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First published online February 24, 2006
doi: 10.1242/10.1242/dev.02275

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RNA-binding proteins SOP-2 and SOR-1 form a novel PcG-like complex in C. elegans

Tingting Zhang^*, Yinyan Sun^*, E Tian^*, Hansong Deng, Yuxia Zhang, Xin Luo, Qingchun Cai, Huayi Wang, Jijie Chai and Hong Zhang

National Institute of Biological Sciences, Beijing, People's Republic of China.

View larger version (78K): [in a new window]   Fig. 1. Ectopic expression of Hox genes and other defects in sor-1 loss-of-function mutants. (A) Expression of egl-5 in a wild-type L2 larva. The expression of egl-5 is restricted to the tail (not shown) and is absent in the head (bracket). (B) Ectopic expression of egl-5 in a sor-1 mutant. egl-5 is ectopically expressed in many head neurons (marked with bar). (C) Expression of mab-5 in a wild-type larva. The expression of mab-5 is confined to the posterior region (arrow). (D) Ectopic expression of mab-5 in a sor-1 mutant. mab-5 is expressed in the head neurons and hypodermal cells. The expression of mab-5::gfp is absent from the tail region (arrow). (E) Normal expression of pkd-2::gfp in all nine pairs of B-type ray neurons (arrow) and in four head neurons (arrowhead) in a wild-type male. PKD-2::GFP marks both the cell body and the axon of neurons. (F) Ectopic expression of pkd-2::gfp in a sor-1(RNAi) male, indicating that ectopic rays are generated in the anterior body region. Five ectopic rays are located between the arrows. (G) sor-1 mutant hermaphrodites have a protruding vulva phenotype (arrow). (H) Partial hermaphrodite-to-male sexual transformation. An ectopic male ray (arrow), as visualized with pkd-2::gfp, is generated in a sor-1 hermaphrodite. This animal also contains a pkd-2::gfp-positive neuron in the head (arrowhead), which is normally expressed in four male-specific head neurons.

View larger version (86K): [in a new window]   Fig. 2. Synergistic effects of sor-1 and sop-2. (A-F) An earlier onset of ectopic expression of egl-5 in sop-2; sor-1 mutants. Lack of expression of egl-5::gfp in the head region in a `pretzel' stage embryo (arrow) in sor-1 (A,B) and sop-2 (C,D) mutants. Ectopic expression of egl-5::gfp in the head region in a `pretzel' stage sop-2; sor-1(RNAi) embryo (E,F) (arrow). egl-5 is expressed in the tail region in all mutant embryos. Nomarski images (A,C,E); Fluorescence images (B,D,F). (G) Expression of egl-5::gfp in the tail of a sor-1(RNAi) mutant (bracket). (H) Expression of egl-5::gfp in the tail of a sop-2 mutant (bracket). (I) Dramatically expanded expression domain of egl-5::gfp in the tail of a sop-2; sor-1(RNAi) mutant (bracket). (J) Ectopic expression of egl-5::gfp in the middle body region in a sop-2; sor-1(RNAi) mutant (marked with a bar). Irregular fluorescence particles are gut autofluorescence.

View larger version (51K): [in a new window]   Fig. 3. Molecular structure of sor-1 and its predicted gene product. (A) sor-1 was mapped genetically to the region of LG III between unc-32 and sma-3. sor-1 is SL1 trans-spliced. The intron/exon boundaries were confirmed by sequencing the cDNAs yk526e9 and yk336g5 and the RT-PCR products. (B) The protein sequence of SOR-1. Mutant residues in sor-1(bp1), sor-1(bp2) and sor-1(bp3) are shown in red. (C) Direct RNA binding by SOR-1. Various SOR-1 protein fragments were incubated with the radiolabeled RNA (5'UTR of egl-5, C08C3 nucleotides 41280-41840) and binding was assessed by EMSA. Protein-RNA complex is marked by bracket. (D) Binding of SOR-1(443-530) to RNA at a titrated protein concentration (ng/µl). Binding of RNA by SOR-1 is competed by unlabeled ssRNA (10x), dsRNA (10x) and partially by tRNA (100x), but not by DNA (10x and 100x refer to mass excess). The ssRNA, dsRNA and DNA competitors are derived from T08D10 (nucleotides 7800-8408). Similar results were obtained using several other competitors, including the ones derived from Y110A7A (nucleotides 59588-60325) and Y113G7B (nucleotides 81290-82026). Thirty ng/µl of proteins (final concentration) were used for each RNA-binding reaction unless otherwise noted for the titration experiments.

View larger version (103K): [in a new window]   Fig. 4. Colocalization of SOR-1 and SOP-2 in nuclear bodies. (A) Expression of sor-1::gfp in the hypodermal nuclei of adult animals. SOR-1::GFP is distributed throughout the nuclei with accumulation in distinct nuclear speckles. (B) Expression of SOR-1 in an early embryo. Polyclonal antibodies against SOR-1 coupled with rhodamine-conjugated goat anti-rabbit IgG were used to detect expression. SOR-1 is localized in distinct nuclear bodies (arrow). (C-E) Localization of SOP-2::GFP (C) and SOR-1 (D) into nuclear bodies. (E) The superimposable confocal images of SOR-1 (red) and SOP-2::GFP (green) shows that they colocalize (yellow). (F,G) Unaltered localization of SOP-2 into nuclear bodies in sor-1(RNAi) mutant embryos (F). Absence of staining of SOR-1 in sor-1(RNAi) mutant embryos confirms the specificity of the antibody (G). (H-J) The merged images of SOP-2 (H) and DAPI (I) shows that SOP-2(bx91) is localized in cytoplasm in sop-2(bx91) mutant embryos (J). (K-M) The merged images of SOR-1 (K) and DAPI (L) shows that SOR-1 is localized in cytoplasm in sop-2(bx91) mutant embryos (M). (B-M) Single confocal sections.

View larger version (26K): [in a new window]   Fig. 5. Direct interaction between SOR-1 and SOP-2. (A) GST-SOP-2 fusion proteins were incubated with 35S-labeled SOR-1 (amino acids 1-503) to map the interaction domain in SOP-2. Only the N-terminal domain (amino acids 1-217) of SOP-2 bound SOR-1. Smaller fragments of SOP-2 were used to further map the minimal interaction domain. (B) The N terminus of SOR-1 interacts with SOP-2. GST-SOR-1 fusion proteins were incubated with 35S-labeled SOP-2. Twenty percent of the 35S-labeled protein used in the binding reactions was shown. No interaction was detected between the 35S-labeled protein and GST alone or GST fused to the C terminus of SOR-1 but binding was detected between SOP-2 and the N terminus of SOR-1. Smaller fragments of SOR-1 were used to map the interaction domain further. (C) Schematic representation of protein interaction domains of SOR-1 and SOP-2 (connected by arrow). SOP-2 also contains a protein interaction SAM domain, which mediates its self-association (Zhang et al., 2003). (D) Interaction between N terminus of SOP-2 (residues 58-140) and SOR-1 (residues 48-200). The chromatogram of gel filtration is shown on the top and the fractions from the complex are shown at the bottom. Q indicates proteins purified through ion exchange column.

View larger version (28K): [in a new window]   Fig. 6. Lack of orthologs of SOR-1 and SOP-2 in C. briggsae. (A) Schematic representation of sor-1 and its neighboring genes in C. elegans and in C. briggsae. ZK1236.4 corresponds to a transposon in the genome. CBG18140 does not have a clear homolog in C. elegans. Although sor-1 (ZK1236.3) shows some similarity with CBG18142, the latter does not appear to be the ortholog of sor-1 (see Results). (B) Schematic representation of sop-2 and its neighboring genes in C. elegans and in C. briggsae. CBG20941 does not have a clear homolog in C. elegans. sre-52 and sre-54 encode a class of 7 TM chemoreceptors (sre family). (C) Model for the putative SOP-2/SOR-1 complex-mediated transcriptional repression. SOR-1, SOP-2 and perhaps other unidentified components form an RNA-binding complex, which may further recruit chromatin remodeling activity. The RNA components could be involved in targeting the SOP-2/SOR-1 complex to target loci and/or maintaining the integrity of the complex and the distinct nuclear bodies formed by SOR-1 and SOP-2.