Agranat et al. 10.1073/pnas.0710576105. |
Fig. 8. hUpf2 and hUpf3 are not found associated with supraspliceosomes. Supraspliceosomes prepared from HeLa cell nuclei as described in refs. 1 and 2 were fractionated in a sucrose gradient. The 200S fractions were pooled, dialyzed, concentrated, and refractionated in a second sucrose gradient. Aliquots from each fraction were run on a polyacrylamide/SDS gel. Western blotting was performed with anti-hUpf3a and anti-hUpf2. The same antibodies recognize the respective proteins in a NS from which the first gradient was prepared.
1. Spann P, Feinerman M, Sperling J, Sperling R (1989) Isolation and visualization of large compact ribonucleoprotein particles of specific nuclear RNAs. Proc Natl Acad Sci USA 86:466-470.
2. Sperling R, Sperling J (1998) in RNP Particles, Splicing and Autoimmune Diseases, ed Schenkel J (Springer, Berlin), pp 29-47.
Fig. 9. Cross-linking of oligomers containing ADAR1 and hUpf1 is not dependent on RNA. HeLa cell nuclear extract either subjected to RNase A or not treated was cross-linked by DMS for 20 min. (A and B) It was then Western blotted with anti-ADAR1 antibodies (A), and later reprobed with anti-hUpf1 antibodies (B). Non-cross-linked samples are shown for comparison. (C and D) An analogous experiment with RNase V1 treatment. (E) RT-PCR of actin RNA extracted from the HeLa cell nuclear extract, shown in A and B and in C and D, treated and untreated with RNase A (Left) and RNase V1 (Right).
Fig. 10. Two-dimensional gel electrophoretic analysis of non-cross-linked HeLa cell NE. (A) First-dimension SDS/PAGE analysis of untreated NE (−) and cross-linked NE (+). (B and C) HeLa cell NE was run on an SDS/PAGE gel without cross-linking. The gel was soaked in 3% b-mercaptoethanol and electrophoresed on a second identical gel. Western blotting was performed with anti-ADAR1 and anti-hUpf1 as marked on the right. (D) A merge of B and C. The arrows mark the direction of migration of the first and second dimension of the gel.
SI Materials and Methods
Preparation of Splicing Complexes and Supraspliceosomes.
Supraspliceosoems were prepared from HeLa cells (CILBIOTECH) as described in refs. 1 and 2. Nuclear supernatants enriched in supraspliceosomes were prepared from clean cell nuclei and fractionated on either 15-45% (vol/vol) sucrose or 10-45% (vol/vol) glycerol gradients. Centrifugations were carried out at 4°C in an SW41 rotor run at 41 krpm for 90 min (or an equivalent w2t). Fractions sedimenting at 200S (supraspliceosomes), at 70S (where native spliceosomes sediment), and at the top of the gradient were pooled and analyzed. When a second fractionation of supraspliceosomes was required, two to three gradient fractions corresponding to the 200S region of the gradient were combined, dialyzed, concentrated, and refractionated on a second sucrose gradient (1, 2).Western Blot Analysis.
For Western blot analysis, aliquots were applied directly to the wells of a 8 or 6% polyacrylamide/SDS gel and analyzed as described in ref. 3. hUpf1 was probed with rabbit polyclonal antibodies (kindly provided by J. Lykke-Andersen and J. Mendell) diluted 1:2,000, and visualized with horseradish peroxidase conjugated to affinity-pure goat anti-rabbit IgG F(ab)′ fragment or IgG (H+L) diluted 1:10,000. hUpf2 and hUpf3a were probed with the respective rabbit polyclonal antibodies (kindly provided by J. Lykke-Andersen) diluted 1:2,000, and visualized with horseradish peroxidase conjugated to affinity-pure goat anti-rabbit IgG F(ab)′ fragment or IgG (H+L) diluted 1:10,000. ADAR1 was probed with mAb 15.8.6 (kindly provided by K. Nishikura) diluted 1:2,000 and visualized with horseradish peroxidase conjugated to affinity-pure goat anti-mouse IgG F(ab)′ fragment or IgG (H+L), diluted 1:3,000. Sm proteins were probed with anti-Sm mAb Y12 (4) and visualized with horseradish peroxidase conjugated to affinity-pure goat anti-mouse IgG F(ab)′ fragment diluted 1:3,000. Actin was probed with anti-actin goat antibodies I19 (Santa Cruz) diluted 1:500 and visualized with horseradish peroxidase conjugated to affinity-pure donkey anti-goat IgG (H+L) diluted 1:10,000. All dilutions were done in net buffer (50 mM Tris•HCl, pH 7.5, 150 mM NaCl, and either 0.1% Triton or 0.05% Nonidet P-40).Immunoprecipitations.
Indirect IPs were performed as described in ref. 5 by binding the sample (either HeLa cell NE, or gradient fractions) to either anti-ADAR1 WI9 antibodies (kindly provided by K. Nishikura), anti-hUpf1 antibodies or anti-Sm mAb Y12, coupled either to protein G-Sepharose beads (Sigma) (for anti-Sm) or to protein A-agarose beads (Boehringer Mannheim) (for anti-hUpf1 and anti-ADAR1). As controls, we analyzed samples incubated with beads that had not been treated with the relevant antibody, and beads carrying the relevant antibody without prior incubation with the sample. The immunoprecipitated proteins were released by treatment with SDS-containing gel sample buffer.RT-PCR.
Total RNA was treated with RNase-free DNase I (Promega), and cDNA was synthesized with dT15 primer by using Moloney murine leukemia virus reverse transcriptase (Promega). PCRs contained cDNA synthesized from 0.1 to 0.7 mg of total RNA. For PCR analyses of ADAR1, PISD, TXNRD2, SARS, SDHA, and hUpf1, 20-ml aliquots containing 10 pmol of the relevant primer pair and 1.0 unit of TaqDNA polymerase (Promega) were used. For PCR analysis of actin, DAP3, and MGC10471, 25-ml aliquots containing 9 pmol of the relevant primer pair and 1.25 units of AmpliTaq DNA polymerase (Roche Molecular Biochmicals) were used. All gene transcripts were also analyzed using the Lamda Biotech 2´ Taq Master Mix. Amplification was carried out in a DNA Programmable Thermal Controller (PTC-100; MJ Research). PCR was carried out for 30 cycles for all genes except for actin (20 cycles) and the annealing temperatures were 60°C for all transcripts except for TXNRD2 (59°C).The following primer pairs were used for the PCRs: actin, sense, 5′-CAAGGCCAACCGCGAGAAGATGAC-3′, and antisense, 5′-AGGAAGGAAGGCTGGAAGAGTGC-3′; ADAR1: sense, 5′-GAAAATCGCGGTCTCCACT-3′, and antisense, 5′-TCCAAACCTGGGTCTGAGTT-3′; hUpf1: sense, 5′-AGGCCATCGACTCCCCGGTGTCTTT-3′, and antisense, 5′-GTAGAAGATGTTGGATGGGAAGGCG-3′; MAP3K14: sense, 5′-GGATGTGGGAACCCTTACCT-3′, and antisense, 5′-GCTTTGAGAGGCCTTTGATG-3′; TXNRD2: sense, 5′-GCTGGAGTATGGCTGTGTGGGG-3′, and antisense, 5′-TGCGCAGCTTGACTACCTCCTC-3′; PISD: sense, 5′-GGGCGTCCCCATGCGTAAGG-3′, and antisense, 5′-CTCGCCACCTCTTGTCTGCACC-3′; MGC10471: sense, 5′-ACAACTGAGACCCCCAAGTG-3′, and antisense, 5′-GGGATCGCTCACTCACTCTC-3′; DAP3: sense, 5′-AGCGCTTCCTGAACCAGATA-3′, and antisense, 5′-TCCCCAAAGAGCATTGATTC-3′; SDAH: sense, 5′-TGGGAACAAGAGGGCATCTG-3′, and antisense, 5′-CCACCACTGCATCAAATTCATG-3′; SARS: sense, 5′-CTGGCCTGTCTACCTGCTTC-3′, and antisense, 5′-AGAACTCCTCTGCGGTGGTA-3′.
1. Spann P, Feinerman M, Sperling J, Sperling R (1989) Isolation and visualization of large compact ribonucleoprotein particles of specific nuclear RNAs. Proc Natl Acad Sci USA 86:466-470.
2. Sperling R, Sperling J (1998) in RNP Particles, Splicing and Autoimmune Diseases, ed Schenkel J (Springer, Berlin), pp 29-47.
3. Yitzhaki S, Sperling J (1998) Concentrating dilute protein solutions for gel electrophoresis. Biotechniques 24:762-764,766.
4. Lerner EA, Lerner MR, Janeway CA, Jr, Steitz JA (1981) Monoclonal antibodies to nucleic acid-containing cellular constituents: probes for molecular biology and autoimmune disease. Proc Natl Acad Sci USA 78:2737-2741.
5. Yitzhaki S, Miriami E, Sperling R, Sperling J (1996) Phosphorylated Ser/Arg-rich proteins: Limiting factors in the assembly of 200S large nuclear ribonucleoprotein particles. Proc Natl Acad Sci USA 93:8830-8835.