10.1101/sqb.2006.71.060 Access the most recent version at doi: 2006 71: 95-100 Cold Spring Harb Symp Quant Biol C.P. HUNTER, W.M. WINSTON, C. MOLODOWITCH, et al. Caenorhabditis elegans Systemic RNAi in References http://symposium.cshlp.org/content/71/95.refs.html This article cites 24 articles, 8 of which can be accessed free at: service Email alerting click here the box at the top right corner of the article or Receive free email alerts when new articles cite this article - sign up in http://symposium.cshlp.org/subscriptions go to: Cold Spring Harbor Symposia on Quantitative Biology To subscribe to Copyright 2006, Cold Spring Harbor Laboratory Press Cold Spring Harbor Laboratory Press on November 2, 2011 - Published by symposium.cshlp.org Downloaded from

GFP driven by 700 bp of sid-1 upstream sequences is detected in embryos and all larval stages. In adults, GFP is detected in all nonneuronal cells, perhaps explaining the refractoriness of neurons to systemic RNAi. To determine where SID-1 is localized within these cells, we examined the localization of a rescuing GFP-tagged SID-1 (SID- 1C::GFP). SID-1C::GFP rescued the systemic RNAi defect, suggesting that expression and localization would be representative of SID-1. SID-1C::GFP localized throughout cells with strong enrichment at the periphery of cells, consistent with plasma membrane association (Fig. 2B, upper panel). SID-1C::GFP was detected at a much reduced level compared to the promoter fusion con- struct and was detected in a reduced subset of cell types however, sid-1 function is detected in cell types in which the fusion protein was not detected (e.g., body wall muscle). Curiously, SID-1C::GFP is most highly expressed in the cells and tissues that are directly exposed to the 96 HUNTER ET AL. Figure 2. SID-1 structure, function, localization, and conservation. (A) Topology of SID-1 as determined by ��-gal folding assay. The dark blue portions were demonstrated to be extracellular or intracellular constraining the orientation of the respective TM domains. The location and orientation of the light blue and red portions are undetermined. (B) Full-length functional SID-1::GFP (top panel) is enriched at the cell periphery (arrows) compared to GFP (bottom panel). Bar, 10 ��m. (C) Injection of gfp dsRNA into a single intes- tinal cell (arrowhead) in a sid-1 mutant animal that expresses nuclear-localized GFP in all cells demonstrates cell-autonomous RNAi. (D) Alignment of carboxy-terminal invertebrate and vertebrate SID-1 TM domains (gray). (Red) Amino acids that are identical among a majority of proteins (blue) amino acids that are conserved among vertebrates. SID-1 homologs: (Ce) C. elegans (Cb) C. briggsae (Ce*) C. elegans ZK721.1 (Sp) Strongylocentrotus purpuratus (Tc) Tribolium castaneum (Am) Apis mellifera (Mm) Mus muscu- lus (Hs) Homo sapiens (Tn) Tetraodon nigroviridis (Dd) Dictyostelium discoideum (Tc���) the two Tribolium homologs are similar but distinct. (Reprinted in part, with permission, from Feinberg and Hunter 2003 [�� AAAS].) Figure 1. Direct visualization of systemic RNAi. (A) Two separate transgenes direct GFP expression in pharynx and body wall mus- cle. (B) The addition of a third transgene that expresses a gfp hairpin to produce dsRNA in the pharynx silences the GFP reporter in the pharynx (autonomous RNAi) and silences GFP in anterior body wall muscles (systemic RNAi). (C) A sid-1 mutation in the back- ground of the transgenes described for panel B. (ph) Pharynx muscle (bm) body wall muscle. Insets are differential interference con- trast images. (Reprinted in part, with permission, from Winston et al. 2002 [�� AAAS].) A B C B C A D 095-100_Hunter_Symp71.qxd 2/6/07 4:19 PM Page 96 Cold Spring Harbor Laboratory Press on November 2, 2011 - Published by symposium.cshlp.org Downloaded from