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

A Grand Challenge To understand how life works

How do we go from DNA to function?

Find a tractable systems: Model organisms

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

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

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

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

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

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

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

Anatomy of a worm (from www.wormatlas.org)

Anatomy of a worm

Wild-type

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

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

dpy-11 Dumpy worms are shorter.

Lifecycle of a worm (from www.wormatlas.org)

Identifying hermaphrodites Clear patch on L4

A problem: Creating mutations in specific genes is very hard

Can we go directly from sequence to function?

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

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

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

How can dsRNA turn of genes? Gene OFF

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

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-/-

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

RNAi functions in many different organisms dsRNA

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 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 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.

How can an siRNA turn of genes? Gene OFF

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 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

Transcripts are bound by Slicer

Gene transcripts are sliced Gene function stopped

dsRNA silences genes via Dicer and Slicer

RNAi lets us test gene function!

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 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)‏

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.

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

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

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

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

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

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

Least well understood - Arabidopsis as a model….

DNA complexes with histones to form chromatin

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

Model for siRNA-dependent initiation of heterochromatic silencing by RITS

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

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

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). 

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

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

RNAi