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The RNA world

ribosomes. splicing. translation. Non-coding RNAs. RNA modification. transcription. chromatin. The RNA world. Messenger RNA. Proteins. microRNAs (miRNAs). - 19-24 nt long. - Encoded in the genome as hairpin precursor RNA. production dependent on the RNAseIII enzymes Drosha

justin-jenkins
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The RNA world

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  1. ribosomes splicing translation Non-coding RNAs RNA modification transcription chromatin The RNA world MessengerRNA Proteins

  2. microRNAs (miRNAs) - 19-24 nt long - Encoded in the genome as hairpin precursor RNA • production dependent on the RNAseIII enzymes Drosha • and Dicer - incorporated into an RNAi-induced silencing complex (RISC)

  3. History of mi/si RNAs

  4. Overview of the mi/siRNA pathways in plants and animals

  5. Genetic structure of miRNA genes 1. exonic 2. intronic

  6. Structure of miRNA precursors (pre-miR) microRNAs can be derived from both arms of the duplex

  7. Proteins involved in the miRNA pathway

  8. Drosha and Dicer cleavage Dicer and Drosha leave two bp Overhang after cleavage

  9. Structure of miRNA-mRNA duplexes Nt 2-8 of microRNA (“seed”) are crucial for target recognition

  10. Gene regulatory function of miRNAs Translational repression is the primary mode of microRNA function in vertebrates

  11. miRNAs might inhibit translation by different mechanisms

  12. Degradation of miRNA target mRNAs in processing bodies Exact mechanism of translational inhibition is still controversial

  13. microRNA inhibition can be reversible Only a few examples of dynamic microRNA regulation

  14. Regulation of miRNA expression

  15. A large cluster of miR genes on human chr.14 Herve Seitz et al. Genome Res. 2004; 14: 1741-1748

  16. miR cluster on Chr.14 forms a transcriptional unit that is induced by neuronal activity 4 0h 3 0.5h 1h Re;ative mRNA levels 2h 2 3h 4h 6h 1 0 GTL2 miR- RTL1 GS- miR- miR- GB-AW GS- miR- miR- miR- 540 17148 341 370 9477 329 134 541 GTL2 RTL1 snoRNA miR cluster Cortical neurons, 4DIV, treated with 55mM KCl, n=3 S. Khudayberdiev

  17. Regulation of pre-miRNA processing

  18. Inhibitor of processing in non-neural tissues

  19. Adenosine-to-Inosine editing NH3 H2O deaminase adenosine inosine Inosine pairs with cytidine (uridine, adenosine)

  20. ADARs • = Adenosine deaminases acting on RNA • specifically recognize dsRNA • particularly abundant in the nervous system • predominantly nuclear expression  editing of primary transcripts • two major isoforms: ADAR1 and ADAR2 • ADAR1-/- mice die at E12 • J. C. Hartneret al., J. Biol. Chem.279, 4894 (2004)

  21. miR-376 cluster is highly edited in the brain Human Mouse Mouse miRNA-376 cluster Chromosome 14 Chromosome 12 Two highly edited adenosines  at positions (+4) and (+44) [in some miR376 members frequent editing at (-1)]

  22. A-to-I editing in ADAR knockout mice ADAR1 -/- editing of (+44) site is eliminated ADAR2 -/- editing of (-1) site in 376a, b, c and editing of (+4) site in pri-miR 376a is almost eliminated editing of (+44) site is higher in pri-miR-376b and 376c -/-  (+44) site is selectively edited by ADAR1  (-1) and (+4) sites are mainly edited by ADAR2

  23. Does editing effect miRNA function?  Luciferase-Assay with randomly selected targets • unedited miR-376a targets: • arginine/serine rich splicing factor 11 • solute carrier family 16-A1 • threonine/tyrosine kinase • edited miR-376a targets: • phosphoribosyl-pyrophosphate- • synthetase 1 • zinc finger protein 513 • sorting nexin 19

  24. Editing changes miR target pools Relative Luciferase Assay

  25. Many microRNAs are enriched in the nervous system

  26. microRNA function in the nervous system

  27. Sensory neuron specification in the nematode C. elegans

  28. miRNAs are part of the gene regulatory network in sensory neuron specification

  29. miRNAs in neuronal fate decisions in vertebrates

  30. miR-124 function in neuronal specification Neural progenitor Neuron REST bHLH REST NRSE smRNA Neuronal genes miR-124a miR-124a Neuronal genes Non-neuronal genes Non-neuronal genes

  31. Synapse Formation Axon Outgrowth Axon Outgrowth Post-mitotic neural development Synapse Formation Synaptic Plasticity

  32. miR-132 promotes neurite outgrowth Gain-of-function Loss-of-function

  33. Axon Presynaptic terminal PSD Dendritic Spine Dendrite Synapses as sites of information storage

  34. Local synthesis of synaptic proteins nucleus soma dendrite

  35. Dendritic transport of microRNAs

  36. Northern blot P15 brain miR-134 U6 snRNA miR-134 is localized near synaptic sites within dendrites miR-134 miR-134 mismatch synapsin synapsin merge merge Hippocampal neurons, 14DIV

  37. miR-134 Spine volume Spine width/length Spine density 0.7 0.6 * 1.4 0.5 * 1.2 1 Relative spine volume (GFP=1) 0.4 0.8 Spine density (spines/micron) 0.3 0.6 0.2 0.4 0.2 0.1 0 0 GFP miR-134 GFP miR-134 Let-7c 2’O-me-134 2’O-me-134 2’O-me-control 2’O-me-control miR-134 inhibits dendritic spine growth Hippocampal neuron, 21DIV control

  38. The role of microRNAs in dendritic protein synthesis and spine morphology

  39. Multiple mechanisms regulate microRNA function at the synapse

  40. microRNAs in neuronal disease

  41. Tourette Syndrome (mental retardation) SNP in the 3’UTR of Slitrk1 mRNA

  42. SNP in SLITRK1 affects miR function

  43. microRNAs in viral infection in the brain

  44. HSV expresses the viral miR-LAT microRNA

  45. microRNAs and Fragile-X syndrome

  46. Small non-coding RNAs other than microRNAs

  47. BC1 ncRNA regulates local mRNA translation in neurons

  48. snoRNAs and splicing of brain mRNAs

  49. Summary of ncRNA function at the synapse

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