Syncrip/hnRNP Q is required for activity-induced Msp300/Nesprin-1 expression and new synapse formation.

Memory and learning involve activity-driven expression of proteins and cytoskeletal reorganization at new synapses, requiring posttranscriptional regulation of localized mRNA a long distance from corresponding nuclei. A key factor expressed early in synapse formation is Msp300/Nesprin-1, which organizes actin filaments around the new synapse. How Msp300 expression is regulated during synaptic plasticity is poorly understood. Here, we show that activity-dependent accumulation of Msp300 in the postsynaptic compartment of the Drosophila larval neuromuscular junction is regulated by the conserved RNA binding protein Syncrip/hnRNP Q. Syncrip (Syp) binds to msp300 transcripts and is essential for plasticity. Single-molecule imaging shows that msp300 is associated with Syp in vivo and forms ribosome-rich granules that contain the translation factor eIF4E. Elevated neural activity alters the dynamics of Syp and the number of msp300:Syp:eIF4E RNP granules at the synapse, suggesting that these particles facilitate translation. These results introduce Syp as an important early acting activity-dependent regulator of a plasticity gene that is strongly associated with human ataxias

Molecular and physiological roles of long 3′ UTR mRNA isoforms in neurons

The brain is an organ where the greatest proportion of genes are expressed compared to any other part of the body. To add even more complexity, gene expression in the brain is subject to various layers of regulation through RNA processing mechanisms including alternative splicing (AS) and alternative cleavage and polyadenylation (APA). These RNA processing mechanisms contribute to increased transcriptome diversity in the brain. APA often induces the synthesis of mRNA isoforms that harbor the same protein-coding sequence but different length 3′ untranslated regions (3′ UTRs) from a single gene. Alternative 3′ UTRs regulate gene expression post-transcriptionally by modulating transcript stability, translation efficiency, or subcellular localization. In Chapter 1, we reviewed all of the reported functions of 3′ UTRs in the nervous system. Despite the fact 3′ UTR is highly regarded in gene regulation, evidence of impacts of long 3′ UTR loss on in vivo animal is scarce. To study the physiological relevance of long 3′ UTR mRNA isoforms, we have driven our attention to the Calm1 gene. Calm1 is one of the three genes that encode Calmodulin which is required for proper neural development and function. In Chapter 2, we found that the expression of the long 3′ UTR mRNA isoform of Calm1 was necessary for mouse nervous system development and function. Disruption of the Calm1 long 3′ UTR isoform impaired dorsal root ganglion axon development in mouse embryos and neuronal activation upon novel environment exposure in young adult mice. Our results presented direct evidence for the physiological importance of the Calm1 long 3′ UTR mRNA isoform in vivo. To screen molecular and cellular functions of long 3′ UTRs in a fast and efficient manner, establishing an in vitro cell system is warranted. In Chapter 3, we presented mouse embryonic stem cell (mESC)-derived neurons as a suitable cell-culture system. The transcriptomic profile of the mESC-derived neurons closely resembled the profile in the mouse cerebral cortex, showing the suitability of using this system for studying long 3′ UTRs. The mESC system is amenable to genetic manipulation via CRISPR-Cas9, thus providing as good avenue for fast generation of long 3′ UTR isoform knockout lines. As a proof of principle, a workflow for the generation of Myosin phosphatase Rho interacting protein (Mprip) long 3′ UTR isoform knockout cell lines, differentiation into glutamatergic neurons, and confirmation of the long 3′ UTR expression abolishment is presented. Taking advantage of the convenient culture cell system we have established, we next aimed to explore more functions of long 3′ UTRs. A recent discovery in our lab suggested that APA and AS are closely linked RNA processing mechanisms in which long 3′ UTRs modulate upstream AS. In Chapter 4, we explored the coupling events between AS and APA in mouse neurons using Pull-a-Long-Seq (PL-Seq) pipeline, which presents a particular utility in quantifying the coordination of tandem 3′ UTR APA events with upstream cassette exon AS. PL-Seq performed on the Endonuclease V (Endov) gene reveals that expression of its long 3′ UTR in neurons is preferentially associated with an exon skipping event located far upstream of the terminal exon

Identification of novel post-transcriptional features in olfactory receptor family mRNAs.

Olfactory receptor (Olfr) genes comprise the largest gene family in mice. Despite their importance in olfaction, how most Olfr mRNAs are regulated remains unexplored. Using RNA-seq analysis coupled with analysis of pre-existing databases, we found that Olfr mRNAs have several atypical features suggesting that post-transcriptional regulation impacts their expression. First, Olfr mRNAs, as a group, have dramatically higher average AU-content and lower predicted secondary structure than do control mRNAs. Second, Olfr mRNAs have a higher density of AU-rich elements (AREs) in their 3'UTR and upstream open reading frames (uORFs) in their 5 UTR than do control mRNAs. Third, Olfr mRNAs have shorter 3' UTR regions and with fewer predicted miRNA-binding sites. All of these novel properties correlated with higher Olfr expression. We also identified striking differences in the post-transcriptional features of the mRNAs from the two major classes of Olfr genes, a finding consistent with their independent evolutionary origin. Together, our results suggest that the Olfr gene family has encountered unusual selective forces in neural cells that have driven them to acquire unique post-transcriptional regulatory features. In support of this possibility, we found that while Olfr mRNAs are degraded by a deadenylation-dependent mechanism, they are largely protected from this decay in neural lineage cells

Activity-regulated RNA editing in select neuronal subfields in hippocampus

RNA editing by adensosine deaminases is a widespread mechanism to alter genetic information in metazoa. In addition to modifications in non-coding regions, editing contributes to diversification of protein function, in analogy to alternative splicing. However, although splicing programs respond to external signals, facilitating fine tuning and homeostasis of cellular functions, a similar regulation has not been described for RNA editing. Here, we show that the AMPA receptor R/G editing site is dynamically regulated in the hippocampus in response to activity. These changes are bi-directional, reversible and correlate with levels of the editase Adar2. This regulation is observed in the CA1 hippocampal subfield but not in CA3 and is thus subfield/celltype-specific. Moreover, alternative splicing of the flip/flop cassette downstream of the R/G site is closely linked to the editing state, which is regulated by Ca(2+). Our data show that A-to-I RNA editing has the capacity to tune protein function in response to external stimuli

Doctor of Philosophy

dissertationNicotinic acetylcholine receptors are ligand-gated ion channels. These receptors play important roles in physiological as well as pathophysiological processes. The present work was aimed at studying three questions that were centered on pharmacology, expression and physiology of nicotinic receptors. The first chapter describes the results of work that was aimed at understanding the molecular determinants of interaction between α-conotoxin BuIA and complementary subunits of nicotinic acetylcholine receptors. Proline 6 of BuIA was found to be a major determinant of binding to nAChR β2 subunit. Coupling between proline 6 and the residue at 59th position of the β subunit was found to be equal to 2.4 kcal/mol. This work paves the ground for creating selective ligands that discriminate between α3β2 and α3β4 receptors. The second chapter describes the dependence of expression of human α9-containing nicotinic acetylcholine receptors in the Xenopus laevis oocyte expression system on the 5'leader sequence of the α9 subunit. The human α9 subunit was determined to be the limiting factor in the functional expression of α9-containing receptors. The inclusion of the 5'leader from alfalfa mosaic virus before the α9 coding sequence facilitated the expression of human α9 homomeric receptors by ~70 fold and human α9α10 receptors by ~80 fold. As a result, a vector was created that allowed high iv expression levels of α9-containing nAChRs; this advance allows reliable testing of new compounds that target human α9-containing receptors. The third chapter describes results of work aimed at understanding the interaction between rat nicotinic α9α10 and purinergic receptors. Comparison of currents from coactivation of receptors to the predicted currents gave inconclusive results. Comparison of agonist sensitivities for purinergic receptors when receptors are expressed alone and when they are coinjected revealed ~1.6-fold difference in sensitivity, with P2X4 receptors less sensitive to ATP when α9α10 receptors are coexpressed. Interactions between rat α9α10 nicotinic receptor and purinergic P2X7 receptors were also examined and yielded negative results

Dopamine perturbation of gene co-expression networks reveals differential response in schizophrenia for translational machinery.

The dopaminergic hypothesis of schizophrenia (SZ) postulates that positive symptoms of SZ, in particular psychosis, are due to disturbed neurotransmission via the dopamine (DA) receptor D2 (DRD2). However, DA is a reactive molecule that yields various oxidative species, and thus has important non-receptor-mediated effects, with empirical evidence of cellular toxicity and neurodegeneration. Here we examine non-receptor-mediated effects of DA on gene co-expression networks and its potential role in SZ pathology. Transcriptomic profiles were measured by RNA-seq in B-cell transformed lymphoblastoid cell lines from 514 SZ cases and 690 controls, both before and after exposure to DA ex vivo (100 μM). Gene co-expression modules were identified using Weighted Gene Co-expression Network Analysis for both baseline and DA-stimulated conditions, with each module characterized for biological function and tested for association with SZ status and SNPs from a genome-wide panel. We identified seven co-expression modules under baseline, of which six were preserved in DA-stimulated data. One module shows significantly increased association with SZ after DA perturbation (baseline: P = 0.023; DA-stimulated: P = 7.8 × 10-5; ΔAIC = -10.5) and is highly enriched for genes related to ribosomal proteins and translation (FDR = 4 × 10-141), mitochondrial oxidative phosphorylation, and neurodegeneration. SNP association testing revealed tentative QTLs underlying module co-expression, notably at FASTKD2 (top P = 2.8 × 10-6), a gene involved in mitochondrial translation. These results substantiate the role of translational machinery in SZ pathogenesis, providing insights into a possible dopaminergic mechanism disrupting mitochondrial function, and demonstrates the utility of disease-relevant functional perturbation in the study of complex genetic etiologies

Post-transcriptional regulation of microRNA biogenesis and localization in mammalian neurons

The remarkable cognitive capabilities of our brain require a complex and dynamic network of neurons that is able to quickly and precisely react to changes. Adapting to an ever-changing environment and the storage of information requires that sensory information is transformed into long-lasting structural changes. At the molecular level, highly sophisticated and tightly regulated gene expression programs are necessary to alter synaptic connections in the brain without disrupting the existing network. MicroRNAs are important regulators in this neuronal network as they are able to precisely regulate local gene expression. These small non-coding RNAs bind complementary sequences in target mRNAs, thereby repressing their translation into protein. This plays an important role in activity-dependent synapse development, where local protein synthesis in dendrites is required to implement long-lasting changes in synaptic strength. The latter serves as the molecular basis for learning and memory processes. This cumulative dissertation presents two studies that investigate how neuronal micro- RNAs are regulated at the level of biogenesis and localization. The first publication "The DEAH-box helicase DHX36 mediates dendritic localization of the neuronal precursor-microRNA-134" investigates the transport of miRNA-134 to its final destination in the synapto-dendritic compartment. The study describes that already the precursor (pre-miRNA) is located at the synapse and identifies DHX36 as a protein that specifically binds pre-miR-134 and is important for its transport. Knockdown of DHX36 further shows that the localization of pre-miR-134 to the dendrite is of functional importance. The absence of DHX36 leads to elevated expression of known miR-134 targets, accompanied by an increase in dendritic spine volume. The second study presented in this thesis entitled "The nuclear matrix protein Matr3 regulates processing of the synaptic microRNA-138-5p" investigates the expression of microRNA-138. Two distinct precursor forms are known for miR-138, pre-miR-138-1 and pre-miR-138-2. In our study, we demonstrate that pre-miR-138-2 is the primary source for mature miR-138 in neurons. Using pulldown assays we identify the nuclear matrix protein Matrin-3 (Matr3) as a specific interactor of the hairpin structure of both the primary and precursor form of miR-138-2 (pri-/pre-miR-138-2). Knockdown of its expression demonstrates an inhibitory function of Matr3 in the nuclear processing of pri-miR-138-2, resulting in decreased mature miR-138 levels. In summary, this thesis describes novel post-transcriptional regulatory mechanisms that control the expression and sub-cellular localization of two neuronal microRNAs, miR- 134 and miR-138. Both microRNAs have important roles in synaptic plasticity and a precise regulation of their expression is crucial for maintaining a stable and functional neuronal network. A further understanding of the regulation of these microRNAs and their downstream processes is an important step to gain insight into the complex regulatory processes involved in learning and memory, as well as into malfunctions of these systems that occur in neurological diseases

Control of translation elongation in health and disease.

Regulation of protein synthesis makes a major contribution to post-transcriptional control pathways. During disease, or under stress, cells initiate processes to reprogramme protein synthesis and thus orchestrate the appropriate cellular response. Recent data show that the elongation stage of protein synthesis is a key regulatory node for translational control in health and disease. There is a complex set of factors that individually affect the overall rate of elongation and, for the most part, these influence either transfer RNA (tRNA)- and eukaryotic elongation factor 1A (eEF1A)-dependent codon decoding, and/or elongation factor 2 (eEF2)-dependent ribosome translocation along the mRNA. Decoding speeds depend on the relative abundance of each tRNA, the cognate:near-cognate tRNA ratios and the degree of tRNA modification, whereas eEF2-dependent ribosome translocation is negatively regulated by phosphorylation on threonine-56 by eEF2 kinase. Additional factors that contribute to the control of the elongation rate include epigenetic modification of the mRNA, coding sequence variation and the expression of eIF5A, which stimulates peptide bond formation between proline residues. Importantly, dysregulation of elongation control is central to disease mechanisms in both tumorigenesis and neurodegeneration, making the individual key steps in this process attractive therapeutic targets. Here, we discuss the relative contribution of individual components of the translational apparatus (e.g. tRNAs, elongation factors and their modifiers) to the overall control of translation elongation and how their dysregulation contributes towards disease processes

The axonal transcript Tp53inp2 mediates the development of the sympathetic nervous system

Nerve Growth Factor (NGF) is a neurotrophin essential for the survival of sympathetic and sensory neurons. Localisation of mRNA in axons of NGF- dependent neurons supports growth and maintains axon integrity, however how localised transcripts regulates most axonal functions remains unknown. To characterise the 3’UTR of transcripts localised in sympathetic neuron axons, we performed a 3’end RNA-Seq on mRNA isolated from either axons or cell bodies of neurons cultured in compartmentalised chambers. We identified Tp53inp2 as the most abundant transcripts in axons, accounting for almost one third of the reads. Interestingly, despite the abundance of its RNA, the protein for Tp53inp2 is not detectable within axons of sympathetic neurons. We observe that Tp53inp2 is not actively translated, held in a strictly repressed state mediated by its UTRs. Deletion of Tp53inp2 in sympathetic neurons in vivo and in vitro affects both cell survival and axon growth, suggesting a critical role for Tp53inp2 in neuronal development, despite the lack of translation. That this phenotype can be rescued by transfecting a non- translatable form of the transcript, suggests that instead Tp53inp2 acts as an atypical non-coding RNA, whose function is mediated through interaction with the NGF receptor TrkA. We conclude that Tp53inp2 mRNA regulates sympathetic neuron survival and axon growth in a coding-independent manner by interacting with TrkA receptor and enhancing axonal NGF- dependent signalling