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

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Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.

Transcript of Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant...

Page 1: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.

Changes to Syllabus:

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

FastPlant lab delayed:Oct. 4 and 5.

Page 2: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.
Page 3: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change 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

Page 4: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.
Page 5: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.
Page 6: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.
Page 7: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.
Page 8: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change 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

Page 9: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.
Page 10: Changes to Syllabus: Quizzes put back: Change Oct. 3 to Oct. 17 Change Nov. 7 to Nov. 14 FastPlant lab delayed: Oct. 4 and 5.

RNA interference:

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

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

Gene Silencing

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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).

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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

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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

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

GFP expressed in nuclei After adding dsRNA for GFP

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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?

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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

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

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Mechanism of RNAi

Dicer binds dsRNAAnd cleaves makingsiRNA

siRNAs direct Risc to copy homologous mRNA

Dicer binds shortstretch of dsRNA and cleaves it.

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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

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

Protection against viruses

Keep Transposable elements inactive

Gene regulation

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Gene regulation by small RNAs

SiRNAs degrade mRNAto stop gene expression quickly

StRNAs prevent translation to stop gene expression quickly

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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

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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

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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.

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Imprinting is another form of epigenetic gene regulation

ICR – imprintingControl region

Differential methylationleads to differential expression of Maternal and paternal alleles

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

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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

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RDR6

DCL4

AGO

AGO

CUAUAACCGCGCCGAGUUAGU

DCL2

v-siRNA*

RISCv-siRNA

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

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

|||||||||||

viral RNA

RDR1

v-siRNA duplex

Antiviral RNAi - Redundancy of DCL, RDR Factors

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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

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

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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

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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

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

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

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Trans-acting siRNA gene

HYL1