The RNA world

Messenger RNA

Proteins

Non-coding RNAs

splicing

ribosomes

translation

transcriptionRNA modification

chromatin

- Encoded in the genome as hairpin precursor RNA

microRNAs (miRNAs)

- 19-24 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 genes1. 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 bpOverhang 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: 1741-1748

A large cluster of miR genes on human chr.14

0

1

2

3

4

GTL2 miR-540

RTL1 GS-17148

miR-341

miR-370

GB-AW GS-9477

miR-329

miR-134

miR-541

Re;a

tive m

RN

A l

evels

0h0.5h1h2h3h4h6h

miR clusterGTL2 RTL1 snoRNA

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

NH3

adenosine

inosine

deaminase

H2O

Inosine pairs with cytidine (uridine, adenosine)

ADARs

Mouse miRNA-376 cluster

Chromosome 14 Chromosome 12

miR-376 cluster is highly edited in the brain

Human Mouse

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 genes miR-124a

Non-neuronal genes

REST

NRSE smRNA

Neuronal genes

bHLH

miR-124a

Non-neuronal genes

Neural progenitor Neuron

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-function Loss-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-134 miR-134 mismatch

synapsin synapsin

merge merge

Hippocampal neurons, 14DIV

miR-134 U6 snRNA

Northern blotP15 brain

miR-134 inhibits dendritic spine growth

control

Hippocampal neuron, 21DIV

miR-134

0

0.2

0.4

0.6

0.8

1

1.2

1.4

*

*

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

GFP

miR

-134

2’O

-me-

134

2’O

-me-

cont

rol

Sp

ine

den

sity

(sp

ines

/mic

ron

)

Spine volume Spine width/length Spine density

GFP

miR

-134

2’O

-me-

134

2’O

-me-

cont

rol

Let-7

c

Rel

ativ

e sp

ine

volu

me

(GF

P=

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