the rna world
DESCRIPTION
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 PresentationTRANSCRIPT
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The RNA world
Messenger RNA
Proteins
Non-coding RNAs
splicing
ribosomes
translation
transcriptionRNA modification
chromatin
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- 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)
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History of mi/si RNAs
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Overview of the mi/siRNA pathways in plants and animals
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Genetic structure of miRNA genes1. exonic
2. intronic
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Structure of miRNA precursors (pre-miR)
microRNAs can be derived from both arms of the duplex
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Proteins involved in the miRNA pathway
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Drosha and Dicer cleavage
Dicer and Drosha leave two bpOverhang after cleavage
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Structure of miRNA-mRNA duplexes
Nt 2-8 of microRNA (“seed”) are crucial for target recognition
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Gene regulatory function of miRNAs
Translational repression is the primary mode of microRNA function in vertebrates
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miRNAs might inhibit translation by different mechanisms
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Degradation of miRNA target mRNAs in processing bodies
Exact mechanism of translational inhibition is still controversial
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microRNA inhibition can be reversible
Only a few examples of dynamic microRNA regulation
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Regulation of miRNA expression
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Herve Seitz et al. Genome Res. 2004; 14: 1741-1748
A large cluster of miR genes on human chr.14
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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
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Regulation of pre-miRNA processing
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Inhibitor of processing in non-neural tissues
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Adenosine-to-Inosine editing
NH3
adenosine
inosine
deaminase
H2O
Inosine pairs with cytidine (uridine, adenosine)
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ADARs
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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)]
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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
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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
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Editing changes miR target pools
Relative Luciferase Assay
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Many microRNAs are enriched in the nervous system
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microRNA function in the nervous system
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Sensory neuron specification in the nematode C. elegans
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miRNAs are part of the gene regulatory network in sensory neuron
specification
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miRNAs in neuronal fate decisions in vertebrates
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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
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Synaptic Plasticity
Post-mitotic neural development
Axon Outgrowth
Synapse Formation
Axon Outgrowth
Synapse Formation
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miR-132 promotes neurite outgrowth
Gain-of-function Loss-of-function
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Presynaptic terminal
Axon
Dendrite
PSD
Dendritic Spine
Synapses as sites of information storage
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Local synthesis of synaptic proteins
nucleus
soma
dendrite
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Dendritic transport of microRNAs
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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
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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)
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The role of microRNAs in dendritic protein synthesis and spine morphology
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Multiple mechanisms regulate microRNA function at the synapse
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microRNAs in neuronal disease
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Tourette Syndrome (mental retardation)
SNP in the 3’UTR of Slitrk1 mRNA
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SNP in SLITRK1 affects miR function
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microRNAs in viral infection in the brain
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HSV expresses the viral miR-LAT microRNA
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microRNAs and Fragile-X syndrome
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Small non-coding RNAs other than microRNAs
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BC1 ncRNA regulates local mRNA translation in neurons
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snoRNAs and splicing of brain mRNAs
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Summary of ncRNA function at the synapse