Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction

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

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

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

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?

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

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

~200 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.

Expression of double stranded RNA leads to chromatin remodeling

Centromeres are transcriptionally inactive. They have condensed chromatinRepeated sequences lead to production of double stranded RNAsThe dsRNAs maintain the condensed state of the centromeres

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