Email updates

Keep up to date with the latest news and content from BMC Cell Biology and BioMed Central.

Open Access Research article

Masked mRNA is stored with aggregated nuclear speckles and its asymmetric redistribution requires a homolog of mago nashi

Thomas C Boothby and Stephen M Wolniak*

Author Affiliations

Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA

For all author emails, please log on.

BMC Cell Biology 2011, 12:45  doi:10.1186/1471-2121-12-45

Published: 13 October 2011

Additional files

Additional file 1:

Table S1. Numbers of independent trials and representative photographs for experiments presented. Column 1 lists the experiment(s). Column 2 lists the figure(s) with representative images from specific experiment(s). Column 3 lists the total number of photographs taken for each experiment(s). Column 4 lists the total number of independent trials for each experiment(s).

Format: DOCX Size: 60KB Download file

Open Data

Additional file 2:

Figure S1. Differential fluorescent labeling of DNA and RNA via dual DAPI/PY staining. Microspores fixed at 30 minutes of development. PY (green) (a, c, e, g, i, k) and DAPI (blue) (b, d, f, h, j, l) detected via 488 nm and UV illumination respectively. a-b, Microspore stained with PY (green). c-d, Microspore stained with DAPI (blue). e-f, Microspore double-stained with DAPI (blue) and PY(green). g-h, Microspore pretreated with RNase and double-stained with both DAPI (blue) and PY (green). i-j, DNase pretreated samples were double-stained with DAPI (blue) and PY (green). k-l, DNase and RNase treated samples were double-stained with DAPI (blue) and PY (green). Bar = 25 μm.

Format: TIFF Size: 4.9MB Download file

Open Data

Additional file 3:

Movie S1. Pyronin Y labeling reveals discreet aggregations of subnuclear RNA after 2 weeks of dehydration. Microspores collected and fixed after 2 weeks without watering were sectioned and double stained with DAPI and PY. Subnuclear PY (green) signal was detected in successive confocal slices and these were rendered as a 3D model. Movie is in .mov format playable with QuickTime.

Format: MOV Size: 94KB Download file

Playing the movie within this page requires QuickTime and JavaScript. Read more

Open Data

Additional file 4:

Movie S2. Pyronin Y labeling reveals a partial coalescence of RNA aggregates after 4 weeks of dehydration. Microspores collected and fixed after 4 weeks without watering were sectioned and double stained with DAPI and PY. Subnuclear PY (green) signal was detected in successive confocal slices and these were rendered as a 3D model. Movie is in .mov format playable with QuickTime.

Format: MOV Size: 116KB Download file

Playing the movie within this page requires QuickTime and JavaScript. Read more

Open Data

Additional file 5:

Movie S3. Pyronin Y labeling reveals total coalescence of RNA aggregates after 6 weeks of dehydration. Microspores collected and fixed after 6 weeks without watering were sectioned and double stained with DAPI and PY. Subnuclear PY (green) signal was detected in successive confocal slices and these were rendered as a 3D model. Movie is in .mov format playable with QuickTime.

Format: MOV Size: 159KB Download file

Playing the movie within this page requires QuickTime and JavaScript. Read more

Open Data

Additional file 6:

Figure S2. RNA is not detectable within the nuclei of microspores after the first division. a-c, Microspore fixed and sectioned after 1.5 hours of development. a, DAPI (blue). b, Pyronin Y (green). c, merge of a and b. Bar = 25 μm.

Format: TIFF Size: 2.8MB Download file

Open Data

Additional file 7:

Movie S4. Spermidine additions cause the partial dissociation of aggregated nuclear RNA. Microspores were incubated with 10 mM SPD for 4 hours, fixed, sectioned and double stained with DAPI and PY. Subnuclear PY (green) and DAPI (blue) signals were detected in successive confocal slices and these were rendered as a 3D model. Movie is in .mov format playable with QuickTime.

Format: MOV Size: 180KB Download file

Playing the movie within this page requires QuickTime and JavaScript. Read more

Open Data

Additional file 8:

Movie S5. Spermidine additions cause the total dissociation of aggregated nuclear RNA. Microspores were incubated with 10 mM SPD for 4 hours, fixed, sectioned and double stained with DAPI and PY. Subnuclear PY (green) and DAPI (blue) signals were detected in successive confocal slices and these were rendered as a 3D model. Movie is in .mov format playable with QuickTime.

Format: MOV Size: 126KB Download file

Playing the movie within this page requires QuickTime and JavaScript. Read more

Open Data

Additional file 9:

Movie S6. 'Short' FISH probes detect foci of subnuclear masked transcripts that are distinct from chromatin. FISH against masked SPDS (red) was conducted on 20 μm sections taken from microspores fixed after 30 minutes after hydration. Probes were detected using avidin bound TexasRex. Sections were counterstained with DAPI (blue). Movie is in .mov format playable with QuickTime.

Format: MOV Size: 218KB Download file

Playing the movie within this page requires QuickTime and JavaScript. Read more

Open Data

Additional file 10:

Figure S3. Traditional FISH probes fail to detect masked SPDS transcripts within the nuclei of maturing microspores. a-c, Traditional biotinylated probes directed against SPDS transcript (red). Pre-prothallial (a), mid-prothallial (b), and late-prothallial (C) division microspore. The prothallial nucleus denoted by "p" in b and c. Bar = 5 μm.

Format: TIFF Size: 3.1MB Download file

Open Data

Additional file 11:

Figure S4. Defects in asymmetric division are not requisite for symmetric distribution of subnuclear material in Mv-Mago knockdowns. a-c, microspore subjected to Mv-Mago RNAi and fixed after 5 hours of development. (a) DAPI (blue), (b) phase contrast, (c) 4G3 labeling of U2B" (red). d-f, representative microspore subjected to Mv-Mago RNAi and fixed after 4 hours of development. (d) DAPI (blue), (e) phase contrast, (f) masked centrin transcripts (red) detected with 25mer biotinylated FISH probes. Spermatogenous cells denoted by "sp" and jacket cells denoted by "jk." Bar = 25 μm.

Format: TIFF Size: 5.5MB Download file

Open Data