Overview
1. The molecular basis of condensate identity
Biomolecular condensation is a key organizing principle in all cells. Condensates are concentrated assemblies of interacting proteins and nucleic acids that mediate diverse biological functions. While condensates are dynamic entities, freely exchanging components and displaying properties of liquid-like droplets, condensates maintain specific compositional identities. We know very little about how the molecular identities of condensates are defined.
A useful system for studying the molecular origins of condensate identity is the nuclear paraspeckle. Paraspeckles regulate post-transcriptional gene expression, and alterations to paraspeckle morphology and abundance are associated with enhanced proliferation and motility of cancer cells. Paraspeckles comprise multiple, different RNA-binding proteins arranged in distinct core and shell layers, the organization of which is mediated by a long noncoding RNA called NEAT1. It is not known how NEAT1 governs layer formation. We are working to uncover the RNA motif grammar of NEAT1 that directs combinatorial protein binding to assemble layers. We have developed methods to build “paraspeckles in a test tube” from a minimal set of purified proteins and synthetic NEAT1 fragments. To complement our in vitro platform, we are developing tools to visualize the de novo assembly of paraspeckles in living cells.
Image: Fluorescence microscopy image of core-shell droplets reconstituted in vitro from two paraspeckle proteins, each labeled in green and pink, and a short fragment of NEAT1.
2. mRNA nuclear export control
Transcription, the process of producing RNA from DNA, is a central stage in the control of gene expression. Transcription is inherently noisy for many genes, occurring in “bursts” or turning off and on unpredictably. However, mRNA abundance in the cytoplasm is relatively predictable, indicating that transcriptional noise is buffered via regulated mRNA export from nucleus to cytoplasm. Surprisingly little is known about how mRNAs are retained in the nucleus post-transcriptionally.
The paraspeckle is an important mRNA nuclear export regulator. While paraspeckles interact with and affect the nuclear export of mRNAs from more than 1000 different genes, little is known about how paraspeckles select their mRNA targets. We are working to uncover the “code” of mRNA localization to paraspeckles using a variety of approaches, including reporter screening, transcriptomics, live-cell mRNA imaging, and high-throughput RNA-binding studies.
3. mRNA transport over long distances
mRNA transport and localized protein production is a universal and essential requirement in all cells from bacteria to human neurons. mRNAs are transported by RNA-binding proteins, which recognize and “package” specific mRNA targets into transport complexes. While RNA-protein complexes are typically thought to couple directly to molecular motors for long-distance transport, these complexes can also “hitchhike” on the cytoplasmic surfaces of membrane-bound organelles such as endosomes and lysosomes. Hitchhiking-mediated mRNA transport is critical for axon maintenance and survival. While membrane-bound transport carriers appear to be selective for specific mRNA targets, it is not known how such carriers, or their membrane-associated RNA-binding proteins, select their cargoes.
We are working to understand how membrane-bound RNA binding proteins recognize and co-assemble with mRNA targets into membrane-associated transport complexes. We have developed a suite of tools to reconstitute and visualize protein-RNA condensation on membrane surfaces. To complement these in vitro toolsets, we are developing approaches to image and track hitchhiking-based mRNA transport in living cells.
Image: TIRF microscopy image of in vitro condensates assembled on a planar membrane surface. The RNA-binding protein Whi3 (green) forms two-dimensional condensates on the membrane which recruit and cluster the RNA CLN3 (pink) at the condensate edges.
4. Translation at membrane surfaces
The translation of an mRNA message into a protein product is tightly regulated, with a variety of control knobs that tune the rates of translation initiation and elongation. For example, bound protein roadblocks or secondary structural features can modulate preinitiation complex scanning and ribosome translocation, with important consequences for protein folding and stability. mRNAs frequently localize to the surfaces of membrane-bound organelles for translation, including the endoplasmic reticulum and lysosome, even when the encoded protein is not secreted or membrane-embedded. While membrane localization is well understood to dramatically impact the conformational heterogeneity and biological functions of proteins, very little is known about how membrane recruitment impacts mRNA conformation and translation. We are working to dissect the biophysical mechanisms by which membrane association alters translation dynamics. Toward this goal, we are developing tools to monitor real-time mRNA translation on membrane surfaces both in vitro and in living cells.
