SID-1 Functions in Multiple Roles To Support Parental RNAi in Caenorhabditis elegans

Abstract

Systemic RNA interference (RNAi) in Caenorhbaditis elegans requires sid-1, sid-3, and sid-5 Injected, expressed, or ingested double-stranded RNA (dsRNA) is transported between cells, enabling RNAi in most tissues, including the germline and progeny (parental RNAi). A recent report claims that parental RNAi also requires the yolk receptor rme-2 Here, we characterize the role of the sid genes and rme-2 in parental RNAi. We identify multiple independent paths for maternal dsRNA to reach embryos and initiate RNAi. We showed previously that maternal and embryonic sid-1 contribute independently to parental RNAi. Here we demonstrate a role for embryonic sid-5, but not sid-2 or sid-3 in parental RNAi. We also find that maternal rme-2 contributes to but is not required for parental RNAi. We determine that parental RNAi by feeding occurs nearly exclusively in adults. We also introduce 5-ethynyluridine to densely internally label dsRNA, avoiding complications associated with other labeling strategies such as inhibition of normal dsRNA trafficking and separation of label and RNA. Labeling shows that yolk and dsRNA do not colocalize following endocytosis, suggesting independent uptake, and, furthermore, dsRNA appears to rapidly progress through the RAB-7 endocytosis pathway independently of sid-1 activity. Our results support the premise that although sid-1 functions in multiple roles, it alone is central and absolutely required for inheritance of silencing RNAs.

Keywords: Caenorhabditis elegans; RNAi; double-stranded RNA; yolk.

Figure 1

sid-1-dependent and -independent silencing in progeny of dsRNA injected parents. (A–D) Time course of fraction of progeny with the Unc-22 phenotype laid after unc-22 dsRNA gonad injection into wild-type or sid-1 mutant hermaphrodites. (E) Fraction of progeny with Unc-22 phenotype following unc-22 dsRNA gonad or PC injection into sid-1^−/− mutant hermaphrodites crossed to wild-type or sid-1 mutant males. Error bars in (A–D) represent SE from two experiments with 10 injected hermaphrodites each. Error bars in (E) represent SD from 4, 6, and 3 injected hermaphrodites, left to right. ** P < 0.01 by Welch’s t-test.

Figure 2

Maternal RME-2-dependent inherited silencing. (A) Fraction of progeny sensitive to unc-22 silencing among the self-progeny or indicated cross-progeny of hermaphrodites PC-injected with unc-22 dsRNA; n = 6, 5, 3, 4, and 12 injected hermaphrodites respectively. (B) Fraction of progeny sensitive to unc-22 silencing after wild-type parents were exposed to feeding RNAi at the given periods of time after hatching; n = 30 treated parents for each group. (C) Sensitivity to unc-22 feeding RNAi in progeny after treating (i) wild-type parents as adults, (ii) rme-2 mutant parents as adults, or (iii) rme-2 mutant parents as L4 larvae. Because rme-2 mutants have severely reduced fecundity, the results from each individual parent are presented separately for clarity, with silenced progeny represented in black bars and nonsilenced progeny in gray bars. All error bars represent SD. **** P < 0.00001 by t-test. n.s., not significant.

Figure 3

Maternal and zygotic Sid-dependence of inherited silencing. (A) Fraction of unc-22 silenced cross progeny from sid-1; sid-2 double-mutant hermaphrodites first PC-injected with unc-22 dsRNA and then crossed to either wild-type, sid-1; sid-2 double mutant, or sid-2 single mutant males. (B) Fraction of unc-22 silenced cross progeny from sid-1; sid-3 double-mutant hermaphrodites first PC-injected with unc-22 dsRNA and then crossed to wild-type, sid-1; sid-3 double mutant, or sid-3 single mutant males. (C) Schematic of the injection, cross, and unc-22 silencing scoring of cross progeny from sid-1; sid-5 double mutant hermaphrodites first PC-injected with unc-22 dsRNA and then crossed to wild-type males. sid-5 is X-linked, thus hermaphrodite progeny are heterozygous and males are hemizygous. Error bars in (A and B) represent SD from four, seven, and five injected hermaphrodites in (A) and one, three, and three injected hermaphrodites in (B). Three injected hermaphrodites in (C). n.s. = not significant.

Figure 4

Visualizing 5EU labeled functional dsRNA. (A) Injected 5EU dsRNA injected into only one gonad arm produces >50% affected progeny; n = 8 injected hermaphrodites. (B) Localization of PC injected Cy5::5EU heteroduplex dsRNA. Cy5 fluorescence and 5EU detection colocalize in the pseudocoelom (i) and a coelomocyte (cc) (ii; white arrowheads). (C) Independent localization of PC injected 5EU and Cy5 labeled dsRNA. Images in (B) and (C) represent portions of dissected and partially flattened adult hermaphrodites. Thick dotted lines mark the boundary of the animal, and thinner dotted lines mark structures such as the gonad (gon) or intestinal cells (int) as landmarks for orientation. (D, E) Cy5 and 5EU signal in embryos collected from adults injected with the dsRNA species described in (B) and (C), respectively. The two Cy5 images are overexposed, revealing diffuse autofluorescence and no detectable RNA. Bars, 10 μm.

Figure 5

Colocalization of 5EU-labeled dsRNA and VIT-2::GFP on and within oocytes. (A) PC-injected 5EU-labeled dsRNA colocalized with GFP-labeled yolk at the surface of developing oocytes (white arrowheads), but not intracellularly (notched arrowheads). More proximal oocyte (top) contains more VIT-2::GFP. (B) Maximum z-projections of VIT-2::GFP and 5EU-labeled dsRNA in wild-type and sid-1^−/− embryos show little colocalization. Bars, 10 μm.

Figure 6

Colocalization of dsRNA and GFP::RAB-7. (A) 5EU foci detected in embryos after pseudocoelomic 5EU-labeled dsRNA injection colocalizes with GFP:: RAB-7 (notched arrowheads) but is also found outside of RAB-7 structures (open arrowheads) in both wild-type (upper row) and sid-1 mutant (lower row) embryos. Bars, 10 μm. (B) Model for three inherited dsRNA transport pathways. (1) DsRNA injected directly into the syncytial germline can silence the resulting progeny without SID-1. Some injected dsRNA exits the gonad to the PC and is then directly or indirectly via yolk (2) endocytosed into developing oocytes via LDL receptor super-family member RME-2, or (3) this PC dsRNA can also be directly transported into oocytes via SID-1.