Intrinsically disordered regions and RNA binding domains contribute to protein enrichment in biomolecular condensates in Xenopus oocytes.

Proteins containing both intrinsically disordered regions (IDRs) and RNA binding domains (RBDs) can phase separate in vitro, forming bodies similar to cellular biomolecular condensates. However, how IDR and RBD domains contribute to in vivo recruitment of proteins to biomolecular condensates remains poorly understood. Here, we analyzed the roles of IDRs and RBDs in L-bodies, biomolecular condensates present in Xenopus oocytes. We show that a cytoplasmic isoform of hnRNPAB, which contains two RBDs and an IDR, is highly enriched in L-bodies. While both of these domains contribute to hnRNPAB self-association and phase separation in vitro and mediate enrichment into L-bodies in oocytes, neither the RBDs nor the IDR replicate the localization of full-length hnRNPAB. Our results suggest a model where the additive effects of the IDR and RBDs regulate hnRNPAB partitioning into L-bodies. This model likely has widespread applications as proteins containing RBD and IDR domains are common biomolecular condensate residents.

Hitting the mark: Localization of mRNA and biomolecular condensates in health and disease.

Subcellular mRNA localization is critical to a multitude of biological processes such as development of cellular polarity, embryogenesis, tissue differentiation, protein complex formation, cell migration, and rapid responses to environmental stimuli and synaptic depolarization. Our understanding of the mechanisms of mRNA localization must now be revised to include formation and trafficking of biomolecular condensates, as several biomolecular condensates that transport and localize mRNA have recently been discovered. Disruptions in mRNA localization can have catastrophic effects on developmental processes and biomolecular condensate biology and have been shown to contribute to diverse diseases. A fundamental understanding of mRNA localization is essential to understanding how aberrations in this biology contribute the etiology of numerous cancers though support of cancer cell migration and biomolecular condensate dysregulation, as well as many neurodegenerative diseases, through misregulation of mRNA localization and biomolecular condensate biology. This article is categorized under: RNA Export and Localization > RNA Localization RNA in Disease and Development > RNA in Disease RNA in Disease and Development > RNA in Development.

Multivalent interactions with RNA drive recruitment and dynamics in biomolecular condensates in Xenopus oocytes.

RNA localization and biomolecular condensate formation are key biological strategies for organizing the cytoplasm and generating cellular polarity. In Xenopus oocytes, RNAs required for germ layer patterning localize in biomolecular condensates, termed Localization bodies (L-bodies). Here, we have used an L-body RNA-binding protein, PTBP3, to test the role of RNA-protein interactions in regulating the biophysical characteristics of L-bodies in vivo and PTBP3-RNA condensates in vitro. Our results reveal that RNA-protein interactions drive recruitment of PTBP3 and localized RNA to L-bodies and that multivalent interactions tune the dynamics of the PTBP3 after localization. In a concentration-dependent manner, RNA becomes non-dynamic and interactions with the RNA determine PTBP3 dynamics within these biomolecular condensates in vivo and in vitro. Importantly, RNA, and not protein, is required for maintenance of the PTBP3-RNA condensates in vitro, pointing to a model where RNA serves as a non-dynamic substructure in these condensates.

Regulation of spatially restricted gene expression: linking RNA localization and phase separation.

Subcellular restriction of gene expression is crucial to the functioning of a wide variety of cell types. The cellular machinery driving spatially restricted gene expression has been studied for many years, but recent advances have highlighted novel mechanisms by which cells can generate subcellular microenvironments with specialized gene expression profiles. Particularly intriguing are recent findings that phase separation plays a role in certain RNA localization pathways. The burgeoning field of phase separation has revolutionized how we view cellular compartmentalization, revealing that, in addition to membrane-bound organelles, phase-separated cytoplasmic microenvironments - termed biomolecular condensates - are compositionally and functionally distinct from the surrounding cytoplasm, without the need for a lipid membrane. The coupling of phase separation and RNA localization allows for precise subcellular targeting, robust translational repression and dynamic recruitment of accessory proteins. Despite the growing interest in the intersection between RNA localization and phase separation, it remains to be seen how exactly components of the localization machinery, particularly motor proteins, are able to associate with these biomolecular condensates. Further studies of the formation, function, and transport of biomolecular condensates promise to provide a new mechanistic understanding of how cells restrict gene expression at a subcellular level.

L-bodies are RNA-protein condensates driving RNA localization in Xenopus oocytes.

Ribonucleoprotein (RNP) granules are membraneless compartments within cells, formed by phase separation, that function as regulatory hubs for diverse biological processes. However, the mechanisms by which RNAs and proteins interact to promote RNP granule structure and function in vivo remain unclear. In Xenopus laevis oocytes, maternal mRNAs are localized as large RNPs to the vegetal hemisphere of the developing oocyte, where local translation is critical for proper embryonic patterning. Here we demonstrate that RNPs containing vegetally localized RNAs represent a new class of cytoplasmic RNP granule, termed localization-bodies (L-bodies). We show that L-bodies contain a dynamic protein-containing phase surrounding a nondynamic RNA-containing phase. Our results support a role for RNA as a critical component within these RNP granules and suggest that cis-elements within localized mRNAs may drive subcellular RNA localization through control over phase behavior.

Organizing the oocyte: RNA localization meets phase separation.

RNA localization is a key biological strategy for organizing the cytoplasm and generating both cellular and developmental polarity. During RNA localization, RNAs are targeted asymmetrically to specific subcellular destinations, resulting in spatially and temporally restricted gene expression through local protein synthesis. First discovered in oocytes and embryos, RNA localization is now recognized as a significant regulatory strategy for diverse RNAs, both coding and non-coding, in a wide range of cell types. Yet, the highly polarized cytoplasm of the oocyte remains a leading model to understand not only the principles and mechanisms underlying RNA localization, but also links to the formation of biomolecular condensates through phase separation. Here, we discuss both RNA localization and biomolecular condensates in oocytes with a particular focus on the oocyte of the frog, Xenopus laevis.

Using the Xenopus Oocyte Toolbox.

The Xenopus oocyte is a unique model system, allowing both the study of complex biological processes within a cellular context through expression of exogenous mRNAs and proteins, and the study of the cell, molecular, and developmental biology of the oocyte itself. During oogenesis, Xenopus oocytes grow dramatically in size, with a mature oocyte having a diameter of ∼1.3 mm, and become highly polarized, localizing many mRNAs and proteins. Thus, the mature oocyte is a repository of maternal mRNAs and proteins that will direct early embryogenesis prior to zygotic genome transcription. Importantly, the Xenopus oocyte also has the capacity to translate exogenous microinjected RNAs, which has enabled breakthroughs in a wide range of areas including cell biology, developmental biology, molecular biology, and physiology. This introduction outlines how Xenopus oocytes can be used to study a variety of important biological questions.

Whole-Mount Immunofluorescence for Visualizing Endogenous Protein and Injected RNA in Xenopus Oocytes.

Asymmetric distribution of mRNA and protein is a hallmark of cell polarity in many systems. The Xenopus laevis oocyte provides many technical advantages to studying such polarity. Thousands of oocytes at different stages of maturity can be harvested from a single ovary and, owing to their relatively large size, even the youngest oocytes can be manually microinjected. Microinjection of fluorescently labeled RNA combined with immunofluorescence of endogenous proteins can provide insight into the cytoplasmic interactions contributing to polarity. Here, we present an updated method to image endogenous protein and microinjected RNA in X. laevis oocytes.

Modeling microtubule-based transport and anchoring of mRNA.

Localization of messenger RNA (mRNA) at the vegetal cortex plays an important role in the early development of Xenopus laevis oocytes. While it is known that molecular motors are responsible for the transport of mRNA cargo along microtubules to the cortex, the mechanisms of localization remain unclear. We model cargo transport along microtubules using partial differential equations with spatially-dependent rates. A theoretical analysis of reduced versions of our model predicts effective velocity and diffusion rates for the cargo and shows that randomness of microtubule networks enhances effective transport. A more complex model using parameters estimated from fluorescence microscopy data reproduces the spatial and timescales of mRNA localization observed in Xenopus oocytes, corroborates experimental hypotheses that anchoring may be necessary to achieve complete localization, and shows that anchoring of mRNA complexes actively transported to the cortex is most effective in achieving robust accumulation at the cortex.

Analysis of Active Transport by Fluorescence Recovery after Photobleaching.

Fluorescence recovery after photobleaching (FRAP) is a well-established experimental technique to study binding and diffusion of molecules in cells. Although a large number of analytical and numerical models have been developed to extract binding and diffusion rates from FRAP recovery curves, active transport of molecules is typically not included in the existing models that are used to estimate these rates. Here we present a validated numerical method for estimating diffusion, binding/unbinding rates, and active transport velocities using FRAP data that captures intracellular dynamics through partial differential equation models. We apply these methods to transport and localization of mRNA molecules in Xenopus laevis oocytes, where active transport processes are essential to generate developmental polarity. By providing estimates of the effective velocities and diffusion, as well as expected run times and lengths, this approach can help quantify dynamical properties of localizing and nonlocalizing RNA. Our results confirm the distinct transport dynamics in different regions of the cytoplasm, and suggest that RNA movement in both the animal and vegetal directions may influence the timescale of RNA localization in Xenopus oocytes. We also show that model initial conditions extracted from FRAP postbleach intensities prevent underestimation of diffusion, which can arise from the instantaneous bleaching assumption. The numerical and modeling approach presented here to estimate parameters using FRAP recovery data is a broadly applicable tool for systems where intracellular transport is a key molecular mechanism.

Using in vivo imaging to measure RNA mobility in Xenopus laevis oocytes.

RNA localization in the Xenopus oocyte is responsible for the establishment of polarity during oogenesis as well as the specification of germ layers during embryogenesis. However, the inability to monitor mRNA localization in live vertebrate oocytes has posed a major barrier to understanding the mechanisms driving directional transport. Here we describe a method for imaging MS2 tagged RNA in live Xenopus oocytes to study the dynamics of RNA localization. We also focus on methods for implementing and analyzing FRAP data. This protocol is optimized for imaging of the RNAs in stage II oocytes but it can be adapted to study dynamics of other molecules during oogenesis. Using this approach, mobility can be measured in different regions of the oocyte, enabling the direct observation of molecular dynamics throughout the oocyte.

Directional transport is mediated by a Dynein-dependent step in an RNA localization pathway.

Cytoplasmic RNA localization is a key biological strategy for establishing polarity in a variety of organisms and cell types. However, the mechanisms that control directionality during asymmetric RNA transport are not yet clear. To gain insight into this crucial process, we have analyzed the molecular machinery directing polarized transport of RNA to the vegetal cortex in Xenopus oocytes. Using a novel approach to measure directionality of mRNA transport in live oocytes, we observe discrete domains of unidirectional and bidirectional transport that are required for vegetal RNA transport. While kinesin-1 appears to promote bidirectional transport along a microtubule array with mixed polarity, dynein acts first to direct unidirectional transport of RNA towards the vegetal cortex. Thus, vegetal RNA transport occurs through a multistep pathway with a dynein-dependent directional cue. This provides a new framework for understanding the mechanistic basis of cell and developmental polarity.

Taking a cellular road-trip: mRNA transport and anchoring.

mRNA localization is a crucial mechanism for post-transcriptional control of gene expression used in numerous cellular contexts to generate asymmetric enrichment of an encoded protein. This process has emerged as a fundamental regulatory mechanism that operates in a wide range of organisms to control an array of cellular processes. Recently, significant advances have been made in our understanding of the mechanisms that regulate several steps in the mRNA localization pathway. Here we discuss the progress made in understanding localization element recognition, paying particular attention to the role of RNA structure. We also consider the function of mRNP granules in mRNA transport, as well as new results pointing to roles for the endocytic pathway in mRNA localization.

Molecular motors: directing traffic during RNA localization.

RNA localization, the enrichment of RNA in a specific subcellular region, is a mechanism for the establishment and maintenance of cellular polarity in a variety of systems. Ultimately, this results in a universal method for spatially restricting gene expression. Although the consequences of RNA localization are well-appreciated, many of the mechanisms that are responsible for carrying out polarized transport remain elusive. Several recent studies have illuminated the roles that molecular motor proteins play in the process of RNA localization. These studies have revealed complex mechanisms in which the coordinated action of one or more motor proteins can act at different points in the localization process to direct RNAs to their final destination. In this review, we discuss recent findings from several different systems in an effort to clarify pathways and mechanisms that control the directed movement of RNA.

Visualization of mRNA localization in Xenopus oocytes.

Visualization of in vivo mRNA localization provides a tool for understanding steps in the mechanism of transport. Here we detail a method of fluorescently labeling mRNA transcripts and microinjecting them into Xenopus laevis oocytes followed with imaging by confocal microscopy. This technique overcomes a significant hurdle of imaging RNA in the frog oocyte while providing a rapid method of visualizing mRNA localization in high resolution.

Preparation of a highly active cell-free translation system from immature Xenopus laevis oocytes.

Understanding mechanisms of post-transcriptional control of gene expression has come under much scrutiny in recent years. A key question in this field is how the translation of specific mRNAs is activated or repressed both spatially and temporally in a given cell. In oocytes of the frog Xenopus laevis a number of mRNAs are localized early in oogenesis and subsequently translated at later stages. We have developed a highly active cell-free translation system from oocytes in the early stages of oogenesis that is applicable to the study of translation and translational control of both endogenous and exogenous mRNAs.

Visualizing RNA localization in Xenopus oocytes.

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Multiple kinesin motors coordinate cytoplasmic RNA transport on a subpopulation of microtubules in Xenopus oocytes.

RNA localization is a widely conserved mechanism for generating cellular asymmetry. In Xenopus oocytes, microtubule-dependent transport of RNAs to the vegetal cortex underlies germ layer patterning. Although kinesin motors have been implicated in this process, the apparent polarity of the microtubule cytoskeleton has pointed instead to roles for minus-end-directed motors. To resolve this issue, we have analyzed participation of kinesin motors in vegetal RNA transport and identified a direct role for Xenopus kinesin-1. Moreover, in vivo interference and biochemical experiments reveal a key function for multiple motors, specifically kinesin-1 and kinesin-2, and suggest that these motors may interact during transport. Critically, we have discovered a subpopulation of microtubules with plus ends at the vegetal cortex, supporting roles for these kinesin motors in vegetal RNA transport. These results provide a new mechanistic basis for understanding directed RNA transport within the cytoplasm.

PTB/hnRNP I is required for RNP remodeling during RNA localization in Xenopus oocytes.

Transport of specific mRNAs to defined regions within the cell cytoplasm is a fundamental mechanism for regulating cell and developmental polarity. In the Xenopus oocyte, Vg1 RNA is transported to the vegetal cytoplasm, where localized expression of the encoded protein is critical for embryonic polarity. The Vg1 localization pathway is directed by interactions between key motifs within Vg1 RNA and protein factors recognizing those RNA sequences. We have investigated how RNA-protein interactions could be modulated to trigger distinct steps in the localization pathway and found that the Vg1 RNP is remodeled during cytoplasmic RNA transport. Our results implicate two RNA-binding proteins with key roles in Vg1 RNA localization, PTB/hnRNP I and Vg1RBP/vera, in this process. We show that PTB/hnRNP I is required for remodeling of the interaction between Vg1 RNA and Vg1RBP/vera. Critically, mutations that block this remodeling event also eliminate vegetal localization of the RNA, suggesting that RNP remodeling is required for localization.