2. 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 - Horvitz
8. Genetics - Brenner

6. Gene silencing and RNA viruses share potential to produce dsRNA
Fire and Mello (1998, Nature) used C. elegans
to prove that dsRNA inhibits
expression of endogenous genes
homologous to that dsRNA

7. Double stranded GFP RNA interferes with
expression of GFP in transgenic C. elegans
GFP expressed in nuclei After adding dsRNA for
GFP
Fire and Mello (1998, Nature)
Transgenic C. elegans express GFP in cell nuclei.
Adding tiny amounts of GFP dsRNA into gut stops expression of GFP
in animals of the next generation.

8. Features of RNAi Induced by dsRNA with homology to exons
Target sequence specific
Catalytic: very small amounts of ds RNA are sufficient
Spreads: injection into gut silences genes in embryos
Lasts: Silencing continues as the embryo matures
Small RNAs produced – homologous to original dsRNA

9. Mechanism of RNAi Dicer binds dsRNA and cleaves making siRNA
siRNAs bind to ARGONAUTE to make RISC complex. The RISC complex
binds homologous
RNAs and cleaves them.
Amplification and spread
Verdel et al 2009 Fig 1.

10. Amplification RDRP copies mRNA making more ds RNA.
Dicer cuts that generating
more siRNA
More RDRP is activated and
more dsRNA is made.

11. Current guess: certain classes of small RNAs transported between cells
Spread to other cells
Current guess: certain classes of small RNAs
transported between cells

12. Block transcription of homologous genes
Dicer binds dsRNA
and cleaves making
siRNA
siRNAs bind to ARGONAUTE to make RITS complex.
The RITS complex
binds homologous
DNA or RNA and promotes chromatin condensation.
Verdel et al 2009 Fig 1.

13. Stable block to transcription Verdel et al 2009 Fig 2.
Nuclear RITS complex directs methylation of histones on target genes while they are being expressed.
RITS complex
Alternative models for RITS complex methylating histones on target locus
Genomic DNA is wrapped around histone proteins to make nucleosomes. Methylation of histones increases the condensation of chromosomal DNA making it less accessible to transcription factors
DNA also gets methylated. Methylation patterns are copied when DNA is replicated
Stable block to transcription
Verdel et al 2009 Fig 2.

14. Chromatin condensation blocks transcription
RITS complex recruits other chromosome binding proteins to promote
chromatin condensation
RITS complex
Verdel et al 2009 Fig 3.

15. In plants, a special RNA polymerase produces transcripts in condensed regions of DNA such as centromeres initiating and maintaining chromatin condensation
Verdel et al 2009 Fig 4a.

16. Centromeres have condensed chromatin and highly methylated DNA.
In plants, Pol IV is recruited to transcribe repeated sequences in centromeres.
The production of double stranded RNAs maintains the
condensed state of the centromeres.
Blue are common repeats and orange are retrotransposons
Short RNA homologous to common repeats can be isolated
In Arabidopsis, small RNA production depends on functional PolIV

17. Mechanisms of RNAi Dicing Dicer Base pairing RISC/Argonaut
Slicing RISC/Argonaut
Amplification
RdRp
Spread to other cells
Small dsRNA
Block transcription of homologous genes
RITS

18. Sources of dsRNA Ds RNA viruses Transposable element inverted repeats
Transcripts copied by RdRp
Transcripts produced by PolIV (plants)
miRNAs
Overlapping mRNA transcripts

19. 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
~200 MIRNA genes**
DCL1
HEN1, HYL1
57 unique miRNAs from 26 families
HASTY
miRNA Precursor Transcript
RISC
components
AGO1
miRNA*
AGO1
RISCmiRNA
Target
AAA
AGO1
**Cumulative data from Bartels, Carrington, Chen, Weigel, Zhu, others
Cleaved target
AAA

20. Overlapping transcripts on opposite strands
can generate small RNAs
Controlled expression of the opposing transcripts
lead to RNA mediated silencing
Regulate protein levels

21. of salt stress response
Small RNA regulation
of salt stress response
Salt stress induces ROS
and increases in proline.
High proline protects cells
against water loss.
Increased ROS leads to
induction of an SRO5
transcript that overlaps
the P5CDH transcript
As a result, RNAi is initiated
against P5CDH transcripts.
P5CDH protein levels drop
and proline accumulates
Borsani et al 2005 Cell 123, 1279

22. Summary Small double stranded RNAs regulate gene expression by:
directing degradation of specific transcripts
preventing translation of specific transcripts
promoting chromatin condensation of specific
target genes
Double stranded RNA can be used to mimic mutations in
C. elegans and in other organisms.
Sequence-specific targets
Effect will be carried on in the progeny of
treated animals

23. 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?

24. RNAi affects oocytes of treated C. elegans F1 carriers of RNAi effect
Ref: Tabara et al 1999 Cell 99, On class web page.

25. A real genetic screen for C. elegans
genes essential for RNAi

26. 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

28. Different mutants had
specific phenotypes
for activation of
transposable elements
and silencing of transgenes

31. Functions of genes in RNAi decifered
Rde4 = Initiating RNA binding protein
Rde1 = Argonaut
Rde2 = germline silencing
Mut7 = endonuclease

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