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

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

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.

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

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.

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

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

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.

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.

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

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.

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

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

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

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

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

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

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

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?

RNAi affects oocytes of treated C. elegans F1 carriers of RNAi effect Ref: Tabara et al 1999 Cell 99, 123-132. On class web page.

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

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

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

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

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