Lecture 2: mrna localisation and decay in cells and in tissues
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1 Lecture 2: mrna localisation and decay in cells and in tissues Prof. Ilan Davis, Department of Biochemistry. Senior Research Fellow, Jesus College
2 References Molecular Biology of the Cell, Sixth Edition Authors: Alberts et al. Chapter 21: Development of Multicellular Organisms Principles of Neurobiology, 1st Edition Author: Liqun Luo ISBN: For movies: (click on student and register for resources)
3 Content of lectures 2 1. The human cell atlas 2. All cells same DNA, different gene expression 3. Dogma: transcription regulates development 4. Postranscriptional-regulation 5. RNA enzymes 6. The RNA world hypothesis 7. mrna localisation more generally 8. Example of transport and local translation 9. mrna localisation and synaptic plasticity 10.smFISH and the neuromuscular junction 11. mrna stability: synthesis and decay 12. measuring mrna stability: 4sU incorporation
4
5 The concept behind the human cell atlas Examples scrna sequencing Disaggregate tissues into individual cells and then sequence all mrnas in each cell Single molecule RNA FISH Determine the distribution of many key mrnas spatially within the same tissue
6 Different cells contain the same DNA Each cell type expresses a different set of genes
7 Different cells contain different proteins Proteins common to both tissues Proteins only present in one tissue
8 Regulation of gene expression by transcription factors (the orthodox view)
9 Central dogma
10 Multiple levels of regulation of gene expression Post-transcriptional regulation (at level of RNA) is still under appreciated in development
11 An RNA enzyme The three-dimensional structure of the original ribozyme, the self-splicing intron of Tetrahymena (13). Green and blue ribbons indicate the path of the RNA backbone in the two major domains of the RNA, and the red star marks the active site. Figure by Feng Guo
12 How did life arise on our planet? The RNA world hypothesis
13 + Repression of translation unlocalised mrna mrna degredation Export mrna sorting Localisation By MT motors - Anchoring by actin?
14 cis-acting RNA signals and transacting proteins link the mrna to molecular motor for transport MTs kinesin actin myosin MTs Linkers? Staufen She2+3 BicD Egalitarian dynein/ dynactin mrna
15 Functions of asymmetric cytoplasmic mrna localisation 1) Target proteins to their site of function. 3) Define the axes of the embryo. 2) Segregate cytoplasmic determinants during asymmetric cell division 4) Promote or Restrict protein diffusion in a syncytial embryo (morphogenetic gradients)
16 The Importance of RNA localisation Essential for many fundamental processes: Cell polarity Developmental patterning Neural development Learning and memory Hamilton et al 2012, Biophysics for the life sciences Chapter 11
17 mrna localization in Xenopus oocytes and mesoderm induction An1-4 mrna Vg-1, Vg-T mrna (and many others) animal Dug Melton Yoel Israeli Kim Mowry vegetal
18 mrna localization targets actin to its site of function at the leading edge actin mrna and protein Robert H Singer Zip code 3 UTR Oligonucleotides - disrupt localization and motility Zip code binding protein (ZBP)
19 mrna localisation controls mating type switching in budding yeast Kim Nasmyth Robert H Singer Ira Herskovich Ralf Jansen Ash1 mrna and yeast mating type switch She mutants She 1: Myo4 is a TYPE V MYOSIN She 2: dsrna-binding protein binds to the localization signals She 3: adaptor that bridges the myosin heavy chain and She2 M D
20 Specifying stem cell identity and state in asymmetrically dividing neural stem cells Chris Doe Andrea Brand Juergen Knoblich Prospero - a conserved transcription factor that regulates stem cell renewal and differentiation in Drosophila neural stem cells and in a number of different vertebrate stem cell models.
21 Drosophila larval brain DNA NSC Mitotic cells ~104 neurons, originate from several hundred NSCs (neuroblasts)
22 Much is known about LTP and synaptic plasticity at the molecular level Dlg CamKII Scaffolding Vesicles Membranes Neurotransmitters Ion receptors Calcium channels Calcium binding Signalling Actin Microtubules Translation machinery Calabrese et al DOI: /physiol
23 Regulation of protein availability by localised translation of mrna? mrna Memory and learning Neurodegeneration Not clear how prevalent No convincing examples
24 How does mrna arrive at the axon termini? Diffusion? / Active transport? Kinesin Dynein cell body and dendrites mrna Motorneuron axon Muscle
25 single molecule fluorescent in situ hybridisation (smfish) Intron Exon 3 UTR Technical transformation: 1) High signal to noise 2) Deep penetration 3) Gentle hybridisation conditions 4) Multiplexed with many protein markers 5) Most mrna molecules detected Famine et al., Science Raj et al., Nature Methods Little et al., PLoS Biology Trcek et al., Nature Comm Abbaszadeh & Gavis, Methods
26 DNA Neuron dlg mrna Titlow and Davis 26
27 BRAIN Axons NERVE NMJ NMJ (synapse) Sun et al um 100 um Mushroom bodies protein mrna Titlow and Davis Sun et al., 1999
28 Assay to measure plasticity in the NMJ K + stimulation (90mM) CNS segmental nerves Titlow and Davis No Dlg accumulation at ghost boutons
29 What determines the levels of mrna in the cytoplasm? DNA Transcription mrna Decay d[rna] dt = synthesis - decay x [RNA] (first order decay and zeroth order synthesis)
30 How to measure synthesis and decay of mrna? Rutkowski et al. (2015) Widespread disruption of host transcription termination in HSV-1 infection Nature Communications 6: 7126
31 Animating synthesis and decay Fast synthesis Slow decay
32 Hypothesis: decay rate has a dramatic affect on mrna distribution in the cell Fast decay Slow decay
33 Thanks Any questions?
34 Copyright Notice Staff and students of this university are reminded that copyright subsists in a number of diagrams in this presentation, and the work from which it was taken. This Digital Copy has been made under the terms of a CLA licence which allows you to: - access and download a copy; - print out a copy. The Digital Copy and any digital or printed copy supplied to or made by you under the terms of this Licence are for use in connection with this Course of Study. You may retain such copies after the end of the course, but strictly for your own use. All copies (including electronic ones) shall include this Copyright Notice and shall be destroyed and/or deleted if and when required by the University. Except as provided for by copyright law, no further copying, storage or distribution (including by ) is permitted without the consent of the copyright holder. The author (which term includes artists and other visual creators) has moral rights in the work and neither staff nor students may cause, or permit, the distortion, mutilation or other modification of the work, or any other derogatory treatment of it, which would be prejudicial to the honour or reputation of the author. Course of Study: Molecular and Cellular Biochemistry, Year 3 Name of Designated Person authorising scanning: Prof. Ilan Davis All textbooks and individual references to primary or review papers in this lecture 34
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