Human HeLa and retinal pigment epithelial RPE-1 cells were maintained in DMEM and DMEM/F-12, respectively, supplemented with 10% fetal calf serum. Mouse NIH3T3 cells were maintained in DMEM supplemented with 10% calf serum.
Antibodies and Plasmids
Rabbit anti-PSPC1 was described previously (Fox et al., 2005
). P54NRB/NONO monoclonal antibody was made against a peptide at the C terminus of the PSPC1 protein with 50% identity to P54NRB/NONO (Absolutions Monoclonal Antibody Service, Perth, Australia). This monoclonal recognizes P54NRB/NONO in mouse and human cells (data not shown). An SC35 monoclonal was obtained from Sigma-Aldrich (St. Louis, MO). pCRII-TOPO-hNEAT1 (1-3729 nt) was made by RTPCR of hNEAT1 from HeLa cDNA and TOPO cloning (Invitrogen, Paisley, United Kingdom). pCDNA3-mNEAT1 (1-3177 nt) was a kind gift of John Hutchinson, Harvard Medical School, Boston, MA (Hutchinson et al., 2007
DNA Probes for EM-ISH
NEAT1 probes were biotin-labeled polymerase chain reaction (PCR)-amplified DNA fragments. We mixed 0.5 μg each of two adjacent or slightly overlapping 1- to 1.5-kb DNA fragments and then biotinylated them by nick-translation, except for human D1 probe in which 1 μg of a single 1.49-kb amplicon was used.
The human 5′-NEAT1 probe consisted of 1492-base pair and 1494-base pair DNA fragments corresponding to nt 230-1721 and 1751-3244 of the 22743 base pairs NEAT 1/MENε/β sequence (GQ859162) amplified with pCRII-TOPO-hNEAT1 DNA as a template. D1, D2 and 3′-end human NEAT1 probes were amplified from 100 ng of human genomic DNA, as follows: a 1491-base pair fragment (nt 7257-8748) for the D1 probe, fragments of 1299 base pairs and 1162 base pairs (nt 12841-14160 and 14735-15897) for D2 and fragments of 1087 base pairs (nt 20260-21346) and 1036 base pairs (nt 21647-22682) for the 3′-end NEAT1 probe. The mouse 5′-end NEAT1 probe was with 1511 and 1623 base pairs DNA fragments (nt 224-1734 and to nt 1481-3082 of NR_003513.2) amplified from 10 ng of pCDNA3-mNEAT1.
A 324-base pair fragment (nt 303-616 of HU1-1 sequence J00318.1) amplified from human genomic DNA was used as U1 snRNA probe. Amplicons were biotinylated by nick-translation for 3 h at 15°C in reactions containing 1 μg of DNA; 0.02 mM dATP, dCTP, and dGTP; and 0.05 mM biotin-16-dUTP.
Fixation and Embedding for Electron Microscopy
Cells fixed in 1.6% glutaraldehyde were dehydrated in ethanol and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate.
For embedding in Lowicryl K4M, cells were fixed 1 h either in 4% paraformaldehyde or in 1.6% glutaraldehyde at 4°C and dehydrated in methanol. Polymerization was at −30°C for 5 d under UV light. Ultrathin sections were stained with 4% uranyl acetate.
Antibodies and DNA probes used in this study were routinely tested on thin sections obtained with both fixatives. Because results obtained with both fixatives were comparable and to preserve the structures, the data shown and quantified in this study were from glutaraldehyde-fixed thin sections.
Localization of Proteins by Immunoelectron and Fluorescence Microscopy
For immuno-EM localization of the P54NRB/NONO and PSPC1 proteins, Lowicryl-embedded thin sections were incubated at room temperature for 1 h with the primary antibodies diluted 1/25 in phosphate-buffered saline (PBS) and for 30 min with anti-mouse or anti-rabbit antibodies coupled to 10-nm gold particles (BBInternational, Cardiff, United Kingdom). For immunofluorescence (IF), HeLa cells were fixed in 4% paraformaldehyde/PBS for 5 min at 4°C, permeabilized in 1% Triton-X-100/PBS for 15 min, and incubated with primary (rabbit anti-PSPC1, 1:500; mouse anti-P54NRB/NONO, 1:500; or mouse anti-SC35, 1:1000) and secondary anti-mouse-fluorescein isothiocyanate and/or anti-rabbit tetramethylrhodamine B isothiocyanate antibodies diluted at 1:250 (Jackson ImmunoResearch Laboratories, West Grove, PA). Cells were 4,6-diamidino-2-phenylindole (DAPI) stained, mounted in Vectashield (Vector Laboratories, Burlingame, CA), and imaged with an inverted Ti-E microscope (Nikon, Tokyo, Japan).
In Situ Hybridization at the Ultrastructural Level
Hybridization was performed as described previously (Visa et al., 1993
; Souquere et al., 2009
), for 3 h at 37°C or at 65°C for U1 snRNA and NEAT1
probes, respectively. Hybrids were detected with a goat anti-biotin antibody conjugated to 10-nm gold particles (BBInternational). Detection of DNA requires NaOH treatment of the thin sections before hybridization (Puvion-Dutilleul and Puvion, 1991
; Puvion-Dutilleul and Pierron, 1992
). This treatment was omitted in all our experiments. Specificity of RNA detection was controlled by a 89 or a 97% decrease of the U1 snRNA nuclear labeling when sections were pretreated either with RNase A (from 9.6 ± 3.4 to 1.1 ± 0.8 gold particles/μm2
; n = 18 and 14, respectively) or were pretreated with protease and subsequently with RNase (0.3 ± 0.3 gold particles/μm2
; n = 15) as in Supplemental Figure 1C.
Protease digestion was performed before hybridization with 0.2 mg/ml protease (from Streptomyces griseus, P6911, Sigma-Aldrich) in distilled water for 15 min at 37°C. RNase treatment was with 1 mg/ml RNase A (BDH, Poole, Dorset, United Kingdom) in 10 mM Tris-HCl buffer, pH 7.3, for 1 h at 37°C.
Thin sections were analyzed with a Tecnai Spirit (FEI, Hillsboro, OR) transmission electron microscope. Digital images were taken with a SIS MegaviewIII charge-coupled device camera (Olympus, Tokyo, Japan).
Gold particle density after EM-ISH was established on 17–28 cell profiles containing at least one IGAZ/PSP of a size ≥0.05 μm2. Approximately 125 μm2 of surrounding nucleoplasm were considered for each sample. Surface areas were determined with analySIS (Olympus Soft Imaging Solutions, Münster, Germany). Gold particles were counted by eye. Calculations and standard deviations were obtained with Excel (Microsoft, Redmond, WA).
To analyze gold particles distribution within IGAZ/PSP, four layers were delineated by progressive down-scaling of the external contour to 71, 50, and 25%. This generated four regions from the periphery to the interior with relative surface areas of 50, 25, 18.75, and 6.25%, respectively. Gold particles were counted and expressed as percentage of gold particles per area as shown in , with no corrections for differences in surface areas.
Figure 3. Localization of various regions of NEAT1_v2 in IGAZ/PSPs of HeLa cells by EM-ISH. (A) Schematic representation of the NEAT1 transcripts and of the probes used for EM-ISH (in red). Location of repeats is indicated by orange boxes (repeat masker scores (more ...)
IGAZ/PSP dimensions were determined on glutaraldehyde/Lowicryl thin sections after immunolabeling with an antibody or by EM-ISH with the human or mouse 5′-NEAT1 probes. Long and short axis of the fibrillar region of the IGAZ/PSP was measured in HeLa and NIH3T3 cells by using analySIS. A two-tailed Student's test was used to compare human and murine short axis (Sx).