Fig 1.
Schematic overview of nuclear mRNA export.
The primary steps in mRNA export are shown. (1) Recruitment of the NXF-1–NXT-1 (NXF1-p15) heterodimer to mRNA (2) nascent mRNP-NXF-1 is docked to the nuclear pore, (3) followed by translocation (4) and cytoplasmic release (5) at the cytoplasmic filaments.
Fig 2.
nxf-1(t2160ts) mutant shows unattached Pun pharynx.
(A) Schematic overview of pharyngeal morphogenesis during WT and nxf-1(t2160ts) embryogenesis. (B) Differential interference contrast (DIC) image of comma, 1.5-fold and 2-fold embryos showing WT embryo elongation with pharynx attached to the buccal cavity and similar images of nxf-1(t2160ts) mutant embryos in which the pharynx failed to reach the buccal cavity (unattached phenotype, Pun). White arrows point to the basement membrane surrounding the developing pharynx. (C) Genomic organization of the nxf-1 gene and NXF-1 protein domains. Red arrowhead indicates the t2160ts mutation. (D) mRNA visualized by FISH with a poly-dT oligonucleotide conjugated with Cy3. Strong accumulation of poly(A)mRNA in the nxf-1(t2160ts) nucleus contrasts with the WT nucleus where no signal accumulation was distinguishable, and mRNA concentrates in the cytoplasm. mRNA accumulation is measured as relative fluorescent units (RFU) in the cellular nucleus and cytoplasm of FISH-stained embryos.
Table 1.
Phenotypes observed in nxf-1 mutants and after RNAi of mRNA export factors and EJC core proteins.
Fig 3.
NXF-1 is concentrated in the nucleus.
(A) (top panel) NXF-1::GFP expression in the early two-cell stage embryos (A1, A1') during cell division of the AB blastomere (A2, A2'), and in the four-cell stage (A3, A3'). Bottom panel shows quantification of the NXF-1 diffused into the cytoplasm during cell division. (B) NXF-1 expression in C. elegans adult somatic cells. Pictures show details of the head (B1, B1'), tail (B2, B2'), gonad (B3, B3') and oocytes (B4, B4'). White arrows indicate cellular nuclei. Black arrowheads indicate cytoplasmic granules. A detail of the localization of NXF-1 in the nuclear envelope of gonadal nuclei is shown in B3, B3' inset. Scale bar: 10μm.
Fig 4.
nxf-1(t2160ts) Pun pharynxes show normal tissue differentiation but failed arcade polarization.
(A) Representative three-fold WT and nxf-1(t2160ts) embryos expressing pMYO-2::GFP reporter in pharyngeal muscle and (B) RIC-19::GFP expression in M1 and M2 neurons in three-fold embryos. (C) WT and nxf-1(t2160ts) embryos expressing PHA-4::GFP and (D) CDH-3::GFP reporter in the arcade cells, lateral epidermal cells and the seam cells. (E) Representative images of Ppha-4::membrane–GFP reporter expression and (F) PAR-6::GFP shows a clear cell polarization in WT but a mislocalized expression in nxf-1(t2160ts) embryonic epithelia. Corresponding DIC (left) and fluorescence images (right) are paired for each embryo. Anterior is left, dorsal is up.
Fig 5.
mRNA export components NXF-1/NXF1, NXT-1/NXT1 and HEL-1/UAP56 are required for C. elegans foregut tubulogenesis and epidermal morphogenesis where EJC core components RNP-4/Y14 and MAG-1/Mago-nashi are essential for C. elegans epidermal morphogenesis but dispensable for pharyngeal morphogenesis.
Worms expressing AJM::GFP reporter in the L4 stage were fed with RNAi clones of hel-1, nxf-1, nxt-1, mag-1 and rnp-4. RNAi clones of nxf-1 and nxt-1 were diluted with L4440 at a 1:1 concentration for a “milder” RNAi effect. Worms were grown at 15°C and images were taken the next day. Embryos depleted of nxf-1, nxt-1 and hel-1 show pharyngeal and hypodermal defects (asterisks). WT, rnp-1(RNAi) and mag-1(RNAi) arcade cells (between white lines) expressed AJM::GFP, illustrating that they are epithelialized.
Fig 6.
nxf-1(t2160ts) gonads show a reduced mitotic region with fewer cells in the M phase.
(A) Representative pictures of the α-pH3 immunostained gonads. N2 (WT) and nxf-1(t2160ts) worms were synchronized. Gonads were dissected, fixed, immunostained with α-pH3 and counterstained with DAPI. (B) Mitotic cell quantification in the gonadal mitotic region of WT and nxf-1(t2160ts) background. (C) pH3 positive cell quantification in the gonadal mitotic region of WT and nxf-1(t2160ts) background. The number of pH3 positive cells and mitotic cells was manually counted in Z-Stack. Student’s t-test; ***P<0.0001. n = 20 gonads. Scale bar: 10μm.
Fig 7.
Analysis of the embryonic cell cycle progression in nxf-1(t2160ts) mutant compared to a WT embryo under the same conditions.
(A) Representative examples of cell lineages within the AB blastomere descendants from a WT embryo and the same cells from an nxf-1(t2160ts) mutant embryo. (B) Cell division takes significantly longer in the mutant than in the WT. As a consequence, nxf-1(t2160ts) develops slower than a WT embryo recorded under the same conditions. (C) Duration of each cellular generation for WT and nxf-1 (t2160ts) mutant embryos (n = 12 cells from 3 different WT and nxf-1 (t2160ts) embryos).
Table 2.
Up- and down-regulated pathways determined by KEGG enrichment analysis.
Significant pathways have been selected according to their q-value. Genes, within each category, showing significantly altered expression are shown in the right-hand column.
Fig 8.
F-actin visualized by Phalloidin-iFluor 488 staining.
(A) F-actin is apically enriched in arcade cells in WT worms. White squares indicate the anterior end and arcade cells. (B) Images of phalloidin stained embryos were taken at different focal planes. Epidermal fluorescence was measured at the upper levels of the embryo whereas pharyngeal and intestinal fluorescence were measured at the center of the embryo. The histogram shows the decrease in F-actin staining in the nxf-1(t2160ts) mutant compared to the WT (n = 14) on different regions of the embryo (pharynx, intestine and epidermis). F-actin is significantly reduced throughout the nxf-1(t2160ts) embryos. Student’s t-test; *p<0.05, **p<0.01. n = 20. Scale bar: 10μm.
Table 3.
NXF-1 and its protein partners co-immunoprecipitated by LC-MS/MS (Liquid Chromatography-Mass Spectrometry/Mass Spectrometry).