Supplemental Material

This article contains the following supporting material:

• Supplementary data S1 - Sub-nuclear localization of RBII-48, RBII-78 and RBII-36 on rat brain sections. In situ hybridization was performed with Cy3-labeled oligonucleotide probes (red) complementary to RBII-48, RBII-78 and RBII-36 mature C/D RNAs. Nucleoli are visualized by a Cy5-labeled oligonucleotide directed against U3 snoRNA used as a nucleolar marker (green). The white arrow indicates the elongated, extra-nucleolar signal.
• Supplementary data S2 - Representative nuclear RNA tracks at the transcription site. In some cells, track RNA signals can be multi-branched (panels e/e' and g/g') or they can display a less obvious linear form (panels f/f' and h/h'). Note that in most cases, RNA signals fill up and adopt the same shape of the poor DAPI-stained regions of the nucleus, as clearly observed in panels c/c', d/d', f/f', g/g'. The specificity of the RNA signals detected by the spliced-specific Cy3-labelled probe is demonstrated by its absence in the mouse embryonic fibroblasts (panels a/a'; no Bsr sequence homology in the mouse genome) and in Ref52, an immortalized rat fibroblast cell line that does not express Bsr gene (panels b/b'). Bar: 5 μm.
• Supplementary data S3 - Do nuclear dot-like RNA signals correspond to single RNA molecules? The structural organisation of Bsr punctuated signals is puzzling. They might correspond either to clusters of multiple spliced Bsr RNAs in an unknown nuclear structure, or they might be single RNA molecules diffusing through the nucleoplam. To distinguish between these two possibilities, the number of hybridized Cy3-labelled spliced-probes within 285 individual nuclear dots in 8 different nuclei was quantified by using the method described in (Femino et al., 1998), see also below). We reasoned that a large number of probes (i.e. largely exceeding 85, the largest theoretical number of hybridized probes) will formally exclude the single RNA molecule hypothesis. We found that 48% of the nuclear dots do not contain more than 10 probes, with a range of 1 to 124 probes hybridized per dot. These results are consistent with the notion that each nuclear dot might represent a single (or a few) RNA molecule(s).
  
  Fluorescence signal quantification method. Digital images from a series of 10 to 14 focal planes were acquired with a 0.2 or 0.3 µm-step through a hybridized cell. Images were restored with the deconvolution algorithm of the Huygens Professional Software (Scientific Volume Imaging BV, Hilversrum, The Netherlands). Fluorescence dot quantification was performed with the Imaris software (Bitplane AG, Zurich, Switzerland). Images were threshold to remove the background noise (typical threshold was of ~7% of the maximum intensity of the image). The total fluorescence intensity of each dot was then calculated and converted into the number of molecules of hybridized probes by use of the following calibration procedure set up by (Femino et al., 1998). Briefly, solutions of probe molecules dispersed in 3µl of mounting medium with final concentrations ranging from 0.25ng/µl to 4ng/µl were mounted between a slide and a coverslip. Knowing the molecular weight of the probe and the area of the coverslip, the concentration unit was converted form ng/µl into the number of molecules of probes per pixel. For each probe solution, three single plane images were taken under the same conditions as the hybridized cells. The average gray level per pixel was given by the Metamorph software (Universal Imaging). The calibration curve was obtained by plotting the gray level per pixel against the number of molecules per pixel and used to determine the number of molecules of probes hybridized for each Bsr dot signal.
• Supplementary data S4 - The intra-cellular location of Bsr RNAs upon drug treatments. A) REFs were treated (+) or not (-) by actinomycinD (AMD, 5µg/ml for 300 min), leptomycin B (LMB, 10nM for 300 min), 2D-glucose 6mM + sodium azide (2Dglc+azide; 10mM for 300 min), arsenite (0.5 mM for 30 min), cycloheximide (CHX; 20µg/ml for 240 min), puromycin (200µg/ml for 240 min) or 5,6-dichloro-1-β-D-ribobenzimidazole (DRB, 25µg/ml for 180 min.). Spliced Bsr RNAs were detected with the Cy3-labelled spliced oligonucleotide probe and the percentage of cells with cytoplasmic signals was estimated by counting a minimum of 400 cells for each treatment. Since control values can differ from one experiment to another (see text), the untreated controls were set to 1. B) A representative AMD-treated cell (right) that displays nearly complete relocation of spliced Bsr RNAs in the cytoplasm. An untreated cell is shown as control (left). Note: increased background signals were routinely observed after AMD treatment. Bars: 5 μm.
• Supplementary data S5 - The intra-cellular fate of spliced Bsr RNAs in cultured primary hypothalamic neurons. (A) spliced Bsr RNAs form multiple dispersed nucleoplasmic dot-like signals and a single, large nuclear RNA track per nucleus (top), with in a small population of neurons (5-8%), a few dot-like signals in the dendritic compartments (bottom). To further confirm the specificity of these relatively weak cytoplasmic RNA signals, successive RNA FISH were carried out with Cy3-labeled spliced probes first (left) and then with Alex488-labeled probes (middle) designed to recognise the internal part of Bsr exons. (B) Bsr RNAs associated with SGs in arsenite-treated neurons (0.5 mM for 3h). Bsr RNAs are detected with a Cy3-labeled spliced-RNA probe while SGs (or dendrites) are visualized by a Cy5-labeled polydT probe. The white arrows indicate the position of Bsr dot-like signals. Two different focal planes of the same cell are shown. The contrasts have been enhanced to highlight Bsr dots. Bars: 5 μm.