Crosstalk between the actin cytoskeleton and Ran-mediated nuclear transport

BACKGROUND: Transport of macromolecules into and out of the nucleus is a highly regulated process. The RanGTP/RanGDP gradient controls the trafficking of molecules exceeding the diffusion limit of the nuclear pore across the nuclear envelope. RESULTS: We found genetic interaction between genes establishing the Ran gradient, nuclear transport factor 2 (ntf-2), Ran GTPase activating protein (Sd), and the gene encoding Drosophila Profilin, chickadee (chic). The severe eye phenotype caused by reduction of NTF2 is suppressed by loss of function mutations in chic and gain of function mutations in Sd (RanGAP). We show that in chic mutants, as in Sd-RanGAP, nuclear export is impaired. CONCLUSION: Our data suggest that Profilin and the organization of the actin cytoskeleton play an important role in nuclear trafficking

Melanotic Mutants in Drosophila: Pathways and Phenotypes

Mutations in >30 genes that regulate different pathways and developmental processes are reported to cause a melanotic phenotype in larvae. The observed melanotic masses were generally linked to the hemocyte-mediated immune response. To investigate whether all black masses are associated with the cellular immune response, we characterized melanotic masses from mutants in 14 genes. We found that the melanotic masses can be subdivided into melanotic nodules engaging the hemocyte-mediated encapsulation and into melanizations that are not encapsulated by hemocytes. With rare exception, the encapsulation is carried out by lamellocytes. Encapsulated nodules are found in the hemocoel or in association with the lymph gland, while melanizations are located in the gut, salivary gland, and tracheae. In cactus mutants we found an additional kind of melanized mass containing various tissues. The development of these tissue agglomerates is dependent on the function of the dorsal gene. Our results show that the phenotype of each mutant not only reflects its connection to a particular genetic pathway but also points to the tissue-specific role of the individual gene

Zfrp8/PDCD2 Interacts with RpS2 Connecting Ribosome Maturation and Gene-Specific Translation

<div><p>Zfrp8/PDCD2 is a highly conserved protein essential for stem cell maintenance in both flies and mammals. It is also required in fast proliferating cells such as cancer cells. Our previous studies suggested that Zfrp8 functions in the formation of mRNP (mRNA ribonucleoprotein) complexes and also controls RNA of select Transposable Elements (<i>TE</i>s). Here we show that in Zfrp8/PDCD2 knock down (KD) ovaries, specific mRNAs and <i>TE</i> transcripts show increased nuclear accumulation. We also show that Zfrp8/PDCD2 interacts with the (40S) small ribosomal subunit through direct interaction with RpS2 (uS5). By studying the distribution of endogenous and transgenic fluorescently tagged ribosomal proteins we demonstrate that Zfrp8/PDCD2 regulates the cytoplasmic levels of components of the small (40S) ribosomal subunit, but does not control nuclear/nucleolar localization of ribosomal proteins. Our results suggest that Zfrp8/PDCD2 functions at late stages of ribosome assembly and may regulate the binding of specific mRNA-RNPs to the small ribosomal subunit ultimately controlling their cytoplasmic localization and translation.</p></div

Gene expression and RNA processing in <i>Zfrp8</i> KD ovaries.

<p>(A) Quantitative RT-PCR of fold accumulation of select transcripts in young ovaries. (B-D) Exon-intron structures of <i>Pino</i> and <i>RpL36</i> loci and typical structure of pre-rRNA (according to FlyBase). Fragments/primer pairs used for qRT-PCR are show as navy blue rectangles. Primer pares are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147631#pone.0147631.s008" target="_blank">S3 Table</a>. (E) qRT-PCR of total RNA from control and <i>Zfrp8</i> KD ovaries showed no accumulation of non-spliced transcripts (nst) and un-processed pre-rRNA. Fragments for spliced transcripts are labeled "st". (A and E) Fold accumulation of the transcripts in controls (<i>nos-GAL4</i>/+ and <i>UAS-Zfrp8 RNAi</i>/+) and in <i>Zfrp8</i> KD (nos-<i>GAL4/UAS-Zfrp8 RNAi</i>) are shown relative to <i>w118</i> controls (mean ± SD; n≥ 3, normalized to <i>Rp49/RPL32</i>,<i>GAPDH2</i>,<i>and RpS2</i>).</p

Zfrp8 regulates cytoplasmic levels of RpS2, RpS11 and RpS13 in somatic cells.

<p>(A-B') <i>tj-GAL4</i> driven GFP-RpS18 expression shows similar levels and distribution in control (A-A') and <i>Zfrp8</i> KD(s) follicle cells. (C-D’) Similarly, RFP-RpL36 (red) levels are not changed in <i>Zfrp8</i> KD(s) follicle cells (D-D’) compared to controls (C-C’). (E-F") <i>Zfrp8</i> KD causes the reduction in cytoplasmic levels of GFP-RpS2 (green) in the follicle cells(F-F‴). Compare the protein levels in germaria and consecutive egg chambers to similar stages in control (E-E"). Lack of Zfrp8 also cause accumulation of GFP-RpS2 in nuclear clusters (arrow, compare E" and F"). (G-J") GFP-RpS13 (green, G-H") and GFP-RpS11 (green, I-J") were localized in the cytoplasm and nucleoli in follicle cells of control ovaries (G-G", I-I"). Cytoplasmic levels of both proteins were reduced in <i>Zfrp8</i> KD follicle cells, however the protein accumulation in nuclei and nucleoli was not affected (compare H" and G", J" and I"). DAPI (DNA, blue), size bar is 20μm.</p

Zfrp8 affects cytoplasmic localization of select transcripts.

<p>(A-C') Levels and localization of <i>TAHRE</i> transcripts (FISH, red) were low in control ovaries (A-A"), and strongly increased in the cytoplasm of <i>Armi</i>^<i>1</i><i>/Armi</i>^{<i>72</i>.<i>1</i>} ovaries (B-B', arrow). In <i>Zfrp8 KD</i> ovaries <i>TAHRE</i> RNA was present at similar levels in both nuclei and cytoplasm (D-D', arrows). (D-I') FISH with <i>Pino</i> (D-E), <i>sta</i> (F-G') and <i>RpL36</i> (H-I") probes. Levels of <i>Pino</i> RNA is somewhat increased in <i>Zfrp8 KD</i> ovaries (E-E'), and significantly elevated in nurse cell nuclei (arrows in D' and E'). (F-G') <i>sta</i> transcripts did not show changes in levels or localization in <i>Zfrp8</i> KD. (H-I') <i>RpL36</i> transcript showed increased nuclear accumulation in <i>Zfrp8</i> KD ovaries (arrows, in H' and I'). Additional images of ovarioles including germaria are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147631#pone.0147631.s004" target="_blank">S4 Fig</a>. DNA blue (DAPI), size bar is 20μm.</p

Graphic model of Zfrp8/PDCD2 function.

<p>Graphic model of Zfrp8/PDCD2 function.</p

Tet protein function during Drosophila development

<div><p>The TET (Ten-eleven translocation) 1, 2 and 3 proteins have been shown to function as DNA hydroxymethylases in vertebrates and their requirements have been documented extensively. Recently, the Tet proteins have been shown to also hydroxylate 5-methylcytosine in RNA. 5-hydroxymethylcytosine (5hmrC) is enriched in messenger RNA but the function of this modification has yet to be elucidated. Because Cytosine methylation in DNA is barely detectable in Drosophila, it serves as an ideal model to study the biological function of 5hmrC. Here, we characterized the temporal and spatial expression and requirement of Tet throughout Drosophila development. We show that Tet is essential for viability as Tet complete loss-of-function animals die at the late pupal stage. Tet is highly expressed in neuronal tissues and at more moderate levels in somatic muscle precursors in embryos and larvae. Depletion of Tet in muscle precursors at early embryonic stages leads to defects in larval locomotion and late pupal lethality. Although Tet knock-down in neuronal tissue does not cause lethality, it is essential for neuronal function during development through its affects upon locomotion in larvae and the circadian rhythm of adult flies. Further, we report the function of Tet in ovarian morphogenesis. Together, our findings provide basic insights into the biological function of Tet in Drosophila, and may illuminate observed neuronal and muscle phenotypes observed in vertebrates.</p></div