Week 3.Fire.RNAi.paper.Journal.Club.SP23(1) (1)

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Week 3 Lab Journal Club “ Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans” Andrew Fire, SiQun Xu, Mary K. Montgomery, Steven A. Kostas, Samuel E. Driver, & Craig Mello Nature Vol 391 19 February 1998 pp. 806-811 Learning Objectives Antisense mechanism of RNA interference RNA interference in endogenous gene function Requirement for double-stranded RNA for effective interference Phenotypes of null mutants of unc-22, fem-1 and hlh-1 in C. elegans. Experimental technique to introduce dsRNA into the worms Ectopic transcripts Using GFP-tagged proteins to measure dsRNA interference in worms. Using the phenotypes of unc-22, fem-1 and hlh-1 to measure dsRNA interference. Worm transfer Procedure This week we will transfer 10 adult worms from the 6 cm plates you have grown to 10 cm NGM agar plates to obtain a large number of gravid adults in preparation for the egg prep and the beginning of the RNAi experiment next week. Please transfer your worms and then take part in the class discussion of the Fire et al. paper on double- stranded RNA interference of specific gene expression. The lab report below is due before the week 3 lab section meeting begins.

Name______Raghu Chinta_______________________ __________ Sec._Monday___ TA____________Emily_____________________ Due before the Week 3 lab section meeting begins. Lab Report 3 “ Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans” Andrew Fire, SiQun Xu, Mary K. Montgomery, Steven A. Kostas, Samuel E. Driver, & Craig Mello Nature Vol 391 19 February 1998 pp. 806-811 1. What is a null mutant? A mutation in which a gene loses its function, which leads to it not being transcribed into RNA and translated into the corresponding protein. 2. What are the phenotypes of the following null mutants in the Fire et al. paper: Null Mutant Phenotype unc-22 severe twitching fem-1 no sperm unc-54 paralyzed hlh-1 lumpy-dumpy larvae 3. In the past, geneticists exposed organisms to chemical mutagens or X-rays in the hope of finding mutations in biochemical pathways, cell structures, or regulatory systems. Since the advent of whole genome sequencing and gene annotation, it is possible to find a specific gene by its sequence. Knowing the sequence of a particular gene would allow manipulation of the expression of that gene by more direct methods that random mutagenesis. One of those methods is using antisense RNA to interfere with translation of the mRNA of a gene. Explain the model of hybridization between injected antisense RNA and endogenous messenger RNA which results in interference with gene expression. RNA to be injected is prepared using bacteriophage RNA polymerases on the target gene on DNA plasmids. The antisense RNA is designed to have a sequence complementary to the specific target mRNA of interest. This RNA is introduced into the cell where they encounter endogenous target mRNA and because of the complementary sequences between the antisense RNA and target mRNA, base-pairing can occur, forming a dsRNA (double-stranded RNA). This dsRNA is seen as foreign by cellular machinery. In response to the presence of dsRNA, the cell activates the RNAi pathway which involves the enzyme Dicer that cuts the dsRNA into smaller RNA fragments called siRNAs. These siRNAs can then prevent the translation of the corresponding protein by either degrading or repressing the target mRNA. 4. In the beginning of this paper, the authors refer to an earlier paper in 1991, Fire, A., Albertson, D., Harrison, S. & Moerman, D. Production of antisense RNA leads to effective and specific inhibition of gene expression in C. elegans muscle. Development 113 , 503-514 (1991). In the 1991 paper, the Fire lab reports successfully using antisense RNA to interfere with gene function. In this 1998 paper we study now, two observations are made. Both antisense and sense RNA can cause the same interference, and the interference persists to the next generation of worms, when most endogenous mRNAs are rapidly degraded in the worm embryo. The authors explain that antisense and sense RNA is synthesized by RNA polymerases from the target gene on DNA plasmids. They also say that although these RNA polymerases are very specific, they can sometimes make random or ectopic transcripts. What is an ectopic transcript?

An ectopic transcript is an RNA transcript that is produced from a gene that is found in a location where it is not expected to be located. 5. If an antisense RNA were synthesized and a variety of ectopic transcripts were also made at the same time, could any of these form dsRNA? Yes, it is possible for any free antisense RNA and ectopic transcripts to form dsRNA if they have complementary sequences that allow them to base-pair with each other. The formation of dsRNA would depend on the specific sequences of the antisense RNA and the ectopic transcripts. 6. How did the authors separate sense and antisense RNA transcripts from the random ectopic transcripts in the same preparation? The RNA transcripts were purified by electrophoresis on low-gelling-temperature agarose. 7. Below is a portion of Table 1 of the Fire paper, “Potent and Specific Genetic Interference by double- stranded RNA in Caenorhabditis elegans. ” What evidence is presented here that the genetic interference by double stranded RNA occurs in the cytoplasmic compartment of the cells? Explain. Table 1 Effects of Sense, Antisense and mixed RNAs on progeny of injected animals Gene F1 Phenotype wild type hermaphrodite fem-1 null mutant( fem-1 gene deleted) female (no sperm) RNAi Experiment Gene Segment Size(in Kb) Injected RNA F1 Phenotype fem1A Exon 10 531 sense hermaphrodite(99%) antisense hermaphrodite(99%) sense+antisense female (72%) fem1B Intron 8 556 sense+antisense hermaphrodite(99%) 8. What other parts of a gene are ineffective in genetic interference as dsRNA? The promoter region appears to not affect interference. 9. Please examine the experiments that follow and determine what F1 phenotype was observed and how it was observed as the result of injection of dsRNA. For example, if the red eye gene of Drosophila were inhibited by dsRNA injection into the fly larvae, the phenotype would be white eyes and the level of the phenotype would be lack of production of pigment in an eye cell. Experiment/Group of Experiments F1 Phenotype Observation Table 1 unc-22 twitching muscle defects/impaired motility fem-1 no sperm Female unc-54 embryonic/larval arrest defective muscle cells hih-1 lumpy-dumpy larvae defective muscle cells

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Table 1 & Fig. 2 myo-3::NLS:gfp:lacZ Nuclear localization of myo myo-3::MtLS:gfp Mitochondrial localization of myo Fig. 3 mex-3 mRNA detection Table 2 Injection of ds RNA into gonad or body cavity 10. Which of these experiments shows dsRNA interference in the earliest stage of the Central Dogma information transmission stages? Table 1 and Figure 2 The completed lab report can be uploaded in the Student Assessment section of the Canvas website.

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