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