Changes to Syllabus:

Quizzes put back:Change Oct. 3 to Oct. 17Change Nov. 7 to Nov. 14

FastPlant lab delayed:Oct. 4 and 5.

Advantages of C. elegans:

1. rapid life cycle

2. hermaphrodite

3. prolific reproduction

4. transparent

5. only ~1000 cells

6. laser ablation

7. complete cell lineage map

8. genetics

Advantages of C. elegans:

1. rapid life cycle

2. hermaphrodite

3. prolific reproduction

4. transparent

5. only ~1000 cells

6. laser ablation

7. complete cell lineage map

8. genetics

RNA interference:

A type of gene regulation Involving small RNA molecules and induced by double stranded RNA

Adding a Chalcone synthase gene from Petunia fused to a strong viral promoterto transgenic Petunia interfered with expressionof the native homologous’gene.

Gene Silencing

Gene Silencing

1.More common from strong promoters. 2. More common from inverted repeat inserts

(could make a double stranded RNA).

3. Induced by RNA viruses (have double stranded RNA replication intermediates).

Tobacco plant expressing GFP proteinInfected with RNA virus with GFP geneVirus infection travels through veinsGFP expression inhibited starting at veins.

RNA viruses can block expression of a transgene if a copy of the transgene has been added

Gene silencing and RNA virusesshare potential to produce dsRNA

Fire and Mello used C. elegans to prove that dsRNA inhibits expression of endogenous genes homologous to that dsRNA

Double stranded GFP RNA interferes with expression of GFP in transgenic C. elegans

GFP expressed in nuclei After adding dsRNA for GFP

Double stranded GFP RNA interferes with expression of GFP in transgenic C. elegans

GFP expressed in nuclei After adding dsRNA for GFP

How would you design a genetic screen to identify the genes involved in the RNAi mechanism?

Features of RNAi

Induced by dsRNA with homology to exons

Catalytic: very small amounts of ds RNA are sufficient

Spreads: injection into gut silences genes in embryos

Small RNAs produced

The real genetic screen for C. elegans genes essential for RNAi

Mechanism of RNAi

Dicer binds dsRNAAnd cleaves makingsiRNA

siRNAs direct Risc to copy homologous mRNA

Dicer binds shortstretch of dsRNA and cleaves it.

Catalysis: RdRP copies mRNA making more ds RNA. Dicer cuts that generating more siRNA

More RdRP is activated and more dsRNA is made.

Spread: dsRNA transported to other cells.Not in Drosophila or mammals

What is the function of RNAi mechanism in non-transgenic organisms?

Protection against viruses

Keep Transposable elements inactive

Gene regulation

Gene regulation by small RNAs

SiRNAs degrade mRNAto stop gene expression quickly

StRNAs prevent translation to stop gene expression quickly

MIRNA gene

HASTY

5’

3’

CC UU C C AUGAGAGAGU CU GAUAUUGGC UGGUUCA UCAGAU |:|||||||| || ||||||||| :||:||| |||||| 44nt loop UGCUCUCUCA GA CUAUAACCG GCCGAGU AGUCUA U CU C U

C C 5' UAUUGGC UGGUUCA UCACU 3' ||||||| ||||||| |||3' CUAUAACCG GCCGAGU AGU 5' C U

5’

3’

CC UU C C AUGAGAGAGU CU GAUAUUGGC UGGUUCA UCAGAU |:|||||||| || ||||||||| :||:||| |||||| 44nt loop UGCUCUCUCA GA CUAUAACCG GCCGAGU AGUCUA U CU C U

AAA

Pol II

DCL1

HEN1, HYL1

C C 5' UAUUGGC UGGUUCA UCACU 3' ||||||| ||||||| |||3' CUAUAACCG GCCGAGU AGU 5' C U

AGO1

AGO1

CUAUAACCGCGCCGAGUUAGU

UAUUGGCCUGGUUCACUCCACU

AGO1

CUAUAACCGCGCCGAGUUAGU

AAA ..... .............

AAA

DCL1

miRNA*

RISCcomponents

RISCmiRNA

Target

Cleaved target

57 unique miRNAs from 26 families

~100 MIRNA genes**

**Cumulative data from Bartels, Carrington, Chen, Weigel, Zhu, others

m7GpppN AAAAA

5’ Cap

Introns

Poly(A) tailmiRNA Precursor

Transcript

MIRNA genes are PolII genes that encode a hairpin shaped mRNA.Dicer cleaves the double stranded portion to makea short dsRNA.That combines with the RISC complex and directs cleavage of a specific target mRNA

HYL1

miRNAs and Targets in Animals

C. elegans 114Drosophila 78Zebrafish 362Mouse 245Human 321

Registered miRNAs

Fig. 3 from Lewis, Burge and Bartel (2005) Cell 120, 15-20

May target 1/3 of all genes

Development - e.g. timing, stem cell function, differentiationCell and organ identityCancer - mis-regulation, deletion, duplication of MIRNA genes

HerpesvirusesSV40

Transcriptional gene silencing is initiated by RNA directed methylation of promoter regions

dsRNA homologous to promoters leads to methylation and inactivation by recruitment of chromatin remodeling enzymes.

Imprinting is another form of epigenetic gene regulation

ICR – imprintingControl region

Differential methylationleads to differential expression of Maternal and paternal alleles

RNA is a gene regulator as well as a carrier of information

Plant miRNA Targets

Animal miRNA Targets

Single sites

miRNA:target high complementary

Usually in coding region

Multiple sites, combinatorial

miRNA:target low complementary

3’ UTR region

microRNA Targets in Plants and Animals

-------lin-14 mRNA 3’UTR

AAA

AGGAAUACAGGGAGCCAGGCA || |||||||||||||||||3' ACCGUAUGUCCCUCGGUCCGU 5

-

ARF10 mRNA

miR160

UUGCAC-UCUCAGGGA |::||| ||||||||| 3’ AGUGUG AGAGUCCCU

A C U 5’ C C CU lin-4 miRNA

AAA

RDR6

DCL4

AGO

AGO

CUAUAACCGCGCCGAGUUAGU

DCL2

v-siRNA*

RISCv-siRNA

|||||||||||||||||||||||||||||||||||

||||||||||| ||||||||||| |||||||||||

|||||||||||

viral RNA

RDR1

v-siRNA duplex

Antiviral RNAi - Redundancy of DCL, RDR Factors

MIRNA gene

HASTY

5’

3’

CC UU C C AUGAGAGAGU CU GAUAUUGGC UGGUUCA UCAGAU |:|||||||| || ||||||||| :||:||| |||||| 44nt loop UGCUCUCUCA GA CUAUAACCG GCCGAGU AGUCUA U CU C U

C C 5' UAUUGGC UGGUUCA UCACU 3' ||||||| ||||||| |||3' CUAUAACCG GCCGAGU AGU 5' C U

5’

3’

CC UU C C AUGAGAGAGU CU GAUAUUGGC UGGUUCA UCAGAU |:|||||||| || ||||||||| :||:||| |||||| 44nt loop UGCUCUCUCA GA CUAUAACCG GCCGAGU AGUCUA U CU C U

AAA

Pol II

DCL1

HEN1, HYL1

C C 5' UAUUGGC UGGUUCA UCACU 3' ||||||| ||||||| |||3' CUAUAACCG GCCGAGU AGU 5' C U

AGO1

AGO1

CUAUAACCGCGCCGAGUUAGU

UAUUGGCCUGGUUCACUCCACU

AGO1

CUAUAACCGCGCCGAGUUAGU

AAA ..... .............

AAA

DCL1

miRNA*

RISCcomponents

RISCmiRNA

Target

Cleaved target

Pol II

RDR6, SGS3

HEN1

AGO

AGO

CUAUAACCGCGCCGAGUUAGU

AGO

CUAUAACCGCGCCGAGUUAGU

AAA ..... .............

AAA

DCL4

ta-siRNA*

RISCcomponents

RISCta-siRNA

Target

Cleaved target

|||||||||||||||||||||||||||||||||||

||||||||||| ||||||||||| |||||||||||

|||||||||||

Trans-acting siRNA gene

Vazquez et al. (2004) Mol. Cell 16, 69-79; Peragine et al. (2004) Genes Dev. 18, 2368-2379

HYL1Poethig, Vaucheret/ D. Bartel labs

MIR gene

HASTY

5’

3’

CC UU C C AUGAGAGAGU CU GAUAUUGGC UGGUUCA UCAGAU |:|||||||| || ||||||||| :||:||| |||||| 44nt loop UGCUCUCUCA GA CUAUAACCG GCCGAGU AGUCUA U CU C U

C C 5' UAUUGGC UGGUUCA UCACU 3' ||||||| ||||||| |||3' CUAUAACCG GCCGAGU AGU 5' C U

5’

3’

CC UU C C AUGAGAGAGU CU GAUAUUGGC UGGUUCA UCAGAU |:|||||||| || ||||||||| :||:||| |||||| 44nt loop UGCUCUCUCA GA CUAUAACCG GCCGAGU AGUCUA U CU C U

AAA

Pol II

DCL1

HEN1, HYL1

C C 5' UAUUGGC UGGUUCA UCACU 3' ||||||| ||||||| |||3' CUAUAACCG GCCGAGU AGU 5' C U

AGO1

AGO1

CUAUAACCGCGCCGAGUUAGU

UAUUGGCCUGGUUCACUCCACU

AGO1

CUAUAACCGCGCCGAGUUAGU

AAA ..... .............

AAA

DCL1

miRNA*

RISCcomponents

RISCmiRNA

Target

Cleaved target

Pol II

RDR6, SGS3

HEN1

AGO

AGO

CUAUAACCGCGCCGAGUUAGU

AGO

CUAUAACCGCGCCGAGUUAGU

AAA ..... .............

AAA

DCL4

ta-siRNA*

RISCcomponents

RISCta-siRNA

Target

Cleaved target

|||||||||||||||||||||||||||||||||||

||||||||||| ||||||||||| |||||||||||

|||||||||||

Trans-acting siRNA gene

HYL1