Transcriptomic characterization of membraneless organelles with scalable resolution - PROJECT SUMMARY Membraneless organelles (MLOs) are prevalent in eukaryotic cells, with a typical size of 1-200 nm to several microns. Through phase separation, MLOs can be assembled under native condition or upon external stimuli, based on either scaffold proteins or scaffold RNAs. “Client” biomolecules, including both RNAs and proteins are further recruited to these MLOs to form localized proteome and transcriptome. MLOs spatially organize RNA- protein interactions and coordinate biochemical reactions. They are widely involved in processing essential cellular RNAs, assembling ribonucleoprotein complexes, regulating RNA metabolism, and responding to cellular stress. Changes in the morphology or the residing RNA and protein components of MLOs are often found to be associated with aging, infection and various human diseases. Despite the essential biological functions of MLOs and potentials of targeting MLOs for therapeutics, adaptable and sensitive methods for efficient transcriptomic characterization of MLOs, in both native and pathogenic samples, are still lacking. Many MLOs contain internal sun-domains or display layered structures. However, the functions of intra-MLO organization in most MLOs are unclear, presenting a gap in our understanding of MLO biology. Do transcripts differentially occupy different subdomains in MLO? Does localization to different subdomains differentially impact the RNA metabolism? These are outstanding questions that have never been addressed, due to the lack of tools with the capability of transcriptomic characterization at subdomain resolution. This proposal aims to fill the critical technical gaps by providing a new platform for transcriptomic mapping of MLOs and intro-MLO organization. A key innovation of our methods is the targeted in situ reverse transcription, with an engineered reverse transcriptase, which can be localized to a specific MLO through a protein targeting module (via protein-antibody interaction), or an RNA targeting module (via RNA-MS2 coat protein interaction). Our new methods have several key advantages: (1) with enhanced and controllable spatial resolution to allow subdomain characterization; (2) highly modular to allow broad characterization of MLOs with either marker proteins or RNAs, and of different physical dimensions; (3) highly adaptable to different sample types by avoiding any requirement of genetic manipulation; and (4) highly efficient for the potential application to tissue samples including clinical samples, and even potentially to the single-cell level. Taking advantage of the new methods, together with super-resolution imaging, we will for the first time perform sub-organelle transcriptomic characterization of nuclear speckles and nucleoli, two prominent nucleus-localized MLOs. These results will reveal differential intra-organelle localization of transcripts and provide critical functional insights for internal organization of these MLOs.
