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. 2010 Apr;137(8):1305-14.
doi: 10.1242/dev.044255. Epub 2010 Mar 10.

Perinuclear P granules are the principal sites of mRNA export in adult C. elegans germ cells

Ujwal Sheth  1 , Jason Pitt, Shannon Dennis, James R Priess
Affiliations

Perinuclear P granules are the principal sites of mRNA export in adult C. elegans germ cells

Ujwal Sheth et al. Development. 2010 Apr.

Abstract

Germline-specific granules of unknown function are found in a wide variety of organisms, including C. elegans, where they are called P granules. P granules are cytoplasmic bodies in oocytes and early embryos. Throughout most of the C. elegans life cycle, however, P granules are associated with clusters of nuclear pore complexes (NPCs) on germ cell nuclei. We show that perinuclear P granules differ from cytoplasmic P granules in many respects, including structure, stability and response to metabolic changes. Our results suggest that nuclear-associated P granules provide a perinuclear compartment where newly exported mRNAs are collected prior to their release to the general cytoplasm. First, we show that mRNA export factors are highly enriched at the NPCs associated with P granules. Second, we discovered that the expression of high-copy transgenes could be induced in a subset of germ cells, and used this system to demonstrate that nascent mRNA traffics directly to P granules. P granules appear to sequester large amounts of mRNA in quiescent germ cells, presumably preventing translation of that mRNA. However, we did not find evidence that P granules normally sequester aberrant mRNAs, or mRNAs targeted for destruction by the RNAi pathway.

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Figures

Fig. 1.
Fig. 1.
Perinuclear P granules are asymmetric. (A) One arm of the adult hermaphrodite gonad showing nuclei (black) and P granules (green). (B) A single P granule as described in the text; compare with F. env, nuclear envelope; Pg-NPCs, P granule-associated nuclear pore complexes. (C-E) Examples of small, possibly newly formed, P granules in the mitotic zone showing nuclear pores (small black arrows), presumptive crests (white arrows) and the base (black arrowhead). (F-H) Perinuclear P granules in the pachytene zone (F,G) and in a late oogonium (H); note that NPCs are not clustered in late oogonia and oocytes. (I) Detached, cytoplasmic P granule in a young oocyte. Arrows and arrowheads are the same as in C-E. Scale bars: 500 nm.
Fig. 2.
Fig. 2.
Perinuclear P granules are persistent structures with dynamic components. (A,A′) P granules (numbered) on three pachytene germ nuclei filmed for 20 minutes. (B-B′) Photobleaching of a single perinuclear P granule (arrow); the elapsed time shown is 79 seconds. (C) Quantified fluorescence recovery curve for the P granule photobleached in B (black diamonds) compared with a single-exponential best-fit curve (white circles). (D) Dissected, normal gonad showing PGL-1::GFP in P granules and in the cytoplasm. (E) Gonad 5 hours after injection with α-amanitin showing PGL-1::GFP; clumps of PGL-1 persist in the distal mitotic germ cells but PGL-1 is lost from pachytene P granules. (F) High magnification of germ nuclei from the gonad in panel E; inset shows the same magnification of pachytene germ nuclei in an α-amanitin-injected ama-1(m188) control gonad. (G) Pachytene nuclei 5 hours after injection of cycloheximide. (H) The left panel shows pachytene-specific loss of PGL-1::GFP in an nxf-1(RNAi) gonad; the right panel shows the nuclear envelope staining (NPP-9) in the same gonad. Asterisk indicates the distal tip of the gonad. Scale bars: 2.5 μm in A,B; 25 μm in D,E,H; 5 μm in F,G.
Fig. 3.
Fig. 3.
DDX-19 is enriched at the base of P granules. (A) Gonad and intestine co-stained for NPP-9 and DDX-19 as indicated. (B) Eight-plane projection of gonad co-stained for NPP-9 and PGL-1 as indicated. (C-E) Single-plane merged images of dashed boxes in c, d and e are shown at high magnification in C (rotated), D and E, respectively. (F) Germ nucleus from a fourth larval stage (L4) gonad co-stained for DDX-19, PGL-1 and NPP-9 as indicated. (G) Adult oogonia showing detached P granules (green), NPP-9 (blue) and DDX-19 (red); inset shows a detached P granule at high magnification. Arrows indicate examples of detached P granules. Scale bars: 25 μm in A,B; 5 μm in C-E,G; 2.5 μm in F.
Fig. 4.
Fig. 4.
DDX-19 is concentrated at Pg-NPCs. (A) Surface view of germ nuclei in a gonad stained for NPP-9, PGL-1 and DDX-19 as indicated. (B) Merged images as indicated of a single germ nucleus (outlined) from A, stained as indicated. In all panels, arrows indicate large patches of NPP-9 associated with P granules (Pg-NPCs) and arrowheads indicate small foci of NPP-9 (NPCs). Scale bars: 2.5 μm.
Fig. 5.
Fig. 5.
NXF-1 is concentrated at Pg-NPCs. (A) Gonad and intestine (inset) co-stained for NPP-9 and NXF-1 as indicated. (B) Surface view of a single pachytene germ nucleus (outlined) stained for NXF-1 and PGL-1 as indicated; note extensive association of the two proteins (arrow). (C) Optical cross-section of a single pachytene nucleus stained for NXF-1, PGL-1 and DNA as indicated. Note NXF-1 localization to the base of P granules (arrow) and in the nucleolus (arrowhead). (D) Optical cross-section of a single pachytene nucleus stained for NXF-1, NPP-9 and DNA as indicated. Scale bars: 25 μm in A; 2.5 μm in B-D.
Fig. 6.
Fig. 6.
Newly exported mRNA traffics through P granules. (A-G) Panels show in situ hybridization detection with either alkaline phosphatase (A) or fluorescence (all others) of tissues/nuclei after recovery from heat shock for the times (t) indicated. Worms contained a high-copy array of a hsp16.2::gfp::unc-54 3′UTR transgene; nascent mRNA was visualized with an in situ probe against gfp mRNA. (A) Gonad and intestine. The transgene is expressed in all intestinal nuclei, somatic nuclei in the gonad (arrowhead) and germ nuclei within the expression zone (bracket). (B) High magnification of a pachytene nucleus in the expression zone showing P granules, mRNA and DNA as indicated; the arrowhead indicates the presumptive site of transgene (TG) expression. (C) Surface view of the gonad immediately after heat shock. (D) Optical section through the gonad core 30 minutes after heat shock; transgene-derived mRNA is abundant in the core underlying the expression zone. (E) Germ nuclei 15 minutes after heat shock showing nascent mRNA and the P granule marker GLH-1 as indicated. The arrow points to one example of perinuclear foci of mRNA coinciding with P granules. (F) High-magnification surface view of a single pachytene nucleus stained for the induced mRNA and GLH-1 as indicated. Arrows indicate the same as in E. (G) Optical cross-section through a pachytene nucleus showing mRNA (green) in P granules (red). Merge is in yellow. Scale bars: 25 μm in A,C,D; 2.5 μm in B,E-G.
Fig. 7.
Fig. 7.
Perinuclear mRNA localization requires P granules. (A) Intestinal cell 15 minutes after heat shock, showing little or no mRNA (green) adjacent to the NPP-9 zone (red). (B) Germ cell in the same animals showing perinuclear foci of RNA (green, left panel) adjacent to the NPP-9 zone (red) and coincident with P granules (green, right panel). (C,C′) Single pachytene germ cell from a heat-shocked, glh-1(RNAi) animal. Staining as indicated for transgenic mRNA, NPP-9 and DNA. Note the very low level of perinuclear mRNA (arrow; shown at high magnification in C′). (D,D′) Germ cell from mock-treated control animal stained as indicated and showing a high level of perinuclear mRNA (arrow; shown at high magnification in D′). Scale bars: 25 μm in A,C,D; 2.5 μm in B,E-G.
Fig. 8.
Fig. 8.
mRNAs targeted for degradation are not enriched in P granules. (A,B) FISH analysis of hsp16.2::gfp::pos-1 3′UTR expression on worms exposed to dsRNA for the pos-1 3′UTR. (A) Pachytene nuclei; double-headed arrow indicates an example of colocalized nascent mRNA (green) and P granules (red). (B) Low magnification view of the same gonad in A, showing that the induced mRNA has entered the gonad core and moved downstream to oogonia. (C-E) Panels show wild-type (WT) or mutant mex-3 mRNA detected by in situ hybridization in the strains indicated. mex-3(zu155) mRNA with a premature stop codon is degraded in the most mature oocytes, where it normally is translated (D, arrowhead), but persists when the NMD pathway is inhibited [smg-2(RNAi)] (E). (F-I′) FISH of the indicated mRNA in WT or mutant gonads (F-I); high magnification of the respective regions indicated by arrowheads are shown in F′-I′. Adjacent mRNA (green) and PGL-1 (red) signals are indicated in F′ for perinuclear P granules (arrowhead) and cytoplasmic P granules (arrows). Scale bars: 2.5 μm in A; 25 μm in B-I; 5 μm in F′-I′.
Fig. 9.
Fig. 9.
FG-repeat proteins in P granules. (A) Diagram showing FG repeats (vertical bars) in the nucleoporin NPP-9 and in four additional C. elegans proteins encoded by gonad-enriched mRNAs. (B,C) In situ hybridization for sense (B) and antisense (C) ddx-19(exon4) mRNA. (D,E) In situ hybridization for sense (D) and antisense (E) f58g11.2 mRNA. (F)Two-cell embryo showing colocalization of F58G11.2::GFP with cytoplasmic P granules (arrow). (G) Fifty-cell embryo showing colocalization of F58G11.2::GFP with P granules (arrow). Scale bars: 25 μm.
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