RNA granules in flux: dynamics to balance physiology and pathology.

The life cycle of an mRNA is a complex process that is tightly regulated by interactions between the mRNA and RNA-binding proteins, forming molecular machines known as RNA granules. Various types of these membrane-less organelles form inside cells, including neurons, and contribute critically to various physiological processes. RNA granules are constantly in flux, change dynamically and adapt to their local environment, depending on their intracellular localization. The discovery that RNA condensates can form by liquid-liquid phase separation expanded our understanding of how compartments may be generated in the cell. Since then, a plethora of new functions have been proposed for distinct condensates in cells that await their validation in vivo. The finding that dysregulation of RNA granules (for example, stress granules) is likely to affect neurodevelopmental and neurodegenerative diseases further boosted interest in this topic. RNA granules have various physiological functions in neurons and in the brain that we would like to focus on. We outline examples of state-of-the-art experiments including timelapse microscopy in neurons to unravel the precise functions of various types of RNA granule. Finally, we distinguish physiologically occurring RNA condensation from aberrant aggregation, induced by artificial RNA overexpression, and present visual examples to discriminate both forms in neurons.

RGS4 RNA Secondary Structure Mediates Staufen2 RNP Assembly in Neurons.

RNA-binding proteins (RBPs) act as posttranscriptional regulators controlling the fate of target mRNAs. Unraveling how RNAs are recognized by RBPs and in turn are assembled into neuronal RNA granules is therefore key to understanding the underlying mechanism. While RNA sequence elements have been extensively characterized, the functional impact of RNA secondary structures is only recently being explored. Here, we show that Staufen2 binds complex, long-ranged RNA hairpins in the 3'-untranslated region (UTR) of its targets. These structures are involved in the assembly of Staufen2 into RNA granules. Furthermore, we provide direct evidence that a defined Rgs4 RNA duplex regulates Staufen2-dependent RNA localization to distal dendrites. Importantly, disrupting the RNA hairpin impairs the observed effects. Finally, we show that these secondary structures differently affect protein expression in neurons. In conclusion, our data reveal the importance of RNA secondary structure in regulating RNA granule assembly, localization and eventually translation. It is therefore tempting to speculate that secondary structures represent an important code for cells to control the intracellular fate of their mRNAs.

Visualizing RNA granule transport and translation in living neurons.

In polarized cells, such as neurons, the synthesis of an mRNA does not ensure its proper cellular expression. Most mature transcripts require the association with RNA-binding proteins, resulting in the formation of RNA granules, which are then transported within the cytoplasm along the cytoskeleton and delivered to their proper subcellular locations, where they can be locally translated. Here we review current microscopy methods that have been developed to visualize RNA granule formation, transport and translation at the single cell level with a special emphasis on the MS2 and SunTag systems. They include the labeling of mRNAs and RNA-binding proteins in living cells or even the detection of newly synthesized proteins in situ.

Synaptically-targeted long non-coding RNA SLAMR promotes structural plasticity by increasing translation and CaMKII activity.

Long noncoding RNAs (lncRNAs) play crucial roles in maintaining cell homeostasis and function. However, it remains largely unknown whether and how neuronal activity impacts the transcriptional regulation of lncRNAs, or if this leads to synapse-related changes and contributes to the formation of long-term memories. Here, we report the identification of a lncRNA, SLAMR, which becomes enriched in CA1-hippocampal neurons upon contextual fear conditioning but not in CA3 neurons. SLAMR is transported along dendrites via the molecular motor KIF5C and is recruited to the synapse upon stimulation. Loss of function of SLAMR reduces dendritic complexity and impairs activity-dependent changes in spine structural plasticity and translation. Gain of function of SLAMR, in contrast, enhances dendritic complexity, spine density, and translation. Analyses of the SLAMR interactome reveal its association with CaMKIIα protein through a 220-nucleotide element also involved in SLAMR transport. A CaMKII reporter reveals a basal reduction in CaMKII activity with SLAMR loss-of-function. Furthermore, the selective loss of SLAMR function in CA1 disrupts the consolidation of fear memory in male mice, without affecting their acquisition, recall, or extinction, or spatial memory. Together, these results provide new molecular and functional insight into activity-dependent changes at the synapse and consolidation of contextual fear.

RNA granules in neuronal plasticity and disease.

RNA granules are dynamic entities controlling the spatiotemporal distribution and translation of RNA molecules. In neurons, a variety of RNA granules exist both in the soma and in cellular processes. They contain transcripts encoding signaling and synaptic proteins as well as RNA-binding proteins causally linked to several neurological disorders. In this review, we highlight that neuronal RNA granules exhibit properties of biomolecular condensates that are regulated upon maturation and physiological aging and how they are reversibly remodeled in response to neuronal activity to control local protein synthesis and ultimately synaptic plasticity. Moreover, we propose a framework of how neuronal RNA granules mature over time in healthy conditions and how they transition into pathological inclusions in the context of late-onset neurodegenerative diseases.

RNA supply drives physiological granule assembly in neurons.

Membraneless cytoplasmic condensates of mRNAs and proteins, known as RNA granules, play pivotal roles in the regulation of mRNA fate. Their maintenance fine-tunes time and location of protein expression, affecting many cellular processes, which require complex protein distribution. Here, we report that RNA granules-monitored by DEAD-Box helicase 6 (DDX6)-disassemble during neuronal maturation both in cell culture and in vivo. This process requires neuronal function, as synaptic inhibition results in reversible granule assembly. Importantly, granule assembly is dependent on the RNA-binding protein Staufen2, known for its role in RNA localization. Altering the levels of free cytoplasmic mRNA reveals that RNA availability facilitates DDX6 granule formation. Specifically depleting RNA from DDX6 granules confirms RNA as an important driver of granule formation. Moreover, RNA is required for DDX6 granule assembly upon synaptic inhibition. Together, this data demonstrates how RNA supply favors RNA granule assembly, which not only impacts subcellular RNA localization but also translation-dependent synaptic plasticity, learning, and memory.

Pumilio2 and Staufen2 selectively balance the synaptic proteome.

Neurons have the capacity to adapt to environmental stimuli, a phenomenon termed cellular plasticity. The underlying processes are controlled by a network of RNA-binding proteins (RBPs). Their precise impact, however, is largely unknown. To address this important question, we chose Pumilio2 (Pum2) and Staufen2 (Stau2), which both regulate synaptic transmission. Surprisingly, even though both RBPs dynamically interact with each other in neurons, their respective impact on the transcriptome and proteome is highly selective. Although Pum2 deficiency leads to reduced translation and protein expression, Stau2 depletion preferentially impacts RNA levels and increases protein abundance. Furthermore, we show that Pum2 activates expression of key GABAergic synaptic components, e.g., the GABAA receptor scaffold protein Gephyrin. Consequently, Pum2 depletion selectively reduced the amplitude of miniature inhibitory postsynaptic currents. Together, our data argue for an important role of RBPs to maintain proteostasis in order to control distinct aspects of synaptic transmission.

Live cell imaging reveals 3'-UTR dependent mRNA sorting to synapses.

mRNA transport restricts translation to specific subcellular locations, which is the basis for many cellular functions. However, the precise process of mRNA sorting to synapses in neurons remains elusive. Here we use Rgs4 mRNA to investigate 3'-UTR-dependent transport by MS2 live-cell imaging. The majority of observed RNA granules display 3'-UTR independent bidirectional transport in dendrites. Importantly, the Rgs4 3'-UTR causes an anterograde transport bias, which requires the Staufen2 protein. Moreover, the 3'-UTR mediates dynamic, sustained mRNA recruitment to synapses. Visualization at high temporal resolution enables us to show mRNA patrolling dendrites, allowing transient interaction with multiple synapses, in agreement with the sushi-belt model. Modulation of neuronal activity by either chemical silencing or local glutamate uncaging regulates both the 3'-UTR-dependent transport bias and synaptic recruitment. This dynamic and reversible mRNA recruitment to active synapses would allow translation and synaptic remodeling in a spatially and temporally adaptive manner.

Meet the players: local translation at the synapse.

It is widely believed that activity-dependent synaptic plasticity is the basis for learning and memory. Both processes are dependent on new protein synthesis at the synapse. Here, we describe a mechanism how dendritic mRNAs are transported and subsequently translated at activated synapses. Furthermore, we present the players involved in the regulation of local dendritic translation upon neuronal stimulation and their molecular interplay that maintain local proteome homeostasis. Any dysregulation causes several types of neurological disorders including muscular atrophies, cancers, neuropathies, neurodegenerative, and cognitive disorders.

Interactome of two diverse RNA granules links mRNA localization to translational repression in neurons.

Transport of RNAs to dendrites occurs in neuronal RNA granules, which allows local synthesis of specific proteins at active synapses on demand, thereby contributing to learning and memory. To gain insight into the machinery controlling dendritic mRNA localization and translation, we established a stringent protocol to biochemically purify RNA granules from rat brain. Here, we identified a specific set of interactors for two RNA-binding proteins that are known components of neuronal RNA granules, Barentsz and Staufen2. First, neuronal RNA granules are much more heterogeneous than previously anticipated, sharing only a third of the identified proteins. Second, dendritically localized mRNAs, e.g., Arc and CaMKIIα, associate selectively with distinct RNA granules. Third, our work identifies a series of factors with known roles in RNA localization, translational control, and RNA quality control that are likely to keep localized transcripts in a translationally repressed state, often in distinct types of RNPs.