pubmed.ncbi.nlm.nih.gov

PGL proteins self associate and bind RNPs to mediate germ granule assembly in C. elegans - PubMed

  • ️Sat Jan 01 2011

PGL proteins self associate and bind RNPs to mediate germ granule assembly in C. elegans

Momoyo Hanazawa et al. J Cell Biol. 2011.

Abstract

Germ granules are germ lineage-specific ribonucleoprotein (RNP) complexes, but how they are assembled and specifically segregated to germ lineage cells remains unclear. Here, we show that the PGL proteins PGL-1 and PGL-3 serve as the scaffold for germ granule formation in Caenorhabditis elegans. Using cultured mammalian cells, we found that PGL proteins have the ability to self-associate and recruit RNPs. Depletion of PGL proteins from early C. elegans embryos caused dispersal of other germ granule components in the cytoplasm, suggesting that PGL proteins are essential for the architecture of germ granules. Using a structure-function analysis in vivo, we found that two functional domains of PGL proteins contribute to germ granule assembly: an RGG box for recruiting RNA and RNA-binding proteins and a self-association domain for formation of globular granules. We propose that self-association of scaffold proteins that can bind to RNPs is a general mechanism by which large RNP granules are formed.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.

PGL proteins autonomously form globular granules. (A–D) Immunofluorescence images of CHO cells expressing C. elegans P-granule components. (A) GST::PGL-3, (B) GST::MEX-3, (C) GST::GLH-1, (D) coexpression of PGL-3::6×His and GST::PGL-1. (E–H) Transgenic C. elegans expressing GFP-tagged proteins. (E and F) GFP::PGL-3 ectopically expressed via the pes-10 (E) or myo-2 (F) promoter. (G and H) GFP::GLH-1 ectopically expressed via the pes-10 (G) or myo-2 (H) promoter. (E and G) An ∼50-cell embryo. (F and H) Adult pharynx. Bars, 10 µm.

Figure 2.
Figure 2.

PGL proteins recruit other P-granule components in CHO cells. (A and B) SYTOX staining of DNA and RNA as shown without (A) or with (B) ribonuclease A treatment. (C–F) Immunofluorescence images of CHO cells. (C) Endogenous poly(A)-binding protein (PABP) in a cell not expressing PGL-3. (D) A cell expressing PGL-3::6×His; PGL-3::6×His, and endogenous PABP is shown. (E) A cell coexpressing PGL-3::6×His and GST::MEX-3. (F) a cell coexpressing PGL-3::6×His and GST::GLH-1. Bar, 10 µm.

Figure 3.
Figure 3.

PGL proteins function as the scaffold for P-granule assembly. (A) Schematic representation of a 4-cell stage C. elegans embryo. (B–H) Immunofluorescence images of 4-cell stage C. elegans embryos. Maximum projections of confocal Z-series images that cover whole embryos are shown. In each image, the protein that was detected is indicated. Bar, 10 µm. (B, D, and F) Control wild-type embryos. (C, E, and G) pgl-1(RNAi);pgl-3(RNAi) or pgl-1(RNAi);pgl-3(bn104, RNAi) embryos. (H) glh-1(RNAi);glh-4(gk225) embryo. PGLs: both PGL-1 and PGL-3 detected by the mixtures of monoclonal antibodies KT3 (anti-PGL-3) and KT4 (anti-PGL-1). GFP::PGL-3ΔRGG and GFP::MEX-3 were detected by anti-GFP. POS-1 and GLH-1 were detected by anti-POS-1 and anti-GLH-1, respectively.

Figure 4.
Figure 4.

PGL-3 has two distinct domains for self-interaction and recruitment of RNA and RNA-binding proteins. In each image, the protein that was detected is indicated. (A) Summary of the structure–function analysis of PGL proteins in CHO cells and C. elegans embryos. N.A., not applicable. N.D., not determined. *, 50% of cells formed small amorphous aggregates. (B–G) Immunofluorescence images of CHO cells. (B) A cell expressing GST::PGL-3; endogenous PABP was colocalized with GST::PGL-3 granules. (C) A cell expressing GST::PGL-3ΔRGG; endogenous PABP was excluded from GST::PGL-3ΔRGG granules. (D) A cell expressing GST::PGL-3(1–318). (E) A cell expressing GST::PGL-3(Δ160-319). (F) A cell coexpressing GST::PGL-3(1–318) and PGL-3::6×His; GST::PGL-3(1–318) were recruited to PGL-3::6×His granules. (G) A cell coexpressing GST::PGL-3(Δ160-319) and PGL-3::6×His; GST::PGL-3(Δ160-319) were not recruited to PGL-3::6×His granules. (H and I) pgl-1(RNAi);pgl-3(bn104) 4-cell stage embryo expressing GFP::PGL-3 (H) or GFP::PGL-3ΔRGG (I), stained by anti-GFP and anti-POS-1. (J–L) Wild-type C. elegans 4-cell stage embryos expressing GFP-tagged PGL-3 variants, stained by anti-GFP and anti-PGL-1 (KT4 mAb). (J) GFP::PGL-3, (K) GFP::PGL-3(1–318), (L) GFP::PGL-3(Δ160-319). Bars, 10 µm.

Figure 5.
Figure 5.

A two-step model of P-granule formation. (1) PGL proteins bind to diverse small mRNPs through RGG boxes. (2) PGL proteins self-aggregate through direct interaction between self-interaction domains. (3) P granules are assembled as large RNPs.

Similar articles

Cited by

References

    1. Amiri A., Keiper B.D., Kawasaki I., Fan Y., Kohara Y., Rhoads R.E., Strome S. 2001. An isoform of eIF4E is a component of germ granules and is required for spermatogenesis in C. elegans. Development. 128:3899–3912 - PMC - PubMed
    1. Arkov A.L., Wang J.Y., Ramos A., Lehmann R. 2006. The role of Tudor domains in germline development and polar granule architecture. Development. 133:4053–4062 10.1242/dev.02572 - DOI - PubMed
    1. Brangwynne C.P., Eckmann C.R., Courson D.S., Rybarska A., Hoege C., Gharakhani J., Jülicher F., Hyman A.A. 2009. Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science. 324:1729–1732 10.1126/science.1172046 - DOI - PubMed
    1. Brenner S. 1974. The genetics of Caenorhabditis elegans. Genetics. 77:71–94 - PMC - PubMed
    1. Decker C.J., Teixeira D., Parker R. 2007. Edc3p and a glutamine/asparagine-rich domain of Lsm4p function in processing body assembly in Saccharomyces cerevisiae. J. Cell Biol. 179:437–449 10.1083/jcb.200704147 - DOI - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources