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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 - PowerPoint PPT Presentation
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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