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? 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: May 1974

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

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

Identify mutants Deduce the function of mutated 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

Anatomy of a worm (from

Anatomy of a worm

Wild-type

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

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

Dumpy worms are shorter. dpy-11

Lifecycle of a worm (from

Identifying hermaphrodites Clear patch on L4

A problem: Creating mutations in specific genes is very hard

Can we go directly from sequence to function? Function?

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

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 geneTurns 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 Negative controluninjected mex-3B antisense RNAmex-3B dsRNA Antisense RNA Weak effect dsRNA Strong effect Double-stranded RNA was a contaminant in antisense experiments

How can dsRNA turn of genes? Gene OFF dsRNA

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

GFP – germline transgene Dicer is required for RNAi GFP dsRNA wild-type dcr-1 -/-

Dicer is an evolutionarily conserved nuclease

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

How can an siRNA turn of genes? Gene OFF siRNA

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

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! Function? RNAi

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.

Organisms vary in their ability to take up foreign dsRNA and use it in the RNAi pathway. The effects of RNA interference can be both systemic and heritable in plants and C. elegans, although not in Drosophila or mammals. In plants, RNAi is thought to propagate by the transfer of siRNAs between cells through plasmodesmata (channels in the cell walls that enable communication and transport).[32] Heritability comes from methylation of promoters targeted by RNAi; the new methylation pattern is copied in each new generation of the cell.[62] A broad general distinction between plants and animals lies in the targeting of endogenously produced miRNAs; in plants, miRNAs are usually perfectly or nearly perfectly complementary to their target genes and induce direct mRNA cleavage by RISC, while animals' miRNAs tend to be more divergent in sequence and induce translational repression.

microRNAs regulate gene expression without cleavage

small temporal RNAs (stRNAs) stRNA precursor stRNA Translational repression RNA or protein level L1 stageL2 stageL3 stageL4 stageAdult stage LIN-14, LIN-28 proteinsLIN-41 proteinLIN-29 protein let-7 RNA lin-4 RNA Dicer lin-4 miRNA represses translation of lin-14 (L1 to L2 molt)

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

Model for translational repression M7 GpppG AAAAAA RISC RISC recognizes a target

Model for translational repression M7 GpppG AAAAAA RISC DCP GW other Other proteins are also recruited – either along with RISC or later

Model for translational repression M7 GpppG 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

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

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

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