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.

3. 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

8. 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

10. RNA interference:
A type of gene regulation
Involving small RNA molecules
and induced by double stranded RNA

11. Adding a Chalcone synthase
gene from Petunia fused to
a strong viral promoter
to transgenic Petunia
interfered with expression
of the native homologous’
gene.
Gene Silencing

12. Gene Silencing 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).

13. RNA viruses can block expression of a transgene
if a copy of the transgene has been added
Tobacco plant expressing GFP protein
Infected with RNA virus with GFP gene
Virus infection travels through veins
GFP expression inhibited starting at veins.

14. Gene silencing and RNA viruses share potential to produce dsRNA
Fire and Mello used C. elegans
to prove that dsRNA inhibits
expression of endogenous genes
homologous to that dsRNA

15. Double stranded GFP RNA interferes with
expression of GFP in transgenic C. elegans
GFP expressed in nuclei After adding dsRNA for
GFP

16. 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?

17. 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

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

19. Mechanism of RNAi Dicer binds dsRNA And cleaves making siRNA
siRNAs direct Risc to
copy homologous
mRNA
Dicer binds short
stretch of dsRNA
and cleaves it.

20. 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

21. What is the function of RNAi mechanism in non-transgenic organisms?
Protection against viruses
Keep Transposable elements inactive
Gene regulation

22. Gene regulation by small RNAs
SiRNAs degrade mRNA
to stop gene expression
quickly
StRNAs prevent translation
to stop gene expression
quickly

23. miRNA Precursor Transcript
MIRNA genes are PolII genes that encode
a hairpin shaped mRNA.
Dicer cleaves the double stranded portion to make
a short dsRNA.
That combines with the RISC complex and directs
cleavage of a specific target mRNA
MIRNA gene
Pol II
AAA
DCL1
HYL1
~100 MIRNA genes**
DCL1
HEN1, HYL1
57 unique miRNAs from 26 families
HASTY
RISC
components
AGO1
miRNA Precursor Transcript
miRNA*
AGO1
RISCmiRNA
Target
AAA
AGO1
**Cumulative data from Bartels, Carrington, Chen, Weigel, Zhu, others
Cleaved target
AAA

24. miRNAs and Targets in Animals
Fig. 3 from Lewis, Burge and Bartel (2005) Cell 120, 15-20
May target 1/3 of all genes
Registered miRNAs
C. elegans 114
Drosophila 78
Zebrafish 362
Mouse 245
Human 321
Herpesviruses
SV40
Development - e.g. timing, stem cell function, differentiation
Cell and organ identity
Cancer - mis-regulation, deletion, duplication of MIRNA genes

25. 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.

26. Imprinting is another form of epigenetic gene regulation
ICR – imprinting
Control region
Differential
methylation
leads to
differential
expression of
Maternal and
paternal alleles

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

28. microRNA Targets in Plants and Animals
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
ARF10 mRNA
lin-14 mRNA
3’UTR
AAA - - - - - - - -
AAA
UUGCAC-UCUCAGGGA
|::||| |||||||||
3’ AGUGUG AGAGUCCCU
A C U 5’
C C CU
AGGAAUACAGGGAGCCAGGCA
|| |||||||||||||||||
3' ACCGUAUGUCCCUCGGUCCGU 5
miR160
lin-4 miRNA

29. Antiviral RNAi - Redundancy of DCL, RDR Factors
v-siRNA duplex
AGO
v-siRNA*
AGO
RISCv-siRNA

30. Trans-acting siRNA gene
MIRNA gene
Trans-acting siRNA gene
Pol II
Pol II
Poethig, Vaucheret/ D. Bartel labs
AAA
DCL1
HYL1
RDR6, SGS3
DCL1
HEN1, HYL1
DCL4
HEN1
HASTY
RISC
components
AGO1
RISC
components
AGO
miRNA*
ta-siRNA*
AGO1
RISCmiRNA
AGO
RISCta-siRNA
Target
Target
AAA
AAA
AGO1
AGO
Cleaved target
Cleaved target
AAA
AAA
Vazquez et al. (2004) Mol. Cell 16, 69-79; Peragine et al. (2004) Genes Dev. 18,

31. Trans-acting siRNA gene
MIR gene
Trans-acting siRNA gene
Pol II
Pol II
AAA
DCL1
HYL1
RDR6, SGS3
DCL1
HEN1, HYL1
DCL4
HEN1
HASTY
RISC
components
AGO1
RISC
components
AGO
miRNA*
ta-siRNA*
AGO1
RISCmiRNA
AGO
RISCta-siRNA
Target
Target
AAA
AAA
AGO1
AGO
Cleaved target
Cleaved target
AAA
AAA