Browsing by Department "Ibt-Ctcr-Translational Cancer Research"

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Global Changes In the 3UTRome Toggle Responsiveness To Growth Factors
Ibt-Ctcr-Translational Cancer Research; TAMHSC; https://hdl.handle.net/20.500.14641/679; DHHS-NIH-Eunice Kennedy Shriver National Institute of Child Health & Human Development
SUMMARY RNA binding proteins like the Pumilio family (PUFs) exert repression on 3'UTRs, and hence mRNA stability and translation. Alternative polyadenylation (APA) causes 3'UTR length to vary. Coordinated large-scale shortening of 3'UTRs through APA can lead to evasion from 3'UTR- mediated repressive signals and may be a key regulatory regime during development. Yet most identified cases of 3'UTR-mediated PUF repression - and APA to evade repression - were found ad hoc, between interacting gene pairs. Thus, a gap exists in our understanding of how these interactions are orchestrated at the global level of the 3'UTRome and APA. EGF patterns the C. elegans vulva. Of six equipotent vulval precursor cells (VPCs), the three closest to the EGF source are induced to form the vulva, while the distal three remain uninduced. Remarkably, this event occurs with 99.8% fidelity. Three redundant PUFs are expressed specifically in the uninduced cells, suggesting that distal VPCs enact a PUF- and 3'UTR-dependent program to become non-responsive to signal. In the germline, the same PUFs repress ERK/MAP kinase; this same mechanism may be adopted by non-responsive VPCs. Germline immunoprecipitation (IP) of one PUF identified many potential target 3'UTRs. From this dataset, we identified multiple target mRNAs from genes in each vulval signaling cascade (EGFR/Ras/Raf/MEK/ERK, Notch/CSL, PI3K/PDK/Akt, and EGFR/Ras/ RalGEF/Ral). We hypothesize that the redundant PUFs collectively repress mRNAs of all four identified signaling cascades to demarcate signal-non-responsive from signal-responsive cells. Our central hypothesis is that switching from proximal (short 3'UTR) to distal (long 3'UTR) polyadenylation sequence (PAS) usage governs switching from signal-responsiveness to non- responsiveness. We will systematically test this hypothesis by joining bottom-up and top-down specific aims. We propose to: 1) test 3'UTRs from the PUF IP list of vulval signaling genes for ability to mediate PUF-dependent reporter repression in non-responsive cells, 2) survey the VPC 3'UTRome and global proximal-to-distal PAS switching, and 3) validate select candidates by altering endogenous 3'UTRs and polyadenylation signals via CRISPR/Cas9 genome editing and deleting associated PUFs. We will ascertain the contribution to developmental fidelity by APA, and the changes in repressive access points in the 3'UTRs caused by APA.
Optogenetic Toolkit for Remote Control of Voltage-Gated Calcium Channels
Ibt-Ctcr-Translational Cancer Research; TAMHSC; https://hdl.handle.net/20.500.14641/616; DHHS-NIH-National Institute of General Medical Science
Project Summary/Abstract: Owing to the successful use of engineered microbial opsins to remotely control neuronal excitability at high spatiotemporal resolution, considerable new insights into the causal relationship between circuit activity and neuropsychiatric diseases have been obtained through optogenetic approaches. Existing optogenetic tools are primarily designed to manipulate the flow of ions, such as sodium, potassium and chloride, that support an electrogenic role in the brain of mammals. By contrast, calcium ion passing through voltage gated calcium (CaV) channels not only alters membrane potential but also functions as an indispensable messenger to regulate neuronal gene expression, synaptic transmission, neurite outgrowth and memory formation. CaV channels are also essential for cardiac and smooth contractility. CaV channels serve as emerging and attractive therapeutic targets for neuropsychiatric and cardiovascular diseases. However, noninvasive tools to directly and selectively photo-modulate the flow of calcium ion in neurons and cardiomyocytes are still limited. Another technical roadblock that faces the current in vivo optogenetics and optical neuromodulation is the inability of most existing tools to stimulate deep and wide within the brain without the use of invasive indwelling fiber optic probes. To tackle these two technical challenges, we propose to optically inhibit CaV channel activity by engineering light sensitivities into key CaV negative regulators, and in parallel, to develop bio-compatible nano-antennae-upconversion nanoparticles (UCNPs) as a “cordless” optogenetic platform to capture and convert low power, tissue-penetrant, near infrared radiation (NIR) into blue light. This nanoantenna will act as a light-delivery transducer to modulate voltage-gated calcium channels, as well as calcium-dependent activities in excitable tissues, with precise spatiotemporal control. We propose two specific aims to advance our platform to excitable cells by using neuronal cells as proof-of-concept. In Aim 1, we will develop new optogenetic tools to photo-control CaV channel function, and characterize the capacity of our first-generation UCNPs to act as “nanoantenna”. In Aim 2, we will develop next generation UCNPs with high photoconversion efficiencies and enhanced biocompatibility. We anticipate that our NIR light-stimulable optogenetic platform will enable remote and noninvasive control of cell activities in excitable tissues, and permit modulation of their intricate inter- cellular interactions under both physiological and pathological conditions at large scale. Given the wide distribution and close involvement of CaV channels in multiple diseases, the techniques and tools that we develop can be broadly extended to interrogate other types of tissues across multiple systems. In summary, the proposed early-stage, cordless optogenetic technology is anticipated to overcome many of the limitations of current fiber optics-based optogenetic approaches, and will enable new and broad applications in both biomedical research and human health.