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Genetics, Vol. 178, 801-814, February 2008, Copyright © 2008
doi:10.1534/genetics.107.081588
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Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045
4 Corresponding author: Department of Molecular Biosciences, 5041 Haworth Hall, 1200 Sunnyside Ave., University of Kansas, Lawrence, KS 66045.
E-mail: timmons{at}ku.edu
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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We previously reported a role for C. elegans ATP-binding cassette (ABC) proteins in RNAi, with emphasis on the analysis of one member of this family, haf-6. We also identified an additional eight transporter genes that are similarly required for RNAi: abt-1, pgp-4, pgp-11, haf-2, mrp-1, pmp-1, wht-1(C05D10.3), and wht-3(C16C10.12) (SUNDARAM et al. 2006). These initial assays were limited in number and were restricted to observations of RNAi activities in germ line and muscle. However, the genome of C. elegans harbors 61 different ABC transporter genes that are collectively expressed in a wide array of tissues (ZHAO et al. 2004). Thus, we considered that the narrow scope of our initial set of assays might have limited our ability to identify the complete set of ABCRNAi genes—in particular, those ABC transporter mutants that might have somatic RNAi defects in nonmuscle tissue.
Here we set out to identify additional C. elegans ABC transporters that could be classified as ABCRNAi genes by a more thorough assessment of RNAi activities in all available ABC transporter mutant strains. Of the 61 ABC transporter genes, mutations in 49 different genes are represented. We tested each mutant strain for RNAi defects using additional assays, and we performed complementation testing between nonallelic mutants. Our complementation testing utilized ABC transporter mutants and mutants defective for mut-7 or rde-2. Because deletion alleles were used in each of the complementation tests, any noncomplementation interactions that are observed likely represent cases of combined haplo-insufficiency. Second-site noncomplementation (SSNC) is often an indicator of physical interactions between proteins (STEARNS and BOTSTEIN 1988; CAMPBELL et al. 1995; HAARER et al. 2007); in other cases the interactions reveal functional requirements from multiple pathways that contribute to a given process (YOOK et al. 2001; JASPERSEN et al. 2004). In either case, a noncomplementation result implies a common function or influence on a particular process (HAWLEY and GILLILAND 2006). Our results allowed us to classify an additional gene, haf-9, as an ABCRNAi gene. We also reveal common and distinct features related to RNAi defects for each of the mutant strains.
ABC transporters are classified into subfamilies on the basis of relative homologies, as well as the number and positioning of conserved transmembrane and ABC domains. The ABCRNAi subset of transporter genes consists of diverse subfamilies (A, B, C, D, and G), and both full transporter and half molecules are represented in this group. A genetic interaction with mut-7 and rde-2 is a unifying feature of this diverse group of genes; thus, the ABCRNAi subset can be defined on the basis of function, rather than subfamily classification. The genetic interactions with mut-7 and rde-2, as well as other common phenotypes, point to an ability for ABCRNAi transporters to influence RNAi mechanisms that ultimately affect chromosome function and integrity.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Mutants used in phenotypic analyses:
VC1046 [abce-1(gk481)], VC712 [mrp-4(ok1095) X], VC561 [abcf-1(ok830) V], GH534 [mrp-4(cd8) X], VC544 [abcf-2(ok771) III], RB1070 [mrp-6(ok1027) X] RB1645 [abch-1(ok2034) II], RB1269 [mrp-8(ok1360) III], VC572 [abcx-1(ok865) II], NL132 [pgp-1(pk17) IV], FX703 [abt-1(tm703) IV], VC134 [pgp-2(gk114) I], RB842 [abt-2((ok669) I], NL131 [pgp-3(pk18) X], FX502 [abt-2(tm502) I], VC21 [pgp-4(gk16) X], FX1050 [abt-4(tm1050) V], RB959 [pgp-5(ok856) X], RB817 [abt-4(ok633) V], FX663 [pgp-5(tm663) X], RB1765 [abt-5(ok2268) I], RB1047 [pgp-6 (ok994) X], RB1151 [cft-1(ok1180) V], VC286 [pgp-7(ok528)/szT1;+/szT1 I], MT4983 [ced-7(n1996) III], FX830 [pgp-9(tm830) V], MT4984 [ced-7(n1997 III], FX996 [pgp-10(tm996) X], MT8886 [ced-7(n2024) III], RB1045 [pgp-10(ok991) X], FX843 [haf-1(tm843) IV], FX333 [pgp-11(tm333) II], RB867 [haf-1(ok705) IV], VC26 [pgp-12(gk19) X], VC16 [haf-2(gk13) II], RB894 [pgp-13(ok747) X], VC1186 [haf-3(gk549) V], RB1041 [pgp-15(ok987) X], RB1107 [haf-3(ok1086) V], RB908 [pmp-1(ok773) II], VC449 [haf-4(gk240) I], RB1108[pmp-3(ok1087) V], RB1080 [haf-4(ok1042) I], FX968 [pmp-3(tm968) V], VC252 [haf-5(gk155) III], RB675 [pmp-4(ok396) IV], VC287 [haf-5(gk161) III], VC189 [pmp-4(ok396) IV], XX193 [haf-6(ne335) I; him-5(e1490) V], XX194 [haf-6(ne335) I; ccIs8160(rpL28::GFP); him-5(e1490) V], XX195 [haf-6(ne335) I; cIs8160(rpL28::GFP)], XX364 [haf-6(ne335) I], FX688 [wht-1(tm688) III], VC118 [haf-7(gk46) V], RB1007 [wht-3(ok927) III], VC15 [haf-8(gk12) IV], RB1033[wht-3(ok962) III], VC32 [haf-9(gk23) I], FX714 [wht-3(tm714) III], NL147 [mrp-1(pk89) X], RB1058 [wht-4(ok1007) II], NL147[mrp-1(ut153) X], RB932 [wht-5(ok806) IV], RB1713 [mrp-2(ok2157) X], VC629 [wht-6(ok882) III], RB1028 [mrp-3(ok955) X], RB933 [wht-7(ok812) III], VC692 [wht-9(ok1044) III].
Other strains, outcrossing, and DNA sequence verification:
To help prevent or monitor for accidental introduction of RNAi-related mutations that may be present in the background of wild-type N2 populations, we maintain wild-type strains as subtypes. These subtypes have passed our tests for brood size and sensitivity to pop-1 and unc-22 foods and lack temperature-sensitive Sterility or Him phenotypes. Many of the phenotypes here were obtained using deletion alleles, and in most cases recapitulating the effects of the mutation using RNAi is not a comparable methodology. For example, in our complementation tests, the animals tested are trans-heterozygotes and RNAi would likely induce a null phenotype; therefore, the null mutations and RNAi would produce dissimilar genetic conditions. We repeated experiments using more than one mutant subtype; these were generated by outcrossing the mutant strains to different N2 subtypes. Each N2 subtype, and the corresponding mutant subtype, would be expected to harbor different sets of background mutations.
XX1181 [mut-7(pk204) III; dpy-4(e1166) unc-17(e245) IV] was generated from crosses of PD2027 [dpy-4(e1166) unc-17(e245) IV] and NL917 [mut-7(pk204) III]. XX1187 [rde-2(ne221) I; dpy-4(e1166) unc-17(e245) IV] was generated from crosses of PD2027 [dpy-4(e1166) unc-17(e245) IV] and WM29 [rde-2(ne221) I]. XX1569 and XX1570 [mut-7(pk204) III] are independent F2 sibling subtypes from a cross of XX1181 and NL917. XX1571-3 [rde-2(ne221) I] subtypes were similarly derived from crosses of XX1187 and WM29.
Strains XX1824–1826 harbor the mut-7(pk201) mutation. XX1824 [mut-7(pk201); yyEx62.2-rde-2 cosmid] was derived from a cross between XX1570 mutants and XX506 transgenic animals. XX1825 [mut-7(pk201); yyEx75.1-haf-6 cDNA (G562R)] was derived from a cross using XX1570 and XX752. XX1826 [mut-7(pk201); yyEx97.4-haf-6 cDNA (wild type)] was derived from a cross using XX1570 and XX1318. Strains XX1827–1829 harbor the rde-2(pk1657) mutation. XX1827 [rde-2(pk1657); yyEx62.2-rde-2 cosmid] was derived from a cross between WM29 mutants and XX506 transgenic animals. XX1828 [rde-2(pk1657); yyEx75.1-haf-6 cDNA (G562R)] was derived from a cross using WM29 and XX752. XX1829 [rde-2(pk1657); yyEx97.4-haf-6 cDNA (wild type)] was derived from a cross using WM29 and XX1318.
NL3643 [unc-22(st136) IV] was outcrossed to wild-type males (XX171, an N2 strain from the lab of Andrew Fire) five times before it was used in additional crosses. The unc-22(st136) allele harbors a transposon insertion, and this outcrossed strain (XX366) has not displayed Mutator activity under any growth condition.
The ABCRNAi mutants identified previously [abt-1(tm703), haf-2(gk13), haf-6(ne335), mrp-1(pk89), pgp-4(gk16), pgp-11(tm333), pmp-1(ok773), wht-1/C05D10.3(tm688), wht-3/C16C10.12(ok962), and the newly identified haf-9(gk23)] were tested for RNAi activity as unoutcrossed lines and again after three rounds of outcrossing to wild-type males (XX171). Outcrossed lines were established from cloned individuals, and extensive PCR analyses were performed on the cloned adult and on the ensuing population to ensure homozygosity for each deletion. Most strains that displayed noncomplementing interactions were subjected to additional rounds of outcrossing using wild-type XX937 males (a selected subtype from an N2 stock obtained from the Caenorhabditis Genetics Center, DR subclone of CB origin) or XX366, a transposon-insertion line that does not display Mutator activity.
We sequenced DNA prepared from each of the haf-6, rde-2, and mut-7 noncomplementing strains, and in each mutant we verified that the other two genes were wild type in sequence. DNA sequences were obtained from genomic DNA as well as from RT–PCR products. cDNAs were easily generated, indicating gene expression, and the splicing patterns were as expected. (Both rde-2 mRNA isoforms were observed in all mutant strains.) We sequenced an rde-2 cDNA 1737 bp in length from the following strains: XX194 [haf-6(ne335) I; him-5(e1490) V], NL917 [(mut-7(pk201) III] (260 bp from the middle of this cDNA were not read), and NL1820 [mut-7(pk720) III]. We sequenced a haf-6 cDNA 2000 bp in length from XX1569 [mut-7(pk204) III], XX1571 [rde-2(ne221) I], NL1820 [mut-7(pk720) III], and NL3531 [rde-2(pk1657) I]. A total of 2943 bp of mut-7 cDNA were sequenced in XX194 [haf-6(ne335) I; him-5(e1490) V], XX1571 [rde-2(ne221) I], and NL3531 [rde-2(pk1657) I]. Sequence information from genomic DNA isolated from each mutant strain validated our results derived from cDNAs and provided information in gaps.
Feeding strains and feeding-based assays for RNAi defects:
Feeding plates harboring bacteria engineered to express dsRNA were prepared using the HT115(DE3) host bacteria as described (TIMMONS et al. 2001; HULL and TIMMONS 2004; SUNDARAM et al. 2006). Plasmids transformed into this bacterial strain were derived from L4440 vector (TIMMONS and FIRE 1998). pop-1 food targets the TCF/LEF1 transcription factor and produces sterility in young animals reared on this food; unc-22 food (TIMMONS and FIRE 1998) targets a gene expressed in muscle and induces a Twitching phenotype. Other feeding strains were obtained from the MRC Geneservice RNAi library (KAMATH et al. 2003). Male stocks were established for each mutant strain by rearing animals at 20° on feeding strains with plasmids targeting him genes. mut-7 and rde-2 animals are Him, and we observed that mutant males sire progeny only when the males arose by means unrelated to the mut-7/rde-2 defect. When rde-2 or mut-7 males are produced via RNAi phenocopy of Him pathway genes, matings can be successful. RNAi assays were performed as described (TIMMONS et al. 2001; HULL and TIMMONS 2004; SUNDARAM et al. 2006) at 15°, 20°, and 25°.
Generation of transgene lines and transgene-based rescue of RNAi:
Transgene lines were established as complex arrays to better ensure expression in the germ line (KELLY et al. 1997). Plasmid DNA sequences were linearized before injecting. unc-25::gfp sequences were used as a dominant transformation marker; bacteriophage lambda DNA was used as carrier DNA. Cosmid F21C3 harbors wild-type rde-2 genomic sequence and provides for rescue of the RNAi defects in rde-2 mutants. Plasmid pLT417 harbors let-858 promoter::let-858 5'-UTR::haf-6 cDNA::let-858 3'-UTR sequences. The haf-6 coding region in pLT417 harbors a G562R substitution that affects a conserved amino acid in the ATP-binding cassette domain, and this plasmid provides partial rescue of RNAi defects in haf-6 mutants. Plasmid pLT372 is identical to pLT417 except that the haf-6 cDNA sequence is wild type; this plasmid rescues the RNAi defects in haf-6 mutants (SUNDARAM et al. 2006). Transgene arrays were generated in wild type and crossed into mutant backgrounds. Strains were verified for homozygosity by RNAi behavior (of nontransgenic siblings), temperature-sensitive sterility phenotypes, PCR, and/or DNA sequencing, as appropriate to the strain. RNAi activity was assessed by placing animals as L2/L3 larvae on pop-1 food. Absence of live progeny is indicative of an intact RNAi response.
Complementation testing:
Conclusions from our complementation tests were based on two or more independent crosses that were tested using different preparations of pop-1 feeding plates. Crosses were performed using one hermaphrodite per mating on standard OP50-seeded plates with no obvious contamination. The presence of 50% males in the progeny indicated a successful cross. RNAi activity was assessed by noting the number of F2 progeny produced per F1 animal placed on pop-1 food. The assays were performed under identical conditions in an incubator set at 20°. We did not assess RNAi activity in trans-heterozygous males. In Figure 2A, mut-7 and rde-2 mutants were used as the hermaphrodite parent with haf-6 males to generate trans-heterozygotes; reciprocal crosses with haf-6 as parental hermaphrodite produced progeny with normal RNAi activity. Complementation testing was performed using all combinations of rde-2(ne221), mut-7(pk204) (both harbor point mutations that result in a premature stop codon and a S812L substitution, respectively), rde-2(pk1657), and mut-7(pk720) (both harbor large deletions). In Tables 2 and 3, conclusions were drawn using data from at least two sets of independent genetic crosses and the progeny from each cross was tested on different preparations of pop-1 food. The F1 cross-progeny were placed as L2/L3 larvae on plates and the number of F2 animals per F1 adult was tabulated. We used the following classification scheme in our assessments: –, 0–5 F2's per F1 trans-heterozygote (indicative of wild-type RNAi activity); w, 6–20 F2's per F1; +, 21–50 F2's per F1; and ++, >50 F2's per F1 (indicative of strong RNAi defects). Trans-heterozygous combinations that displayed – and w results were considered complementary (C); combinations that produced + and ++ results were considered noncomplementary (NC); allele sets that produced + results were considered weak (W); and sets that displayed wide experiment-to-experiment variations (– and ++) were labeled inconsistent (I). Strains that displayed noncomplementation were subjected to three rounds of additional outcrossing and were retested for RNAi defects. A weak result could reflect a maternally supplied protein with a short half-life in the germ line. Alternatively, a weak RNAi defect might indicate that the paternal gene is transcriptionally activated relatively late in the critical period. Such conditions may also contribute to inconsistent results as well.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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In earlier experiments, most of the ABC transporter mutants we tested displayed normal RNAi activities, except for the nine mutants we now refer to as ABCRNAi mutants. The fact that only about one-sixth of the ABC transporter genes, and not all ABC transporter genes, are required for efficient RNAi leads one to speculate that there is some degree of specificity with regard to how ABC transporters might affect RNAi function. The goal of the experiments reported here is to address the question of specificity by determining if additional ABC transporters are required for efficient RNAi, functions that may have been missed in previous examinations. Because the RNAi defects in ABC transporter mutants were revealed using feeding protocols, and because RNAi feeding protocols have some degree of experiment-to-experiment variability, we initially retested all available ABC transporter mutants. We first simply repeated our previous observations using pop-1 and unc-22 foods. All tests were again conducted at three different temperatures (15°, 20°, and 25°) to uncover potential heat or cold sensitivities, and the experiments were performed on the original strains as well as strains that had been subjected to additional outcrossings in our lab (see MATERIALS AND METHODS). The results obtained did not differ from our previous reports (SUNDARAM et al. 2006 and data not shown).
We next utilized a more comprehensive set of feeding strains, targeting a wider array of genes expressed mostly in the soma (Table 1). Again, this more comprehensive set of assays did not reveal additional ABCRNAi mutants; RNAi activity was observed in all of the strains except those already designated as ABCRNAi (data not shown). Because all the ABCRNAi transporter mutants display RNAi defects when bacterial feeding strains are used to target genes expressed in the germ line, this broader set of data allows us to speculate that ABCRNAi transporter functions are particularly required in the germ line.
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RNAi defects in haf-6 mutants are dosage dependent with respect to the amount of dsRNA introduced (SUNDARAM et al. 2006), and we considered that the SSNC results may indicate dosage sensitivity with respect to HAF-6, MUT-7, and RDE-2 proteins as well. This hypothesis is supported by the observation that the RNAi defects in mut-7 and rde-2 mutants can be partially rescued by extra copies of haf-6(+) DNA (Figure 2B). We also observed an ability of rde-2 sequences to partially rescue the RNAi defects in haf-6 mutants. Wild-type rde-2 sequences do not provide rescue of RNAi defects in mut-7(pk204) (Figure 2B); however, these mutants harbor a version of MUT-7 that cannot bind RDE-2. Thus, rescue of RNAi defects in mut-7(pk204) animals by overexpression of HAF-6 may occur independently of an RDE-2/MUT-7 protein complex.
All ABCRNAi mutants display genetic interactions with mut-7 and rde-2 mutants:
We reasoned that we might use complementation testing with mut-7 or rde-2 alleles as an assay to help reveal additional ABC transporter genes that are required for RNAi. We therefore extended our complementation testing to all available ABC transporter mutant strains. The vast majority of the ABC transporter alleles in these experiments harbor deletions; whereas both deletions and point mutations in mut-7 and rde-2 alleles are available and were used in our assays. The mut-7 point mutations displayed similar genetic behavior to the deletion strains, and this was the case for rde-2 as well. Because the results we obtained using mut-7 strains were similar to those obtained using rde-2 mutants, we refer to the use of either of these two mutant strains, their activities, and their genetic behaviors as mut-7/rde-2.
The RNAi defects in mut-7/rde-2 mutants were complemented by all the ABC transporter mutants that display wild-type RNAi activity, with the exception of haf-9 (Tables 2 and 3 and supplemental Tables 1 and 2 at http://www.genetics.org/supplemental/). Therefore, we now include haf-9 in our list of ABCRNAi genes, despite the fact that homozygous mutants with a single-gene defect in haf-9 are wild type for RNAi activity. Our collective results imply that the SSNC assays are a more sensitive indicator for RNAi function than feeding-based RNAi assays. This additional set of data points to specificity with regard to the ability of ABC transporters to influence RNAi mechanisms, as >75% of the 49 ABC transporter mutants tested complement the RNAi defects in mut-7/rde-2. In addition, we hypothesize that the RNAi defects in ABC transporter mutants are specific to the mut-7/rde-2 branch of the RNAi pathway, as a number of strains with defects in other RNAi pathways, such as the RNA-induced silencing complex (RISC) pathway or pathways that facilitate dsRNA trafficking, complemented the RNAi defects in mut-7 and rde-2 (Table 2 and supplemental Table 1). Thus not only are the SSNC tests sensitive assays for the detection of RNAi-related functions for ABC transporters, but also the assays help narrow the ABCRNAi functions to a particular branch of the RNAi network (LEE et al. 2006; YIGIT et al. 2006).
Long-lasting parent-of-origin effects influence whether RNAi phenotypes are observed in trans-heterozygous progeny:
In the sections above, we demonstrated SSNC interactions between ABCRNAi and mut-7/rde-2 mutants: trans-heterozygous progeny displayed RNAi defects. We also observed a parent-of-origin effect in those SSNC interactions. In complementation tests using mut-7/rde-2 as the parental hermaphrodite and ABCRNAi mutants as parental males, RNAi defects were observed in trans-heterozygous progeny (Table 3 and supplemental Table 2). However, in most reciprocal crosses utilizing mut-7 or rde-2 as the parental male, the resulting trans-heterozygous progeny displayed a wild-type level of RNAi activity (Table 3 and supplemental Table 2). In control experiments, a parent-of-origin effect is not revealed in simple crosses utilizing wild-type males and hermaphrodites that are homozygous for mut-7 (or rde-2): wild-type levels of RNAi activity are displayed in both sets of genetically identical progeny produced from reciprocal crosses. Thus, a dependency on maternal MUT-7/RDE-2 is revealed only when the animal is also heterozygous for an ABCRNAi transporter gene.
The RNAi defects in trans-heterozygotes are surprisingly long lasting. Animals were placed on feeding plates as young larvae, and RNAi activities were assessed later in development, in the germ line of the ensuing adult. mut-7 and rde-2 mutations are recessive, and RNAi activity can be provided from paternal sources in animals that are singly heterozygous for mut-7 or rde-2. However, in trans-heterozygous (mut-7/rde-2)/+; +/ABCRNAi progeny that originate from oocytes lacking maternal MUT-7 or RDE-2, paternal mut-7/rde-2 does not provide rescue of RNAi defects. In these trans-heterozygous progeny, the maternal load of any ABCRNAi transporters should be inherited at wild-type levels. How the genetic condition of heterozygosity for an ABCRNAi gene prevents paternal rescue of mut-7/rde-2 is a question that remains to be answered.
Some combinations of ABCRNAi mutations also fail to complement:
We extended our analyses to see if pairs of different ABCRNAi mutants would complement one another (Table 3). We anticipated that we might gain information regarding redundancy with respect to ABC transporter functions in RNAi or results that might indicate heterodimerization partners for the half transporters. We observed a few pairs of noncomplementing ABCRNAi transporter alleles and several instances where the SSNC results were parent-of-origin dependent (Table 3). In general, most combinations of ABC transporter mutants were complementary, highlighting the specificity of these interactions and ruling out the possibility of a common background mutation in each of the strains.
Most of our noncomplementation experiments utilized pop-1 food. We considered that the RNAi defects we observed in trans-heterozygous animals might reflect an ability of this genetic condition to influence the pop-1 pathway. To address this question, we retested noncomplementing pairs of alleles using cks-1 food and again observed noncomplementation. cks-1 is a conserved cell-cycle regulator required for cyclin-dependent kinase activity and proper cell division (POLINKO and STROME 2000; SHIRAYAMA et al. 2006), confirming that the defect in trans-heterozygotes resides in the RNAi pathway and not in the pop-1 pathway (supplemental Table 2).
Conditional phenotypes are observed in ABCRNAi mutants:
The SSNC interactions we uncovered led us to investigate whether the ABCRNAi genes, like mut-7 or rde-2, are required for endogenous RNAi-associated processes. We first looked to see if ABCRNAi mutants shared common phenotypes with mut-7 and rde-2. Unlike mut-7 and rde-2, the ABCRNAi mutants do not have a high rate of chromosome nondisjunction, as they do not display a high incidence of males (Him) phenotype under any of the environmental conditions we tested, including elevated temperature, the presence of exogenous dsRNAs or heavy metal cations, or starvation. However, we did observe additional phenotypes in some ABCRNAi transporter mutants that are indicative of defects in RNAi mechanisms. We report three main observations.
First, the brood sizes are reduced in some mutants reared under conditions of elevated temperature (Figure 3A). While most mutants have normal fertility, haf-6 and haf-9 mutants produce fewer progeny at elevated temperatures. mut-7 and rde-2 mutants also have reductions in brood sizes, and it is widely appreciated that both rde-2 and mut-7 stocks will eventually stop producing progeny when reared at 25° for several generations.
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Finally, mut-7 and rde-2 mutants display an increased rate of transposon mobilization (Mutator activity), and some ABCRNAi mutants display temperature-sensitive Mutator activity as well (Table 4). Strains with Mutator activity include haf-6(ne335), haf-9(gk23), wht-1(tm688), and mrp-1(pk89). While mut-7 and rde-2 animals display Mutator activity at all temperatures, all the ABCRNAi mutants are temperature sensitive for this phenotype. We hypothesize that this collective set of temperature-sensitive phenotypes in haf-6 and other ABCRNAi mutants reflects endogenous roles for ABCRNAi transporters in cellular processes that also require the function of MUT-7/RDE-2, roles that ultimately affect chromosome integrity.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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61 ABC transporter proteins that collectively reside on most intracellular membranes. Because ABC transporter-dependent movement of substrates is essential for subcellular organelle biogenesis and function, and because substrates can be transported between organelles (NUNES et al. 2005; SCHAHEEN et al. 2006; SCHROEDER et al. 2007), it is conceivable that alterations in ABC transporter function might simply affect cell physiology in a more general manner, with indirect effects on RNAi function. If we assume this possibility is correct, then we would expect to observe RNAi defects in multiple tissues in most of the ABC transporter mutants, yet this is not what we observed. RNAi defects were observed in a limited subset of the mutants tested.
ABCRNAi transporters have specific effects on RNAi functions:
Evidence of specificity with regard to RNAi phenotypes is provided by the following observations: only the ABCRNAi subset of transporters displays defects in RNAi, with the exception of haf-9 mutants. While this is a functional definition that we applied on the basis of our results, this definition applies only to 30% of the 49 transporter mutants tested. Furthermore, only the ABCRNAi subset of transporter mutants displays SSNC interactions with mut-7/rde-2.
The fact that the RNAi functions of ABCRNAi transporters are required in germ-line tissue provides additional evidence of specificity. Even though several of the ABCRNAi genes are expressed in various somatic tissues (ZHAO et al. 2004), all ABCRNAi transporters are required for efficient RNAi in the germ line. Moreover, our extensive set of RNAi assays has not revealed an ABC transporter gene that is required for RNAi solely in somatic cells. mut-7 and rde-2, which interact genetically with ABCRNAi mutants, are also required for efficient RNAi in the germ line, and the corresponding mutants display phenotypes indicative of germ-line defects (meitoic nondisjunction, cosuppression, and high rates of transposon mobilization). By contrast, some ABC transporters are expressed in the germ line, yet are not required for RNAi. For example, ced-7 is required in the germ line for engulfment of apoptotic corpses (WU and HORVITZ 1998), yet ced-7 mutants are wild type with respect to the RNAi activities assessed in this report. Finally, some ABCRNAi mutants display temperature-sensitive phenotypes in the germ line, phenotypes that are similar to those observed in mut-7/rde-2 mutants. These temperature-sensitive phenotypes include Mutator activity, reductions in brood size, and RNAi defects in response to ingestion of unc-22 food. While germ-line tissue is clearly the focus of ABCRNAi function, we cannot rule out additional trafficking roles between germ-line and somatic tissues.
Finally, within the RNAi network of mechanisms (LEE et al. 2006; YIGIT et al. 2006), evidence of specificity is observed with respect to ABCRNAi transporter function. This is revealed in SSNC assays using mut-7/rde-2, genes that are required in RNAi-related processes that affect chromosome as well as miRNA functions. By contrast, ABCRNAi transporters do not interact genetically with genes that are required in other RNAi-related processes, including genes that facilitate systemic silencing by dsRNA trafficking (sid-1) or genes that are required in dsRNA processing and RISC-based mechanisms (rde-1 and rde-4) (TABARA et al. 2002; FEINBERG and HUNTER 2003; YIGIT et al. 2006). Additionally, even though ABCRNAi transporter mutants display temperature-sensitive defects in transposon silencing, they do not interact genetically with other mutants that display temperature-sensitive transposon silencing defects (rsd-6) (HAN et al., unpublished results). Similarly, while mut-7/rde-2 mutants have increased rates of nondisjunction, ABCRNAi transporter mutants did not interact with other mutants that display dsRNA-inducible nondisjunction phenotypes (rsd-2) (HAN et al., unpublished results). It may be the case that SSNC interactions are observed between pairs of genes that normally produce limited product. RSD-2, RSD-6, SID-1, RDE-1, and RDE-4 may not be limiting in quantity, and therefore the corresponding mutants would not be expected to display SSNC with ABCRNAi mutants. On the other hand, Him and Mutator phenotypes can be induced in rsd-2 and rsd-6 mutants (HAN et al., unpublished results), which is consistent with the possibility that RSD-2 and RSD-6 proteins are limiting.
The common phenotypes and genetic interactions between ABCRNAi transporters and mut-7/rde-2 imply common functions that affect chromosome function and transcriptional silencing:
In addition to roles in chromosome function and integrity, mut-7 and rde-2 are also required for cosuppression and silencing of transgenes in the germ line (TABARA et al. 1999; DERNBURG et al. 2000; KETTING and PLASTERK 2000; KIM et al. 2005). Both these phenomena are observed when transgenes are introduced into C. elegans, and both phenomena have similar features that are suggestive of transcriptional-level silencing. Cosuppression refers to an ability of a transgene to silence gene expression from the transgene itself, as well as from endogenous genes with sequence homology to the transgene. While specific sequences may be more prone to cosuppression, the exact nature of the triggering molecule is not known. Silencing of transgene expression is frequently observed for sequences that express in the germ line. Both cosuppression and transgene silencing require chromatin factors and RNAi, and the transgenes that are silenced in the germ line display histone methylation patterns reminiscent of heterochromatin (KELLY et al. 2002; ROBERT et al. 2005). Both transgene silencing and cosuppression require mut-7 and rde-2, but not rde-1 (DERNBURG et al. 2000).
Here, we described an unusual temperature-sensitive and method-of-delivery-dependent RNAi defect that we observe in mut-7, rde-2, and haf-6 mutants, and we postulate that this phenotype also reflects impairments in transcriptional silencing. Upon ingestion of unc-22 food, these mutants display RNAi defects when the experiments are conducted at elevated temperatures. Normally, the unc-22 gene is an effective RNAi target in RNAi experiments using wild-type animals, and unc-22 sequences have been noted to induce cosuppression as well (FIRE et al. 1991). Taken together, we postulate that unc-22 sequences have a tendency to trigger transcriptional silencing responses in these assays and that unc-22 is a particularly amenable RNAi target due to the combined activities of RISC and transcriptional silencing mechanisms. We further hypothesize that mut-7, rde-2, and haf-6 are required to maintain transcription-level silencing at elevated temperatures. On a related note, we recently reported temperature-sensitive defects in transposon silencing in rsd-6 mutants, and rsd-6 also displays temperature-sensitive RNAi defects when reared on unc-22 food (W. HAN, unpublished results). Finally, we point out that chromosome-related events such as chromosome disjunction, transposon mobilization, and recombination are intrinsically sensitive to environmental conditions (HILDRETH and ULRICHS 1969; ROSE and BAILLIE 1979; HASHIDA et al. 2006). Thus the temperature-sensitive response to unc-22 food that we observe in mutants is likely a hallmark for genes with RNAi activities that may ultimately affect transcriptional silencing.
Precisely how ABCRNAi transporter and mut-7/rde-2 functions are integrated is not known; however, we can invoke at least two models on the basis of the genetic and transgene rescue data (Figure 4). In a parallel pathways model, HAF-6 and RDE-2/MUT-7 activities are predicted to reside in different pathways that converge to a common RNAi function, with a threshold level of activity from both pathways required for full RNAi effect. Alternatively, in a protein complex model, both ABCRNAi and RDE-2/MUT-7 function as a multiprotein complex that is effectual only when threshold numbers of complexes are complete. Because overexpression of one protein can rescue the RNAi defect in a mutant that is deficient for the second protein (Figure 2B), the former model is strongly favored; however, the latter model cannot be confidently eliminated. Indeed, RDE-2 and MUT-7 proteins physically associate (TOPS et al. 2005).
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The maternally deposited MUT-7/RDE-2 products are particularly long lived:
We noted that maternally deposited MUT-7/RDE-2 products were particularly long lived, as revealed in the SSNC tests, and the maternal requirement for MUT-7/RDE-2 is observed only when animals are heterozygous for ABCRNAi (Table 3). We propose that the set-aside nature of germ-line progenitors and asymmetric cell divisions during embryogenesis allows for preferential segregation of maternal MUT-7/RDE-2 gene products into germ-line progenitor cells. We further speculate that the RNAi defects observed in adult trans-heterozygotes provides evidence of a long-lived nature of maternal MUT-7/RDE-2.
The presence of functional, plasma membrane-associated ABC transporters has important applications in stem cell biology, as ABC transporters serve as phenotypic markers for stem cell populations, and expression of particular transporters can influence developmental decisions of stem cells (BUNTING 2002; LIN et al. 2006). It is interesting to speculate that specific transcriptional or chromosome-related functions that could affect differentiation are afforded by ABC transporters acting through specific RNAi mechanisms in germ-line progenitors and in cultured stem cells.
A network of activity is revealed through analysis of noncomplementing pairs of ABCRNAi mutants:
A few pairs of ABCRNAi mutants were noncomplementing: some pairs of mutants failed to complement in reciprocal crosses, whereas SSNC in other sets of mutants was dependent upon the parental configuration of alleles (Table 3 and Figure 5, left section). In examples where SSNC is observed in reciprocal crosses, the results may reflect requirements for both transporters in one germ line. Alternatively, one transporter may be required in the male germ line. A SSNC that displays a parent-of-origin effect in reciprocal crosses might reflect functional redundancy for ABCRNAi genes in one gonad. Because some transporters may function as homo- and/or heterodimers, and because ABCRNAi function may not be restricted to germ-line tissue, it is not possible to draw firm conclusions regarding functional redundancy, tissue-specific requirements, or dimerization partners using the existing data. Nonetheless, we did note a pattern emerging from these data (Figure 5, right section).
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Environmental conditions may influence gene expression via two conserved and interrelated networks:
Environmental conditions such as elevated temperature can influence chromosome-associated activities such as transcription, transgene silencing, recombination, meiotic chromosomal disjunction, and transposon mobilization. The additional phenotypes we observed when ABCRNAi mutants were reared at elevated temperatures (Mutator and unc-22 RNAi defects) imply an ability for ABCRNAi transporters to protect chromosome-related functions and influence transcription in suboptimal environments. On a related note, but also mRNA regulation by miRNAs has been shown to be influenced by cellular stressors (BHATTACHARYYA et al. 2006a,b; PETERS and MEISTER 2007). Precisely how RNAi mechanisms respond to environmental influences is not known. Interestingly, in addition to a 3'–5' exonuclease domain, the MUT-7 protein also has similarity to conserved proteins of unknown function that are found in several hyperthermophilic archaea and other bacteria, raising the possibility that this domain might have functional roles that are influenced by temperature. Regions similar to this conserved domain are also found in 3'–5' exonuclease domain proteins from paramecium and humans (supplemental Figure 1 at http://www.genetics.org/supplemental/).
We speculate that a network of ABCRNAi functions may allow for multiple means of influencing mut-7/rde-2, and ultimately chromosome function and integrity, in various environmental conditions. ABC transporters and RNAi pathway components appear to be functionally connected as part of an ancient cellular mechanism that responds to the environment.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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FOOTNOTES |
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2 Present address: BIO5 Institute, University of Arizona, Tuscon, AZ 85721.
3 Present address: Combined MD/PhD Training Program, University of Minnesota, Minneapolis, MN 55455.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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BHATTACHARYYA, S. N., R. HABERMACHER, U. MARTINE, E. I. CLOSS and W. FILIPOWICZ, 2006a Relief of microRNA-mediated translational repression in human cells subjected to stress. Cell 125: 1111–1124.[CrossRef][Medline]
BHATTACHARYYA, S. N., R. HABERMACHER, U. MARTINE, E. I. CLOSS and W. FILIPOWICZ, 2006b Stress-induced reversal of microRNA repression and mRNA P-body localization in human cells. Cold Spring Harbor Symp. Quant. Biol. 71: 513–521.[CrossRef][Medline]
BROOKS, D. R., P. J. APPLEFORD, L. MURRAY and R. E. ISAAC, 2003 An essential role in molting and morphogenesis of Caenorhabditis elegans for ACN-1, a novel member of the angiotensin-converting enzyme family that lacks a metallopeptidase active site. J. Biol. Chem. 278: 52340–52346.
BRUNSCHWIG, K., C. WITTMANN, R. SCHNABEL, T. R. BURGLIN, H. TOBLER et al., 1999 Anterior organization of the Caenorhabditis elegans embryo by the labial-like Hox gene ceh-13. Development 126: 1537–1546.[Abstract]
BUNTING, K. D., 2002 ABC transporters as phenotypic markers and functional regulators of stem cells. Stem Cells 20: 11–20.
CAMPBELL, R. B., D. A. SINCLAIR, M. COULING and H. W. BROCK, 1995 Genetic interactions and dosage effects of Polycomb group genes of Drosophila. Mol. Gen. Genet. 246: 291–300.[CrossRef][Medline]
DERNBURG, A. F., J. ZALEVSKY, M. P. COLAIACOVO and A. M. VILLENEUVE, 2000 Transgene-mediated cosuppression in the C. elegans germ line. Genes Dev. 14: 1578–1583.
DESHPANDE, R., T. INOUE, J. R. PRIESS and R. J. HILL, 2005 lin-17/Frizzled and lin-18 regulate POP-1/TCF-1 localization and cell type specification during C. elegans vulval development. Dev. Biol. 278: 118–129.[CrossRef][Medline]
EDENS, W. A., L. SHARLING, G. CHENG, R. SHAPIRA, J. M. KINKADE et al., 2001 Tyrosine cross-linking of extracellular matrix is catalyzed by Duox, a multidomain oxidase/peroxidase with homology to the phagocyte oxidase subunit gp91phox. J. Cell Biol. 154: 879–891.
EISENMANN, D. M., J. N. MALOOF, J. S. SIMSKE, C. KENYON and S. K. KIM, 1998 The beta-catenin homolog BAR-1 and LET-60 Ras coordinately regulate the Hox gene lin-39 during Caenorhabditis elegans vulval development. Development 125: 3667–3680.[Abstract]
FEINBERG, E. H., and C. P. HUNTER, 2003 Transport of dsRNA into cells by the transmembrane protein SID-1. Science 301: 1545–1547.
FIRE, A., D. ALBERTSON, S. W. HARRISON and D. G. MOERMAN, 1991 Production of antisense RNA leads to effective and specific inhibition of gene expression in C. elegans muscle. Development 113: 503–514.[Abstract]
FRAND, A. R., S. RUSSEL and G. RUVKUN, 2005 Functional genomic analysis of C. elegans molting. PLoS Biol. 3: e312.[CrossRef][Medline]
FRASER, A. G., R. S. KAMATH, P. ZIPPERLEN, M. MARTINEZ-CAMPOS, M. SOHRMANN et al., 2000 Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408: 325–330.[CrossRef][Medline]
FUKUSHIGE, T., M. G. HAWKINS and J. D. MCGHEE, 1998 The GATA-factor elt-2 is essential for formation of the Caenorhabditis elegans intestine. Dev. Biol. 198: 286–302.[Medline]
FUKUSHIGE, T., M. J. HENDZEL, D. P. BAZETT-JONES and J. D. MCGHEE, 1999 Direct visualization of the elt-2 gut-specific GATA factor binding to a target promoter inside the living Caenorhabditis elegans embryo. Proc. Natl. Acad. Sci. USA 96: 11883–11888.
HAARER, B., S. VIGGIANO, M. A. HIBBS, O. G. TROYANSKAYA and D. C. AMBERG, 2007 Modeling complex genetic interactions in a simple eukaryotic genome: actin displays a rich spectrum of complex haploinsufficiencies. Genes Dev. 21: 148–159.
HASHIDA, S. N., T. UCHIYAMA, C. MARTIN, Y. KISHIMA, Y. SANO et al., 2006 The temperature-dependent change in methylation of the Antirrhinum transposon Tam3 is controlled by the activity of its transposase. Plant Cell 18: 104–118.
HAWLEY, R. S., and W. D. GILLILAND, 2006 Sometimes the result is not the answer: the truths and the lies that come from using the complementation test. Genetics 174: 5–15.
HILDRETH, P. E., and P. C. ULRICHS, 1969 A temperature effect on nondisjunction of the X chromosomes among eggs from aged Drosophila females. Genetica 40: 191–197.[CrossRef][Medline]
HULL, D., and L. TIMMONS, 2004 Methods for delivery of double-stranded RNA into Caenorhabditis elegans. Methods Mol. Biol. 265: 23–58.[Medline]
JASPERSEN, S. L., B. J. HUNEYCUTT, T. H. GIDDINGS, JR., K. A. RESING, N. G. AHN et al., 2004 Cdc28/Cdk1 regulates spindle pole body duplication through phosphorylation of Spc42 and Mps1. Dev. Cell 7: 263–274.[CrossRef][Medline]
KAMATH, R. S., A. G. FRASER, Y. DONG, G. POULIN, R. DURBIN et al., 2003 Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421: 231–237.[CrossRef][Medline]
KELLY, W. G., S. XU, M. K. MONTGOMERY and A. FIRE, 1997 Distinct requirements for somatic and germline expression of a generally expressed Caernorhabditis elegans gene. Genetics 146: 227–238.[Abstract]
KELLY, W. G., C. E. SCHANER, A. F. DERNBURG, M. H. LEE, S. K. KIM et al., 2002 X-chromosome silencing in the germline of C. elegans. Development 129: 479–492.[Medline]
KETTING, R. F., and R. H. PLASTERK, 2000 A genetic link between co-suppression and RNA interference in C. elegans. Nature 404: 296–298.[CrossRef][Medline]
KETTING, R. F., T. H. HAVERKAMP, H. G. VAN LUENEN and R. H. PLASTERK, 1999 Mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD. Cell 99: 133–141.[CrossRef][Medline]
KIM, J. K., H. W. GABEL, R. S. KAMATH, M. TEWARI, A. PASQUINELLI et al., 2005 Functional genomic analysis of RNA interference in C. elegans. Science 308: 1164–1167.
LEE, R. C., C. M. HAMMELL and V. AMBROS, 2006 Interacting endogenous and exogenous RNAi pathways in Caenorhabditis elegans. RNA 12: 589–597.
LIN, R., S. THOMPSON and J. R. PRIESS, 1995 pop-1 encodes an HMG box protein required for the specification of a mesoderm precursor in early C. elegans embryos. Cell 83: 599–609.[CrossRef][Medline]
LIN, R., R. J. HILL and J. R. PRIESS, 1998 POP-1 and anterior-posterior fate decisions in C. elegans embryos. Cell 92: 229–239.[CrossRef][Medline]
LIN, T., O. ISLAM and K. HEESE, 2006 ABC transporters, neural stem cells and neurogenesis–a different perspective. Cell Res. 16: 857–871.[CrossRef][Medline]
MCKEOWN, C., V. PRAITIS and J. AUSTIN, 1998 sma-1 encodes a betaH-spectrin homolog required for Caenorhabditis elegans morphogenesis. Development 125: 2087–2098.[Abstract]
MOERMAN, D. G., G. M. BENIAN, R. J. BARSTEAD, L. A. SCHRIEFER and R. H. WATERSTON, 1988 Identification and intracellular localization of the unc-22 gene product of Caenorhabditis elegans. Genes Dev. 2: 93–105.
NATARAJAN, L., B. M. JACKSON, E. SZYLEYKO and D. M. EISENMANN, 2004 Identification of evolutionarily conserved promoter elements and amino acids required for function of the C. elegans beta-catenin homolog BAR-1. Dev. Biol. 272: 536–557.[CrossRef][Medline]
NUNES, F., M. WOLF, J. HARTMANN and R. J. PAUL, 2005 The ABC transporter PGP-2 from Caenorhabditis elegans is expressed in the sensory neuron pair AWA and contributes to lysosome formation and lipid storage within the intestine. Biochem. Biophys. Res. Commun. 338: 862–871.[CrossRef][Medline]
PETERS, L., and G. MEISTER, 2007 Argonaute proteins: mediators of RNA silencing. Mol. Cell 26: 611–623.[CrossRef][Medline]
POLINKO, E. S., and S. STROME, 2000 Depletion of a Cks homolog in C. elegans embryos uncovers a post-metaphase role in both meiosis and mitosis. Curr. Biol. 10: 1471–1474.[CrossRef][Medline]
PRAITIS, V., E. CICCONE and J. AUSTIN, 2005 SMA-1 spectrin has essential roles in epithelial cell sheet morphogenesis in C. elegans. Dev. Biol. 283: 157–170.[CrossRef][Medline]
ROBERT, V. J., T. SIJEN, J. VAN WOLFSWINKEL and R. H. PLASTERK, 2005 Chromatin and RNAi factors protect the C. elegans germline against repetitive sequences. Genes Dev. 19: 782–787.
ROGALSKI, T. M., G. P. MULLEN, M. M. GILBERT, B. D. WILLIAMS and D. G. MOERMAN, 2000 The UNC-112 gene in Caenorhabditis elegans encodes a novel component of cell-matrix adhesion structures required for integrin localization in the muscle cell membrane. J. Cell Biol. 150: 253–264.
ROSE, A. M., and D. L. BAILLIE, 1979 The effect of temperature and parental age on recombination and nondisjunction in Caenorhabditis elegans. Genetics 92: 409–418.
RUAL, J. F., J. CERON, J. KORETH, T. HAO, A. S. NICOT et al., 2004 Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library. Genome Res. 14: 2162–2168.
SCHAHEEN, L., G. PATTON and H. FARES, 2006 Suppression of the cup-5 mucolipidosis type IV-related lysosomal dysfunction by the inactivation of an ABC transporter in C. elegans. Development 133: 3939–3948.
SCHROEDER, L. K., S. KREMER, M. J. KRAMER, E. CURRIE, E. KWAN et al., 2007 Function of the Caenorhabditis elegans ABC transporter PGP-2 in the biogenesis of a lysosome-related fat storage organelle. Mol. Biol. Cell. 18: 995–1008.
SHIRAYAMA, M., M. C. SOTO, T. ISHIDATE, S. KIM, K. NAKAMURA et al., 2006 The conserved kinases CDK-1, GSK-3, KIN-19, and MBK-2 promote OMA-1 destruction to regulate the oocyte-to-embryo transition in C. elegans. Curr. Biol. 16: 47–55.[CrossRef][Medline]
SIEGFRIED, K. R., A. R. KIDD, 3RD, M. A. CHESNEY and J. KIMBLE, 2004 The sys-1 and sys-3 genes cooperate with Wnt signaling to establish the proximal-distal axis of the Caenorhabditis elegans gonad. Genetics 166: 171–186.
SIMMER, F., C. MOORMAN, A. M. VAN DER LINDEN, E. KUIJK, P. V. VAN DEN BERGHE et al., 2003 Genome-wide RNAi of C. elegans using the hypersensitive rrf-3 strain reveals novel gene functions. PLoS Biol. 1: E12.[Medline]
STEARNS, T., and D. BOTSTEIN, 1988 Unlinked noncomplementation: isolation of new conditional-lethal mutations in each of the tubulin genes of Saccharomyces cerevisiae. Genetics 119: 249–260.
STREIT, A., R. KOHLER, T. MARTY, M. BELFIORE, K. TAKACS-VELLAI et al., 2002 Conserved regulation of the Caenorhabditis elegans labi
