Exploring Gene Function in C. elegans:Mutations and RNA Interference
Carolina Biological Supply Company
Bruce NashDolan DNA Learning CenterCold Spring Harbor Laboratory
Craig Mello
• He was awarded the 2006 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
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: 71-94 May 1974
Why Worms?
ProfileSoil nematodeGenome size: 100 MbNumber of chromosomes: 6Generation time: about 2 daysFemale reproductive capacity: 250 to 1000 progeny
Special characteristicsStrains Can Be FrozenHermaphroditeKnown cell lineage pattern for all 959 somatic cellsOnly 302 neuronsTransparent bodyCan be characterized geneticallyAbout 70% of Human Genes have related genes in C. elegans
=~
Worms have gut, muscle, skin and a nervous system like us
About 40% of worm genes are very related
to human genes
How do geneticists study gene function?
Dumpy mutant
The wild type gene must maintain normal body shape.
Wild type
• 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
Examining, growing and caring for worms
An experiment showed that the antisense model didn’t make “sense”
First noticed that sense RNA was as effective as antisense RNA for suppressing gene expression in worm
Guo S, and Kemphues KJ.1995
Antisense RNA
Turns off gene Turns off gene????
Sense RNA
Double-stranded RNA causes silencing:RNA Interference!
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
Negative control uninjected
mex-3B antisense RNA mex-3B dsRNA
Antisense RNA
Weak effect
dsRNA
Strong effect
Double-stranded RNA was a contaminant in antisense experiments
Cell free extract
RNAi?
Biochemistry to the rescueRNAi in vitro...
Hannon Lab, Zamore Lab, Tuschl Lab, Sharp Lab
dsRNA
RNAi functions in many different organisms
Dicer
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.
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.
Dicer cuts dsRNA into short RNAs
Dicer
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.
Dicer cuts dsRNA into short RNAs
Dicer
Slicer uses siRNAs to slice transcripts
2004
Song et al. Solved the crystal structure of pyrococcus Argonaute, showing it is Slicer
Slicer
Slicer
siRNA
2004
Song et al. Solved the crystal structure of pyrococcus Argonaute, showing it is Slicer
Slicer uses siRNAs to slice transcripts
2004
Song et al. Solved the crystal structure of pyrococcus Argonaute, showing it is Slicer
Slicer uses siRNAs to slice transcripts
2004
Song et al. Solved the crystal structure of pyrococcus Argonaute, showing it is Slicer
Slicer uses siRNAs to slice transcripts
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)
RNAi by feeding is simple
Feeding worms bacteria that express dsRNAs or soaking worms in dsRNA sufficient to induce silencing (Gene 263:103, 2001; Science 282:430, 1998)
• Simply feed C. elegans bacteria expressing double-stranded RNA complementary to the gene you want to silence!
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.
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.
small temporal RNAs (stRNAs)
stRNA precursor
stRNA
Translational repression
RN
A o
r p
rote
inle
vel
L1 stage L2 stage L3 stage L4 stage Adult stage
LIN-14, LIN-28 proteins LIN-41 protein LIN-29 protein
let-7 RNAlin-4 RNA
Dicer
• lin-4 miRNA represses translation of lin-14 (L1 to L2 molt)
Model for translational repression
M7GpppG AAAAAA.........RISC
DCP GW
other
Other proteins are also recruited – either along with RISC or later
Model for translational repression
M7GpppG AAAAAA.........RISC
DCP GW
other
decapping to P-bodies
block translation?(e.g. Filipowicz)
de-adenylation?(e.g. Giraldez, Belasco, Rivas)
RISC may block through multiple mechanisms
Regulation of gene expression at the level of chromatin
Sequence-independentlinker 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
dsRNA-mediated silencing in various organisms:Multiple mechanisms that are Dicer-dependant
Meister & Tuschl, 2004
Genome wide transcription?
• 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).
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.
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
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