Supplemental Research Data

Supplemental Research data

Supplemental Materials

Strains and Alleles.

UF5 (gqIs2[ppk-1 gbp-2::gfp]) is a kind gift from Tamara Chessa and Dr David Weinkove. This strain is partially sterile. RNAi against ppk-1 induces strong sterility defects, indicating that the sterility of UF5 is probably a consequence of the presence of multiple copies of ppk-1 phenocopying a loss-of-function allele of the gene.

AZ212 (unc-119(ed3) ruIs32[pAZ132:pie-1::gfp::H2B] III), SS579 (pgl-1(bn101) IV), SS580 (pgl-1(bn102) IV) (Kawasaki et al. 1998), MH1157 (him-5(e1490) V; egl-13(ku194) X) (Hanna-Rose and Han 1999; Cinar et al. 2003), MT1078 (egl-13(n483) X) and RB1090 (hpl-2(ok1061) III) were provided by the C.elegans genetic center (CGC: RB1090 (hpl-2(ok1061) III) was generated by the C. elegans gene knockout consortium ( It removes genomic DNA between positions 11940 and 13701 (according to the cosmid clone K01G5); in addition 14 nucleotides are inserted between the deletion ends. This rearrangement is predicted to remove completely or partially the last exon of the respective isoforms of hpl-2. FX329 (rha-1(tm329)) and FX1021 (K10D2.3(tm1021)) were provided by the National BioResources Project ( Tm329 removes genomic sequences between positions 19,358 and 20,416 (according to the cosmid clone T07D4) which encode a region containing a dsRNA-binding domain of RHA-1. Tm1021 removes genomic sequences between positions 7322 and 7965; 9 nucleotides are inserted between the deletion ends and a truncated protein missing most of the functional domains of K10D2.3 is encoded as a consequence of this rearrangement. Pk1673, a deletion allele of ppw-2 was recovered from a deletion library (as described in (Jansen et al. 1997)). Its sequencing reveals that it removes genomic sequences between positions 58,075 and 61,216 (according to the YAC clone Y110A7A).

The construction and characterization of strain NL3700 is described in (Sijen and Plasterk 2003).

SupplementalReferences

Dudley, N.R., Labbe, J.C., and Goldstein, B. 2002. Using RNA interference to identify genes required for RNA interference. Proc Natl Acad Sci U S A99: 4191-6.

Hanna-Rose, W. and Han, M. 1999. COG-2, a sox domain protein necessary for establishing a functional vulval-uterine connection in Caenorhabditis elegans. Development126: 169-79.

Jansen, G., Hazendonk, E., Thijssen, K.L., and Plasterk, R.H. 1997. Reverse genetics by chemical mutagenesis in Caenorhabditis elegans. Nat Genet17: 119-21.

Sijen, T. and Plasterk, R.H. 2003. Transposon silencing in the Caenorhabditis elegans germ line by natural RNAi. Nature426: 310-4.

Tijsterman, M., May, R.C., Simmer, F., Okihara, K.L., and Plasterk, R.H. 2004. Genes required for systemic RNA interference in Caenorhabditis elegans. Curr Biol14: 111-6.

Winston, W.M., Molodowitch, C., and Hunter, C.P. 2002. Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1. Science295: 2456-9.

Table S1. Requirements to establish germline co-suppression

(A) A single copy transgene, pie-1::gfp::H2B (pie-1::gfp), stably expressing the marker gene gfp in the germ line is silenced in animals containing extrachromosomal (Ex) or integrated (Is) arrays carrying multiple complete or truncated () gfp sequences under the control of promoter regions of ubiquitous expressed genes. (B) If the repetitive gfp sequences are not promoter-driven, co-suppression is not induced. Number of lines exhibiting indicated phenotype is indicated. + No silencing; - complete and strong silencing, heritable to progeny (see Figure 3A).

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Argonaute family

RNA binding

Helicase

PolyA Pol Like

K10D2.3

K04F10.6

HP-like

SET

HMG box

SNF2

Exportin/importin

Histone-like

Deacetylases

Acetyltransferase

Other chroma prot

Diverse

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Table S2: candidate approach to identify cde genes by RNAi

One hundred and eighty-six candidates were selected because of their similarity with cde genes identified in the genome-wide approach (designed as ‘+*’ in the ‘results’ column of this table) or because they look like genes involved in silencing processes in other species. Ten candidates were scored as positive (designed as ‘+’ in this table) using both NL3847 and NL3864. They were considered as cde genes. Re-expression was also observed when feeding NL3847 with food targeting dcr-1 (+/-).

Table S3. Classification, description and functional characterization of cde genes

Class 0: Previously characterized mutator/cde genes (5)

PolyQ domain

PAZ/PIWI domains

RNA helicase

Unknown

Unknown

Class I: Undetermined biological process (12)

Cons. domains

No domain

Class II: Various biological processes (19)

Metabolism

Y40B10A.6

Signalling

Chromosome seg.

Class III: RNA and Protein metabolism (15)

M03F8.3 b

Argonaute family

Class IV: Chromatin structure and transcription (13)

T12E12.2

Trans/DNA bind.

Other DNA bind.

a genes also involved in transposon silencing.

b Genes also involved in miRNA processing and function.

c Genes also involved in RNAi (this study and (Dudley et al. 2002; Vastenhouw et al. 2003; Chen et al. 2005)).

cde genes were identified in genome-wide (GW) or pre-selected candidates approach (CA) RNAi screens and fall into 5 classes. The putative function or encoded functional domain(s) are given (Domains/function). The strength of the cde phenotype in F1 after RNAi targeting is indicated (column “Screen”: S = more than 70% of cde worms, M = between 70% and 40% of cde worms, W = less than 40% of cde worms). Developmental phenotypes (“Phe” column) were scored (Ste = sterility, Dev= development defects and Mes = maternal effect sterility). Orthologs/paralogs of these genes have been identified in Caenorhabditis elegans (Ce), Caenorhabditis briggsae (Cb), Homo sapiens (Hs), Mus musculus (Mm), Drosophila melanogaster (Dm), Danio rerio (Dr), Arabidopsis thaliana (At), Schizosaccharomyces pombe (Sp) and Saccharomyces Cerevisiae (Sc).

Supplemental Figure (Legend)

Figure S1: Requirements to establish cosuppression. (A) A single copy transgene, pie-1::gfp::H2B (pie-1::gfp), stably expressing the marker gene gfp in the germline is silenced in animals containing extrachromosomal (Ex) or integrated (Is) arrays carrying multiple complete or truncated () gfp sequences under the control of promoter regions of ubiquitous expressed genes. If the repetitive gfp sequences are not promoter-driven, cosuppression is not induced. Repetitive gfp copies under the control of promoter-containing regions of the somatically expressed genes, unc-54, myo-3, hsp-16.48 and hsp-16.2 fail to induce germline cosuppression Number of lines exhibiting indicated phenotype is indicated. + No silencing; - complete and strong silencing, heritable to progeny (see Fig. 3A). -/+ incomplete silencing (only a fraction of animals carrying the extra-chromosomal array exhibit silencing of pie-1::gfp::H2B), not heritable to progeny. (B) and (C) example of a line carrying the pie-1::gfp::H2B transgene in combination with an unc-54::gfp::unc-54 array. pie-1::gfp::H2B is not cosuupressed by the array (B) although it is strongly expressed in the soma (C). In addition to these observation, we showed that cosuppression was still induced when pie-1::gfp::H2B and the dpy-30::gfp::unc-54array were crossed into rsd-6(pk3300) (Tijsterman et al. 2004) or sid-1(pk3321)(Winston et al. 2002)background (data not shown), in which it has been demonstrated that RNAi-inducing RNA signals cannot spread from the soma to the germ line. Taken together, these observations suggest that only RNA signals expressed in the germline efficiently trigger cosuppression.

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