Mapping and engineering RNA-driven architecture of the multiphase nucleolus
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ABSTRACT:
PROVIDER: PRJNA1257780 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR33397100_1.fastq.gz | Fastqsanger.gz | |||
| SRR33397100_2.fastq.gz | Fastqsanger.gz | |||
| SRR33397101_1.fastq.gz | Fastqsanger.gz | |||
| SRR33397101_2.fastq.gz | Fastqsanger.gz | |||
| SRR33397102_1.fastq.gz | Fastqsanger.gz |
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Project description:Mapping and engineering RNA-driven architecture of the multiphase nucleolus
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Project description:Biomolecular condensates are key features of intracellular compartmentalization. As the most prominent nuclear condensate in eukaryotes, the nucleolus is a multiphase liquid-like structure where ribosomal RNAs (rRNAs) are transcribed and processed, undergoing multiple maturation steps to form the small and large ribosomal subunits (SSU and LSU). However, how rRNA processing is coupled to the layered nucleolar organization is poorly understood due to a lack of tools to precisely monitor and perturb nucleolar rRNA processing dynamics. Here, we developed two complementary approaches to spatiotemporally map rRNA processing and engineer de novo nucleoli. Using sequencing in parallel with imaging, we found that rRNA processing steps are spatially segregated, with sequential maturation of rRNA required for its outward movement through nucleolar phases. By generating synthetic nucleoli in cells through an engineered rDNA plasmid system, we show that defects in SSU processing can alter the ordering of nucleolar phases, resulting in inside-out nucleoli and preventing rRNA outflux, while LSU precursors are necessary to build the outermost layer of the nucleolus. These findings demonstrate how rRNA is both a scaffold and substrate for the nucleolus, with rRNA acting as a programmable blueprint for the multiphase architecture that facilitates assembly of an essential molecular machine.
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Project description:Biomolecular condensates are key features of intracellular compartmentalization. As the most prominent nuclear condensate in eukaryotes, the nucleolus is a multiphase liquid-like structure where ribosomal RNAs (rRNAs) are transcribed and processed, undergoing multiple maturation steps to form the small and large ribosomal subunits (SSU and LSU). However, how rRNA processing is coupled to the layered nucleolar organization is poorly understood due to a lack of tools to precisely monitor and perturb nucleolar rRNA processing dynamics. Here, we developed two complementary approaches to spatiotemporally map rRNA processing and engineer de novo nucleoli. Using sequencing in parallel with imaging, we found that rRNA processing steps are spatially segregated, with sequential maturation of rRNA required for its outward movement through nucleolar phases. Furthermore, by generating synthetic nucleoli in cells through an engineered rDNA plasmid system, we show that defects in SSU processing can alter the ordering of nucleolar phases, resulting in inside-out nucleoli and preventing rRNA outflux, while LSU precursors are necessary to build the outermost layer of the nucleolus. These findings demonstrate how rRNA is both a scaffold and substrate for the nucleolus, with rRNA acting as a programmable blueprint for the multiphase architecture that facilitates assembly of an essential molecular machine.
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Project description:Biomolecular condensates are key features of intracellular compartmentalization. As the most prominent nuclear condensate in eukaryotes, the nucleolus is a layered multiphase liquid-like structure and the site of ribosome biogenesis. In the nucleolus, ribosomal RNAs (rRNAs) are transcribed and processed, undergoing multiple maturation steps that ultimately result in formation of the ribosomal small subunit (SSU) and large subunit (LSU). However, how rRNA processing is coupled to the layered nucleolar organization is poorly understood due to a lack of tools to precisely monitor and perturb nucleolar rRNA processing dynamics. Here, we developed two complementary approaches to spatiotemporally map rRNA processing and engineer de novo nucleoli. Using sequencing in parallel with imaging, we found that rRNA processing steps are spatially segregated, with sequential maturation of rRNA required for its outward movement through nucleolar phases. Furthermore, by generating synthetic de novo nucleoli through an engineered rDNA plasmid system in cells, we show that defects in SSU processing can alter the ordering of nucleolar phases, resulting in inside-out nucleoli and preventing rRNA outflux, while LSU precursors are necessary to build the outermost layer of the nucleolus. These findings demonstrate how rRNA is both a scaffold and substrate for the nucleolus, with rRNA acting as a programmable blueprint for the multiphase architecture that facilitates assembly of an essential molecular machine.
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Project description:This SuperSeries is composed of the SubSeries listed below. Gene positioning in nuclear space is central to regulation, with repressive chromatin domains often contacting the nuclear lamina or nucleoli. The nucleolus undergoes structural changes in different cellular states, potentially altering genome organization. Yet, how nucleolar states influence 3D-genome architecture remains underexplored, largely due to the lack of methods able to map nucleolar-associated domains (NADs) in single cells and stressed nucleoli. We developed NoLMseq, a technique combining laser-capture microdissection and DNA sequencing to map NADs in single cells. NoLMseq uncovered unexplored features of chromosome organization around nucleoli, including NAD heterogeneity in mouse embryonic stem cells, yielding two major populations with distinct chromatin and developmental states. NADs predominantly contact nucleoli monoallelically, with contact frequency correlating with gene expression and chromatin states. Under nucleolar stress, NoLMseq revealed extensive chromosome reorganization, highlighting the importance of nucleolus integrity in genome organization. Thus, NoLMseq provides a critical tool to study 3D-genome responses to nucleolar states in health and disease.
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