2017 — 2021 |
Canzio, Daniele |
K99Activity Code Description: To support the initial phase of a Career/Research Transition award program that provides 1-2 years of mentored support for highly motivated, advanced postdoctoral research scientists. R00Activity Code Description: To support the second phase of a Career/Research Transition award program that provides 1 -3 years of independent research support (R00) contingent on securing an independent research position. Award recipients will be expected to compete successfully for independent R01 support from the NIH during the R00 research transition award period. |
Coupling Promoter Choice and Alternative Rna Splicing in the Mammalian Protocadherin Gene Cluster @ Columbia University Health Sciences
TITLE: ?Coupling promoter choice and alternative RNA splicing in the mammalian Protocadherin gene cluster? PROJECT SUMMARY: Eukaryotic gene expression is regulated by a complex network of functional coupling between the processes of transcription initiation, elongation and RNA processing. The mammalian Protocadherin (Pcdh) gene cluster provides a remarkable and fundamentally important system to study the underlying mechanisms of this coupling process. The proteins encoded by the Pcdh gene cluster play an essential role in neural circuit assembly by providing individual neurons with a unique cell surface ?code? that forms the basis of self-recognition. The Pcdh cell surface code is generated by a complex process of stochastic promoter choice, alternative RNA splicing and combinatorial assembly of Pcdh cis-dimers at the cell surface. The transcriptional process involves ?stochastic? activation of individual Pcdh gene promoters through long-range enhancer-promoter DNA looping (which requires the DNA binding protein CTCF), transcription of as much as 250,000 base pairs of DNA, followed by splicing of a promoter proximal 5' splice site to a distant 3' splice site. Although significant progress has been made in understanding the genomic DNA organization, single neuron expression, and chromosome domain configuration of the clustered Pcdh, the mechanisms by which transcriptional initiation and elongation, and RNA processing are coupled remain unknown. Recent studies have revealed a remarkable organization of highly conserved RNA duplex structures in the Pcdh pre-mRNAs, and a striking pattern of convergent transcription at Pcdh promotors. These preliminary observations have lead to a model in which these RNA secondary structures regulate 5' splice site choice, and a novel mechanism for promoter choice. Aim 1 of this proposal is to Determine the architecture of Pcdh ? and ? RNA precursors in mammalian cells and investigate their functions, and Aim 2 is to Determine the role of CTCF in regulating transcription and processing of Pcdh? RNAs. A variety of approaches will be used to accomplish these aims, including the in vivo analysis of RNA secondary structures using chemical probes and RNASeq methods, single molecule visualization methods to image the translocation of RNA polymerase as it proceeds through the gene cluster, and gene editing methods to identify regulatory elements required for transcription and splicing. The proposed studies are poised to reveal novel and exciting regulatory mechanisms governing eukaryotic gene regulation. Moreover, as Pcdh proteins play a central role in neural circuit assembly, and they have been implicated in neurological diseases, understanding the details of Pcdh gene expression will not only provide fundamental insights into novel mechanisms of gene expression, but also lead to a better understanding of the genetic basis of neurological diseases, such as autism. Thus, the proposed research is of direct relevance to human health.
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2021 |
Canzio, Daniele |
DP2Activity Code Description: To support highly innovative research projects by new investigators in all areas of biomedical and behavioral research. |
How Do Neurons Recognize Self From Non-Self? @ University of California, San Francisco
PROJECT SUMMARY This essay aims to understand a fundamental property of neurons: their ability to self-recognize and self- avoid. Self-avoidance is an essential aspect of a neuron's function as it ensures that branches from the same cell minimize their overlap while maximizing their interactions with branches from other cells. In mammals, at the core of this process is the generation of sufficient Protocadherin (Pcdh) protein isoform diversity such that essentially every neuron in the brain is differentially barcoded at its surface and therefore appears different to other neurons. The generation of Pcdh protein isoform diversity requires complex mechanisms of Pcdh transcriptional and pre-mRNA splicing such that distinct Pcdh mRNA molecules - bearing a different 5' end (variable exon) but an identical 3' end (constant exons) - are expressed in individual cells. Understanding how different Pcdh mRNA molecules are produced represents a long-term mystery in the field of neuroscience. Answering this fundamental mystery is key in illuminating the process of neuronal self-avoidance and represents the first essential step toward dissecting how dysregulation of this Pcdh mediated self-avoidance can lead to severe neurological disorders, such as for instance autism spectrum disorder and schizophrenia. Despite their critical function in the brain, however, limited progress has been made in understanding how Pcdh mRNAs are transcribed and properly spliced as general models of gene expression regulation have failed to recapitulate this complex mechanism and as the tools required to study it directly in vivo have lagged behind. In this proposal, we aim to (i) test a paradigm-shifting hypothesis of Pcdh RNA transcription and splicing based on alternative trans-splicing of variable and constant exons encoded in tandem on the same DNA strand - a mechanism that we propose to be regulated by the 3D genome topology of the Pcdh locus - and (ii) design technological innovations that will allow precise manipulation of the Pcdh gene cluster in vivo to test our hypothesis directly in neurons. These studies have the potential to illuminate the complex mechanism of the generation of Pcdh isoform diversity and its role in neuronal self-avoidance and wiring of healthy and disease brains. The findings from these studies are also poised to open up a new class of regulatory mechanisms of RNA processing reactions, previously unobserved and uncharacterized in mammals but that we speculate are utilized by cells to overcome challenging problems of pre-mRNA splicing associated with complex gene architectures.
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