advantages of c. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/1.jpg)
![Page 2: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/2.jpg)
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 3: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/3.jpg)
![Page 4: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/4.jpg)
![Page 5: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/5.jpg)
![Page 6: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/6.jpg)
RNA interference:
A type of gene regulation Involving small RNA molecules and induced by double stranded RNA
![Page 7: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/7.jpg)
Adding a Chalcone synthase gene from Petunia fused to a strong viral promoterto transgenic Petunia interfered with expressionof the native homologous’gene.
Gene Silencing
![Page 8: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/8.jpg)
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).
![Page 9: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/9.jpg)
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
![Page 10: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/10.jpg)
Double stranded RNA is common to Viruses that can silence host gene expression
And to transgenes that can silence host gene expression
![Page 11: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/11.jpg)
Transgenes insert into chromosomal DNA randomly
or
![Page 12: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/12.jpg)
Transgenes often integrate in complexes
![Page 13: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/13.jpg)
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
![Page 14: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/14.jpg)
Double stranded GFP RNA interferes with expression of GFP in transgenic C. elegans
GFP expressed in nuclei After adding dsRNA for GFP
![Page 15: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/15.jpg)
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
![Page 16: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/16.jpg)
Mechanism of RNAi
Dicer binds dsRNAAnd cleaves makingsiRNA
siRNAs direct Risc to bind homologous mRNA and cleave it.
![Page 17: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/17.jpg)
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
![Page 18: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/18.jpg)
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?
![Page 19: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/19.jpg)
How would you set up a genetic screen to investigate the mechanism of RNAi?
Design a screen for mutants in genes required for RNAi
Easy to induce RNAi in C. elegans by feeding them E. coli engineered to express a transgene in both directions – sense and antisense
![Page 20: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/20.jpg)
A real genetic screen for C. elegans genes essential for RNAi
![Page 21: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/21.jpg)
A real genetic screen for C. elegans genes essential for RNAi
![Page 22: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/22.jpg)
Candidate mutants isolated after feeding E. coli producing double stranded RNA
How could double stranded RNA be made in E. coli?
Checked candidate mutants by microinjection into gut
The listed mutants passed the secondary test
![Page 23: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/23.jpg)
![Page 24: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/24.jpg)
Different mutants had Specific Phenotypes For activation of Transposable elements and silencing of transgenes
![Page 25: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/25.jpg)
![Page 26: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/26.jpg)
![Page 27: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/27.jpg)
Functions of genes in RNAi decifered
Rde4 = Initiating RNA binding protein
Rde1 = Argonaut
Rde2 = ?
Mut7 = endonuclease
![Page 28: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/28.jpg)
What is the function of RNAi mechanism in non-transgenic organisms?
Protection against viruses
Keep Transposable elements inactive
Gene regulation
![Page 29: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/29.jpg)
Gene regulation by small RNAs
SiRNAs degrade mRNAto stop gene expression quickly
StRNAs prevent translation to stop gene expression quickly
![Page 30: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/30.jpg)
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
![Page 31: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/31.jpg)
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
![Page 32: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/32.jpg)
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.
![Page 33: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/33.jpg)
Centromeres are transcriptionally inactive. They have condensed chromatinRepeated sequences lead to production of double stranded RNAsThe dsRNAs maintain the condensed state of the centromeres
![Page 34: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/34.jpg)
Transcriptional Gene silencing requires dsRNA, DNA methyltransferases, histone methylation and histone deacetylation.
RNA directed DNA methylation
DNA methylases associate with histone modification enzymes leading to changes in histone methlyation patterns and histone deacetylation.Condensed chromatin results.
Histone methylation
![Page 35: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/35.jpg)
RNA is a gene regulator as well as a carrier of information
![Page 36: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/36.jpg)
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.
![Page 37: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/37.jpg)
Imprinting is another form of epigenetic gene regulation
ICR – imprintingControl region
Differential methylationleads to differential expression of Maternal and paternal alleles
![Page 38: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/38.jpg)
Establishment of differential methylation
CTCF zinc finger proteinProtects ICR in femalegermline
Denovo methylases Cannot methylate H19
Igf2 insulin-related growth factorRepressed in extraembryonic tissuesand in some embryo tissues
Imprinting
![Page 39: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/39.jpg)
RNA directed methylases also affect methylation of genes in germline.
May be important to imprinting
![Page 40: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/40.jpg)
RNA can mediate differential expression of genes on same chromosome
Mechanism not like RNAi because it works in cis.
Like X inactivation
Extraembryonic tissues:
Igf2r and distant, linked genes Maternal ICR is methylated,Paternal alleles are not expressed.
Paternal X chromosome Not expressed.
![Page 41: Advantages of C. elegans : 1. rapid life cycle 2. hermaphrodite 3. prolific reproduction](https://reader035.vdocument.in/reader035/viewer/2022062801/5681438a550346895db00885/html5/thumbnails/41.jpg)
RNA directed regulation of gene expression is more common than we thought.