 nucleotides in length (snRBPs), identifying informative distinctions from the total RNA-binding proteome determined in parallel. Strikingly, the snRBPs include most glycolytic enzymes from yeast. With further methodological developments using silica matrices, 12 tRNAs were identified as specific binders of the glycolytic enzyme GAPDH. We show that tRNA engagement of GAPDH is carbon source-dependent and regulated by the RNA polymerase III repressor Maf1, suggesting a regulatory interaction between glycolysis and RNA polymerase III activity. We conclude that snRIC2C and other 2C-derived methods greatly facilitate the study of RBPs, revealing previously unrecognised interactions.

molecular biology↗

bioRxiv2022

The RNA-binding protein landscapes differ between mammalian organs and cultured cells

System-wide approaches have unveiled an unexpected breadth of the RNA-bound proteomes of cultured cells. Corresponding information regarding RNA-binding proteins (RBPs) of mammalian organs is still missing, largely due to technical challenges. Here, we describe ex vivo eRIC (enhanced RNA interactome capture) to characterize the poly(A)RNA-bound proteomes of three different mouse organs. The resulting organ atlases encompass more than 1300 RBPs active in brain, kidney or liver. Nearly a quarter (291) of these had formerly not been identified in cultured cells, with more than 100 being metabolic enzymes. Remarkably, RBP activity differs between organs independent of RBP abundance, suggesting organ-specific levels of control. Similarly, we identify systematic differences in RNA binding between animal organs and cultured cells. The pervasive RNA binding of enzymes of intermediary metabolism in organs points to tightly knit connections between gene expression and metabolism, and displays a particular enrichment for enzymes that use nucleotide cofactors. We describe a generically applicable refinement of the eRIC technology and provide an instructive resource of RBPs active in intact mammalian organs, including the brain.

molecular biology↗

bioRxiv2021

Transcriptional profiling of early differentiation of primary human mesenchymal stem cells into chondrocytes

Articular cartilage has only very limited regenerative capacities in humans. Tissue engineering techniques for cartilage damage repair are limited in the production of hyaline cartilage. Mesenchymal stem/stromal cells (MSCs) are multipotent stem cells and can be differentiated into mature cartilage cells, chondrocytes, which could be used for repairing damaged cartilage. Chondrogenesis is a highly complex, relatively inefficient process lasting over 3 weeks in vitro. In order to better understand chondrogenic differentiation, especially the commitment phase, we have performed transcriptional profiling of MSC differentiation into chondrocytes from early timepoints starting 15 minutes after induction to 16 hours and fully differentiated chondrocytes at 21 days. Transcriptional gene regulatory networks (GRN) were identified using time-course clustering and upstream-regulator predictions which revealed that cells start epigenetic reprogramming as early as 2 hours after induction and commit to differentiation within 4-6 hours. After that they adapt their gene expression to cater for differentiation specific protein production. These results suggest that interventions to improve the frequency and efficiency of differentiation should target early processes.

molecular biology↗

bioRxiv2020

RNA regulates Glycolysis and Embryonic Stem Cell Differentiation via Enolase 1

Cells must coordinate their metabolism and fate trajectories (1, 2), but the underlying mechanisms are only beginning to be discovered. To understand why the glycolytic enzyme enolase 1 (ENO1) binds RNA (3-6), we studied this phenomenon in vitro, in human cells, and during mouse embryonic stem cell differentiation. We find specific cellular RNA ligands that inhibit ENO1s enzymatic activity in vitro. Increasing the concentration of these ligands in cultured cells inhibits glycolysis. We demonstrate that pluripotent stem cells expressing an ENO1 mutant that is hyper-inhibited by RNA are severely impaired in their glycolytic capacity and in endodermal differentiation, whereas cells with an RNA binding-deficient ENO1 mutant display disproportionately high endodermal marker expression. Our findings uncover ENO1 riboregulation as a novel form of metabolic control. They also describe an unprecedented mechanism involved in the regulation of stem cell differentiation. One Sentence SummaryRNA directly regulates enzyme activity to control metabolism and stem cell fate

molecular biology↗

bioRxiv2018

The Human RNA-Binding Proteome and Its Dynamics During Arsenite-Induced Translational Arrest

Proteins and RNA functionally and physically intersect in multiple biological processes, however, currently no universal method is available to purify protein-RNA complexes. Here we introduce XRNAX, a method for the generic purification of protein-crosslinked RNA, and demonstrate its versatility to study the composition and dynamics of protein-RNA interactions by various transcriptomic and proteomic approaches. We show that XRNAX captures all RNA biotypes, and use this to characterize the sub-proteomes that interact with coding and non-coding RNAs (ncRNAs), and to identify hundreds of protein-RNA interfaces. Exploiting the quantitative nature of XRNAX, we observe drastic remodeling of the RNA-bound proteome during arsenite-induced stress, distinct from autophagy-induced changes in the total proteome. In addition, we combine XRNAX with CLIP-seq to validate the interaction of ncRNA with Lamin B and EXOSC2. Thus, XRNAX is a resourceful approach to study structural and compositional aspects of protein-RNA interactions to address fundamental questions in RNA-biology.

molecular biology↗

bioRxiv2017

The small non-coding vault RNA1-1 acts as a riboregulator of autophagy

Vault RNAs (vtRNA) are small, 88-100nt non-coding RNAs found in many eukaryotes. Although they have been linked to drug resistance, apoptosis and nuclear transport, their function remains unclear. Here we show that a human vtRNA, RNA1-1, specifically binds to the autophagy receptor sequestosome-1/p62. Antisense-mediated depletion of vault RNA1-1 augments, whereas increased vault RNA1-1 expression restricts, autophagic flux in a p62-dependent manner. Bulk autophagy induced by starvation reduces the levels of vault RNA1-1 and the fraction of RNA-bound p62. These findings show that RNAs can act as riboregulators of biological processes by interacting with proteins, and assign a function to a vault RNA.

molecular biology↗