Advanced tools for RNA spatial biology

RNA molecules are central drivers of cellular compartmentalization, nucleating and scaffolding subcellular structures in all kingdoms of life. In humans, such architectural RNAs coordinate chromatin structure at nearly all distance scales, and they scaffold an array of “membraneless organelles” that collectively control cellular metabolic, epigenetic, transcriptional, and stress-signaling programs. Dysregulation of these RNA-scaffolded structures is causally implicated in a broad range of human pathologies, including neurodegenerative diseases like ALS, RNA viral infections like HIV, and nearly all cancers. Yet, most of these structures have long eluded detailed molecular characterization, in part because they are too dynamic and fragile to be analyzed by conventional biochemical approaches.

The Shechner lab seeks to overcome this challenge, elucidating the molecular mechanisms by which RNAs coordinate subcellular architecture, and how these mechanisms are rewired in disease. Our highly multidisciplinary approach combines classical cell biology and biochemistry with cutting-edge chemical biology, synthetic biology, and high-throughput genomics methods. We specialize in the development of powerful, yet “democratized” new tools: methods built from standard parts and straightforward manipulations, and thus readily accessible to most molecular biology labs.

Our prior work included CRISPR-Display, which enables RNA domains to be ectopically localized to targeted genomic sites, and CLING, which uses CRISPR-Display to visualize chromatin dynamics in live cells, and APEX-RIP, which uses in situ proximity-biotinylation to map RNA subcellular localization, transcriptome wide. Read more about these techniques here.

Assembly, disassembly, and misassembly of RNA-scaffolded biomolecular condensates. Using O-MAP, we are probing the long-elusive assembly pathways for several key RNA-centric membraneless organelles, and are interrogating how these pathways are rewired in disease. This will reveal fundamental mechanisms of cellular compartmentalization, and a host of new therapeutic targets.

Parsing the “molecular grammar” of RNA-driven compartmentalization. Although the core physical principals that drive biomolecular condensation are becoming increasingly clear, how cells exploit these principles to build complex and dynamic structures in vivo remain relatively unknown. Such structures are extraordinarily complex—comprising thousands of distinct molecular species—far beyond what has been reconstituted in vitro or visualized in live cells. By combining O-MAP with experimental genomics tools, we aim to elucidate the yet-unknown “molecular grammar” by which cells sort these species into their correct target locale.

Nascent transcripts as drivers of nuclear architecture. We are pursuing the hypothesis that nascent transcripts help to build functionalized microcompartments within the nucleus (e.g. "speckles," "splicing factories," etc), which regulate chromatin architecture and gene expression. We are using O-MAP to achieve an unprecedented molecular dissection of one such compartment, revealing novel genomic interactions, transcripts, and proteins with mechanistic implications for tissue development and disease. Generalizing this approach, we aim to understand more broadly how the nascent transcripts of key developmental genes sculpt nuclear architecture.

Expanding the O-MAP toolkit. We are expanding O-MAP to build a suite of powerful new tools for Spatial Biology. Ongoing projects include: targeting O-MAP to other biopolymers or cellular features; targeting O-MAP to specific subpopulations of transcripts; targeting O-MAP to phenotype-specific populations of cells within complex samples; adapting O- MAP for diagnostic- and target-discovery in clinical isolates and biobank samples.

More details on past projects can be found on our Publications page.
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