rna therapeutics institute (rti) university of massachusetts medical school developmental robustness...
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RNA TherapeuticsInstitute (RTI)
University of Massachusetts Medical School
Developmental Robustness
MicroRNA Pathways In C. elegans Development
microRNAgene
Pri-microRNA
Pre-microRNA miRNP
Inhibition of protein productionfrom mRNA targets
Regulation of Gene Expression by Animal MicroRNAs
Invertebrates: ~150 microRNA genes
Vertebrates: ~1000 microRNA genes
AGO
AGOAGO
An
GW
Effectors
Modulators
NHL
Eumetazoa
sponge
Animals
Plants
RNAi
lancelet
human
fly
beetle
roundworm
sea anenome
~10>100weed
One Pan-Eumetazoan family
1
lancelet
human
fly
beetle
roundworm
sea anenome
mir-100
let-7 mir-125
Hsa-mir-100 AACCCGUAGAUCCGAACUUGUGCel-mir-52 CACCCGUACAUAUGUUUCCGUGCU
How Did/Do MicroRNAs Evolve?
MicroRNAs are Endogenous RNAi
Agents
UGAGGUAGUAGGUUGUAUAGUUUGAGGUAGUAGGUUGUGUGGUUUGAGGUAGUAGGUUGUAUGGUUAGAGGUAGUAGGUUGCAUAGU-UGAGGUAGGAGGUUGUAUAGU-UGAGGUAGUAGAUUGUAUAGUUUGAGGUAGUAGUUUGUACAGU-UGAGGUAGUAGUUUGUGCUGU-
Human let-7-FamilyUGAGGUAGUAGGUUGUAUAGUUUGAGGUAGGCUCA-GUAGAUGCUGAGGUAGUAUGUAAUAUUGUAUGAGGUAGGUGCGAGAAAUGA-
Worm let-7-Family
> 35 novel Pan-Bilaterian families
lancelet
human
fly
beetle
roundworm
sea anenome
mir-100
let-7 mir-125
~150
~800
Fungi mould ~10
Worm
Fly
Human
hsa-miR-1 UGGAAUGUAAAGAAGUAUGUAU cel-miR-1 UGGAAUGUAAAGAAGUAUGUAdme-miR-1 UGGAAUGUAAAGAAGUAUGGAG
Chen and Rajewsky CSHS 2006
Evolutionary fluidity of target gene sets
Explicitly-Homologous
HBL-1
differentiation
let-60/Ras
LIN-41
C. elegans hypodermal lineages
LIN-28
let-7
Evolution of MicroRNA-Target Interactions
Functionally-Analogous
let-7
human cells
normalHmga2
tumor
Mayr, Hemann and Bartel, 2007
Explicitly-Homologous
lin-4 and let- 7 control developmental timing (1993, 1999).••••• Single gene visible phenotypes •••••
Functions of microRNA genes in C. elegans
C. elegans “Heterochronic” Mutants
lin-4(e912)(Chalfie et al, 1980)
Wild type
lin-4- Wild Type
Retarded Development of lin-4 mutants
Adult Alae Formation at L4 MoltLI
N-1
4 ov
er-e
xpre
ssio
n
L4 Molt
L3 Molt
ALAE
lin-4
lin-14 3’ UTR Deletion
2
3
1
4Adult
Embryo
lin-4 and let-7 microRNAs control cell fate progression
Adult
let-7lin-41
lin-41
lin-41 ...U AUU U.. UUAUACAACC CUGCCUC GAUAUGUUGG GAUGGAG let-7 UU AU U
lin-14 ..C ACCAA C.. UCACA CUCAGGGA lin-4 AGUGU GAGUCCCU G A A C ACUC
Larval stag
es
7-Famhbl-1
hbl-1
hbl-1 ..A CUUGU U.. AACUA CC GCUACCUUA UUGAU GG UGAUGGAGUmir-84 AUGUU A
lin-4lin-14
lin-14
Utterly Robust Cell Fate Specification and Execution
lin-4 and let- 7 control developmental timing (1993, 1999).••••• Single gene visible phenotypes •••••
Genetics of microRNA genes in C. elegans
Loss of individual microRNAs causes mutant phenotypes in sensitized genetic backgrounds in C. elegans. Brenner et al, … Abbott. Curr Biol. (2010)
Visible Phenotypesfor 25 of 31
miRNA genes
The microRNA miR-1 regulates a MEF-2-dependent retrograde signal at neuromuscular junctions. Simon et al…Ruvkun, Kaplan, Kim. Cell. (2008)
Stress the worms
Most Caenorhabditis elegans microRNAs are individually not essential for development or viability. Miska, Abbott, Alvarez-Saavedra …Bartel, Ambros, Horvitz. PLoS Genet. (2007)
A drag… No New Phenotypes
Lucky!
The let-7 MicroRNA family … mir-48, mir-84, and mir-241 function together to regulate developmental timing in C. elegans. Abbott et al…Bartel, Horvitz, Ambros. Dev. Cell. (2005)
Redundancy withina microRNA family
Many families of Caenorhabditis elegans microRNAs are not essential for development or viability Ezequiel Alvarez-Saavedra and H. Robert Horvitz Curr Biol. 2010
3 of 15 familiesare essential
Loss of individual microRNAs causes mutant phenotypes in sensitized genetic
backgrounds in C. elegans. Brenner et al, 2010
microRNAs Are Embedded Within Diverse Networks
• Redundancy between microRNA families
• Redundancy with gene regulatory “hubs”
Robustness thru
Redundancy
mir-34
mir-83
mir-1
GonadalMorphogenesis
Fertility
Argonaute
Buffering morphogenetic processes against temperature changes
Distinct MicroRNAs with Convergent Targets
mir-34mir-83
pat-3 mRNA
peb-1 mRNA
PAT-3
Gonadal Morphogenesis
PEB-1
Buffered against
Temperature changes
Samantha Burke & Molly Hammell
B-integrin
Transcription factor
Hermaphrodite Gonad Morphogenesis in C. elegans
Phase 1
Phase 3
Phase 2
From Worm Atlas
Migration Defects in mir-34 (-/-) and mir-83 (-/-) Mutants
Samantha Burke & Molly Hammell
Li et al., Carthew Cell 2009 137(2): 273-282
Drosophila mir-7 Buffers Eye Development Against Temperature Changes
Taking a Cue from Richard Carthew….
Constant 20 ℃
Temperature Oscillations Aggravate mir-34 & mir-83 Phenotypes
15oC – 25oC Oscillations (8 hr)
Samantha Burke & Molly Hammell
mir-83(0)mir-34(+)
mir-83(+)mir-34(+)
mir-83(0)mir-34(0)
mir-83(+)mir-34(0)
214
19
54
% d
efec
tive
gona
dal m
igra
tion
***
Samantha Burke & Molly Hammell
Buffering morphogenetic processes against temperature changes
Distinct MicroRNAs with Convergent Targets
mir-34mir-83
pat-3 mRNA
peb-1 mRNA
PAT-3
Gonadal Morphogenesis
PEB-1
Buffered against
Temperature changes
Samantha Burke & Molly Hammell
B-integrin
Transcription factor
An
AGO
Xantha Karp
HBL-1 mRNA
Modulation of microRNA levels and (lin-4) activity
Reprogramming of HBL-1 microRNA regulation by life history
let-7Family
lin-4
Post-dauer: mir-48, mir-84, mir-241, let-7
lin-4: MAJOR Role
mir-48, mir-84, mir-241, let-7
lin-4: minor roleContinuous: NHL-2
2
3
1
4Adult
Accommodation of Stress and Diapause in C. elegans Development
UnfavorableFavorable
Dauer-interrupted
Larval stag
es
Insulin/IGFDAF-16/Fox0
Dauer larva
Continuous
2
3
1
4Adult
Embryo
lin-4 and let-7 microRNAs control cell fate progression
Adult
let-7lin-41
lin-41
lin-41 ...U AUU U.. UUAUACAACC CUGCCUC GAUAUGUUGG GAUGGAG let-7 UU AU U
lin-14 ..C ACCAA C.. UCACA CUCAGGGA lin-4 AGUGU GAGUCCCU G A A C ACUC
Larval stag
es
7-Famhbl-1
hbl-1
hbl-1 ..A CUUGU U.. AACUA CC GCUACCUUA UUGAU GG UGAUGGAGUmir-84 AUGUU A
lin-4lin-14
lin-14
Utterly Robust Cell Fate Specification and Execution
(Optional) Developmental Quiescence
2
3
1
4Adult
Embryo
Adult
let-7lin-41
lin-41
Larval stag
es
7-Famhbl-1
hbl-1
lin-4lin-14
lin-14
(Optional) Developmental Quiescence
lin-4 and let-7 microRNAs control cell fate progression
Robust Timing of cell fates
Dauer larva
Adu
lt
L1 L2 L4L3
miR-48
miR-84
miR-241
let-7
lin-4
Wild type
LIN-29LIN-41
lin-4
let-7
HBL-1
LIN-14L1
L2
L3
L4
Ad
let-7Fammir-48mir-84
mir-241let-7
Dev
elo
pm
enta
l S
ign
als
MicroRNA pathways
Transcription Factors
microRNAs in C. elegans cell fate progression
Robust Timing of cell fates
LIN-28
Dauer Larva Quiescence
LIN-29LIN-41
lin-4
let-7
LIN-14L1
L2
L3
L4
Ad
let-7Fammir-48mir-84
mir-241let-7
Dev
elo
pm
enta
l S
ign
als
Dosage-Sensitive activity of let-7-Family microRNAs
HBL-1
Wild type
L1
L2
L3
L4
Ad
let-7-Fam -1UGAGGUAGUAGGUUGUAUAGUUUGAGGUAGGCUCA-GUAGAUGCUGAGGUAGUAUGUAAUAUUGUAUGAGGUAGGUGCGAGAAAUGA-
Worm let-7-Family
LIN-28
LIN-29LIN-41
lin-4
let-7
HBL-1
LIN-14
let-7Fammir-48mir-84
mir-241let-7
Dev
elo
pm
enta
l S
ign
als
HBL-1
Adult-specific col-19::GFP
NormalRetarded
L1
L2
L3
L4
Ad
L3
L4
L2
UGAGGUAGUAGGUUGUAUAGUUUGAGGUAGGCUCA-GUAGAUGCUGAGGUAGUAUGUAAUAUUGUAUGAGGUAGGUGCGAGAAAUGA-
Worm let-7-Family
L1
L2
L3
L4
Ad
let-7-Fam -3Wild type
LIN-28
Dosage-Sensitive activity of let-7-Family microRNAs
Retarded phenotype of let-7-Fam -3 mutant
Retarded Adult
Normal Adult
Adult-specific col-19::GFP
let-7-Fam -3let-7-Fam -3
Healthy environmentStressful environment
LIN-29LIN-41
lin-4
let-7
LIN-14L1
L2
HBL-1
Normal Retarded
Dev
elo
pm
enta
l S
ign
als
HBL-1
Post-dauer Development of let-7-Fam -3 mutant
Dauer Larva Quiescence
“Post-dauer” Suppression
Ad
L3
L4
L2
L3
L4
let-7Fammir-48mir-84
mir-241let-7
LIN-28
Adult-expression of col-19::GFP
Wild Type let-7-Fam-3
Continuous Post-dauer Continuous Post-dauer
Post-Dauer Suppression of let-7-Fam -3 Phenotypes
let-7-Fam -3let-7-Fam -3
LIN-29LIN-41
lin-4
let-7
LIN-14L1
L2
Post-dauer Suppression of let-7-Fam -3 Phenotypes
Normal Retarded
Dev
elo
pm
enta
l S
ign
als
High Pheromone Low PheromoneHealthy environmentStressful environment
HBL-1 Dauer Larva Quiescence
Post-Dauer Suppression
Ad
L3
L4
let-7Fammir-48mir-84
mir-241let-7
Mod
LIN-28
Xantha Karp
Post-Dauer Suppression
LIN-29LIN-41
lin-4
let-7
LIN-14L1
L2
HBL-1
Normal Retarded
Dev
elo
pm
enta
l S
ign
als
Mod HBL-1
Retarded
High Pheromone Low Pheromone
let-7-Fam -3Mod(0); let-7-Fam -3let-7-Fam -3
Xantha Karp
Gene products required for Post-dauer Suppression
Dauer Larva Quiescence
Ad
L3
L4
L2
L3
L4
let-7Fammir-48mir-84
mir-241let-7
LIN-28
let-7-Fam -3let-7-Fam -3
LIN-29LIN-41
lin-4
let-7
LIN-14L1
L2
Normal Retarded
Dev
elo
pm
enta
l S
ign
als
High Pheromone Low Pheromone
NHL-2 HBL-1Dauer Larva Quiescence
“Post-Dauer” Suppression
Ad
L3
L4
let-7Fammir-48mir-84
mir-241let-7
NHL-2/TRIM32 is required for Post-dauer Suppression
LIN-28
Xantha Karp
nhl-2(0); let-7-Fam -3nhl-2(0); let-7-Fam -3
LIN-29LIN-41
lin-4
let-7
LIN-14L1
L2
HBL-1
Normal Retarded
Dev
elo
pm
enta
l S
ign
als
NHL-2 HBL-1
Retarded
High Pheromone Low Pheromone
Dauer Larva Quiescence
“Post-Dauer” Suppression
Ad
L3
L4
L2
L3
L4
let-7Fammir-48mir-84
mir-241let-7
NHL-2/TRIM32 is required for Post-dauer Suppression
LIN-28
Xantha Karp
Post-Dauer let-
7-Fam-3
Post-Dauer
nhl-2; let-7-Fam-3
Col-19::GFP expression in Post-Dauer Adults
NHL-2/TRIM32 is required for Post-dauer Suppression
LIN-28
lin-4(0); let-7-Fam -3lin-4(0); let-7-Fam -3
“Post-Dauer” Suppression
LIN-29LIN-41
lin-4
let-7
LIN-14L1
L2
HBL-1
lin-4 microRNA is required for Postdauer Suppression
Normal Retarded
Dev
elo
pm
enta
l S
ign
als
NHL-2 HBL-1
Retarded
High Pheromone Low Pheromone
Xantha Karp
let-7-Fam -3 let-7-Fam -3
Dauer Larva Quiescence
Ad
L3
L4
L2
L3
L4
let-7Fammir-48mir-84
mir-241let-7
L1 L2 Dauer larva
Post Dauer L3
Post Dauer L3
lin-4 is required for down regulation of HBL-1 Between L2 and Post Dauer L3
L1 L2 Dauer larva Post Dauer L3
let-7-Fam-3
WT
lin-4; let-7-Fam-3
WT WT WT
Down Regulated
Not Down Regulated
HBL-1::GFP::UTR-HBL-1
LIN-28
lin-4(0); let-7-Fam -1lin-4(0); let-7-Fam -1
LIN-29LIN-41
lin-4
let-7
LIN-14L1
L2
HBL-1
Dev
elo
pm
enta
l S
ign
als
NHL-2 HBL-1
Retarded
High Pheromone
Xantha Karp
let-7-Fam -1 let-7-Fam -1
Dauer Larva Quiescence
Ad
L3
L4
L2
L3
L4
let-7Fammir-48mir-84
mir-241let-7
Normal
Low Pheromone
L2
Ad
L3
L4
lin-4 function is More Critical after Dauer Larva Quiescence
lin-4; lin-14(n179) mir-84
lin-4; lin-14(n179)
lin-14(n179) mir-84
wild-type
0% 20% 40% 60% 80% 100%
no alaegapped alaeSeries3
lin-4(0); mir-84(0)
mir-84(0)
% Adults expressing larval cell fates
**
**
** P < 0.01
Continuous
Post-Quiescence
Continuous
Post-Quiescence
*All strains contain lin-14(n179)
lin-4 microRNA is More Critical after Dauer Larva Quiescence
Xantha Karp
microRNA abundance
Dauer vs Continuous
Developmental profiles of lin-4 and let-7-Family microRNAs
L2D/L2 Dauer/L2molt PDL3/L3 PDL4/L40
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
lin-4
mir-84
let-7
mir-241mir-48
Dauer/L2m PDL3/L3 PDL4/L4L2D/L2
1.0
Taqman Q-RT/PCR Dauer Traversing vs Continuously-Developing
An
AGO
Xantha Karp
HBL-1 mRNA
Modulation of microRNA levels and (lin-4) activity
Reprogramming of HBL-1 microRNA regulation by life history
let-7Family
lin-4
Post-dauer: mir-48, mir-84, mir-241, let-7
lin-4: MAJOR Role
mir-48, mir-84, mir-241, let-7
lin-4: minor roleContinuous: NHL-2
Insulin/IGFDAF-16/Fox0
Physiological Modulation of microRNA activity
Enhancers and suppressors of let-7-Family loss-of-function mutations
Zhiji Ren, Chris Hammell
col-19 Alae Description
rab-7 enhance enhance endosomal trafficking
vps-11 enhance enhance endosomal trafficking
vps-33.1/33.2 suppress suppress endosomal trafficking
pqn-29 suppress suppress prion like gene
pqn-67 suppress suppress prion like gene
C01A2.4 (CHMP2B) suppress suppress ESCRT complex
nhl-2 enhance enhance miRISC modulator
cgh-1 enhance enhance miRISC essential component
lin-46 enhance enhance scaffolding protein
Bacterial Diet
Physiological Modulation of microRNA activity
Zhiji Ren
MetabolicSignaling
Innate Immunity
Bacterial Quality/Toxicity
mir-48mir-241
Buffering against
food stress
mir-84 let-7 Developmental
cell fate specification
let-7-Family microRNAs in Diet and Development
Developmental Timing, Robustness and Stress Responses
Samantha Burke
Former:
Alexey Wolfson
Anna Zinovyeva
Sungwook Choi Omid Harandi
Current:
Catherine Sterling
Molly Hammell
Xantha Karp
Chris Hammell
Rosalind Lee
Maria Ow
Katie McJunkin
Elizabeth Thatcher
Circulating microRNAs in human plasma and CSF
microRNAs and long noncoding RNAs
LIN-28 Function
Regulation of microRNA stability/turnover
MicroRNAs in diverse stress responses
Zhiji Ren