The complex interplay of RNA-binding proteins and RISC in neurons

An extensive network of RNA-binding proteins (RBPs) is at the center of posttranscriptional gene regulation. Importantly, different RBPs – including microRNA-loaded Argonaute (Ago) – can bind to a single mRNA resulting in antagonistic or cooperative mode of actions, thereby determining the fate and function of an mRNA. Here, I investigated the impact of two different RBPs, HuR and Staufen2 (Stau2), on microRNA/Ago-dependent gene expression homeostasis. The results of my thesis allow me to present a working model how these three RBPs might control neuronal function in a novel RNA-structure dependent manner. HuR protein binds to AU-rich elements within mRNAs. In the case of Regulator of G-protein signaling (Rgs4) mRNA, I find that HuR binding occurs close to a miR-26/RISC binding site, resulting in Rgs4 destabilization. As both binding sites are in close proximity within a predicted RNA hairpin structure, only synergistic action of HuR and miR-26 results in Rgs4 repression. I propose a novel mechanism involving the trifold combination of HuR, miR-26-loaded Ago and RNA secondary structure in governing functional regulation of Rgs4 mRNA in neurons. Certain RBPs such as Stau2 protein bind to double-stranded RNAs (dsRNAs), thereby shaping local and global secondary structures of mRNAs. Based on preliminary data linking Stau2 and the miRNA pathway, I investigated Stau2-dependent expression, localization and function of the miRNA-induced silencing complex (RISC) in neurons. Proteome and small RNA transcriptome analysis in Stau2 deficient primary neurons revealed significant upregulation of several RISC associated proteins, including Ago1/2, while global miRNA levels were unaffected. This upregulation was accompanied by decreased global translation and translocation of Ago2 from Processing-bodies, sites of mRNA storage, to translating polysomes. Phenotypically, depletion of Ago1/2 reduced dendritic branching. This effect could be rescued by simultaneous knockdown of Stau2, suggesting that Ago1/2 and Stau2 functionally counterbalance each other in neurons. I hypothesize that Stau2’s ability to bind to dsRNA stabilizes defined mRNA structures thereby governing association of RISC and mRNAs. Based on Stau2 hiCLIP experiments by our collaborator Jernej Ule, I was able to define a long-range RNA duplex in the 3’-untranslated region of Rgs4 mRNA bound by Stau2 in vivo. This RNA duplex is necessary and sufficient to drive Stau2-dependent ribonucleoprotein particle (RNP) assembly as well as dendritic RNA localization in neurons. Together, the data presented in my thesis support a model, in which balanced expression and interdependent action of RBPs, RISC and RNA structure shapes RNP assembly and gene expression homeostasis, important for neuronal function

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

Forebrain-specific, conditional silencing of Staufen2 alters synaptic plasticity, learning, and memory in rats

Background: Dendritic messenger RNA (mRNA) localization and subsequent local translation in dendrites critically contributes to synaptic plasticity and learning and memory. Little is known, however, about the contribution of RNA-binding proteins (RBPs) to these processes in vivo. Results: To delineate the role of the double-stranded RBP Staufen2 (Stau2), we generate a transgenic rat model, in which Stau2 expression is conditionally silenced by Cre-inducible expression of a microRNA (miRNA) targeting Stau2 mRNA in adult forebrain neurons. Known physiological mRNA targets for Stau2, such as RhoA, Complexin 1, and Rgs4 mRNAs, are found to be dysregulated in brains of Stau2-deficient rats. In vivo electrophysiological recordings reveal synaptic strengthening upon stimulation, showing a shift in the frequency-response function of hippocampal synaptic plasticity to favor long-term potentiation and impair long-term depression in Stau2-deficient rats. These observations are accompanied by deficits in hippocampal spatial working memory, spatial novelty detection, and in tasks investigating associative learning and memory. Conclusions: Together, these experiments reveal a critical contribution of Stau2 to various forms of synaptic plasticity including spatial working memory and cognitive management of new environmental information. These findings might contribute to the development of treatments for conditions associated with learning and memory deficits

The dsRBP Staufen2 governs RNP assembly of neuronal Argonaute proteins

Mature microRNAs are bound by a member of the Argonaute (Ago1-4) protein family, forming the core of the RNA-induced silencing complex (RISC). Association of RISC with target mRNAs results in ribonucleoprotein (RNP) assembly involved in translational silencing or RNA degradation. Yet, the dynamics of RNP assembly and its underlying functional implications are unknown. Here, we have characterized the role of the RNA-binding protein Staufen2, a candidate Ago interactor, in RNP assembly. Staufen2 depletion resulted in the upregulation of Ago1/2 and the RISC effector proteins Ddx6 and Dcp1a. This upregulation was accompanied by the displacement of Ago1/2 from processing bodies, large RNPs implicated in RNA storage, and subsequent association of Ago2 with polysomes. In parallel, Staufen2 deficiency decreased global translation and increased dendritic branching. As the observed phenotypes can be rescued by Ago1/2 knockdown, we propose a working model in which both Staufen2 and Ago proteins depend on each other and contribute to neuronal homeostasis

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

Forebrain-specific, conditional silencing of Staufen2 alters synaptic plasticity, learning, and memory in rats

© The Author(s).[Background]: Dendritic messenger RNA (mRNA) localization and subsequent local translation in dendrites critically contributes to synaptic plasticity and learning and memory. Little is known, however, about the contribution of RNA-binding proteins (RBPs) to these processes in vivo. [Results]: To delineate the role of the double-stranded RBP Staufen2 (Stau2), we generate a transgenic rat model, in which Stau2 expression is conditionally silenced by Cre-inducible expression of a microRNA (miRNA) targeting Stau2 mRNA in adult forebrain neurons. Known physiological mRNA targets for Stau2, such as RhoA, Complexin 1, and Rgs4 mRNAs, are found to be dysregulated in brains of Stau2-deficient rats. In vivo electrophysiological recordings reveal synaptic strengthening upon stimulation, showing a shift in the frequency-response function of hippocampal synaptic plasticity to favor long-term potentiation and impair long-term depression in Stau2-deficient rats. These observations are accompanied by deficits in hippocampal spatial working memory, spatial novelty detection, and in tasks investigating associative learning and memory. [Conclusions]: Together, these experiments reveal a critical contribution of Stau2 to various forms of synaptic plasticity including spatial working memory and cognitive management of new environmental information. These findings might contribute to the development of treatments for conditions associated with learning and memory deficits.This study was supported by grants from the Spanish Ministry of Science and Competitiveness (BFU2014-56692-R to AG and JMD-G) and the Junta de Andalucía (Spain; BIO-122, CVI-02487, and P07-CVI-02686 to AG and JMD-G), from the European Community’s Seventh Framework Program (FP7/2007- 2013 to AG and DB) under grant agreement number 201714 (DEVANX), from the Austrian Science Fund (SFB-F43 to MAK) and the DFG (FOR2333 to MAK; SPP-1738 to MAK and DB). RS is a Boehringer Ingelheim Fonds fellow

Nasal neuron PET imaging quantifies neuron generation and degeneration

Olfactory dysfunction is broadly associated with neurodevelopmental and neurodegenerative diseases and predicts increased mortality rates in healthy individuals. Conventional measurements of olfactory health assess odor processing pathways within the brain and provide a limited understanding of primary odor detection. Quantification of the olfactory sensory neurons (OSNs), which detect odors within the nasal cavity, would provide insight into the etiology of olfactory dysfunction associated with disease and mortality. Notably, OSNs are continually replenished by adult neurogenesis in mammals, including humans, so OSN measurements are primed to provide specialized insights into neurological disease. Here, we have evaluated a PET radiotracer, [11C]GV1-57, that specifically binds mature OSNs and quantifies the mature OSN population in vivo. [11C]GV1-57 monitored native OSN population dynamics in rodents, detecting OSN generation during postnatal development and aging-associated neurodegeneration. [11C]GV1-57 additionally measured rates of neuron regeneration after acute injury and early-stage OSN deficits in a rodent tauopathy model of neurodegenerative disease. Preliminary assessment in nonhuman primates suggested maintained uptake and saturable binding of [18F]GV1-57 in primate nasal epithelium, supporting its translational potential. Future applications for GV1-57 include monitoring additional diseases or conditions associated with olfactory dysregulation, including cognitive decline, as well as monitoring effects of neuroregenerative or neuroprotective therapeutics

Choroid plexus-derived miR-204 regulates the number of quiescent neural stem cells in the adult brain

Regulation of adult neural stem cell (NSC) number is critical for lifelong neurogenesis. Here, we identified a post-transcriptional control mechanism, centered around the microRNA 204 (miR-204), to control the maintenance of quiescent (q)NSCs. miR-204 regulates a spectrum of transcripts involved in cell cycle regulation, neuronal migration, and differentiation in qNSCs. Importantly, inhibition of miR-204 function reduced the number of qNSCs in the subependymal zone (SEZ) by inducing pre-mature activation and differentiation of NSCs without changing their neurogenic potential. Strikingly, we identified the choroid plexus of the mouse lateral ventricle as the major source of miR-204 that is released into the cerebrospinal fluid to control number of NSCs within the SEZ. Taken together, our results describe a novel mechanism to maintain adult somatic stem cells by a niche-specific miRNA repressing activation and differentiation of stem cells