TheC. eleganshomolog of nucleoporin Nup98 is required for the integrity and function of germline P granules

Abstract

All transgenes were driven by the pie-1 promoter (maternal expression), unless otherwise indicated, and were constructed by Gateway cloning (Invitrogen) (Landy, 1989). Coding sequences and predicted 3ЈUTRs were amplified from N2 genomic DNA (npp-7, npp-9, npp-10). Gene structure of npp-10 was analyzed by cDNA sequencing [ends of cDNAs were verified by 5Ј RACE with SL1 primer, and 3Ј RACE with oligo(dT) primer], and the corrected mRNA sequence, including four additional 5Ј exons, was submitted to GenBank (accession number GU174496). The GFP::mCherry construct was generated by PCR fusion. Transgenic lines were generated by microparticle bombardment (Praitis et al., 2001). RNAiRNAi was performed by feeding worms with bacteria expressing doublestranded RNA (Timmons and Fire, 1998 SUMMARYC. elegans P granules are conserved cytoplasmic ribonucleoprotein complexes that are unique to the germline and essential for fertility. During most of germline development, P granules are perinuclear and associate with clusters of nuclear pores. In an RNAi screen against nucleoporins, we have identified a specific nucleoporin essential for P granule integrity and function. The C. elegans homolog of vertebrate Nup98 (CeNup98) is enriched in P granules and associates with the translationally repressed, P granuleenriched mRNA nos-2 (nanos homolog). Loss of CeNup98 causes P granules to disperse in the cytoplasm and to release nos-2 mRNA. Embryos depleted for CeNup98 express a nos-2 3ЈUTR reporter prematurely. In the mouse, Nup98 immunoprecipitates with the germ granule component MVH. Our findings suggest that, in germ cells, the function of Nup98 extends beyond transport at the nuclear pore to include mRNA regulation in the cytoplasm.

Loss of PGL-1 and PGL-3, a family of constitutive germ-granule components, promotes germline apoptosis in C. elegans

“…Ultrastructural analysis of P granules in germ cells of adult hermaphrodite gonads has shown that P granules are tightly associated with nuclear pores on the nuclear envelope (Pitt et al, 2000). In addition, a nuclear pore component, Nup98, has been shown to be essential for the perinuclear association of PGL-1 (Voronina and Seydoux, 2010). Furthermore, FG repeats, a hallmark of many nuclear pore proteins, have been found in another family of P-granule proteins comprising GLH-1, -2 and -4 (Kuznicki et al, 2000).…”

Sm protein down-regulation leads to defects in nuclear pore complex disassembly and distribution in C. elegans embryos

“…OCF3: unc-119(ed3); jjIs1092[pNUT1:npp-1::gfp + unc-119(+)]; ltIs37 [pAA64: pie-1p::mCHERRY::his-58 + unc-119 (+)] (Golden et al, 2009) ; OCF17: unc-119(ed3); ocfIs3[pie-1 prom::GFP::npp-5::pie-1 3′UTR +unc119 (+)]; JH2184: unc-119(ed3); axIs1595 [pie-1 prom::GFP:npp-9::npp-9 3′UTR] (Voronina and Seydoux, 2010); AZ212: unc-119(ed3); [pie-1 prom::GFP::hisH2B::pie-1 3′UTR +unc119 (+)]; OCF22: unc-119(ed3); ocfIs5[pie-1 prom::mCherry::npp-1::pie-1 3′UTR +unc119 (+)]; OCF15: unc-119(ed3); ocfIs2[pie-1 prom::mCherry::sp12::pie-1 3′UTR +unc119 (+)]; OCF25(expressing mCherry::NPP-1,GFP::NPP-5): generated by crossing OCF22 with OCF17; OCF26(expressing mCherry::NPP-1,YFP::LMN-1): generated by crossing OCF4 (Golden et al, 2009) with OCF22 and backcrossing with N2; OCF28(expressing mCherry::NPP-1,GFP::TBA-2): generated by crossing OCF22 with OD57: unc-119(ed3); ltIs37 [pAA64: pie-1p::mCHERRY::his-58 + unc-119 (+)]; ltIs25 [pAZ132; pie-1p::GFP::tba-2 + unc-119 (+)] (McNally et al, 2006); OCF16(expressing GFP::NPP-1, mCherry::SP12): generated by crossing OCF15 with LW1094: unc-119(ed3); jjIs1094[pNUT1:npp-1::gfp + unc-119(+)]; MSN146(expressing CR::SP12, GFP::TBA-2): unc-119(ed3) III; ltIs76 [pAA178: pie-1p::mCHERRY::sp12 + unc-119 (+)]; ltIs25 [pAZ132; pie-1p::GFP::tba-2 + unc-119 (+)]. The nucleoporin-GFP and mCherry fusion proteins used in this study were not tested for rescuing the functionality of mutant alleles and therefore it is not known whether they are functional.…”

Molecular Insights into Intracellular RNA Localization

“…The molecular interactions that lead to the liquid-like behavior of P-granules was addressed by two recent studies that explored the molecular requirements for the formation of P-granules (Updike et al , 2011; Updike and Strome, 2009; Voronina and Seydoux, 2010). Both studies made the important observation that several proteins present in P-granules (the Vasa related proteins GHL-1, GHL-2 and GLH-3) contain repeats of the amino acids phenylalanine and glycine, and these were required in GLH-1 for granule formation (Updike et al, 2011).…”

“…Consistent with a link between the nuclear pore and P-granules, P-granules exhibited a size exclusion of fluorescent dextrans, similar to the nuclear pore (Updike et al, 2011). In addition, RNAi against a subset of nuclear pore proteins disrupted P-granule formation (Voronina and Seydoux, 2010). Furthermore, GFP-fusions of several nuclear pore proteins colocalized with P-granules, and mutation of the C. elegans Nup98 homolog disrupted P-granule formation.…”

“…Furthermore, GFP-fusions of several nuclear pore proteins colocalized with P-granules, and mutation of the C. elegans Nup98 homolog disrupted P-granule formation. Interestingly, the mouse Nup98 also interacted with the mouse Vasa homolog in mouse testes extract, suggesting that nucleoporins and nucleoporin-like interactions may be important for GCG formation in other systems(Voronina and Seydoux, 2010). Taken together, these results suggest that GCG exhibit many properties of nuclear pores and that regulated, homotypic interactions between FG-repeats are important for granule coalessence.…”