1. Exploring Gene Function in C. elegans: Mutations and RNA Interference
Carolina Biological Supply Company
Bruce Nash
Dolan DNA Learning Center
Cold Spring Harbor Laboratory

2. Craig Mello
He was awarded the Nobel Prize for Physiology or Medicine, along with Andrew Z. Fire, for the discovery of RNA interference.
This research was conducted at the University of Massachusetts Medical School and published in 1998

3. A Grand Challenge
To understand how life works

4. How do we go from DNA to function?

5. Find a tractable systems:
Model organisms

6. What makes a good model organism?
Ease of cultivation
Simplicity
Relevant biology
Amenability
(W)hen I embarked on this problem, I decided that what was needed was an experimental organism which was suitable for genetical study and in which one could determine the complete structure of the nervous system.
S. Brenner Genetics 77: May 1974

7. Caenorhabditis elegans
The model organism:
Caenorhabditis elegans
Electron micrograph of a C. elegans hermaphrodite

8. Why Worms? Profile Soil nematode Genome size: 100 Mb
Number of chromosomes: 6
Generation time: about 2 days
Female reproductive capacity: 250 to 1000 progeny
Special characteristics
Strains Can Be Frozen
Hermaphrodite
Known cell lineage pattern for all 959 somatic cells
Only 302 neurons
Transparent body
Can be characterized genetically
About 70% of Human Genes have related genes in C. elegans

9. About 40% of worm genes are very related to human genes~ =
Worms have gut, muscle, skin and a nervous system like us
About 40% of worm genes are very related to human genes

10. Deduce the function of mutated genes
How do geneticists study gene function?
Identify mutants
Deduce the function of mutated genes

11. How do geneticists study gene function?
Wild type
Dumpy mutant
The wild type gene must maintain normal body shape.

12. Examining, growing and caring for worms
Students examine worm behavior and morphology
Students identify stages of worm development
Students examine mutant strains and compare to wild type
Students culture C. elegans

13. Anatomy of a worm
(from

14. Anatomy of a worm

15. Wild-type

16. Identifying hermaphrodites
Clear patch on L4
L4 with clear patch
Adult
with
embryos

17. bli-1
Notice the large clear area on the side of the worm (a blister in the cuticle)

18. dpy-11
Dumpy worms are shorter.

19. Lifecycle of a worm
(from

20. Identifying hermaphrodites
Clear patch
on L4

21. A problem:
Creating mutations in specific genes is very hard

22. Can we go directly from sequence to function?

23. One approach: Antisense RNA turns down specific genes
This can give the same phenotype as a mutant

24. An experiment showed that the antisense model didn’t make “sense”
Sense RNA
Antisense RNA
Turns off gene
Turns off gene????
First noticed that sense RNA was as effective as antisense RNA for suppressing gene expression in worm
Guo S, and Kemphues KJ.
1995

25. Double-stranded RNA causes silencing:
RNA Interference!
Negative control
uninjected
Antisense RNA
dsRNA
Weak effect
Strong effect
mex-3B antisense RNA
mex-3B dsRNA
Double-stranded RNA was a contaminant in antisense experiments
First described RNAi phenomenon in C. elegans by injecting dsRNA into C. elegans, which led to an efficient sequence-specific silencing and coined the term "RNA Interference".
Fire et al.
1998

26. How can dsRNA turn of genes?
Gene OFF

27. Biochemistry to the rescue RNAi in vitro...
Cell free extract
RNAi?
Hannon Lab, Zamore Lab, Tuschl Lab, Sharp Lab

28. Dicer is required for RNAi
wild-type
GFP
dsRNA
Dicer is in fact required for RNAi
Seen most easily in the germline
No apparent defect in soma, but most probably maternal contributions
Since the worms are sterile, can’t look at F2s
GFP – germline transgene
dcr-1-/-

29. Dicer is an evolutionarily conserved nuclease
Assayed activity from
Flies, arabidopsis, tobacco,neurospora,C. elegans, mouse, human and we are now trying pombe in collaboration with Tom Volpe

30. RNAi functions in many different organisms
dsRNA

31. Dicer cuts dsRNA into short RNAs
2001
Bernstein et al.
Cloned Dicer, the RNase III enzyme that is evolutionarily conserved and contains helicase and PAZ domains, as well as two dsRNA-binding domains.

32. Dicer cuts dsRNA into short RNAs
2001
Bernstein et al.
Cloned Dicer, the RNase III enzyme that is evolutionarily conserved and contains helicase and PAZ domains, as well as two dsRNA-binding domains.

33. Dicer cuts dsRNA into short RNAs
siRNA
2001
Bernstein et al.
Cloned Dicer, the RNase III enzyme that is evolutionarily conserved and contains helicase and PAZ domains, as well as two dsRNA-binding domains.

34. How can an siRNA turn of genes?
Gene OFF

35. Slicer uses siRNAs to slice transcripts
2004
Song et al.
Solved the crystal structure of pyrococcus Argonaute, showing it is Slicer

36. Slicer uses siRNAs to slice transcripts
2004
Song et al.
Solved the crystal structure of pyrococcus Argonaute, showing it is Slicer

37. Slicer uses siRNAs to slice transcripts
2004
Song et al.
Solved the crystal structure of pyrococcus Argonaute, showing it is Slicer

38. Slicer uses siRNAs to slice transcripts
2004
Song et al.
Solved the crystal structure of pyrococcus Argonaute, showing it is Slicer

39. Transcripts are bound by Slicer

40. Gene transcripts are sliced
Gene function stopped

41. dsRNA silences genes via Dicer and Slicer

42. RNAi lets us test gene function!

43. C. elegans is amenable to many forms of RNAi treatment
The kit uses RNAi by feeding
Feeding worms bacteria that express dsRNAs or soaking worms in dsRNA sufficient to induce silencing (Gene 263:103, 2001; Science 282:430, 1998)‏

44. RNAi by feeding is simple
Simply feed C. elegans bacteria expressing double-stranded RNA complementary to the gene you want to silence!
Feeding worms bacteria that express dsRNAs or soaking worms in dsRNA sufficient to induce silencing (Gene 263:103, 2001; Science 282:430, 1998)‏

45. The RNAi feeding vector has two T7 RNA polymerase promoters
T7 RNA polymerase is a viral polymerase.
It binds to a specific T7 promoter sequence.
The L4440 vector has two T7 promoters to make RNA from both strands.

46. Inducing RNAi by Feeding
Demonstrate to your students the power of silencing a single gene.
Teach about a powerful method for determining gene function.
Introduce your students to a model organism used for studying many aspects of biology, including development and gene function.
Engage in bioinformatics exercises exploring protein function and C. elegans and human gene relatedness.

48. microRNAs regulate gene expression without cleavage
Let-7 is encoded as a ~70 nt precursor
Processed to 21 nt mature form
Regulate developmental transisitons – inhibiting expression of target genes
TRANSLATIONAL LEVEL, unlike RNAi
Hypothesis : dicer does the cleavage

49. small temporal RNAs (stRNAs)
RNA or protein level
L1 stage
L2 stage
L3 stage
L4 stage
Adult stage
LIN-14, LIN-28 proteins
LIN-41 protein
LIN-29 protein
let-7 RNA
lin-4 RNA
• lin-4 miRNA represses translation of lin-14 (L1 to L2 molt)
stRNA precursor
stRNA
Translational repression
Dicer
Before RNAi well characterized, Ambros and Ruvkun studying heterochronic development
Discover mutation pointing to small non-coding RNA, lin-4
First synthesized as long precursor, processed into a single-stranded short RNA
Short RNA could possibly base pair to 3’ UTR target mRNA, in this case lin-14, and repress translation by unclear mechanisms
Ruvkun lab discover additonal heterochronic gene, let-7, together for a class of small temporal RNAs because temporal expression pattern
Emerge a novel gene regulatory network where stRNAs act as triggers that help orchestrate ordered development
Recent exciting result bridging two intro was discoverd by Mello, Ruvkun and Zamore
Demonstrate Dicer also key enzyme that processes stRNA precursors into mature stRNA

50. RNAi acts to regulate gene expression by two dicer-dependent mechanisms
miRNA
siRNA

51. Model for translational repression
RISC
M7GpppG
AAAAAA
RISC recognizes a target

52. Model for translational repressionGW
DCP
RISC
M7GpppG
AAAAAA
other
Other proteins are also recruited –
either along with RISC or later

53. Model for translational repression
decapping
to P-bodies GW
DCP
RISC
M7GpppG
AAAAAA
other
de-adenylation?
(e.g. Giraldez,
Belasco, Rivas)
block translation?
(e.g. Filipowicz)
RISC may block through multiple mechanisms

54. Least well understood - Arabidopsis as a model….

55. DNA complexes with histones to form chromatin

56. Regulation of gene expression at the level of chromatin
Sequence-independent
linker histones: control DNA compaction and accessibility to trans-acting factors
post-translational modifications of histone tails: control compaction of DNA and serve as docking sites for trans-acting factors
Range: Can act at the level of a single gene, often acts over groups of genes and over larger domains (20-200kb), and can affect gene expression over an entire chromosome

57. Model for siRNA-dependent initiation of heterochromatic silencing by RITS

58. Heterochromatin and epigenetics
Model for maintenance of heterochromatic silencing by RITS
Heterochromatin and epigenetics

59. Multiple mechanisms that are Dicer-dependant
dsRNA-mediated silencing in various organisms:
Multiple mechanisms that are Dicer-dependant
Meister & Tuschl, 2004

60. Genome wide transcription?
Numerous studies, using a variety of techniques, estimate that at minimum 63% of the genome is transcribed into RNA, with most estimates settling at > 90%!
Also of note is that all of this detected RNA has to be stable enough in the cell to be detected and that it is estimated that ~20% never leaves the nucleus as noted in these papers.
Kapranov, P. et al. Science 316, 1484–1488 (2007). 
Cheng, J. et al. Science 308, 1149–1154 (2005). 
Bertone, P. et al. Science 306, 2242–2246 (2004). 
Birney, E. et al. Nature 447, 799–816 (2007). 

61. Moral of Story Lots of RNA!
Cells seem to regulate transcription, largely in cases of differentiation, development, and response to stress
Little else known
Conveniently, differentiation and responses to stressors is rather key to immunology

62. Types of small RNA (does not encode long ncRNA’s… a whole different kind of monster)

63. RNAi