METHOD DETAILS

Cells (6–7 million) were collected and suspended in cold RIPA buffer with HALT Protease Inhibitor (1:100)(Thermo Fisher, 78429). Tubes were incubated on ice for 30 minutes and mixed by vortex every 10 minutes. The lysates were spun in a pre-chilled microcen-trifuge (21,000 × g, 20 minutes, 4°C). Sample concentration was determined by BCA assay. NuPage Reducing agent and NuPage LDS were added and the samples were incubated at 70°C for 10 minutes before being loaded onto a 10% Bis-Tris gel. The gel was run in MOPS-SDS running buffer (200V, 1 hour, room temperature) and the proteins were transferred from the gel to PVDF membrane using a Novex wet transfer apparatus (30V, 1 hour, 4°C). The membrane was blocked in 5% milk (TBST 20 mM Tris, 150 mM NaCl, 0.1% Tween 20) for 30 minutes and then rinsed in TBST. The membrane was incubated on a rocker with primary antibodies in 5% milk overnight at 4°C. This was followed by washing the membrane three times with TBST, incubated with secondary antibody, and washed five times with TBST. SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher, 34077) was added to the membrane and exposed to film.

100 million cells from each cell line were pelleted and washed in cold PBSW (1x PBS supplemented with Protease Inhibitor Cocktail at 1:100 (Sigma, P8340)) and re-pelleted. To separate the nuclei from the cytoplasmic fraction, 1:50 NP-40 was added to cells and the mixture was shaken and centrifuged (8800 rpm, 10 minutes). The supernatant containing the cytoplasmic fraction was removed and the cells were treated with benzonase to degrade RNA and DNA.

The nuclei were incubated on ice for 90 minutes and vortexed periodically. 500mM NaCl, 2% NP-40, PBSW were added to the nuclear pellet and the mixture was homogenized and spun down. The supernatant was collected and the process was repeated. An additional 250mM NaCl, 1% NP-40, PBSW was added to the pellet, homogenized, incubated on ice, and spun down followed by collecting the supernatant. A final wash of the nuclear pellet was performed with PBSW. Nuclear extracts were collected and stored at −80°C.

Protein A/G beads (SCBT, sc2003) were washed and incubated with FLAG M2 antibody (Sigma, F3165) for 1 hour at room temperature. Then, the antibody-bound protein A/G beads were incubated with 1% BSA in 1x PBS to block non-specific binding sites. Upon the third wash, 25mg of Dimethyl pimelimidate dihydrochloride powder (DMP) (Sigma, D8388-1G) in 1 mL of 200mM N-ethylmaleimide (NEM, Thermo Fisher Scientific) was added to the bound beads and incubated at room temperature for 30 minutes. The reaction was repeated 2 more times. 50mM Glycine/HCl was added after the third wash and the beads were washed extensively with PBSW + 2% NP-40 before immunoprecipitation.

The protein extracts were incubated with Protein A/G beads for 30 minutes at room temperature and then spun to clear the supernatant. The supernatants were incubated with the antibody-bound Protein A/G beads (4°C, overnight) and the bead-antibody-protein complexes were washed three times with IP buffer. 10% SDS was added to the beads and incubated (15 minutes, 37°C) followed by collecting the supernatant. The process was repeated twice with 1% SDS and the washes were combined.

The immunoprecipitated samples were run on an SDS-polyacrylamide gel and stained with Coomassie Blue (Gel Code Blue, Pierce Chemical). Each lane was cut into eight sections for processing. Proteins in each section were reduced with 10mM dithiothreitol (Sigma-Aldrich, D0632) and alkylated with 55mM iodoacetamide (Sigma-Aldrich, I1149) then digested with trypsin. Peptides were extracted from gel slices three times with 60% acetonitrile and 5% formic acid/water. The peptide mixtures were dissolved in 1% formic acid and submitted for liquid chromatography-tandem mass spectrometry (LC-MS/MS) on an Orbitrap mass spectrometer.

RNA was isolated from LUC7-like knockdown and control cell lines as follows. Cells (6–7 million) were collected, pelleted, and kept on ice. High Pure RNA Isolation Kit (Roche, 11828665001) protocol was used following manufacturer’s instructions to purify the RNA.

Ribosomal RNA was depleted using the Illumina RiboZero Plus kit (Illumina, 20040525) and libraries were prepared for high throughput sequencing using the Illumina TruSeq kit according to manufacturer’s protocols. 100bp paired-end sequencing was performed on three biological replicates for LUC7-like knockdown and control cell lines on the Illumina HiSeq2500 at a depth of 60 million reads.

Experiments were performed using the single-end crosslinking immunoprecipitation (seCLIP) protocol (Van Nostrand et al., 2017b). K562 cells with CRISPR-tagged LUC7L, LUC7L2 and LUC7L3 were transferred to 10cm plates for crosslinking using a Stratalinker 2400 with 254 nm light at 400 mJ/cm². Samples were sonicated using the Biorupter (Diagenode) on the “low” setting for 30 s intervals followed by 30 s pauses for 5 minutes at 4°C. DNase I (2 μl, Invitrogen AM2239) and RNase I (10 μl, 1:25 RNase I:PBS solution, Ambion AM2295) were added to the sample and mixed in the Thermomixer (1200 rpm, 37°C, 5 minutes). LUC7-like protein-RNA complexes were immunoprecipitated with V5 antibody (anti-V5, Bethyl, A190-120A) followed by stringent washes using high salt wash buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, in RNase/DNase free H₂O) and wash buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl₂, 0.2% Tween-20, in RNase/DNase free H₂O). Following washing, RNA was dephosphorylated using FastAP and T4 PNK and a 3′ RNA adaptor was ligated onto the RNA. Samples were denatured and resolved using SDS-PAGE on a 4%–12% Bis-Tris gel. The gel was transferred to PVDF membrane in NuPAGE transfer buffer and the region on the membrane corresponding to the respective LUC7-like proteins and 75 kDa above were isolated for RNA extraction. The isolated RNA was reverse transcribed (AffinityScript Kit 600107) followed by the addition of a 5′ linker to the cDNA. The final libraries were amplified using Q5 PCR mix (NEB) and the 175–350bp region corresponding to the library was excised from a 3% low-melting temp agarose gel. The libraries were quantified on the BioAnalyzer D1000 then sequenced on the Illumina HiSeq2500 at a depth of 100–150 million single-end reads for all pooled samples. This provides a depth of 16.7–25 million reads per sample.