Biology subjects

Shechner, D. M.

Publications and source records attributed to Shechner, D. M..

9 recordsLinked to original sources

bioRxiv2026

A Modular Platform for Purification of Organelle-associated Mitochondria Reveals Functional Specialization at Organelle Contact Sites

Mitochondria perform diverse metabolic and signaling functions, yet how these activities are spatially organized within the mitochondrial network of cells remains poorly understood. Organelle contact sites are spatially restricted hubs that regulate mitochondrial metabolism, signaling, and dynamics, and are therefore well positioned to influence mitochondrial specialization. Investigation of contact site-associated mitochondrial populations has been hindered by a lack of methods to isolate these subpopulations. Here, we develop Organelle Contact-dependent Affinity Purification (ORCA), a workflow for the isolation and analysis of subpopulations of intact mitochondria and associated proteins defined by their organelle contacts. ORCA revealed distinct proteomes for mitochondria associated with the endoplasmic reticulum, lysosomes, peroxisomes, and the Golgi apparatus, demonstrating that organelle contacts define biochemically specialized mitochondrial populations. Focused analysis of Golgi-associated mitochondria showed enrichment of mitochondrial ribosomes and increased mitochondrial translation, revealing an unexpected role for Golgi-mitochondria contacts in regulating mitochondrial protein homeostasis. ORCA also identified the previously uncharacterized Golgi protein KIAA0930/GMO1 as an evolutionarily conserved regulator of oxidative phosphorylation at Golgi-mitochondria contacts. Together, our findings establish ORCA as a broadly applicable approach for investigating the spatial organization of intracellular organelles and reveal organelle contacts as key determinants of mitochondrial specialization.

cell biology↗

bioRxiv2025

Xist Repeat A coordinates an assembly of SR proteins to recruit SPEN and induce gene silencing

The lncRNA Xist represents a paradigm to understand the mechanisms of RNA-mediated gene silencing in mammals, which remain largely unresolved. To induce silencing, Xist recruits the RNA-binding protein SPEN through its 5'-proximal Repeat A domain. Yet, how Repeat A recruits SPEN and how SPEN coordinates silencing remain unclear. We report that sequences in Repeat A critical for SPEN recruitment directly bind SR-rich splicing factors. SRSF1, one such factor, is required for optimal SPEN recruitment and its RS-domain recruits SPEN when tethered to Xist. SPEN and SR-protein-binding motifs promote Repeat As association with many proteins, including the m6A machinery and elongating RNA polymerase II. SPEN also represses autosomal genes where its recruitment coincides with SR-protein binding. Our results reveal an unexpectedly essential role for splicing factors in coordinating silencing by Xist and suggest that the sensing of SR-protein-rich assemblies is a general mechanism through which SPEN targets genes for repression.

genetics↗

bioRxiv2024

Nascent transcript O-MAP reveals the molecular architecture of a single-locus subnuclear compartment built by RBM20 and the TTN RNA

Eukaryotic nuclei adopt a highly compartmentalized architecture that influences nearly all genomic processes. Understanding how this architecture impacts gene expression has been hindered by a lack of tools for elucidating the molecular interactions at individual genomic loci. Here, we adapt oligonucleotide-mediated proximity-interactome mapping (O-MAP) to biochemically characterize discrete, micron-scale nuclear neighborhoods. By targeting O-MAP to introns within the TTN pre-mRNA, we systematically map the chromatin loci, RNAs, and proteins within a muscle-specific RNA factory organized around the TTN locus. This reveals an unanticipated compartmental architecture that organizes cis- and trans-interacting chromosomal domains, including a hub of transcriptionally silenced chromatin. The factory also recruits dozens of unique RNA-binding and chromatin-scaffolding factors, including QKI and SAFB, along with their target transcripts. Loss of the cardiac-specific splicing factor RBM20--a master regulator of TTN splicing that is mutated in dilated cardiomyopathy--remodels nearly every facet of this architecture. This establishes O-MAP as a pioneering method for probing single-locus, microcompartment-level interactions that are opaque to conventional tools. Our findings suggest new mechanisms by which coding genes can "moonlight" in nuclear-architectural roles.

genomics↗

bioRxiv2024

DNA O-MAP uncovers the molecular neighborhoods associated with specific genomic loci

The accuracy of crucial nuclear processes such as transcription, replication, and repair, depends on the local composition of chromatin and the regulatory proteins that reside there. Understanding these DNA-protein interactions at the level of specific genomic loci has remained challenging due to technical limitations. Here, we introduce a method termed "DNA O-MAP", which uses programmable peroxidase-conjugated oligonucleotide probes to biotinylate nearby proteins. We show that DNA O-MAP can be coupled with label-free or sample multiplexed quantitative proteomics, targeted chemical perturbations, and next-generation sequencing to quantify DNA-proximal proteins and DNA-DNA interactions at specific genomic loci. Furthermore, we establish that DNA O-MAP is applicable to both repetitive and unique genomic loci of varying sizes, from kilobase HOX gene clusters to megabase alpha-satellite repeats, and that DNA O-MAP can measure proximal molecular effectors in a homolog-specific manner.

systems biology↗

bioRxiv2023

Programmable peroxidase-assisted signal amplification enables flexible detection of nucleic acid targets in cellular and histopathological specimens

In situ hybridization (ISH) is a powerful tool for investigating the spatial arrangement of nucleic acid targets in fixed samples. ISH is typically visualized using fluorophores to allow high sensitivity and multiplexing or with colorimetric labels to facilitate co-visualization with histopathological stains. Both approaches benefit from signal amplification, which makes target detection effective, rapid, and compatible with a broad range of optical systems. Here, we introduce a unified technical platform, termed pSABER, for the amplification of ISH signals in cell and tissue systems. pSABER decorates the in situ target with concatemeric binding sites for a horseradish peroxidase-conjugated oligonucleotide which can then catalyze the massive localized deposition of fluorescent or colorimetric substrates. We demonstrate that pSABER effectively labels DNA and RNA targets, works robustly in cultured cells and challenging formalin fixed paraffin embedded (FFPE) specimens. Furthermore, pSABER can achieve 25-fold signal amplification over conventional signal amplification by exchange reaction (SABER) and can be serially multiplexed using solution exchange. Therefore, by linking nucleic acid detection to robust signal amplification capable of diverse readouts, pSABER will have broad utility in research and clinical settings.

genomics↗

bioRxiv2023

Oligonucleotide-directed proximity-interactome mapping (O-MAP): A unified method for discovering RNA-interacting proteins, transcripts and genomic loci in situ.

Throughout biology, RNA molecules form complex networks of molecular interactions that are central to their function, but remain challenging to investigate. Here, we introduce Oligonucleotide-mediated proximity-interactome MAPping (O-MAP), a straightforward method for elucidating the biomolecules near an RNA of interest, within its native cellular context. O-MAP uses programmable oligonucleotide probes to deliver proximity-biotinylating enzymes to a target RNA, enabling nearby molecules to be enriched by streptavidin pulldown. O-MAP induces exceptionally precise RNA-localized in situ biotinylation, and unlike alternative methods it enables straightforward optimization of its targeting accuracy. Using the 47S pre-ribosomal RNA and long noncoding RNA Xist as models, we develop O-MAP workflows for unbiased discovery of RNA-proximal proteins, transcripts, and genomic loci. This revealed unexpected co-compartmentalization of Xist and other chromatin-regulatory RNAs and enabled systematic characterization of nucleolar-chromatin interactions across multiple cell lines. O-MAP is portable to cultured cells, organoids, and tissues, and to RNAs of various lengths, abundances, and sequence composition. And, O-MAP requires no genetic manipulation and uses exclusively off-the-shelf parts. We therefore anticipate its application to a broad array of RNA phenomena.

molecular biology↗

bioRxiv2017

High-throughput identification of RNA nuclear enrichment sequences

One of the biggest surprises since the sequencing of the human genome has been the discovery of thousands of long noncoding RNAs (lncRNAs)1-6. Although lncRNAs and mRNAs are similar in many ways, they differ with lncRNAs being more nuclear-enriched and in several cases exclusively nuclear7,8. Yet, the RNA-based sequences that determine nuclear localization remain poorly understood9-11. Towards the goal of systematically dissecting the lncRNA sequences that impart nuclear localization, we developed a massively parallel reporter assay (MPRA). Unlike previous MPRAs12-15 that determine motifs important for transcriptional regulation, we have modified this approach to identify sequences sufficient for RNA nuclear enrichment for 38 human lncRNAs. Using this approach, we identified 109 unique, conserved nuclear enrichment regions, originating from 29 distinct lncRNAs. We also discovered two shorter motifs within our nuclear enrichment regions. We further validated the sufficiency of several regions to impart nuclear localization by single molecule RNA fluorescence in situ hybridization (smRNA-FISH). Taken together, these results provide a first systematic insight into the sequence elements responsible for the nuclear enrichment of lncRNA molecules.

genomics↗

bioRxiv2017

Live-cell mapping of organelle-associated RNAs via proximity biotinylation combined with protein-RNA crosslinking

The spatial organization of RNA within cells is a crucial factor in a wide range of biological functions, spanning all kingdoms of life. However, a general understanding of RNA localization has been hindered by a lack of simple, high-throughput methods for mapping the transcriptomes of subcellular compartments. Here, we develop such a method, termed APEX-RIP, which combines peroxidase-catalyzed, spatially restricted in situ protein biotinylation with RNA-protein chemical crosslinking. We demonstrate that, using a single protocol, APEX-RIP can isolate RNAs from a variety of subcellular compartments, including the mitochondrial matrix, nucleus, bulk cytosol, and endoplasmic reticulum (ER), with higher specificity and coverage than do conventional approaches. We furthermore identify candidate RNAs localized to mitochondria-ER junctions and nuclear lamina, two compartments that are recalcitrant to classical biochemical purification. Since APEX-RIP is simple, versatile, and does not require special instrumentation, we envision its broad application in a variety of biological contexts.

biochemistry↗

bioRxiv2016

Chromatin environment, transcriptional regulation, and splicing distinguish lincRNAs and mRNAs

While long intergenic noncoding RNAs (lincRNAs) and mRNAs share similar biogenesis pathways, these transcript classes differ in many regards. LincRNAs are less evolutionarily conserved, less abundant, and more tissue-specific, suggesting that their pre- and post-transcriptional regulation is different from that of mRNAs. Here, we perform an in-depth characterization of the features that contribute to lincRNA regulation in multiple human cell lines. We find that lincRNA promoters are depleted of transcription factor (TF) binding sites, yet enriched for some specific factors such as GATA and FOS relative to mRNA promoters. Surprisingly, we find that H3K9me3--a histone modification typically associated with transcriptional repression--is more enriched at the promoters of active lincRNA loci than at those of active mRNAs. Moreover, H3K9me3-marked lincRNA genes are more tissue-specific. The most discriminant differences between lincRNAs and mRNAs involve splicing. LincRNAs are less efficiently spliced, which cannot be explained by differences in U1 binding or the density of exonic splicing enhancers, but may be partially attributed to lower U2AF65 binding and weaker splicing-related motifs. Conversely, the stability of lincRNAs and mRNAs is similar, differing only with regard to the location of stabilizing protein binding sites. Finally, we find that certain transcriptional properties are correlated with higher evolutionary conservation in both DNA and RNA motifs, and are enriched in lincRNAs that have been functionally characterized.

genomics↗