Extract preparation

Extracts for verification of transient protein expression and for NMD inhibition

Transfected HeLa or 293T cells were collected by trypsinisation 48 hr after transfection, washed twice with PBS and lysed in RIPA buffer (50 mM TRIS-HCl pH 8, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40, 10%

glycerol and complete protease inhibitors [Roche]).

Extracts for immunoprecipitation experiments for paraspeckle proteins

HeLa cells at a confluency of ~70% were collected by trypsinisation, washed twice with PBS and lysed in 10 mM HEPES-KOH, pH 7.9, 100 mM KCl, 5 mM MgCl2, 0.5%

Nonidet P-40, 1 mM DTT (Invitrogen), 100 units/ml RNaseOUT (Invitrogen), 0.2%

vanadyl ribonucleoside complex (Fluka) and complete protease inhibitors.

Extracts for immunoprecipitation experiments to analyse the interaction of SF1 with Malat-1 and Men-ε/β

HeLa cells were collected by trypsinisation at a confluency of ~70%, washed twice with PBS and lysed in Bradford-compatible RIPA (RIPAB) buffer (50 mM TRIS-HCl, pH 8, 100 mM NaCl, 2 mM EDTA, 0.1% SDS, 0.25% sodium deoxycholate, 0.1% NP40 and 10% glycerol), supplemented with complete protease inhibitors (Roche) and 100 u/ml RNaseOUT (DundeeCellProducts).

Extracts from mouse tissues

Mouse tissues were extracted as described above and 100 mg of each tissue were resuspended in 1 ml of ice-cold tissue lysis buffer (TLB) (10 mM Tris, pH 8, 1% Triton X-100, 2 mM EDTA, 10% glycerol, 137 mM NaCl) supplemented with complete protease inhibitors (Roche). Tissues were homogenised in an AxonLab Homogenizer (DispomixDrive, MedicTools) and centrifuged at 1,000 rpm for 1 min at 4°C to remove cellular debris. The supernatants were sonicated for 15 min in 30 sec intervals and centrifuged at 13,000 rpm for 20 min at 4°C. The supernatant was used for Western blotting.

Protein concentrations were measured with the Bradford reagent (Bio-Rad).

C.10. Immunoprecipitations (IP) IP of paraspeckle proteins

Cell extracts (corresponding to 1.5 mg total protein) were pre-cleared by incubation for 1 hr at 4°C with 20 µl of packed Dynabeads Protein G (Invitrogen) rinsed three times with 0.1 M sodium acetate, pH 5.3. Anti-SF1 (1 µg) in 20 µl sodium acetate was coupled to 20 µl of packed Dynabeads for 1 hr at RT, and the beads were washed with

IP buffer (50 mM TRIS-HCl, pH 7.4, 100 mM NaCl, 1 mM MgCl2, and 0.05% NP40) to remove unbound antibody. Following a 1-hr incubation at 4°C of the lysates with anti-SF1-coated beads, the supernatant was removed and the beads were washed six times with IP buffer. Bound proteins were eluted by incubation of the beads in SDS loading buffer for 5 min at 95°C followed by SDS-PAGE and Western blotting. For RNase A treatment, extracts were prepared in RIPAB buffer without addition of RNAse inhibitors. They were then incubated with 0.3 mg/ml RNase A (Sigma) for 15 min at RT prior to IP, and no RNAse inhibitors were used during the IP. For IP of p54^nrb the beads were coated with 1 µg of antibody.

IP to analyse the interaction of SF1 with Malat-1 and Men-ε/β

Cell extracts (corresponding to 6 mg of total protein) were pre-cleared by incubation for 2 hr at 4°C with 80 µl of packed Dynabeads Protein G (Invitrogen) that were washed six times with 0.1 M sodium acetate, pH 5.3. Anti-SF1 (4 µg), anti-PSF (16 µg) or anti-IgG2a (5.6 µg), were coupled to 80 µl of packed Dynabeads each for 2 hr at RT, and the beads were washed three times with RIPAB buffer to remove unbound antibody. Following a 2-hr incubation at 4°C of the lysates with antibody-coated beads, the supernatant was removed and the beads were washed six times with RIPAB buffer.

One quarter of the beads was used for SDS-PAGE and Western blotting, the remainder for RNA isolation.

C.11. SDS-PAGE and Western blotting

Proteins were denatured by incubation in SDS loading buffer for 5 min at 95°C, separated by 7.5% SDS-PAGE (unless stated otherwise) and transferred to nitrocellulose. Membranes were blocked with 5% milk in TN-Tween (20 mM TRIS-HCl, pH 7.5, 150 mM NaCl and 0.05% Tween 20) and probed with primary and secondary antibodies. They were further washed with TN-Tween and antibodies were detected with the SuperSignal kit (Pierce).

C.12. Isolation of RNA, RT-PCR and real-time PCR RNA isolated from HeLa and MCF7 cells

Cells were washed twice in ice-cold PBS and lysed in the appropriate amount of Trizol.

RNA isolated from mouse tissues

Mouse tissues were extracted as described above and 100 mg of each tissue were resuspended in 1 ml of Trizol. Tissues were homogenized in an AxonLab Homogenizer (MedicTools), centrifuged at 1,000 rpm for 1 min and the supernatant was used for RNA isolation.

RNA isolated from IP experiments to test the SF1 - Malat-1, Men-ε/β interaction

After the last wash of the immunoprecipitation, beads were resuspended in 1 ml Trizol and incubated for 10 min at RT. Subsequently, the beads were removed and the supernatant was used for RNA isolation.

Total RNA isolation from cells, mouse tissues and IPs (Dynabeads) with Trizol () was performed according to the manufacturer’s instructions.

Reverse transcription (RT) for HeLa cells, MCF7 cells and mouse tissues

Isolated RNA was treated with 1,000 units of RQ1-DNase (Promega) for 30 min at 37°C and extracted with phenol-chloroform. cDNA was prepared with 1 μg of oligo-dT(12-18) (Sigma), 10 mM dNTPs and 1 μg RNA for 5 min at 65°C, followed by addition of 40 units RNaseOUT, 0.1 M DTT, 5 x MMLV buffer and 200 units of MMLV-RT (Invitrogen) and incubation for 1 hr at 37°C.

The rat and mouse cDNAs used in part D.3.2 were a gift from T. O’Reilly (Novartis, Basel, Switzerland). cDNAs from HeLa cells, undifferentiated and differentiated rat B35 cells, as well as mouse 3T3 cells were provided by G.Tanackovic.

PCR

PCR reactions were performed on cDNA with the Expand high-fidelity PCR system (Roche) for 40 cycles. All primers were purchased at Microsynth. For the radioactive PCR, N1 primer was labeled with γ–³⁵P ATP. Primer details are provided in Table 3.

Table 3. Primers used in this study

Primer Name Target Sequence 5’→3’ Orientation Purpose

N1 SF1 exon1 CGCTGGAACCAAGACACA Sense PCR

E6C SF1 exon6 CTCATCTTCTCCTGGCAAC Anti-sense PCR

E10BN SF1 exon10 CTACCACGGCATGCATGG Sense PCR

E14bc SF1 exon14 TAACTCAGGCTGCTTTGCCAAACCA Anti-sense PCR

MALAT-1 MALAT-1 GGCAGGAGAGACAACAAAGC Sense PCR

MALAT-1 MALAT-1 CCTCGACACCATCGTTACCT Anti-sense PCR MALAT-1 MALAT-1 GGAAGACAGAAGTACGGGAAGGCGAAG Sense FISH-Probe MALAT-1 MALAT-1 CATCACTGAAGCCCACAGGAACAAGTC Anti-sense FISH-Probe

Men-ε/β Men-ε/β GGGCCATCAGCTTTGAATAA Sense PCR

Men-ε/β Men-ε/β CTTGAAGCAAGGTTCCAAGC Anti-sense PCR

Men-β Men-β GCTGAGAAGGAAGGTGCTTG Sense PCR

Men-β Men-β CTGGCTAGTCCCAGTTCAGC Anti-sense PCR

Men-ε/β Men-ε/β TAGTTGTGGGGGAGGAAGTG Sense FISH-Probe

Men-ε/β Men-ε/β TGGCATGGACAAGTTGAAGA Anti-sense FISH-Probe Men-β Men-β GTCTTTCCATCCACTCACGTCTATTT Sense FISH-Probe Men-β Men-β CACCCTAACTCATCTTACAGACCACCAG Anti-sense FISH-Probe

GAPDH_RT GAPDH AGCTTGTCATCAACGGGAAG Sense PCR

GAPDH_RT GAPDH TTTGATGTTAGTGGGGTCTCG Anti-sense PCR

RT and real-time PCR for IP experiments to test the SF1 - Malat-1, Men-ε/β interaction Reverse transcription was performed as above, but both random primer mix (Bio-Rad) and oligo-dT(12-18) (Sigma) were used in this case. Real-time PCR was performed in a 96-well plate (Bio-Rad) in an iCycler (Bio-Rad). Reactions were set up with SYBR Green Supermix (Bio-Rad) according to the manufacturer’s instructions and one fourth of the cDNA made by RT was used per real time PCR reaction. Primer sets for the amplification of Malat-1, Men-ε/β, Men-β and GAPDH (Microsynth) are shown in Table 2.