Biology subjects

Sekaran, T.

Publications and source records attributed to Sekaran, T..

6 recordsLinked to original sources

bioRxiv2026

Identification of RNA Targets of Classical and Non-Canonical RNA-binding Proteins by soniCLIP

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/745202v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@52e3edorg.highwire.dtl.DTLVardef@1f21637org.highwire.dtl.DTLVardef@909ee8org.highwire.dtl.DTLVardef@b0c907_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACTC_FLOATNO C_FIG Crosslinking and immunoprecipitation followed by sequencing (CLIP-seq) is widely used to identify the RNA targets of RNA-binding proteins (RBPs). However, its application to non-canonical RBPs lacking canonical RNA-binding domains and frequently displaying low or transient RNA occupancy, is limited by low signal-to-noise ratios, high input requirements and error-prone ligation steps during library preparation. To overcome these limitations, we developed soniCLIP, a streamlined CLIP-seq workflow that replaces RNase-mediated RNA fragmentation with sonication and uses a ligation-free strategy for library construction. soniCLIP is optimized for reproducible identification of enriched RBP-associated RNA regions from limited starting material. We benchmarked soniCLIP against the widespread eCLIP approach and observed reproducible recovery of known RBP-associated regions and target recovery comparable to ENCODE eCLIP, while requiring only 10% (500 {micro}g) of protein input. We further applied soniCLIP to the glycolytic enzyme and non-canonical RBP pyruvate kinase M2 (PKM2). We identified 197 significantly enriched RNA regions and validated selected targets by RIP-qRT-PCR and in vitro binding assays. By combining reduced input requirements, high reproducibility, a shortened 3.5-day workflow and the elimination of gel-based purification, soniCLIP provides an efficient and robust approach for the identification of RNA targets of canonical and non-canonical RBPs.

molecular biology↗

bioRxiv2026

Muscle LIM Protein is a Non-Canonical RNA-Binding Protein that Engages in RNA-Dependent Cytoskeletal Assemblies

Muscle LIM Protein (MLP) is a key component of the cardiomyocyte Z disc that is essential for sarcomere integrity and cardiac homeostasis. Accordingly, loss of MLP function results in (cardio)myopathy phenotypes in animal models and human patients. In this study, we present the first RNA-binding proteome (RBPome) of human cardiomyocytes using enhanced RNA interactome capture (eRIC). Data integration with existing RBPome datasets identified MLP as an evolutionary conserved and previously unrecognized non-canonical RNA-binding protein (RBP) in cardiomyocytes. Using complementary biochemical approaches, we confirmed direct RNA association of MLP. Moreover, RNA integrity was required for MLP interactions with cytoskeletal proteins and for the formation of higher-order MLP-containing assemblies, revealed by co-immunoprecipitation and RNA-dependent sedimentation profiling. Subcellular fractionation and imaging analyses indicated that MLPs association with cytoskeletal complexes may be weaker and more dynamic compared to core sarcomere proteins like -actinin. Domain mapping identified two glycine-rich regions within MLP as RNA contact sites, and RNA-binding-deficient MLP mutants showed impaired association with cytoskeletal complexes. Together, our findings suggest RNA binding as a regulated property of MLP and uncover an RNA-dependent layer of cytoskeletal organization in cardiomyocytes.

molecular biology↗

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↗

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↗

bioRxiv2020

Planarian CREB-binding protein (CBP) gene family regulates stem cell maintenance and differentiation

The regulation of stem cells plasticity and differentiation is still an open question in developmental biology. CBP (CREB-binding protein)/p300 is a conserved gene family which functions as a transcriptional co-activator and shows an important role in a wide range of cellular processes, such as cell death, DNA damage response and tumorigenesis. Moreover, CBPs have an acetyl transferase activity that is relevant as histone and non-histone acetylation results in changes in chromatin architecture and protein activity that affects gene expression. Many studies have shown the conserved functions of CBP/p300 on stem cell proliferation and differentiation. The planarian Schmidtea mediterranea is an excellent model to study in vivo the molecular mechanism underlying stem cell differentiation during regeneration. We have identified five different Smed-cbp genes in S. mediterranea that show different expression patterns. Functional analyses indicate that Smed-cbp-2 seems to be essential for stem cell maintenance and cell survival. On the other hand, the silencing of Smed-cbp-3 results in the growth of apparently normal blastemas; however, these remain largely depigmented and undifferentiated. Smed-cbp-3 silencing affects the differentiation of several cell lineages including neural, epidermal, digestive and excretory cell types. Finally, we have analyzed the predicted interactomes of CBP-2 and CBP-3 as an initial step to better understand their function on planarian stem cell biology.

developmental biology↗