University of
Massachusetts
Medical School
RNA Therapeutics
Institute (RTI)
MicroRNA Pathways In C. elegans
Development
Developmental
Robustness
Regulation of Gene Expression by Animal MicroRNAs
Invertebrates:
~150 microRNA genes
Vertebrates:
~1000 microRNA genes
microRNA
gene
Inhibition of protein production
from mRNA targets
Pri-microRNA
Effectors
GW
AGO
NHLAGO
Modulators
AGO
Pre-microRNA
miRNP
An
How Did/Do MicroRNAs Evolve?
Hsa-mir-100 AACCCGUAGAUCCGAACUUGUG
Cel-mir-52 CACCCGUACAUAUGUUUCCGUGCU
One Pan-Eumetazoan family
Human let-7-Family
Worm let-7-Family
UGAGGUAGUAGGUUGUAUAGUU
UGAGGUAGUAGGUUGUGUGGUU
UGAGGUAGUAGGUUGUAUGGUU
AGAGGUAGUAGGUUGCAUAGUUGAGGUAGGAGGUUGUAUAGUUGAGGUAGUAGAUUGUAUAGUU
UGAGGUAGUAGUUUGUACAGUUGAGGUAGUAGUUUGUGCUGU-
UGAGGUAGUAGGUUGUAUAGUU
UGAGGUAGGCUCA-GUAGAUGC
UGAGGUAGUAUGUAAUAUUGUA
UGAGGUAGGUGCGAGAAAUGA-
> 35 novel
Pan-Bilaterian families
MicroRNAs are
Endogenous RNAi
Agents
~800
human
lancelet
fly
1
beetle
Eumetazoa
RNAi
roundworm
Animals
sea anenome
sponge
Plants
weed
Fungi
mould
~10
>100
~10
~150
Evolutionary fluidity of target gene sets
Explicitly-Homologous
Worm
Fly
Human
hsa-miR-1 UGGAAUGUAAAGAAGUAUGUAU
cel-miR-1 UGGAAUGUAAAGAAGUAUGUA
dme-miR-1 UGGAAUGUAAAGAAGUAUGGAG
Chen and Rajewsky CSHS 2006
Evolution of MicroRNA-Target Interactions
C. elegans hypodermal lineages
Explicitly-Homologous
Functionally-Analogous
let-60/Ras
let-7
HBL-1
LIN-28
LIN-41
human cells
let-7
differentiation
tumor
Hmga2
normal
Mayr, Hemann and Bartel, 2007
Functions of microRNA genes in C. elegans
lin-4 and let- 7 control developmental timing (1993, 1999).
••••• Single gene visible phenotypes •••••
C. elegans “Heterochronic” Mutants
Retarded Development of lin-4 mutants
Wild Type
lin-4-
LIN-14 over-expression
Adult Alae Formation at L4 Molt
ALAE
lin-4
L3 Molt
L4 Molt
lin-14 3’ UTR Deletion
Utterly
Cell Fate Specification
andprogression
Execution
lin-4
andRobust
let-7 microRNAs
control cell fate
Embryo
Larval stages
lin-14
lin-4
lin-14
hbl-1
7-Fam
hbl-1
lin-41
let-7
lin-41
1
2
3
4
Adult
Adult
lin-14 ..C
ACCAA
C..
lin-41..A
hbl-1
...U
UCACA
CUUGU CUCAGGGA
AUU
U..
lin-4
AACUA
AGUGU
UUAUACAACC
CC
GAGUCCCU
GCUACCUUA
CUGCCUC
UUGAU
GAUAUGUUGG
G
GG
A UGAUGGAGU
GAUGGAG
let-7
mir-84
UU
AUGUU
A
C AU
A
U
ACUC
Genetics of microRNA genes in C. elegans
lin-4 and let- 7 control developmental timing (1993, 1999).
••••• Single gene visible phenotypes •••••
Most Caenorhabditis elegans microRNAs are individually not
essential for development or viability.
Miska, Abbott, Alvarez-Saavedra …Bartel, Ambros, Horvitz. PLoS Genet. (2007)
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)
Many families of Caenorhabditis elegans microRNAs are not
essential for development or viability
Ezequiel Alvarez-Saavedra and H. Robert Horvitz Curr Biol. 2010
The microRNA miR-1 regulates a MEF-2-dependent retrograde
signal at neuromuscular junctions.
Lucky!
A drag… No
New Phenotypes
Redundancy within
a microRNA family
3 of 15 families
are essential
Stress the worms
Simon et al…Ruvkun, Kaplan, Kim. Cell. (2008)
Loss of individual microRNAs causes mutant phenotypes in
sensitized genetic backgrounds in C. elegans.
Brenner et al, … Abbott. Curr Biol. (2010)
Visible Phenotypes
for 25 of 31
miRNA genes
• Redundancy between microRNA families
microRNAs Are Embedded
Within Diverse Networks
mir-34
Gonadal
Morphogenesis
Argonaute
mir-83
• Redundancy with gene regulatory “hubs”
Robustness
thru
Redundancy
Loss of individual microRNAs causes
mutant phenotypes in sensitized genetic
backgrounds in C. elegans.
Brenner et al, 2010
mir-1
Fertility
Distinct MicroRNAs with Convergent Targets
Buffering morphogenetic processes against temperature changes
B-integrin
PAT-3
Buffered against
pat-3 mRNA
mir-83 mir-34
peb-1 mRNA
Gonadal
Morphogenesis
Temperature changes
PEB-1
Transcription factor
Samantha Burke & Molly Hammell
Hermaphrodite Gonad Morphogenesis in C. elegans
Phase 1
Phase 2
Phase 3
From Worm Atlas
Migration Defects in mir-34 (-/-) and mir-83 (-/-) Mutants
***
Wild type
80
60
40
17
20
60
40
20
ir83
(-/
-)
0
(-/
-);
m
ir83
80
m
ir34
m
ir34
m
n=200
(-/
-)
5
0
N
2
0
8
100
% of gonad arms scored
Migration defective
(-/
-)
% of gonad arms scored
100
Samantha Burke & Molly Hammell
100
***
100
Taking a Cue from Richard Carthew….
Drosophila mir-7 Buffers Eye Development Against Temperature Changes
Li et al., Carthew Cell 2009 137(2): 273-282
Temperature Oscillations Aggravate mir-34 & mir-83 Phenotypes
15oC – 25oC Oscillations (8 hr)
r-8
3
r-3
4
(-/
d arms scored
***
Migration defective
80
Wild type
60
60
Samantha Burke & Molly Hammell
N2
***
100
80
12
0
mi
100
6
4
-)
3
r-8
i
m
3
14
20
(-/
r-8
mi
(-/
(-/
-)
(-/
2
-);
mi
4
-)
0
40
mi
r-3
r-3
-)
N2
n=200
19
6054
mi
r-8
mi
r-3
3(
4(
-/-)
-/-)
;m
ir-8
3(
-/-)
0
14
20
N2
5
mi
0
8
mi
r-3
4(
-/-)
17
20
40
Wild type
80
(-/
-)
60
Migration defectiv
mi
r-3
60
% of gonad arms scored
Wild type
80
(-/
% of gonad arms scored
80
40
100***
100 defective
Migration
% of gonad arms scored
***
100
100
d arms scored
Constant 20 ℃
80
64
60
-);
4
***
***
Migration
defe
Wild type
% defective gonadal migration
***
54
14
19
2
mir-83(+)
mir-34(+)
mir-83(0)
mir-34(+)
mir-83(+)
mir-34(0)
Samantha Burke & Molly Hammell
mir-83(0)
mir-34(0)
Distinct MicroRNAs with Convergent Targets
Buffering morphogenetic processes against temperature changes
B-integrin
PAT-3
Buffered against
pat-3 mRNA
mir-83 mir-34
peb-1 mRNA
Gonadal
Morphogenesis
Temperature changes
PEB-1
Transcription factor
Samantha Burke & Molly Hammell
Post-dauer:
mir-48,
mir-84, mir-241, let-7
lin-4: MAJOR Role
Continuous:
lin-4: minor role
NHL-2
mir-48, mir-84, mir-241, let-7
AGO
let-7
Family
lin-4
An
Modulation of microRNA levels and (lin-4) activity
Xantha Karp
Accommodation of Stress and Diapause in C. elegans Development
Favorable
Unfavorable
Larval stages
1
Insulin/IGF
DAF-16/Fox0
2 Dauer
larva
3
4
Adult
Continuous
Dauerinterrupted
Utterly
Cell Fate Specification
andprogression
Execution
lin-4
andRobust
let-7 microRNAs
control cell fate
Embryo
Larval stages
lin-14
lin-4
lin-14
hbl-1
7-Fam
hbl-1
lin-41
let-7
lin-41
1
2
(Optional) Developmental Quiescence
3
4
Adult
Adult
lin-14 ..C
ACCAA
C..
lin-41..A
hbl-1
...U
UCACA
CUUGU CUCAGGGA
AUU
U..
lin-4
AACUA
AGUGU
UUAUACAACC
CC
GAGUCCCU
GCUACCUUA
CUGCCUC
UUGAU
GAUAUGUUGG
G
GG
A UGAUGGAGU
GAUGGAG
let-7
mir-84
UU
AUGUU
A
C AU
A
U
ACUC
lin-4 and let-7 microRNAs control cell fate progression
Embryo
Larval stages
lin-14
lin-4
lin-14
hbl-1
7-Fam
hbl-1
lin-41
let-7
lin-41
1
2
(Optional) Developmental Quiescence
3 Dauer larva
4
Adult
Adult
Robust Timing of
cell fates
microRNAs in C. elegans cell fate progression
Developmental Signals
Wild type
lin-4
LIN-14
LIN-28
L1
L2
miR-48
L3
Dauer Larva Quiescence
miR-84
HBL-1
let-7Fam
mir-48
mir-84
mir-241
let-7
L4
let-7
LIN-41
miR-241
LIN-29
Ad
MicroRNA
pathways
lin-4
Transcription
Factors
let-7
Robust Timing of
cell fates
Dosage-Sensitive activity of let-7-Family microRNAs
Worm let-7-Family
Developmental Signals
UGAGGUAGUAGGUUGUAUAGUU
UGAGGUAGGCUCA-GUAGAUGC
UGAGGUAGUAUGUAAUAUUGUA
UGAGGUAGGUGCGAGAAAUGA-
lin-4
Wild type
-1
let-7-Fam
LIN-14
LIN-28
let-7Fam
L1
L2
L2
L3
L3
L4
L4
Ad
Ad
HBL-1
mir-48
mir-84
mir-241
let-7
let-7
L1
LIN-41
LIN-29
Dosage-Sensitive activity of let-7-Family microRNAs
Worm let-7-Family
Developmental Signals
UGAGGUAGUAGGUUGUAUAGUU
UGAGGUAGGCUCA-GUAGAUGC
UGAGGUAGUAUGUAAUAUUGUA
UGAGGUAGGUGCGAGAAAUGA-
lin-4
LIN-14
LIN-28
L1
L1
L2
L2
L2
L3
L3
L4
L3
L4
L4
Ad
Ad
HBL-1
HBL-1
let-7Fam
mir-48
mir-84
mir-241
let-7
let-7
Wild type
-3
let-7-Fam
LIN-41
LIN-29
Retarded
Normal
Adult-specific col-19::GFP
Adult-specific col-19::GFP
let-7-Fam
-3
Developmental Signals
Stressful environment
lin-4
LIN-14
LIN-28
let-7-Fam
-3
Healthy environment
L1
L2
HBL-1
HBL-1
let-7Fam
L3
L2
mir-48
mir-84
mir-241
let-7
Dauer Larva Quiescence
L3
L4
let-7
LIN-41
LIN-29
L4
Ad
Normal
Retarded
“Post-dauer” Suppression
Wild Type
Post-dauer
Continuous
let-7-Fam-3
Post-dauer
Continuous
Adult-expression of col-19::GFP
let-7-Fam
-3
Developmental Signals
Stressful
environment
High Pheromone
lin-4
LIN-14
LIN-28
let-7Fam
let-7-Fam
-3
Healthy
environment
Low Pheromone
L1
L2
Mod
HBL-1
L3
mir-48
mir-84
mir-241
let-7
Dauer Larva Quiescence
L4
let-7
LIN-41
LIN-29
Ad
Normal
Xantha Karp
Retarded
Post-Dauer Suppression
-3 -3
Mod(0);
let-7-Fam
let-7-Fam
Developmental Signals
High Pheromone
lin-4
LIN-14
LIN-28
let-7Fam
let-7-Fam
-3
Low Pheromone
L1
L2
Mod
HBL-1
HBL-1
L3
L2
mir-48
mir-84
mir-241
let-7
Dauer Larva Quiescence
L3
L4
let-7
LIN-41
LIN-29
L4
Ad
Retarded
Normal
Xantha Karp
Retarded
Post-Dauer Suppression
let-7-Fam
-3
Developmental Signals
High Pheromone
lin-4
LIN-14
LIN-28
let-7Fam
let-7-Fam
-3
Low Pheromone
L1
L2
NHL-2
HBL-1
L3
mir-48
mir-84
mir-241
let-7
Dauer Larva Quiescence
L4
let-7
LIN-41
LIN-29
Ad
Normal
Xantha Karp
Retarded
“Post-Dauer” Suppression
nhl-2(0); let-7-Fam
-3
Developmental Signals
High Pheromone
lin-4
LIN-14
LIN-28
let-7Fam
nhl-2(0); let-7-Fam
Low Pheromone
L1
L2
NHL-2
HBL-1
HBL-1
L3
L2
mir-48
mir-84
mir-241
let-7
Dauer Larva Quiescence
L3
L4
let-7
LIN-41
LIN-29
L4
Ad
Retarded
Normal
Xantha Karp
Retarded
“Post-Dauer” Suppression
-3
Col-19::GFP expression in Post-Dauer Adults
Post-Dauer let7-Fam-3
Post-Dauer
nhl-2; let-7-Fam-3
Developmental Signals
-3 -3
lin-4
LIN-14
LIN-28
let-7Fam
-3 -3
lin-4(0);
let-7-Fam
let-7-Fam
lin-4(0);
let-7-Fam
let-7-Fam
High Pheromone
Low Pheromone
L1
L2
NHL-2
HBL-1
HBL-1
L3
L2
mir-48
mir-84
mir-241
let-7
Dauer Larva Quiescence
L4
L3
let-7
LIN-41
LIN-29
Ad
L4
Retarded
Normal
Retarded
Xantha Karp
“Post-Dauer” Suppression
lin-4 is required for down regulation of HBL-1
Between L2 and Post Dauer L3
L1
WT
L2
Dauer larva
WT
L1
WT
L2
Post Dauer L3
WTPost Dauer L3
let-7-Fam-3
Dauer larva
Post Dauer L3
Down Regulated
HBL-1::GFP::UTR-HBL-1
Not Down Regulated
lin-4; let-7-Fam-3
Developmental Signals
-1 -1
lin-4
LIN-14
LIN-28
let-7Fam
High Pheromone
Low Pheromone
L1
HBL-1
HBL-1
L3
L2
mir-48
mir-84
mir-241
let-7
let-7
lin-4(0);
let-7-Fam
let-7-Fam
L2
L2
NHL-2
LIN-41
Dauer Larva Quiescence
L3
L4
L3
L4
Ad
L4
Ad
LIN-29
Retarded
Xantha Karp
-1 -1
lin-4(0);
let-7-Fam
let-7-Fam
Normal
no alae
gapped alae
wild-type
lin-14(n179) mir-84
Continuous
mir-84(0)
lin-4; lin-14(n179)
Post-Quiescence
**
Continuous
**
lin-4(0); mir-84(0)
lin-4; lin-14(n179) mir-84
Post-Quiescence
0%
50%
% Adults expressing larval cell fates
*All strains contain lin-14(n179)
Xantha Karp
100%
** P < 0.01
Taqman Q-RT/PCR
Dauer Traversing vs Continuously-Developing
1.6
1.4
mir-84
microRNA 1.2
abundance 1.0
1
Dauer vs
Continuous 0.8
let-7
lin-4
mir-48
mir-241
0.6
0.4
0.2
0
L2D/L2
L2D/L2
Dauer/L2molt
Dauer/L2m
PDL3/L3
PDL3/L3
PDL4/L4
PDL4/L4
Insulin/IGF
DAF-16/Fox0
Continuous:
Post-dauer:
mir-48,
mir-84, mir-241, let-7
lin-4: MAJOR Role
lin-4: minor role
NHL-2
mir-48, mir-84, mir-241, let-7
AGO
let-7
Family
lin-4
An
Modulation of microRNA levels and (lin-4) activity
Xantha Karp
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
Physiological Modulation of microRNA activity
Bacterial Diet
Zhiji Ren
let-7-Family microRNAs in Diet and Development
Bacterial Quality/Toxicity
Metabolic
Signaling
Developmental
cell fate
specification
Innate
Immunity
mir-84
let-7
Buffering against mir-48
food stress
mir-241