Supplemental Data. Regulating Gene Expression. through RNA Nuclear Retention

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1 Supplemental Data Regulating Gene Expression through RNA Nuclear Retention Kannanganattu V. Prasanth, Supriya G. Prasanth, Zhenyu Xuan, Stephen Hearn, Susan M. Freier, C. Frank Bennett, Michael Q. Zhang, and David L. Spector Supplemental Experimental Procedures Reporter Constructs For generating reporter constructs containing various CTN-RNA regions the membrane localization signal was PCR amplified from a peyfp-mem vector (BD Biosciences, Clontech, Palo Alto, CA) and cloned upstream of CFP in the pecfp-c1 vector (pmem-cfp-c1 vector). Further, the 3 UTR of mcat2 mrna was PCR amplified from mouse genomic DNA and cloned into the multiple cloning site (MCS) of pmem-cfp-c1 (pmem-cfp-mcat2 3 UTR). For generating the CTN-RNA 3 UTR containing plasmids, the 3 UTR of CTN-RNA spanning from the mcat2 mrna poly(a) site to the CTN-RNA poly(a) site was PCR amplified (the forward primer was designed downstream of the mcat2 poly(a) site to exclude the proximal poly(a) site) and cloned into the MCS of pmem-cfp-mcat2 3 UTR. The PCR amplicon was cloned downstream of the mcat2 3 UTR to generate pmem-cfp-ctnr 3 UTR. As mentioned within the text, for various experiments the MEM-CFP constructs were under the control of CMV, SV2 or CTN-RNA promoters. Construction of Stable Cell Lines NIH-3T3 and C127I cells, grown in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% calf serum and penicillin-streptomycin (Invitrogen, Carlsbad, CA), were transfected with 2-5 µg of desired constructs by electroporation. Stable transformants were selected in 2 mg/ml G418 (Invitrogen, Carlsbad, CA) and isolated colonies were maintained in selection medium. Cells from positive colonies were plated as single cells in conditioned medium and maintained in G418 (0.5 mg/ml) containing medium. Immunofluorescence (IF) Immunofluorescence was performed according to a previously published protocol (Prasanth et al., 2003). For nuclease digestion cells were treated with RNase A (1mg/ml) for 15 min at RT or DNase I (100U/ml) for 1 hr at 37 o C. Primary antibodies were added for 1 h at room temperature: anti-rna polymerase II; H14 [1:20], anti-pml [1:200], anti-b23 [1:400], anti-hp1-α [1:200] anti-sf2/asf [1:50], anti-psp1 [1:100], anti-psf [1:400]). Fixed cells were examined using an Axioplan 2i fluorescence microscope (Carl Zeiss, Thornwood, NY) equipped with Chroma filters (Chroma Technology, Brattleboro, VT), and OpenLab software (Improvision, Boston, MA) was used to collect digital images from an ORCA cooled charge-coupled device camera (Hamamatsu, Bridgewater, NJ). Alternatively, fixed cells were observed using a Deltavision system (Applied Precision, Issaqua, WA) equipped with an Olympus IX-70 microscope and a

2 60X/1.4 N.A. objective lens. Images were deconvolved using SoftWorx and presented as projections of Z-stacks. Immuno-EM Analysis NIH-3T3 cells stably expressing the YFP-PSP1α were grown on thermanox coverslips (Electron Microscopy Sciences, Fort Washington, PA), fixed with 2% formaldehyde and 0.2% glutaraldehyde in PBS and then dehydrated with ethanol by the progressive lowering of temperature followed by embedding and polymerization in Lowcryl K4M (Electron Microscopy Sciences, Fort Washington, PA) at 35 0 C. 20 nm colloidal gold was chemically conjugated to RNase T 1 from Aspergillus oryzae (Sigma, St. Louis, MO) (Cheniclet and Bendayan, 1990). Thin sections collected on nickel grids were first incubated for 1 hr at RT in primary antibody to GFP (rabbit polyclonal 290, Abcam Inc, Cambridge, MA), washed in PBS and incubated for 15 minutes at 37 0 C in a solution of RNAse-gold and goat anti-rabbit antibody conjugated to 5 nm colloidal gold (Amersham Biosciences Corp, Piscataway, NJ). The grids were further washed, air dried and then counterstained with aqueous uranyl acetate. Bromo-UTP and BrdU Incorporation Sense and antisense oligo treated C127I cells were rinsed briefly in glycerol buffer (20 mm Tris- HCl ph 7.4, 5.0 mm MgCl2, 25% glycerol, 0.5 mm phenylmethylsulfonyl fluoride, and 0.5 mm EGTA) followed by permeabilization for 3 min in glycerol buffer supplemented with 0.1% Triton X-100. Cells were rinsed in glycerol buffer and incubated in transcription buffer (100 mm KCl, 50 mm Tris-HCl ph 7.4, 5 mm MgCl2, 0.5 mm EGTA, 25% glycerol, 1.0 mm phenylmethylsulfonyl fluoride, 2.0 mm ATP, 0.5 mm GTP, 0.5 mm CTP, 0.2 mm 5-bromo- UTP, and 1.0 µg/ml RNAsin) for 10 min at 37 C. Cells were washed twice in glycerol buffer and fixed in 2% formaldehyde for 15 min at RT. The bromo-utp was detected by using Alexa-594 conjugated anti-brdu antibodies (Molecular Probes Inc, Eugene, OR). To analyze cell cycle progression, cells were incubated with BrdU (10 µm) for a 10 min pulse and fixed with 2% formaldehyde. Detection of the BrdU labeling was carried out as described previously (Spector et al., 1998). Promoter Analysis To determine the promoter(s) used to transcribe CTN-RNA, total RNA was isolated from various mouse tissues and cell lines and was reverse transcribed using a RT-primer (RT-2 primer) specific to CTN-RNA designed from the unique 3 UTR (Figure S4). These cdnas were further PCR amplified using forward PCR primers from each of the exon 1 variants (1A to 1E) representing promoters A to E respectively and a reverse primer from exon 3 and analyzed by agarose gel electrophoresis.

3 Figure S1. Localization of CTN-RNA Deconvolved images of the localization of CTN-RNA in WT-MEFs. A represents a merge of CTN-RNA and DNA stained by DAPI. Scale bars equal 5 µm.

4 Figure S2. Specificity of the CTN-RNA Probe (A and A ) RNA-FISH analysis using a nick translated pbluescript vector probe in NIH-3T3 cells did not show any specific labeling. (B and B ) Following a pre-treatment of NIH-3T3 cells with RNase A, RNA-FISH using CTN- RNA probes did not show a hybridization signal demonstrating the specificity of the CTN-RNA probe for RNA. DNA was stained with DAPI. The scale bar equals 5 µm.

5 Figure S3. Expression Profile of CTN-RNA in Different Mouse Cell Lines Northern blot analysis using a random labeled probe from the 3 UTR of CTN-RNA in mouse liver and various cell lines; transformed NIH-3T3 fibroblasts, WT-MEFs, embryonic P19 cells and C127I mouse mammary tumor cells. β-actin was used as loading control.

6 Figure S4. Promoter Analysis of CTN-RNA (A) Schematic representation of mcat2 mrna and CTN-RNA. The long arrows represent the position of RT primers used (RT-1 and RT-2). The small arrows represent the primer pairs used to amplify the RT products with specific forward primers for each exon 1 variant (representing each promoter A to E having unique exon 1A to E1 respectively) and common reverse primer (from exon 3). (B and C) Using RT primers from a CTN-RNA specific region (RT-2 primer) followed by PCR with all the exon 1 variant specific primer pairs revealed that promoter A is used by CTN-RNA in all the tissues tested including mouse liver, brain, lungs, skeletal muscle, C127I cells and RAW264.7 macrophage cells (with and without γ-ifn + LPS treatment) (see table).

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8 Figure S5. Characterization of Nuclear CTN-RNA Foci Dual localization studies of CTN-RNA (red: A, B, C & D) with antibodies to RNA polymerase II (green: A ), nucleolar protein B23 (green: B ), HP1α (green: C ) and PML (green: D ) in WT- MEFs did not show any colocalization (see merges A, B, C & D ). Deconvolved images showing dual labeling of CTN-RNA (red: E) and SF2/ASF (green: E ) revealed that some CTN- RNA foci partially overlapped at the periphery of nuclear speckles (merge in E and higher magnification of inset in E ). Scale bar equals 5 µm. Figure S6. RNA Molecules Are Integral Components of Paraspeckles Immuno-EM using an anti-gfp antibody in NIH-3T3 cells stably expressing YFP-PSP1α showed enrichment of YFP-PSP1α (5 nm colloidal gold particles) in paraspeckles (arrow). RNase T1, which has a high affinity for RNA, was chemically conjugated to 20 nm colloidal gold particles and showed predominant enrichment in the YFP-PSP1α positive paraspeckles suggesting the presence of RNA in paraspeckles. Boxed region in Figure A is shown at higher magnification in A. The arrowhead denotes a nucleolus.

9 Figure S7. Paraspeckles Are Sensitive to RNase A Treatment (A) Untreated HeLa cells immunolabeled with PSP1 antibody showed intact paraspeckle labeling. (B) PSP1 antibody labeling following RNase A treatment in HeLa cells showed no PSP1 labeling of paraspeckles. The scale bar equals 5 µm. (C) Untreated NIH-3T3 cells expressing YFP-PSP1α showed the presence of YFP-PSP1α in paraspeckles. (D) YFP-PSP1α expressing NIH-3T3 cells treated with RNAse A showed no YFP-PSP1α localization in paraspeckles. Scale bar equals 5 µm. (E and F) Dual immunolabeling of untreated HeLa cells (E-E ) and DNase I treated cells (F-F ) labeled with PSP1 antibody (red) and a centromere marker antibody AnaC (green). Note that DNase I treatment did not abolish the PSP1 labeling of paraspeckles. DNA was stained with DAPI (blue; A, B, C, D, E & F ).

10 Figure S8. RNase A Treatment Abolishes Paraspeckle Integrity Immuno-EM using anti-gfp antibody in NIH-3T3 cells stably expressing YFP-PSP1α showed GFP positive paraspeckles in untreated cells (see the arrows in Figure A). Pre-treating the cells with RNAse A resulted in the complete disorganization of the paraspeckles (B). The arrowhead in figure B denotes an intact IGC, which is nuclease resistant. Figure S9. PSF Localizes to Paraspeckles Immunolabeling of PSF (Red: A) in WT-MEF cells transiently expressing YFP-PSP1α (green: A ) showed complete colocalization to paraspeckles (yellow labeling in merge: A ). DNA was stained with DAPI. The scale bar equals 5 µm.

11 Figure S10. The Antisense Oligonucleotides Achieve Efficient Knockdown of CTN-RNA in the Presence of IFNγ + LPS in Macrophage Cells (RAW264.7) Significant knockdown of CTN-RNA (AS109 showed ~80%) was achieved in mouse macrophage RAW264.7 cells. Antisense treatment of CTN-RNA in RAW cells, following IFNγ + LPS incubation resulted in significant knockdown of CTN-RNA (~65%). β-actin levels are shown as loading controls. inos levels showed the activation of NO pathway post IFNγ + LPS treatment.

12 Figure S11. CTN-RNA Knockdown Does Not Affect Global Transcription or Cell-Cycle Progression (A and B) In situ transcription analysis in control C127I cells treated with sense oligonucleotides (SENSE 109; A and A ) as well as cells treated with antisense oligonucleotides (AS109; B and B ) showed comparable levels of Br-UTP incorporation. DNA was stained with DAPI. The bar represents 5 µm. (C and D) Cells treated with sense 109 oligo (C and C ) or antisense 109 oligo (D and D ) showed normal cell cycle progression as visualized by BrdU (10 min pulse) incorporation. DNA was stained with DAPI. The bar represents 5 µm. (E and E ) NIH-3T3 cells stably expressing YFP-PSP1α treated with sense (E) and antisense 109 (E ) did not show any difference in the number of YFP-PSP1α positive paraspeckles. The bar denotes 5 µm.

13 Figure S12. Posttranscriptionally Cleaved CTN-RNA Is Translation Competent NIH-3T3 cells were transiently transfected with SV2-MEM-CFP (A-B), SV2-MEM-CFPmCAT2 3 UTR (C-D) and SV2-MEM-CFP-CTN-RNA 3 UTR (E-F). Expression and localization was monitored 24 hours posttransfection in untreated (A, C & E) and cells treated with IFNγ + LPS for 8hrs (B, D & F). DNA is stained with 7-AAD. The scale bar equals 5 µm.

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