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A Newly Appreciated Genetic Modulator

Erick T. Tatro etatro@ucsd.edu June 7, 2012


Psychoneuroimmunology Research Societys 19th Annual Scientic Meeting Educational Short Course: A Tutorial on 21st Century Molecular Biology 5:30 - 5:50 pm

The Basics of MicroRNA

Friday, June 8, 12

Goals
Understand the biology of miR in general
and in context of neurobiology

Integrate concepts to solutions of


studies and trials related to miRs

psychiatric and immunologic illnesses drawing from existing expertise

Interpret and understand experiments,

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Start at the Beginning


Central Dogma of Molecular Biology Information Transfer DNA ! DNA (Replication) DNA ! RNA (Transcription) RNA ! Protein (Translation)

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Start at the Beginning


Central Dogma of Molecular Biology Information Transfer DNA ! DNA (Replication) DNA ! RNA (Transcription) RNA ! Protein (Translation)

!
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Start at the Beginning


Central Dogma of Molecular Biology Information Transfer DNA ! DNA (Replication) DNA ! RNA (Transcription) RNA ! Protein (Translation)

ATCG TAGC

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Start at the Beginning


Central Dogma of Molecular Biology Information Transfer DNA ! DNA (Replication) DNA ! RNA (Transcription) RNA ! Protein (Translation)

ATCG TAGC

!
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UAGC

Start at the Beginning


Central Dogma of Molecular Biology Information Transfer DNA ! DNA (Replication) DNA ! RNA (Transcription) RNA ! Protein (Translation)
UAGC
1st base UUU U UUC UUA UUG CUU C CUC CUA CUG AUU A AUC AUA AUG GUU G GUC GUA GUG (Val/V) Valine (Met/M) Methionine (Ile/I) Isoleucine (Leu/L) Leucine 2nd base U (Phe/F) Phenylalanine UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG (Ala/A) Alanine (Pro/P) Proline (Ser/S) Serine C UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC (Thr/T) Threonine!!!!!!!! AAA AAG GAU GAC GAA GAG (His/H) Histidine (Gln/Q) Glutamine (Asn/N) Asparagine (Lys/K) Lysine (Asp/D) Aspartic acid (Glu/E) Glutamic acid A (Tyr/Y) Tyrosine Stop UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG GGU GGC GGA GGG (Gly/G) Glycine (Ser/S) Serine (Arg/R) Arginine (Arg/R) Arginine G (Cys/C) Cysteine Stop (Trp/W) Tryptophan!!!! 3rd base U C A G U C A G U C A G U C A G

Friday, June 8, 12

Start at the Beginning


Central Dogma of Molecular Biology Information Transfer DNA ! DNA (Replication) DNA ! RNA (Transcription) RNA ! Protein (Translation)
UAGC
1st base UUU U UUC UUA UUG CUU C CUC CUA CUG AUU A AUC AUA AUG GUU G GUC GUA GUG (Val/V) Valine (Met/M) Methionine (Ile/I) Isoleucine (Leu/L) Leucine 2nd base U (Phe/F) Phenylalanine UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG (Ala/A) Alanine (Pro/P) Proline (Ser/S) Serine C UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC (Thr/T) Threonine!!!!!!!! AAA AAG GAU GAC GAA GAG (His/H) Histidine (Gln/Q) Glutamine (Asn/N) Asparagine (Lys/K) Lysine (Asp/D) Aspartic acid (Glu/E) Glutamic acid A (Tyr/Y) Tyrosine Stop UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG GGU GGC GGA GGG (Gly/G) Glycine (Ser/S) Serine (Arg/R) Arginine (Arg/R) Arginine G (Cys/C) Cysteine Stop (Trp/W) Tryptophan!!!! 3rd base U C A G U C A G U C A G U C A G

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Start at the Begining


Central Dogma of Molecular Biology Information Transfer DNA ! DNA (Replication) DNA ! RNA (Transcription) RNA ! Protein (Translation)
Proteins (and RNA) Go on To Carry Out Cellular Functions
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Regulating Function Happens at Each Step


Transcription Structure of the DNA - Histone Modication and Methylation

Naked DNA

The Nucleosome "Beads on a String"

30 nm Fiber Less Active Genes

Isolated Patches Genes Under Active Transcription *Creating RNA*


UAGC

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Regulating Function Happens at Each Step


Transcription Structure of the DNA - Histone Modication and Methylation

Naked DNA

The Nucleosome "Beads on a String"

30 nm Fiber Less Active Genes

Isolated Patches Genes Under Active Transcription *Creating RNA*


UAGC

MicroRNA Transcription is Regulated Just like Other RNAs (e.g. mRNAs)


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Regulating Function Happens at Each Step


Transcription Synthesis of RNA - Cis and Trans Elements

Promoter

Exons

3' UTR

Genomic DNA

Enhancers

Introns

UAGC

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Regulating Function Happens at Each Step


Transcription Synthesis of RNA - Cis and Trans Elements Transcription Factors Example: Glucocorticoid Receptor Androgen Receptor STAT1, STAT2 IFN Response Factors

Genomic DNA

TF's

Pol II

AGAACANNNTGTTCT

Glucocorticoid Response Element (GRE) CRE, ERE, HSE, SRE, HRE, ISRE, etc.

NUCLEUS

UAGC

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Regulating Function Happens at Each Step


Immature mRNA
Ex1 Ex2 Ex3 Ex4 3`UTR Ex5 3`UTR

Splicing, Processing Mature mRNA


5`Cap Coding 3`UTR AAAA

Export
NUCLEUS CYTOPLASM

UAGC

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Regulating Function Happens at Each Step


NUCLEUS

Ribosome Growing Polypeptide


Met Val Leu AAA Phe

CYTOPLASM

Translation
3`UTR AAAA

t-RNA
Coding

5`Cap

-UUU-

Mature mRNA

1st base UUU

2nd base U UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG (Met/M) Methionine (Ile/I) Isoleucine (Leu/L) Leucine (Phe/F) Phenylalanine UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU (Val/V) Valine GCC GCA GCG (Ala/A) Alanine (Pro/P) Proline (Ser/S) Serine C UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC (Thr/T) Threonine!!!!!!!! AAA AAG GAU GAC GAA GAG (His/H) Histidine (Gln/Q) Glutamine (Asn/N) Asparagine (Lys/K) Lysine (Asp/D) Aspartic acid (Glu/E) Glutamic acid A (Tyr/Y) Tyrosine Stop UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG GGU GGC GGA GGG (Gly/G) Glycine (Ser/S) Serine (Arg/R) Arginine (Arg/R) Arginine G (Cys/C) Cysteine Stop (Trp/W) Tryptophan!!!!

3rd base U C A G U C A G U C A G U C A G

UAGC

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Regulating Function Happens at Each Step


This is where microRNAs Act
NUCLEUS

Ribosome Growing Polypeptide


Met Val Leu AAA Phe

CYTOPLASM

Translation
3`UTR AAAA

t-RNA
Coding

5`Cap

-UUU-

Mature mRNA

STOP
1st base UUU 2nd base U UUC UUA UUG CUU C CUC CUA CUG AUU A AUC AUA AUG GUU G GUC GUA GUG (Val/V) Valine (Met/M) Methionine (Ile/I) Isoleucine (Leu/L) Leucine (Phe/F) Phenylalanine UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG (Ala/A) Alanine (Pro/P) Proline (Ser/S) Serine C UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC (Thr/T) Threonine!!!!!!!! AAA AAG GAU GAC GAA GAG (His/H) Histidine (Gln/Q) Glutamine (Asn/N) Asparagine (Lys/K) Lysine (Asp/D) Aspartic acid (Glu/E) Glutamic acid A (Tyr/Y) Tyrosine Stop UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG GGU GGC GGA GGG (Gly/G) Glycine (Ser/S) Serine (Arg/R) Arginine (Arg/R) Arginine G (Cys/C) Cysteine Stop (Trp/W) Tryptophan!!!! 3rd base U C A G U C A G U C A G U C A G

UAGC

miR

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Regulating Function Happens at Each Step


This is where microRNAs Act
NUCLEUS

Ribosome Growing Polypeptide


Met Val Leu AAA Phe

CYTOPLASM

Translation
3`UTR AAAA

t-RNA
Coding

5`Cap

-UUU-

Mature mRNA

STOP
1st base UUU 2nd base U UUC UUA UUG CUU C CUC CUA CUG AUU A AUC AUA AUG GUU G GUC GUA GUG (Val/V) Valine (Met/M) Methionine (Ile/I) Isoleucine (Leu/L) Leucine (Phe/F) Phenylalanine UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG (Ala/A) Alanine (Pro/P) Proline (Ser/S) Serine C UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC (Thr/T) Threonine!!!!!!!! AAA AAG GAU GAC GAA GAG (His/H) Histidine (Gln/Q) Glutamine (Asn/N) Asparagine (Lys/K) Lysine (Asp/D) Aspartic acid (Glu/E) Glutamic acid A (Tyr/Y) Tyrosine Stop UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG GGU GGC GGA GGG (Gly/G) Glycine (Ser/S) Serine (Arg/R) Arginine (Arg/R) Arginine G (Cys/C) Cysteine Stop (Trp/W) Tryptophan!!!! 3rd base U C A G U C A G U C A G U C A G

UAGC

miR Response Element (MRE)


! miR ! ! ! ! KCNMA1 3UTR v.2 CA C C 5' CCACA U U C A 3' GCAGCU GAU AAUCAGAGA :||||| ||| ||:||:||| UGUCGA CUA UUGGUUUCU 3' AGUA U 5' ! ! miR-9 !

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MicroRNAs Modulate Protein Translation Within the RISC at the 3`UTR


RNA - Induced Silencing Complex

STOP

UAGC
miR

Giovanni. Nat Rev Mol Cell Biol. 9, 219-230 (March 2008)


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Many mRNAs Have Multiple MREs in 3`UTR


RISC RISC RISC
5`Cap Coding 3`UTR AAAA

Example: Inwardly Rectifying Potassium Ion Channel

STOP STOP

UAGC

STOP miR miR

miR

Targetscan.org Garcia. Nat Struct Mol Biol., 18:1139-1146 (2011)

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Each miR Can Have 10s or 100s of Targets

STOP

UAGC UAGC

UAGC
miR

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Each miR Can Have 10s or 100s of Targets


Example: miR-9 has 683 Predicted Targets
LDL Receptor Adaptor Protein Ras family (RAB34) Copper Transporter (SLC31A2) Potassium Transporter (KCNMA1)
STOP

_CA_GCGGC__________GAU_GACCAAAG _GU_UGUCG__________CUA_UUGGUUUC_ _CAU__GCA_CUG__________GACCAAAGA _GUA__UGU_GAU__________UUGGUUUCU_ ____ACAGCUG__GUGACCAAAGG ____UGUCGAU__UAUUGGUUUCU _______________ACCAAAGA _______________UGGUUUCU


! ! KCNMA1 3UTR CA C C 5' CCACA U U C A GCAGCU GAU AAUCAGAGA :||||| ||| ||:||:||| UGUCGA CUA UUGGUUUCU 3' AGUA U ! ! miR-9 !

!
miR

UAGC UAGC

UAGC

3'

5' ! !
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Each miR Can Have 10s or 100s of Targets


Resource: Pictar Krek et al, Nature Genetics 37:495-500 (2005) http://pictar.mdc-berlin.de

STOP

UAGC UAGC

UAGC
miR

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MicroRNAs Can Be TissueSpecic


miR-122 - Liver miR-206 - Muscle miR-124a - Central Nervous System Exiqon Corporation Wienholds E., Science. 2005, 309, 310-311
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Summary
MicroRNAs Regulate Function at the Protein
Translation Step

MicroRNAs can be tissue specic or broadly


expressed throughout the organism a target of many microRNAs

MicroRNAs can have many targets; and mRNAs can be


STOP
STOP

STOP

UAGC UAGC

UAGC
miR

UAGC

STOP miR miR

miR

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Transcription and Origin of miRs

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Transcription and Maturation of miRs


Intergenic Transcription Polycistronic miRNA genes Monocistronic miRNA genes Intragenic Genome

pri-miRNA Processing

mRNA

pre-miRNA (~70 nt) Export


NUCLEUS CYTOPLASM

5`Cap

Coding

3`UTR

AAAA

mRNA introns

Pre-processing in the nucleus


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Transcription and Maturation of miRs


NUCLEUS CYTOPLASM

pre-miRNA (~70 nt) Dicer mature miRNA (~22 nt) Gene Regulation

Maturation and function take place in the Cytoplasm

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Genetic Features Highly Conserved Across Species


Distributed throughout Genome (but not on Y Chr)

Guo & Lu. (2010). PLoS ONE 5(6): e11387


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Chromosomal Locations
Experimental Data from Major Depression
and HIV - Frontal Cortex

Distribution of chromosomal distances between dysreg miRs in HIV & MDD Distribution if random
Tatro & Scott. PLOS One. (2010)
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MDD

What does that mean?


Likely cause of dysregulation is at the RNA
transcriptional level (from DNA ! RNA)

Factors that could contribute Activation/suppression by signaling, TFs,


chromatin structure
ATCG TAGC

! ! !

UAGC UAGC UAGC

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MicroRNA Clustering

About 42% of human miRs are Clustered on Genome The polycistronic organization of miRNA genes may have implications for evolution & selection
Altuvia. Nucleic Acids Res. 2005; 33(8): 26972706.
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Hippocampal DG Neurons and microRNAs

MicroRNA and Hippocampal Neurogenesis

and physiological maturation with the development of functional neuronal characteristics and a second selection by synaptic integration into pre-existing hippocampal circuits (Sierra et al., 2010; Encinas and Sierra, 2011). Moreover, AHN generates a whole range of neurogenic cell types that are differentially regulated and may play specic roles in the overall process (Figure 1A). During this slow maturation process, many of the newborn neurons are selected and more than 50% of the newborn GCs die within the rst few weeks after birth (Cameron et al., 1993; Biebl et al., 2000; Kempermann et al., 2003; Hattiangady and Shetty, 2008). The rapid decline during early stages results from active

Although the exact role of newborn neurons in the DG is still under debate, recent data support a functional role for adult-born neurons in learning and memory processes as reviewed in detail elsewhere (Aasebo et al., 2011; Aimone et al., 2011; Koehl and Abrous, 2011; Sahay et al., 2011). We here focus briey on DGdependent memory functions that may decrease with aging. The emerging consensus is that adult-born neurons in the DG play a crucial role in pattern separation, a memory mechanism that permits the differential representation of similar stimuli encoded by hippocampal circuits (Aimone et al., 2009; Sahay et al., 2011; Small et al., 2011). In humans, pattern separation can be assessed by a

2002 by the Authors

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FIGURE 1 | MicroRNas are key regulators in all phases of the adult neurogenesis cascade. Schematic illustration, adapted from (Lucassen et al., 2010), summarizing (A) miRs and targets involved in the regulation of different phases of adult neurogenesis and (B) miRs and targets hypothesized to be involved in the regulation of synaptogenesis during functional integration of

Schouten, Buijink, Lucassen, Fitzsimons. Front Neurosci (2012) 6:25

miR is less well-characterized in the integration phase as opposed to expansion and differentiation phases. (B) Regulation of synaptogenesis by miR-132 and miR-137 has been studied in AHN and in other contexts as well. From these observations, described in the text, we hypothesize that the regulatory network(s) depicted in (B) could be engaged in ne-tuning

MicroRNA and Aging: Evolutionary Development


Differential evolutionary conservation of genomic regions implies miR-mediated processes, though benecial in development, might be detrimental in aging. Analysis of gene expression in PFC through life history.

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MicroRNA and Aging: Evolutionary Development


Analysis of gene expression in PFC through life history.

Somel M et al. Genome Res. 2010;20:1207-1218


2010 by Cold Spring Harbor Laboratory Press

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Differential evolutionary conservation of genomic regions implies miR-mediated processes, though benecial in development, might be detrimental in aging.

MicroRNA and Aging: Evolutionary Development

Somel M et al. Genome Res. 2010;20:1207-1218


2010 by Cold Spring Harbor Laboratory Press

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Example miR involved in Both Inammation and Neuronal Function: miR-132

STOP

miR

UAGC

GR Signaling Blocks BDNFmediated increase in miR-132


Neuroscience. 2010. 165(4):1301-11
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MiR-132 Suppresses p250GAP, inhibitor of Rho GTPases; facilitates NMDA Receptor Signaling & Dendrite Growth

Neuron. 2010. 65(3):373-84.

MicroRNAs can have many targets; and mRNAs can be


a target of many microRNAs and Age

Summary

MicroRNAs Change Expression over Development MicroRNAs involved in Cellular Process in Brain and
Immunity.
STOP
STOP

UAGC

UAGC UAGC

UAGC
miR

STOP miR miR

Copyright 2003 Blue Banana Designs

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Resources
MirBase! ! ! ! ! ! ! ! Targetscan! ! ! ! ! ! ! Pictar! ! ! ! ! ! ! ! ! UCSC Genome Browser!!
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! www.mirbase.org ! www.targetscan.org ! pictar.mdc-berlin.de ! genome.ucsc.edu/