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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Nuclear RNP complex assembly initiates cytoplasmic RNA localization.

Cytoplasmic localization of mRNAs is a widespread mechanism for generating cell polarity and can provide the basis for patterning during embryonic development. A prominent example of this is localization of maternal mRNAs in Xenopus oocytes, a process requiring recognition of essential RNA sequences by protein components of the localization machinery. However, it is not yet clear how and when such protein factors associate with localized RNAs to carry out RNA transport. To trace the RNA-protein interactions that mediate RNA localization, we analyzed RNP complexes from the nucleus and cytoplasm. We find that an early step in the localization pathway is recognition of localized RNAs by specific RNA-binding proteins in the nucleus. After transport into the cytoplasm, the RNP complex is remodeled and additional transport factors are recruited. These results suggest that cytoplasmic RNA localization initiates in the nucleus and that binding of specific RNA-binding proteins in the nucleus may act to target RNAs to their appropriate destinations in the cytoplasm.

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.

Localization of RNAs to the mitochondrial cloud in Xenopus oocytes through entrapment and association with endoplasmic reticulum.

The germ cell lineage in Xenopus is specified by the inheritance of germ plasm, which originates within a distinct "mitochondrial cloud" (MC) in previtellogenic oocytes. Germ plasm contains localized RNAs implicated in germ cell development, including Xcat2 and Xdazl. To understand the mechanism of the early pathway through which RNAs localize to the MC, we applied live confocal imaging and photobleaching analysis to oocytes microinjected with fluorescent Xcat2 and Xdazl RNA constructs. These RNAs dispersed evenly throughout the cytoplasm through diffusion and then became progressively immobilized and formed aggregates in the MC. Entrapment in the MC was not prevented by microtubule disruption and did not require localization to germinal granules. Immobilized RNA constructs codistributed and showed coordinated movement with densely packed endoplasmic reticulum (ER) concentrated in the MC, as revealed with Dil16(3) labeling and immunofluorescence analysis. Vg1RBP/Vera protein, which has been implicated in linking late pathway RNAs to vegetal ER, was shown to bind specifically both wild-type Xcat2 3' untranslated region and localization-defective constructs. We found endogenous Vg1RBP/Vera and Vg1RBP/Vera-green fluorescent protein to be largely excluded from the MC but subsequently to codistribute with Xcat2 and ER at the vegetal cortex. We conclude that germ line RNAs localize into the MC through a diffusion/entrapment mechanism involving Vg1RBP/Vera-independent association with ER.

Conserved and clustered RNA recognition sequences are a critical feature of signals directing RNA localization in Xenopus oocytes.

Although it is widely regarded that the targeting of RNA molecules to subcellular destinations depends upon the recognition of cis-elements found within their 3' untranslated regions (UTR), relatively little is known about the specific features of these cis-sequences that underlie their function. Interaction between specific repeated motifs within the 3' UTR and RNA-binding proteins has been proposed as a critical step in the localization of Vg1 RNA to the vegetal pole of Xenopus oocytes. To understand the relative contributions of repeated localization element (LE) sequences, we used comparative functional analysis of Vg1 LEs from two frog species, Xenopus laevis and Xenopus borealis. We show that clusters of repeated VM1 and E2 motifs are required for efficient localization. However, groups of either site alone are not sufficient for localization. In addition, we present evidence that the X. borealis Vg1 LE is recognized by the same set of RNA-binding proteins as the X. laevis Vg1 LE and is capable of productive interactions with the X. laevis transport machinery as it is sufficient to direct vegetal localization in X. laevis oocytes. These results suggest that clustered sets of cis-acting sites within the LE direct vegetal transport through specific interactions with the localization machinery.

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.

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.