Identifying regulatory elements of mRNA stability and translation in the nervous system

The mechanism from gene to protein, also known as the central dogma, has been one of the fundamental principles of biology. From the start of transcription, to the production of a mature RNA transcript, and to the translation of RNA into protein, regulatory steps control the entire process. Studies have revealed many layers of quality control, especially in regards to mRNA stability, decay, localization and translation. In the nervous system, the idea of mRNA regulation is tempting in regards to certain events, such as those that require quick responses to stimuli. Key experiments have identified ribosomes in dendrites of neurons. RNA binding proteins (RBPs) that localize cytoplasmically and sequences within the untranslated regions of mRNA that mediate binding of these RBPs that regulate its localization and stability. It has become apparent that both cis and trans elements contribute to this tightly controlled network of biology but few studies have examined this interaction in the nervous system of C. elegans. In the following chapters I will describe my studies using the model organism C. elegans to examine RBPs in the nervous system with specific regard to a transcription factor, cebp-1, whose 3'UTR has important roles in controlling its expression during development and adult stages, and two trans factors that may regulate cebp-1 through cebp-1's 3'UT

Identifying regulatory elements of mRNA stability and translation in the nervous system

The mechanism from gene to protein, also known as the central dogma, has been one of the fundamental principles of biology. From the start of transcription, to the production of a mature RNA transcript, and to the translation of RNA into protein, regulatory steps control the entire process. Studies have revealed many layers of quality control, especially in regards to mRNA stability, decay, localization and translation. In the nervous system, the idea of mRNA regulation is tempting in regards to certain events, such as those that require quick responses to stimuli. Key experiments have identified ribosomes in dendrites of neurons. RNA binding proteins (RBPs) that localize cytoplasmically and sequences within the untranslated regions of mRNA that mediate binding of these RBPs that regulate its localization and stability. It has become apparent that both cis and trans elements contribute to this tightly controlled network of biology but few studies have examined this interaction in the nervous system of C. elegans. In the following chapters I will describe my studies using the model organism C. elegans to examine RBPs in the nervous system with specific regard to a transcription factor, cebp-1, whose 3'UTR has important roles in controlling its expression during development and adult stages, and two trans factors that may regulate cebp-1 through cebp-1's 3'UT

Regulatory roles of RNA binding proteins in the nervous system of C. elegans.

Neurons have evolved to employ many factors involved in the regulation of RNA processing due to their complex cellular compartments. RNA binding proteins (RBPs) are key regulators in transcription, translation, and RNA degradation. Increasing studies have shown that regulatory RNA processing is critical for the establishment, functionality, and maintenance of neural circuits. Recent advances in high-throughput transcriptomics have rapidly expanded our knowledge of the landscape of RNA regulation, but also raised the challenge for mechanistic dissection of the specific roles of RBPs in complex tissues such as the nervous system. The C. elegans genome encodes many RBPs conserved throughout evolution. The rich analytic tools in molecular genetics and simple neural anatomy of C. elegans offer advantages to define functions of genes in vivo at the level of a single cell. Notably, the discovery of microRNAs has had transformative effects to the understanding of neuronal development, circuit plasticity, and neurological diseases. Here we review recent studies unraveling diverse roles of RBPs in the development, function, and plasticity of C. elegans nervous system. We first summarize the general technologies for studying RBPs in C. elegans. We then focus on the roles of several RBPs that control gene- and cell-type specific production of neuronal transcripts

Regulatory roles of RNA binding proteins in the nervous system of C. elegans.

Neurons have evolved to employ many factors involved in the regulation of RNA processing due to their complex cellular compartments. RNA binding proteins (RBPs) are key regulators in transcription, translation, and RNA degradation. Increasing studies have shown that regulatory RNA processing is critical for the establishment, functionality, and maintenance of neural circuits. Recent advances in high-throughput transcriptomics have rapidly expanded our knowledge of the landscape of RNA regulation, but also raised the challenge for mechanistic dissection of the specific roles of RBPs in complex tissues such as the nervous system. The C. elegans genome encodes many RBPs conserved throughout evolution. The rich analytic tools in molecular genetics and simple neural anatomy of C. elegans offer advantages to define functions of genes in vivo at the level of a single cell. Notably, the discovery of microRNAs has had transformative effects to the understanding of neuronal development, circuit plasticity, and neurological diseases. Here we review recent studies unraveling diverse roles of RBPs in the development, function, and plasticity of C. elegans nervous system. We first summarize the general technologies for studying RBPs in C. elegans. We then focus on the roles of several RBPs that control gene- and cell-type specific production of neuronal transcripts

Distinct cis elements in the 3′ UTR of the C. elegans cebp-1 mRNA mediate its regulation in neuronal development

The 3' untranslated regions (3' UTRs) of mRNAs mediate post-transcriptional regulation of genes in many biological processes. Cis elements in 3' UTRs can interact with RNA-binding factors in sequence-specific or structure-dependent manners, enabling regulation of mRNA stability, translation, and localization. Caenorhabditis elegans CEBP-1 is a conserved transcription factor of the C/EBP family, and functions in diverse contexts, from neuronal development and axon regeneration to organismal growth. Previous studies revealed that the levels of cebp-1 mRNA in neurons depend on its 3' UTR and are also negatively regulated by the E3 ubiquitin ligase RPM-1. Here, by systematically dissecting cebp-1's 3' UTR, we test the roles of specific cis elements in cebp-1 expression and function in neurons. We present evidence for a putative stem-loop in the cebp-1 3' UTR that contributes to basal expression levels of mRNA and to negative regulation by rpm-1. Mutant animals lacking the endogenous cebp-1 3' UTR showed a noticeable increased expression of cebp-1 mRNA and enhanced the neuronal developmental phenotypes of rpm-1 mutants. Our data reveal multiple cis elements within cebp-1's 3' UTR that help to optimize CEBP-1 expression levels in neuronal development

Inhibition of Axon Regeneration by Liquid-like TIAR-2 Granules

Phase separation into liquid-like compartments is an emerging property of proteins containing prion-like domains (PrLDs), yet the in vivo roles of phase separation remain poorly understood. TIA proteins contain a C-terminal PrLD, and mutations in the PrLD are associated with several diseases. Here, we show that the C. elegans TIAR-2/TIA protein functions cell autonomously to inhibit axon regeneration. TIAR-2 undergoes liquid-liquid phase separation in vitro and forms granules with liquid-like properties in vivo. Axon injury induces a transient increase in TIAR-2 granule number. The PrLD is necessary and sufficient for granule formation and inhibiting regeneration. Tyrosine residues within the PrLD are important for granule formation and inhibition of regeneration. TIAR-2 is also serine phosphorylated in vivo. Non-phosphorylatable TIAR-2 variants do not form granules and are unable to inhibit axon regeneration. Our data demonstrate an in vivo function for phase-separated TIAR-2 and identify features critical for its function in axon regeneration

Gαi/o-coupled receptor signaling restricts pancreatic β-cell expansion.

Gi-GPCRs, G protein-coupled receptors that signal via Gα proteins of the i/o class (Gαi/o), acutely regulate cellular behaviors widely in mammalian tissues, but their impact on the development and growth of these tissues is less clear. For example, Gi-GPCRs acutely regulate insulin release from pancreatic β cells, and variants in genes encoding several Gi-GPCRs--including the α-2a adrenergic receptor, ADRA2A--increase the risk of type 2 diabetes mellitus. However, type 2 diabetes also is associated with reduced total β-cell mass, and the role of Gi-GPCRs in establishing β-cell mass is unknown. Therefore, we asked whether Gi-GPCR signaling regulates β-cell mass. Here we show that Gi-GPCRs limit the proliferation of the insulin-producing pancreatic β cells and especially their expansion during the critical perinatal period. Increased Gi-GPCR activity in perinatal β cells decreased β-cell proliferation, reduced adult β-cell mass, and impaired glucose homeostasis. In contrast, Gi-GPCR inhibition enhanced perinatal β-cell proliferation, increased adult β-cell mass, and improved glucose homeostasis. Transcriptome analysis detected the expression of multiple Gi-GPCRs in developing and adult β cells, and gene-deletion experiments identified ADRA2A as a key Gi-GPCR regulator of β-cell replication. These studies link Gi-GPCR signaling to β-cell mass and diabetes risk and identify it as a potential target for therapies to protect and increase β-cell mass in patients with diabetes