The RNA world Messenger RNA Proteins Non-coding RNAs splicing ribosomes translation transcription RNA modification chromatin

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

History of mi/si RNAs

Overview of the mi/siRNA pathways in plants and animals

Genetic structure of miRNA genes 1. exonic 2. intronic

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

Proteins involved in the miRNA pathway

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

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

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

miRNAs might inhibit translation by different mechanisms

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

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

Regulation of miRNA expression

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

miR clusterGTL2RTL1snoRNA miR cluster on Chr.14 forms a transcriptional unit that is induced by neuronal activity Cortical neurons, 4DIV, treated with 55mM KCl, n=3 S. Khudayberdiev

Regulation of pre-miRNA processing

Inhibitor of processing in non-neural tissues

Adenosine-to-Inosine editing NH 3 adenosineinosine deaminas e H2OH2O Inosine pairs with cytidine (uridine, adenosine)

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. Hartner et al., J. Biol. Chem. 279, 4894 (2004)

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

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

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

Editing changes miR target pools Relative Luciferase Assay

Many microRNAs are enriched in the nervous system

microRNA function in the nervous system

Sensory neuron specification in the nematode C. elegans

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

miRNAs in neuronal fate decisions in vertebrates

REST Neuronal genesmiR-124a Non-neuronal genes REST NRSE smRNA Neuronal genes bHLH miR-124a Non-neuronal genes Neural progenitorNeuron miR-124 function in neuronal specification

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

miR-132 promotes neurite outgrowth Gain-of-functionLoss-of-function

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

Local synthesis of synaptic proteins nucleus soma dendrite

Dendritic transport of microRNAs

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

miR-134 inhibits dendritic spine growth control Hippocampal neuron, 21DIV miR * * GFP miR-134 2’O-me-134 2’O-me-control Spine density (spines/micron) Spine volumeSpine width/lengthSpine density GFP miR-134 2’O-me-134 2’O-me-control Let-7c Relative spine volume (GFP=1)

The role of microRNAs in dendritic protein synthesis and spine morphology

Multiple mechanisms regulate microRNA function at the synapse

microRNAs in neuronal disease

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

SNP in SLITRK1 affects miR function

microRNAs in viral infection in the brain

HSV expresses the viral miR-LAT microRNA

microRNAs and Fragile-X syndrome

Small non-coding RNAs other than microRNAs

BC1 ncRNA regulates local mRNA translation in neurons

snoRNAs and splicing of brain mRNAs

Summary of ncRNA function at the synapse