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First published online 30 January 2008
doi: 10.1242/dev.015438

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The Drosophila hnRNP M homolog Rumpelstiltskin regulates nanos mRNA localization

Roshan A. Jain^* and Elizabeth R. Gavis

Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.

View larger version (36K): [in this window] [in a new window]   Fig. 1. Isolation of Drosophila Rump by tandem RNA-affinity purification. (A) TRAP-tagged RNAs with the streptavidin-binding S1 aptamer and six tandem copies of the MS2 stem-loop (MS2x6). TRAP+2'-3X RNA contains three tandem copies of the nos +2' localization element, whereas the TRAP-ctrl RNA contains transcribed vector sequence. (B) SYPRO Ruby-stained SDS-PAGE gel showing the final RNase eluates from purifications performed in parallel with TRAP+2'-3X and TRAP-ctrl RNAs. The protein band enriched for Rump is marked with an asterisk. The faint band of similar size in the control purification contains contaminating E. coli Hsp70. GST-MCP that elutes non-specifically is marked with an arrowhead. To permit comparison between the two samples, the amount of protein eluate loaded was adjusted according to the pre-elution RNA recovery for each sample. Molecular mass standards are indicated in kDa.

View larger version (65K): [in this window] [in a new window]   Fig. 2. Recognition by Rump correlates with nos RNA localization signal function. (A) UV-crosslinking of MBP-Rump to radiolabeled nos +1 and +2' element RNA probes. (B) UV-crosslinking of MBP-Rump to radiolabeled +2' element RNA in the absence (0) or presence of 200-, 400- and 600-fold molar excess of unlabeled +2' or +1 element RNA. (C) Above is a diagram of the +2' element with the two CGUU motifs (A and D) indicated. The lightly shaded region is highly conserved between D. melanogaster and D. virilis (Bergsten et al., 2001). The portions of the +2' element retained in the deletion mutants (1-5) are indicated by solid bars. C-to-G point mutations in the CGUU motifs were introduced individually (A or D) or in combination (AD) into the full-length +2' element. Beneath is shown UV-crosslinking of MBP-Rump to the indicated radiolabeled RNA probes. (D) Diagram of the nos-tub:nos+2 transgene, which contains only the nos +1 and +2' localization elements. (E,F) In situ hybridization to nos in preblastoderm nosBN embryos carrying (E) the nos-tub:nos+2 and (F) the nos-tub:nos+2(D)G:C transgenes. Because nosBN embryos lack endogenous nos mRNA (Wang et al., 1994), only the transgenic mRNA is detected.

View larger version (38K): [in this window] [in a new window]   Fig. 3. Molecular characterization of Drosophila rump and generation of a rump null mutation. (A) Genomic region surrounding the CG9373/rump locus. The rump transcript is outlined by the thick black line, with the ORF (lighter shading) and three RRMs (darker shading) indicated. The arrowhead marks the P element insertion P{SUPor-P}KG02834 in the first intron and parentheses indicate the limits of the rump1 deletion (dashed line). Transcripts flanking the rump locus are shown in lightest gray. Arrows indicate direction of transcription. (B) Northern blot of total RNA from wild-type (WT) and rump1 ovaries (O) and 0- to 2-hour-old embryos (E), probed for rump and the loading control rp49 (RpL32 - FlyBase). The trace amount of rump detected in early embryos is due to contamination from zygotic rump expression. (C) Immunoblot of total protein from wild-type (WT) and rump1 ovaries, wild-type 0- to 2-hour embryos (E), and cultured Schneider cells (S2). Molecular mass standards are indicated in kDa. After transfer, the membrane was cut below the 37 kDa marker. The top portion was blotted with anti-Rump and the bottom with anti-Snf as a loading control. (D) UV-crosslinking of radiolabeled +2' RNA to 0- to 2-hour embryo (E) or ovarian (O) extract from wild-type or rump1 animals. The position of the p75 binding activity (absent in rump1) is marked with asterisks. UV-crosslinking reactions carried out in parallel were immunoprecipitated with anti-Rump (Rump) or a control anti-β-galactosidase (βgal) antibody.

View larger version (44K): [in this window] [in a new window]   Fig. 4. Rump associates with nos mRNA in vivo. (A) RT-PCR to detect nos in RNA co-immunoprecipitated from wild-type (WT) and rump1 Drosophila ovaries with either anti-Rump or anti-β-galactosidase antibody. Poly(A+) RNA serves as positive control for RT-PCR. Reactions were performed with (+) and without (-) reverse transcriptase and products were visualized with ethidium bromide. Molecular weight standards in the first lane correspond to the 1 kb-Plus Ladder (Invitrogen). Beneath is shown an immunoblot of protein from each IP sample with anti-Rump antibody. (B) RT-PCR for nos (top) and anti-Rump immunoblot (bottom) as in A. Samples from WT (W) and rump1 (r) 0- to 2-hour embryos are indicated. Reactions were carried out with (+) or without (-) reverse transcriptase (RT) using total RNA (T) from the extracts used for IP as a positive control or RNA co-immunoprecipitated with anti-Rump antibody.

View larger version (64K): [in this window] [in a new window]   Fig. 5. Distribution of Rump protein in Drosophila ovaries and embryos. (A,B) Confocal images of stage-10 egg chambers from wild-type (A) and rump1 (B) females stained with anti-Rump antibody (red). The actin cytoskeleton is visualized in green, DNA in blue. nc, nurse cells; fc, follicle cells; oo, oocyte. (C,D) Immunohistochemical staining of preblastoderm wild-type (C) and rump1 (D) embryos with anti-Rump antibody. (E-F') Confocal images showing the posterior poles of blastoderm-stage wild-type (E) and rump1 (F) embryos stained with anti-Rump (red) and anti-Vas (green). DNA is in blue. (E',F') Red channel (Rump) only from E and F. In wild-type embryos, Rump is detected cytoplasmically and in all somatic nuclei, but it is specifically absent from nuclei of pole cells, which are marked by Vas (arrowheads).

View larger version (13K): [in this window] [in a new window]   Fig. 6. Phenotypic analysis of rump. Bar charts show distributions of embryonic cuticle phenotypes for the population of Drosophila embryos that complete embryonic development, binned by the number of abdominal segments produced. For each graph, embryos from different genotypes were collected in parallel under the same conditions and over the same time period. The statistical significance of differences observed between pairs of genotypes was determined using the 2 test. (A) Embryos with eight complete segments (8 seg) were binned separately from those containing partial or entire segment deletions (<8, black) for each genotype. nosBN heterozygotes (nos-/+) do not display segmentation defects (n=210). The rump1 distribution (n=332) differs significantly from nos-/+ or wild-type (not shown) (P<0.001). A single copy of a genomic rump transgene, P[rump], rescues the rump defect (n=483, P<0.001 versus rump1). Removing a single copy of nos from rump1 embryos using a rump1 nosBN recombinant chromosome in trans to rump1 (rump- nos-/rump- +, n=867) increases the percentage of defective embryos beyond that of rump mutants alone (37% versus 11%, P<0.001). Similar results were obtained using the rump1 nosBN recombinant chromosome in combination with either of three deficiencies that delete rump (data not shown). (B) Embryos were binned with: eight complete segments (8 seg); seven complete segments or seven to eight segments with partial deletions (7 seg); six or fewer segments (0-6). tudtux46 heterozygotes (tud-/+) exhibit a low percentage of abdominal defects (n=367). When rump is eliminated (tud-/+; rump-/rump-, n=266), the frequency and severity of abdominal defects are significantly increased (P<0.001).

View larger version (40K): [in this window] [in a new window]   Fig. 7. Rump regulates posterior localization of nos mRNA. The nos-tub:nos+2 transgene (see Fig. 2D) was introduced into either nosBN or rump1 nosBN/Df(rump) nosBN Drosophila females and provides the sole source of maternal nos mRNA. (A) Embryos that develop to produce cuticle were binned as follows: seven or eight partial or complete segments (7-8 seg); four, five or six partial or complete segments (4-6 seg); three or fewer segments (0-3 seg). The nos-tub:nos+2 transgene produces embryos with a range of segmentation (nos-, n=455). When rump is eliminated, segmentation is significantly reduced (nos- rump-, n=220, P<0.001). (B,C) Eve-stained embryos were binned according to the number of Eve stripes observed in the presence (n=153) or absence (n=212) of rump (P<0.001). Embryos representative of seven- and four-stripe bins are shown in C. (D,E) Posterior localization of nos-tub:nos+2 RNA was evaluated by in situ hybridization to 0- to 2-hour embryos and classified as substantial (++), weak or diffuse (+), or undetectable (-) as illustrated in E. Localization is significantly reduced by elimination of rump (nos-, n=382; nos- rump-, n=417, P<0.001).

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