Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 19 (1), 90-7

Tudor Domain ERI-5 Tethers an RNA-dependent RNA Polymerase to DCR-1 to Potentiate endo-RNAi

Affiliations

Tudor Domain ERI-5 Tethers an RNA-dependent RNA Polymerase to DCR-1 to Potentiate endo-RNAi

Caroline Thivierge et al. Nat Struct Mol Biol.

Erratum in

  • Nat Struct Mol Biol. 2013 Feb;20(2):244

Abstract

Endogenous RNA interference (endo-RNAi) pathways use a variety of mechanisms to generate siRNA and to mediate gene silencing. In Caenorhabditis elegans, DCR-1 is essential for competing RNAi pathways-the ERI endo-RNAi pathway and the exogenous RNAi pathway-to function. Here, we demonstrate that DCR-1 forms exclusive complexes in each pathway and further define the ERI-DCR-1 complex. We show that the tandem tudor protein ERI-5 potentiates ERI endo-RNAi by tethering an RNA-dependent RNA polymerase (RdRP) module to DCR-1. In the absence of ERI-5, the RdRP module is uncoupled from DCR-1. Notably, EKL-1, an ERI-5 paralog that specifies distinct RdRP modules in Dicer-independent endo-RNAi pathways, partially compensates for the loss of ERI-5 without interacting with DCR-1. Our results implicate tudor proteins in the recruitment of RdRP complexes to specific steps within DCR-1-dependent and DCR-1-independent endo-RNAi pathways.

Figures

Figure 1
Figure 1. Distinct DCR-1 complexes initiate endo- and exo-RNAi
(a) Gel filtration on wild-type embryonic extract. DRH-1, RDE-4, DCR-1, RRF-3, ERI-5, ERI-1 and DRH-3 proteins were detected by western blot on fractions from a Superose S6 column. The fractionation of molecular weight standards (MW) is indicated. The asterisk (*) labels in DRH-1 (in the low MW fractions) and RDE-4 filtration panels indicate non-specific bands. (b) Immunoprecipitation (IP) of DCR-1, DRH-1 and RDE-4 from wild-type (WT), dcr-1, rde-4 or rde-1 mutant embryos. DCR-1, RDE-1, DRH-1 and RDE-4 proteins were detected in total lysate (LOAD) and IP by western blot. Tubulin was used as a loading control. The asterisk (*) to the right of the RDE-4 panels indicates background signal from the IgG heavy chains used for immunoprecipitation, and co-migrate with RDE-4 around 50kDa. (c) IP of DRH-1 in WT and drh-1 mutant embryos. DRH-1, DCR-1, DRH-3, ERI-5 and ERI-1 were detected by western blot. The asterisk (*) to the right and left of the DRH-1 panel indicate non-specific bands in the loading, and DRH-1 IP lanes, respectively. (d) IP of ERI-5 in WT and eri-5 mutant embryos. DRH-3, DRH-1 and ERI-5 proteins were detected by western blot. The asterisk (*) indicates the non-specific band detected in the input lanes (LOAD) of the DRH-1 blot as in panel c. (e) Interaction map of the proteins detected by MuDPIT analyses in WT embryonic extracts. Proteins in bold (DCR-1, ERI-5, ERI-1 and RDE-4) represent IP targets. See Methods for details on the epitope targeted. Arrowheads indicate interactions detected. The interactions of ERI-5 and ERI-1 in RDE-4 IP included in the diagram were only detected by western. The number of interactions detected exclusively in DCR-1 or ERI-1 MuDPIT experiments are indicated (‘17 or 11 single target hits’ circles) and may reflect divergent functions for these proteins.
Figure 2
Figure 2. ERI-5 promotes the association of an RdRP module to DCR-1 N-terminus
(a, b) IP of DCR-1 and ERI-5 in WT, eri-5, rrf-3 del (deletion mutant, pk1426), rrf-3 pm (point mutant, mg373), eri-3 and eri-1 mutant embryos. DCR-1, RRF-3, DRH-3 and ERI-5 were detected by western blot. Tubulin was used as a loading control. (c) (top) Map of the DCR-1-GST constructs used for the GST pull-down of recombinant (r) ERI-5 or ERI-3. The ability of each DCR-1-GST fusion to interact with rERI-5 or rERI-3 was assessed by western blot (bottom panel) to detect recombinant rERI-5-CBP or rERI-3-FLAG. The results are summarized to the right of the DCR-1 map; “−” denotes weak or no interaction, “+” denotes an interaction. (see Supplementary Fig. 2c for Coomassie Blue gel staining). Percentage (%) of the loading (bottom panel) represents the fraction of rERI-5 and rERI-3 used in the GST pull-down. (d) ERI-3 and ERI-5 bind to DCR-1(272–1045) simultaneously. An increasing amount of rERI-3 was pre-incubated with DCR-1(272–1045) prior to addition of rERI-5 and pull-down of the DCR-1 fragment.
Figure 3
Figure 3. ERI-5 potentiates ERI endo-RNAi small RNA biogenesis
(a) Northern and (b) qRT-PCR analysis of C40A11.10 26-G-RNAs siRNA species (siR26–1) as indicated in WT, eri-5 and rrf-3 (pk1426) mutant embryos. The C40A11.10 probe detected both 26-G- and 22G-RNAs. 5S ribosomal RNA (rRNA) ethidium bromide staining is shown as a loading control in a. The mean of at least three independent experiments is depicted as the ratio of siR26–1 or X-cluster relative to actin. Error bars indicate s.d. (c) Box and whisker plots show the enrichment or depletion of small RNAs targeting 26-G-RNA coding genes (red) and non-annotated 26-G-RNA clusters (yellow) in the indicated mutant. The left panel is an analysis of 26nt antisense reads from embryo small RNA libraries that target the 26-G-RNA loci. The right panel is an analysis of all antisense reads from adult small RNA libraries that target the 26-G-RNA loci. The majority of reads in the adult samples are 22G-RNAs. Values approaching 1 indicate enrichment of small RNA; values approaching 0 indicate depletion. Relative enrichment was calculated as the ratio of mutant per (mutant + wild-type). The top and bottom of each box represent the 75th and 25th percentiles, respectively. The horizontal line within each box represents the median value. The number of loci used to generate box and whisker plots is indicated above each plot and the data are provided in Supplementary Data 1 and 2.
Figure 4
Figure 4. Tandem-tudor domain proteins are required for ERI endo-siRNA biogenesis
(a) Northern and (b) qRT-PCR analysis of C40A11.10 26-G-RNAs (siR26–1) in sel-1 (RNAi) (a negative control, marked with (−)), ekl-1(RNAi), eri-5 and eri-5; ekl-1(RNAi) embryos. The mean of at least three independent experiments is depicted as the ratio of siR26–1 relative to actin. Error bars indicate s.d. (c) qRT-PCR analysis of C40A11.10 26-G-RNAs (siR26–1) in WT, eri-5, eri-4 and double eri-5; eri-4 mutant embryos. The mean of at least three independent experiments is depicted as the ratio of siR26–1 relative to actin. Error bars indicate s.d. (d) IP of EKL-1 and DCR-1 in WT and eri-5 mutant embryos. EKL-1 and DCR-1 proteins were detected by western blot. (e) IP of RRF-3 in WT and eri-5 mutant embryos. DCR-1, RRF-3, EKL-1 and ERI-5 proteins were detected by western blot. Tubulin was used as a loading control.
Figure 5
Figure 5. Roles and paralog organization of RdRP modules in ERI endo-RNAi
(a) IP of EKL-1 in WT and ekl-1(RNAi) (ekl-1 lanes) embryos, and IP of ERI-5 in WT and eri-5 mutant embryos. The RdRPs EGO-1, RRF-1, RRF-3, and the tudor domain EKL-1 and ERI-5 proteins were detected by western blot. Asterisk (*) indicates a non-specific band. (b) Model of the molecular compensation of ERI-5 by EKL-1. Interactions between the RdRP module and the N-terminal helicase domain of DCR-1 couple the generation of dsRNA by RRF-3 with processive DCR-1 activity. In the eri-5 mutant, this coupling is lost and the auto-inhibitory function of the helicase domain predominates, resulting in inefficient 26-G-RNA production. (c) Paralogous RdRP modules function sequentially in ERI endo-RNAi. An RdRP module comprised of RRF-3, DRH-3 and ERI-5 together with DCR-1 function at the initial step to generate 26-G-RNAs, the primary siRNAs of the ERI pathway that program ERGO-1. A paralogous RdRP module comprised of RRF-1, DRH-3 and EKL-1 is responsible for secondary siRNA generation that is independent of DCR-1. This abundant pool of small RNAs programs the WAGO Argonautes to effect endo-RNAi silencing. Paralogous EGO-1 complexes may be involved in this and other RNAi pathways. Some of the ERIC components were omitted from the model for clarity.

Similar articles

See all similar articles

Cited by 21 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback