11 research outputs found
The dynamic localisation of eGFP-Nxt1.
<p>Phase contrast (A, C, E, G, I, K, M, O) and fluorescence images (B, D, F, H, J, L, N, P) of ectopically expressed eGFP-Nxt1(wild type) or eGFP-Nxt1-D126N (D, P). (A–D) Testes tips oriented with younger cells towards the top. When expressed in male germline cells the wild type protein (A, B) localised predominantly to the nucleus, and remained stable as spermatocytes matured. The signal from mutant protein was dramatically weaker, and predominantly cytoplasmic (C, D). In early primary spermatocytes the WT protein localised to an intra-nuclear dot, adjacent to the nucleolus (F) as well as throughout the nucleoplasm. As spermatocytes matured the protein relocated to one or more cytoplasmic puncta, frequently found adjacent to the uniformly labelled nucleus (H). Early elongation spermatids showed cytoplasmic puncta and eGFP-Nxt1 localisation to one face of the nuclear envelope (J). Label persisted until late elongation in spermatid nuclei (L). In the ovarian follicular epithelium the wild type eGFP-tagged protein was predominantly nuclear (N), while the D126N form of the protein was not detected (P).</p
Addition of introns partially restores reporter expression in <i>Nxt1</i> but not <i>comr</i>.
<p>RNA in situ hybridisation reveals CG42355-LacZ reporter construct expression in control testes, and no signal in <i>Nxt1<sup>Z2-0488</sup></i>/<i>Nxt1<sup>DG05102</sup></i> or <i>comr</i> mutant testes. Addition of one intron generates detectable, although weak, expression in <i>Nxt1<sup>Z2-0488</sup></i>/<i>Nxt1<sup>DG05102</sup></i> testes, but not in <i>comr</i> mutant testes.</p
Modelling the potential effect of D126N.
<p>The human Nxt1 protein structure was used to model the <i>Drosophila</i> Nxt1 protein folding (WT) using Swisspdb viewer, and the region containing the mutated residue is shown. The side chain of D126 is indicated with an arrow. Computed H-bonds are shown in green. Running the model with the D126N substitution (arrow) revealed disruption of two computed H-bonds.</p
RNAi of <i>Nxt1</i> in spermatocytes phenocopies the <i>Nxt1<sup>z2-0488</sup></i> defect.
<p>(A) Expression of an <i>Nxt1</i> RNAi hairpin construct in the male germline causes a highly penetrant meiotic arrest phenotype. (B) Q-RT-PCR reveals that expression of <i>Nxt1</i> target genes is reduced in <i>Nxt1</i> RNAi testes (dark bars), and that the effect is similar to that seen in <i>Nxt1<sup>z2-0488</sup></i>/<i>Nxt1<sup>DG05102</sup></i> mutant testes (light bars).</p
Mutation of <i>Nxt1</i> leads to meiotic arrest in testes and failure of head eversion in pupae.
<p>(A) All stages of spermatogenesis are seen in wild type testes by phase contrast microscopy. Large round cells near the apical region (*) are primary spermatocytes, while spermatids elongated up the length of the testis (arrow). In <i>Nxt1<sup>z2-0488</sup></i>/<i>Nxt1<sup>DG05102</sup></i> mutant (B) or <i>Nxt1<sup>z2-0488</sup></i>/<i>Deficiency</i> (C) testes, only stages up to mature primary spermatocytes are present. (D) Hoechst fluorescence and (E) phase contrast images of wild type mature primary spermatocytes reveals a prominent nucleolus and distinct chromosome territories in each nucleus. (F) Hoechst fluorescence and (G) phase contrast imaging of <i>Nxt1</i> mutant spermatocytes reveals that the cells arrest with partially condensed chromatin and a prominent nucleolus. (H) <i>Nxt1</i>/+ pupae had everted spiracles, and distinct head, thorax and abdomen (h, t, a), while <i>Nxt1</i> mutant pupae (I–K) often had only partially everted spiracles, and the thorax was at the extreme anterior of the pupal case. Many mutant pupae were also curved (J).</p
Gene expression defects in <i>Nxt1</i> mutant testes places <i>Nxt1</i> in a novel meiotic arrest class.
<p>Comparison of gene expression defects in Nxt1, <i>aly</i> (<i>aly</i>-class mutant) and <i>nht</i> (<i>can</i>-class mutant) by RNA in situ hybridisation. All test genes were expressed in primary spermatocytes of control testes (A–F), with some transcripts persisting into spermatid elongation stages. No signal was detected in tMAC mutant testes (<i>aly</i>) (A″–F″), while all were detected at basal level (<i>CG11249</i> and <i>ran-like</i>) or higher (<i>CycB</i>, <i>CG15177</i>, <i>djl</i>) in <i>nht</i> testes (A′″–F′″). <i>CycB</i>, <i>CG15177</i> and <i>CG42355</i> expression was detected in <i>Nxt1<sup>z2-0488</sup></i>/<i>Nxt1<sup>DG05102</sup></i> mutant testes (A′″, C′″, F′″) while <i>CG11249</i>, <i>ran-like</i> and <i>djl</i>, were not detected in <i>Nxt1</i> testes. <i>CG3927</i> expression (control) is restricted to primary spermatocytes, and robust expression was detected in all four genotypes (G–G′″). Higher magnification images of the CG3927 staining (H–H′″) show a honey-comb appearance of the signal in all four genotypes, indicating mRNA accumulation predominantly in the cytoplasm rather than the nucleus (arrows). Scale bars are 50 µm. Bar in G′″ applies to A–G′″, bar in H′″ applies to H–H′″.</p
The RNA Export Factor, Nxt1, Is Required for Tissue Specific Transcriptional Regulation
<div><p>The highly conserved, Nxf/Nxt (TAP/p15) RNA nuclear export pathway is important for export of most mRNAs from the nucleus, by interacting with mRNAs and promoting their passage through nuclear pores. <i>Nxt1</i> is essential for viability; using a partial loss of function allele, we reveal a role for this gene in tissue specific transcription. We show that many <i>Drosophila melanogaster</i> testis-specific mRNAs require <i>Nxt1</i> for their accumulation. The transcripts that require <i>Nxt1</i> also depend on a testis-specific transcription complex, tMAC. We show that loss of <i>Nxt1</i> leads to reduced transcription of tMAC targets. A reporter transcript from a tMAC-dependent promoter is under-expressed in <i>Nxt1</i> mutants, however the same transcript accumulates in mutants if driven by a tMAC-independent promoter. Thus, in <i>Drosophila</i> primary spermatocytes, the transcription factor used to activate expression of a transcript, rather than the RNA sequence itself or the core transcription machinery, determines whether this expression requires <i>Nxt1</i>. We additionally find that transcripts from intron-less genes are more sensitive to loss of <i>Nxt1</i> function than those from intron-containing genes and propose a mechanism in which transcript processing feeds back to increase activity of a tissue specific transcription complex.</p></div
<i>Nxt1</i> is required for full expression of tMAC dependent reporter constructs.
<p>(A) Schematic diagram to illustrate reporter construct design. Promoter regions are narrow coloured boxes, transcribed regions are broad boxes. The <i>djl</i> promoter and UTRs are light green; ORF grey; intron dark green. <i>CG42355</i> promoter and UTRs are pale pink; ORF grey; introns dark pink. UAS-djl-LacZ uses 5×UAS (red) and hsp70 minimal promoter to the TSS (orange) as the promoter. All reporter transcripts comprise 5′ UTR (from <i>djl</i> or <i>CG42355</i>) fused to <i>Adh</i> 5′UTR (cyan) LacZ ORF (blue) and SV40 3′UTR (cyan). (B) RNA in situ hybridisation with a <i>LacZ</i> probe reveals reduced expression of reporter constructs in <i>Nxt1<sup>z2-0488</sup></i>/<i>Nxt1<sup>DG05102</sup></i> mutant testes compared to control. (C) q-RT-PCR to test <i>LacZ</i> reporter expression in mutant spermatocytes compared to controls. Expression for each transgene was normalised as 1 in the control, and the relative expression in <i>Nxt1<sup>z2-0488</sup></i> homozygotes (blue) and <i>comr</i> (red) mutants was calculated. (D) q-RT-PCR to test the effect of inclusion of introns on reporter expression in <i>Nxt1<sup>z2-0488</sup></i> homozygote spermatocytes compared to controls. Expression for each transgene was normalised to 1 in the control samples and relative expression in mutant was calculated. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003526#s1" target="_blank">Introduction</a> of two introns into the reporters increased expression in the mutant testes compared to the no intron version. Two separate reporter lines for transgenes are shown.</p
Microarray analysis of <i>Nxt1</i>-dependent gene expression in testes.
<p>(A) Scatter plot showing normalised expression levels in control testes vs. <i>Nxt1<sup>z2-0488</sup></i>/<i>Nxt1<sup>DG05102</sup></i> mutant testes of all probes on the Affymetrix expression arrays. Red dots correspond to probes for genes with introns, blue dots are probes for intron-less genes. (B) FlyAtlas expression data for 485 probes that are 16× or more down-regulated in <i>Nxt1</i> testes compared to control. “Up” indicates probes whose signal is 2× or more higher in the specific tissue than whole fly, “Down” indicates 2× or more lower signal in the specific tissue than whole fly. TA ganglion = Thoracioabominal ganglion. (C) Scatter plots showing log2-transformed fold changes in <i>Nxt1</i>-vs-control and <i>aly</i>-vs.-control pairwise combination. Red indicates intron-containing genes, blue represents intron-less genes. (D) Venn diagram to show that most highly <i>Nxt1</i>-dependent genes (blue) have testis-specific expression (green), and that most highly <i>Nxt1</i>-dependent genes are also highly <i>aly</i>-dependent (red), but not <i>vice versa</i>.</p
Expression of both nascent and mature mRNA of target genes is reduced in <i>Nxt1</i> mutant spermatocytes.
<p>(A) Expression of nascent (unspliced) RNA was quantified using primer pairs a+c, b+c or b+d, while mRNA levels were determined with primer pairs a+sr or sf+d. (B) <i>CG12699</i> and <i>ocn</i> control genes were expressed at broadly similar levels in WT and mutant testes, while expression of both nascent and mature RNA for the Nxt1-dependent test genes <i>CG4907</i>, <i>CG10478</i>, <i>CG11249</i>, <i>CG14546</i>, <i>CG16736</i>, <i>CG17380</i>, <i>CG32487</i>, <i>CG33125</i> and <i>pif2</i> were reduced. For each gene, the expression levels of nascent transcript (dark gray) and mature transcript (light gray) in <i>Nxt1<sup>z2-0488</sup></i>/<i>Nxt1<sup>DG05102</sup></i> are presented as relative levels to those found in the wild-type (arbitrarily assigned as 1).</p