Productive mRNA chromatin escape is promoted by PRMT5 activity

Materials availability

All materials will be available upon request.

Cell culture

A549 (human lung adenocarcinoma), HEK293T (human embryonic kidney), IMR90-hTERT (telomerase-immortalized human fetal lung fibroblasts), and NSC-34 (mouse motor neuron-like hybrid cell line) cell lines were used. All cell lines were validated by STR testing prior to use. All cells were maintained at 37°C in a humidified atmosphere with 5% CO2. A549 and HEK293T were cultured in DMEM (Corning) with 10% fetal bovine serum (Hyclone), 100 I.U./mL penicillin, and 100 μg/mL streptomycin. IMR90-hTERT was cultured in EMEM (Corning) supplemented as above. NSC-34 was cultured in DMEM supplemented with 10% FBS and 1% 200 mM L-glutamine (Gibco). All cells were routinely tested for mycoplasma via PCR.⁶⁶ Sex of the cell lines was not determined.

Cell culture drug treatments

Treatment with small molecule inhibitors dissolved in DMSO maintained final concentration of 0.01% DMSO, with 1 μM GSK591 (Cayman),1 μM MS023 (Cayman), 5 nM Pladienolide B (Cayman), 5 μg/mL Actinomycin D (Sigma-Aldrich), or 100 nM Doxorubicin (Cayman). Cells were cultured in conditions listed above for the desired treatment duration. See key resources table for further info on small molecules used in this study. Cell growth rates were measured using the MTS cellular proliferation assay (Promega) and cell cycle using the Viastain PI Cell Cycle Kit (Revvity Health Sciences Inc) per the manufacturers’ instructions.

Poly(A)-RNA sequencing

RNA was extracted using RNEasy Mini Kit (Qiagen) or Trizol (Invitrogen) for the cofactor knockdown and cellular fractionation experiments, respectively, as per the manufacturer’s protocols. RNA quantification and quality control were accomplished with the Qubit RNA BR Assay Kit (Invitrogen) and the Bioanalyzer 2100 (Agilent Technologies). Unstranded (PRMT inhibition time course) and stranded libraries (PRMT5 inhibition subcellular fractionation) were created by Novogene Genetics US. Barcoded libraries were sequenced on an Illumina platform using 150 nt paired-end libraries generating approximately 30 million paired-end reads (9GB total data) per biological replicate. Reads were trimmed and aligned to the human genome (hg38) or to a custom combined human and Drosophila melanogaster genome (hg38-dm6) using the nf-core/rnaseq pipeline.⁶⁰ Differential abundance analysis for non-spike in studies was performed using DESeq2 with a custom script (https://github.com/Shechterlab/nextflow-rnaseq) and for the spike-in studies with a different script (https://github.com/Shechterlab/DESeq2_with_SpikeInNormalization). Alternative splicing events were determined using Replicate Multivariate Analysis of Transcript Splicing (rMATS, version 4.0.2).⁶¹

Precision Run-On (PRO) nascent transcriptome sequencing

PRO-seq was performed as previously described.⁶⁷ Briefly, approximately 10 million A549 cells were treated in T182 flasks. At the conclusion of the treatment, media was aspirated, and cells were rinsed with 10mL of ice-cold PBS, scraped, and collected into 15mL conical tubes on ice. Cells were collected by centrifugation at 1,000 × g at 4°C for 5 minutes. PBS was aspirated and pellets were resuspended in 10mL ice-cold permeabilization buffer. Cells were incubated in permeabilization buffer for five minutes before being centrifuged at 1,000 × g at four degrees for five minutes. Permeabilization buffer was aspirated and permeabilized cells were resuspended in 100μL freezing buffer and snap frozen before being stored at −80°C. When preparing for the run on step, frozen permeabilized cells were thawed on ice. Run-on master mix was warmed to 37°C and 100μL of 2x run-on master mix was added to 100μL of frozen cells before being placed in a 30°C water bath for five minutes with slight agitation after 2.5 minutes of incubation. After five minutes of run-on, 500uL of Trizol LS was added and samples were vortexed until homogenized to stop the reaction. 130μL of chloroform was added, samples were vortexed for 15 seconds, and tubes were centrifuged at 15,000 × g for five minutes at 4°C. The aqueous phase was transferred to a new Eppendorf tube and RNA was precipitated using 2.5 volumes of 100% ethanol for 10 minutes before being centrifuged at 15,000 × g for 20 minutes at 4°C. The pellet was washed with 75% ethanol, centrifuged again, and ethanol removed completely before resuspending the RNA pellet in 19μL of DEPC-treated water. Sample quality was assessed by nanodrop and Tapestation before proceeding to RNA fragmentation with Ambion RNA fragmentation reagent (Ambion AM8740) for 4 minutes at 72°C before adding stop solution to the reaction. Free NTPs were removed using a p30 column.

Nascent RNA was enriched using streptavidin-coated Dynabeads (Thermo Fisher #65305), washed 2X with 500μL of ice cold highsalt wash buffer using a magnet. Beads were then washed 2X with 500μL binding buffer and 1X with low-salt wash buffer. Buffer was removed and beads were resuspended in 300μL of Trizol to dissociate nascent RNA from the beads. All buffers were made according to Mahat et al.⁶⁷ 60μL of chloroform was added and vortexed. The mixture was centrifuged at 15,000 × g for five minutes at 4°C. The aqueous phase was removed and moved to a separate tube before repeating the nascent RNA enrichment and wash steps. After the second round of purification, nascent RNA was precipitated with 1.25μL glycoblue and 2.5 volumes of 100% ethanol before centrifugation at 15,000 × g for 20 minutes at 4°C. Ethanol was removed, and the RNA pellet was washed with 75% ethanol. RNA was centrifuged again before completely removing the 75% ethanol. Pellets were resuspended in 5μL DEPC treated H₂O and taken directly into library preparation using the NEBNext Ultradirectional II RNA sequencing kit (NEB #E7760S) following the manufacturer’s instructions. Samples were sequenced using an Illumina HiSeq.

Acid extraction of chromatin

This protocol was adapted from our previously published protocol.²⁵ Hypotonic Lysis Buffer (HLB) was made by mixing 10 mM Tris-HCl pH 8.0, 1 mM KCl, 1.5 mM MgCl₂, and 1 mM DTT, and supplemented with protease and phosphatase inhibitors. The Nuclear Lysis Buffer (NLB) was prepared with 10 mM Tris-HCl pH 8.0, 250 mM KCl, 0.1% NP40, and 1 mM DTT, and supplemented with protease and phosphatase inhibitors. Cell pellets were resuspended in HLB and incubated for 30 minutes to promote hypotonic swelling and mechanical lysis, followed by centrifugation to pellet intact nuclei. Nuclei were washed with additional HLB. Nuclei were then resuspended in NLB and rotated for 30 min at 4°C. Chromatin was then collected via centrifugation and washed with NLB.

Chromatin was then resuspended in 0.4N H₂SO₄, incubated for 30 min., then spun at 16,000 × g for 10 min at 4°C to remove nuclear debris. The supernatant, containing histones and acid-soluble proteins, was transferred to a fresh tube and treated with TCA to precipitate histones, which were then pelleted, washed with ice-cold acetone, and air-dried. The histone pellet was dissolved in an appropriate volume of ddH₂O, characterized for concentration, and analyzed further using SDS/PAGE gel.

Histone PTM mass spectrometry

Histones were acid-extracted from chromatin using the above protocol. Histone pellets were dissolved in 50 mM ammonium bicarbonate, pH 8.0, and histones were subjected to derivatization using 5 μL of propionic anhydride and 14 μL of ammonium hydroxide (all Sigma Aldrich) to balance the pH at 8.0. The mixture was incubated for 15 min and the procedure was repeated. Histones were then digested with 1 μg of sequencing grade trypsin (Promega) diluted in 50mM ammonium bicarbonate (1:20, enzyme:sample) overnight at room temperature. Derivatization reaction was repeated to derivatize peptide N-termini. The samples were dried in a vacuum centrifuge.

Prior to mass spectrometry analysis, samples were desalted using a 96-well plate filter (Orochem) packed with 1 mg of Oasis HLB C-18 resin (Waters). Briefly, the samples were resuspended in 100 μl of 0.1% TFA and loaded onto the HLB resin, which was previously equilibrated using 100 μl of the same buffer. After washing with 100 μl of 0.1% TFA, the samples were eluted with a buffer containing 70 μl of 60% acetonitrile and 0.1% TFA and then dried in a vacuum centrifuge.

Samples were resuspended in 10 μl of 0.1% TFA and loaded onto a Dionex RSLC Ultimate 300 (Thermo Scientific), coupled online with an Orbitrap Fusion Lumos (Thermo Scientific). Chromatographic separation was performed with a two-column system, consisting of a C-18 trap cartridge (300 μm ID, 5 mm length) and a picofrit analytical column (75 μm ID, 25 cm length) packed in-house with reversed-phase Repro-Sil Pur C18-AQ 3 μm resin. Peptides were separated using a 30 min gradient from 1–30% buffer B (buffer A: 0.1% formic acid, buffer B: 80% acetonitrile + 0.1% formic acid) at a flow rate of 300 nl/min. The mass spectrometer was set to acquire spectra in a data-independent acquisition (DIA) mode. Briefly, the full MS scan was set to 300–1100 m/z in the orbitrap with a resolution of 120,000 (at 200 m/z) and an AGC target of 5x10e5. MS/MS was performed in the orbitrap with sequential isolation windows of 50 m/z with an AGC target of 2x10e5 and an HCD collision energy of 30.

Histone peptides raw files were imported into EpiProfile 2.0 software.⁵⁹ From the extracted ion chromatogram, the area under the curve was obtained and used to estimate the abundance of each peptide. To achieve the relative abundance of post-translational modifications (PTMs), the sum of all different modified forms of a histone peptide was considered as 100% and the area of the particular peptide was divided by the total area for that histone peptide in all of its modified forms. The relative ratio of two isobaric forms was estimated by averaging the ratio for each fragment ion with different mass between the two species. The resulting peptide lists generated by EpiProfile were exported to Microsoft Excel and further processed for a detailed analysis.

Immunoblots

Whole cell lysates were prepared using RIPA buffer (1% NP-40, 150 mM NaCl, 1mM EDTA, 50 mM Tris-HCl pH 8 at 4°C, 0.25% Sodium Deoxycholate, 0.1% SDS, protease inhibitor, and phosphatase inhibitor). Immunoblots were performed on PVDF (Immobilon, Millipore), detected by ECL chemiluminescence (Lumigen, TMA-6) and imaged with an ImageQuant LAS4000 (GE). Antibody concentrations used are presented in Table S5.

Lentiviral CRISPRi knockdown

A two-plasmid lentiviral approach was used for knockdown of target genes. The lenti_dCas9-KRAB-MeCP2 plasmid was a gift from Andrea Califano (Addgene plasmid # 122205; http://n2t.net/addgene:122205; RRID:Addgene_122205) and pXPR_050 was a gift from John Doench & David Root (Addgene plasmid # 96925; http://n2t.net/addgene:96925; RRID:Addgene_96925).^68,69 The dual fusion of KRAB and MeCP2 to dCa9 was chosen due to its superior silencing of both coding and non-coding genes. Custom sgRNA sequences (Table S6) were designed using the Broad CRISPick software (https://portals.broadinstitute.org/gppx/crispick/public) and then cloned into pXPR_050 as previously described.⁶⁹ Briefly, the pXPR_050 plasmid was linearized using BsmBI and purified by a 1% agarose gel. Linearized plasmid was ligated to annealed CRISPick-designed sgRNA oligos in a 1:10 ratio using T4 ligase, per the manufacturer’s instructions. The ligation reaction was subsequently transformed and cultured overnight in LB broth with appropriate antibiotics. Following plasmid purification, all new plasmids were sequence confirmed.

Lentiviral particles were generated using calcium phosphate transfection. Transfection of 293T cells were accomplished by combining 1 pmol of each of the desired lentiviral plasmid, pCMVR8.74 and VSV.G to a final concentration of 124 mM CaCl₂ and 1x HBS (21 mM HEPES pH 7.1, 5.6 mM D-glucose, 4.8 mM KCl, 136 mM NaCl, 746 μM Na₂HPO₄). pCMVR8.74 was a gift from Didier Trono (Addgene plasmid # 22036; http://n2t.net/addgene:22036; RRID:Addgene_22036) and VSV.G was a gift from Tannishtha Reya (Addgene plasmid # 14888; http://n2t.net/addgene:14888; RRID:Addgene_14888). Calcium phosphate solution was then added to 293T cells and media was changed 6–8 hours later. Following 48h of 293T cell culture in standard conditions, viral supernatant was collected and filtered with 0.45 μm size filters and stored at −80°C. Subsequent transduction of A549 cells was accomplished by combining lentiviral supernatant into polybrene-containing media (4 μg/mL) and centrifuging at 30°C for 2h at 1000 × g. Following spinfection, cells were returned to normal culture conditions and appropriate antibiotic selection was initiated after 24h.

Stable expression of the dCas9-KRAB-MeCP2 construct was transduced in A549 cells followed by Blasticidin (Cayman) selection at 10 μg/mL before subsequent transduction with the sgRNA and selection with Puromycin at 2 μg/mL (Cayman). A list of sgRNAs used in this study are listed in Table S6.

Indirect Immunofluorescence

Cells were seeded on coverslips (Neuvitro) and grown for a minimum of 4 days in the presence of 0.01% DMSO or 1μM of drug treatment. Cells were rinsed with warm PBS (Corning) and fixed in 4% methanol-free formaldehyde (ThermoScientific) for 10 minutes at room temperature, rinsed with PBS, and either stored at 4°C in PBS until use or immediately quenched with 0.1M ultraPure glycine (Invitrogen) and blocked 1 hour in 1% fish skin gelatin in PBS with 0.01% Tween-20. Primary antibodies were prepared in blocking solution and inverted coverslips were incubated on antibodies overnight at 4°C in a humid chamber. Coverslips were rinsed in PBS and incubated with secondary antibody (Thermo: 35552, Invitrogen: A21245 or Invitrogen: A21235) at 1:1000 in blocking buffer containing actin-rhodamine at 1:33 (Invitrogen: R37112) for 1 hour at room temp in a humid chamber protected from light. Coverslips were rinsed in PBS and mounted to glass slides with DAPI prolong gold anti-fade (Thermo: P36930).

Images were acquired with a Plan Apochromatic 60x objective (model BZ-PA60, NA 1.4/0.13 mm) and band-pass filters (Chroma Technology: DAPI: OP-87762/49028-UF1, EGFP: OP-87763/49002-UF1, CY3/TRITC: OP-87764/49004-UF1, CY5: OP-87766/49006-UF1) on a Keyence epi-fluorescence microscope (BZ-X810).

Images for figure visualization were captured as 11-image z-stacks with 0.2μm pitch and presented as a maximal projection. Pseudo-colored images were processed in FIJI (ImageJ2, Version: 2.14.0/1.54f, Build: c89e8500e4) with custom macros to adjust levels equally across all treatments of each experiment.⁶⁵ Profile line plots were completed in FIJI. All quantifications were completed on single z-stack images with custom macros in FI-JI; macros are shared on https://github.com/Shechterlab/DeAngelo_2024. Antibody concentrations used are presented in Table S5.

Immunoprecipitation

After treatment, cells were harvested with trypsin (Corning), washed twice with 4°C PBS, and resuspended in modified RIPA buffer (0.5% NP-40, 150 mM NaCl, 1mM EDTA, 50 mM Tris-HCl pH 8 at 4°C) supplemented with 40 U/mL RNaseOUT (Thermo Fisher Scientific) and protease inhibitor (Thermo Fisher Scientific) at 10x cell pellet volume. Samples were incubated on ice for 10 min followed by sonication for 3x5 sec at 20% amplitude with a 1/8-inch probe-tip sonicator. All subsequent steps were completed at 4°C: Insoluble material was removed by 10,000 × g centrifugation for 10 min and lysates were pre-cleared by incubating with Protein G agarose (Millipore-Sigma, 16–201) for 30 minutes with rotation; supernatant was collected from agarose by centrifugation at 10,000 × g for 10 minutes. Concentrations of the cleared supernatants were normalized by bicinchoninic acid assay (Pierce) to 750 μg per condition (1.5 μg/μL). Primary antibody targeting Tri-methyl Guanosine (MBL Life Science, RN019M, clone 235–1; 5μg) was added and incubated over-night with rotation. Samples were treated with Protein G agarose and rotated for 2 hours, then washed three times by pelleting at 10,000 × g for 30 seconds and resuspending in fresh lysis buffer. Samples were prepared in 1x Laemmli buffer for western blotting with antibodies as specified.

Northern Blotting

RNA was isolated using TRIzol (Thermo) and coprecipitated with GlycoBlue (Thermo). The RNA pellet was resuspended in sample buffer (6.8 M Urea in TBE with 10% glycerol and 0.25% Bromophenol Blue/Xylene Cyanide), heated at 90°C for 3 minutes, followed by loading onto an 8% Urea Gel (National Diagnostics) that was pre-run for 45 minutes at 45 watts. The gel was run for one hour at 45 watts in 1x TBE (100 mM Tris, 100 mM Boric acid, and 0.2 mM EDTA) followed by transfer to nitrocellulose in 0.5x TBE at 30 mAmps for four hours at 4°C. Following transfer, RNA was crosslinked to the membrane at 120,000 μJ/cm2 (UV Stratalinker 1800). 5’ end labeling of snRNA probes was performed using ³²P-ɣ-ATP (PerkinElmer) with T4 PNK reaction (NEB). Unincorporated ³²P-ɣ-ATP was removed using a Microspin G-25 column (Cytiva). Post-transfer hybridization was performed at 37°C in a hybridization oven over-night with gentle agitation in hybridization buffer containing 100 mM NaHPO4 pH 7.2, 750 mM NaCl, 1x Denhardt’s Solution (0.02% BSA, 0.02% Ficoll 400, 0.02% Polyvinylpyrrolidone), 1% Herring sperm DNA, and 7% SDS. The next morning, the hybridization solution was carefully discarded according to institutional protocol and the membrane was washed twice with wash buffer (40 mM NaHPO4 pH 7.2, 2% SDS, 1 mM EDTA). The wash buffer was also discarded according to institutional guidelines. The membrane was left to expose on a Phosphoimager screen (Cytiva) and imaged at 633nm using a Typhoon 9400 Variable Mode Imager.

Cloning and expression of PRMT5-MEP50

Full-length human PRMT5 (residues 2–637, Uniprot O14744) and MEP50 (also known as WDR77, residues 2–341, Uniprot Q9BQA1) genes were each cloned into pECFB1 (a pFastbac1 derivative with a LIC cloning site) and each contained an engineered N-terminal 6xHisTEV site. Bacmids were generated in DH10Bac cells and baculovirus produced in Sf9 cells. Production protein expression was performed by co-infection in HiFive cells and 2 days of growth.

Harvested cells were resuspended in hypotonic buffer (10mM HEPES, pH 7.6, 10 mM KCl, 0.5% NP40, 0.5 mM PMSF, and 2 protease inhibitor tablets) and incubated on ice for 1 hr. Swollen cells were sonicated 4 × 30 sec (1 sec on, 1 sec off @ 25% output). Cell debris was removed by centrifugation at 14,000 × g for 30 min. Tris buffer (pH 8) was added to a final concentration of 50 mM to the clarified cell lysate. Glycerol and b-mercaptoethanol were added to the final concentration of 5% and 10 mM, respectively. Guanidine HCl from a 6M stock was added dropwise on a stir plate to a final concentration of 250 mM.

Pre-equilibrated HisPur Nickel-NTA (Thermo) resin was mixed with the lysate and incubated on a rotary shaker for 30 min. The mixture was then centrifuged at 800 × g for 1 min to remove the clear lysate. Ni-NTA beads were packed into a gravity column, washed with 20 column volumes (CVs) of wash buffer (50 mM Tris, pH 8, 5% glycerol, 10 mM b-mercaptoethanol, and 250 mM KCl), followed by 5 CVs of the same buffer containing 30 mM and 45 mM imidazole; proteins were eluted with 2 CVs of the same buffer containing 300 mM imidazole. Elute containing PRMT5-MEP50 was pooled and applied to a Superdex 200 size-exclusion chromatography. Complex-containing fractions were pooled and stored at 1 mg/mL at −80°C. Protein estimations were carried out using Bradford protein assay reagent using BSA as a standard.

Cloning and recombinant expression of SNRP tail domains and SMN Tudor domain

The tail domains of SNRPB (88–230aa), SNRPD1 (71–118aa) and SNRPD3 (74–125aa) were cloned into pRuth5-GST ligation independent expression vector. Recombinant SNRP tails contained an N-terminally fused GST affinity tag and a C-terminal StrepII tag. SMN Tudor domain was expressed from pET28-MHL, a gift from Cheryl Arrowsmith (Addgene plasmid #61946; RRID:Addgene_61946). Recombinant proteins were expressed in Rosetta2 (DE3) E. coli grown in LB medium containing appropriate antibiotics. Cultures were grown to OD600 ~0.7 at 37°C with agitation before induction with 0.5mM IPTG followed by 16h incubation at 20°C with agitation. Cells were harvested by centrifugation and resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 0.5 M NaCl, 5 mM β-ME, 5 mM imidazole and 5% glycerol). Lysis was performed using an Emulsiflex C3 homogenizer (Avestin) at 4°C with two passages at 12,000psi. The lysate was centrifuged at 14,000 × g for 45 min and the cleared lysate was loaded onto HisPur Ni-NTA (Thermo) gravity-flow column. The column was washed extensively with 20 column volumes (CVs) of lysis buffer, followed by 5 CVs of the same buffer containing 30 mM and 45 mM imidazole; proteins were eluted with 2CVs of the same buffer containing 300mM imidazole. Eluate containing SNRP and SMN proteins were pooled, concentrated using spin concentrators, and dialyzed against buffer containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT, and 10% glycerol. SNRPB was further purified using a Strep-TactinXT (IBA Biosciences) column and bound SNRPB was eluted with buffer containing 50 mM biotin. Pure proteins were concentrated to ~5 mg/mL and stored at −80°C. Protein estimations were carried out using Bradford protein assay reagent using BSA as standard.

Methylation of SNRP tails, Electrophoretic mobility shift assays and SMN pulldown

PRMT5-MEP50 mediated Arg methylation of SNRP proteins was carried out in 50 mM Tris buffer, pH 7.8, containing 50 mM NaCl, 1 mM EDTA, 10% glycerol and 5 mM β-ME for 12–14h at 30°C. Typical reaction mixture contained 100–400 μM SAM, SNRP: PRMT5-MEP50 was incubated in a mass ratio of 50:1 substrate; reactions also contain 2 μg Tm0936 SAH-deaminase to minimize the SAH product inhibition of PRMT5.

For electrophoretic mobility shift assays, 30-basepair dsDNA or 30-mer ssRNA of varying sequences were mixed with SNRP/Argmethylated SNRP proteins (and SMN where applicable), incubated for 10 min at room temperature. The reaction mixtures were then subjected to PAGE on 12% TBE gels (29:1) for 45 min at 150V in cold room. Gels were stained with gel red and imaged on BioRad imager.

For SMN pulldown experiment, 100 μg each of SNRPB, SNRPD1 and Arg-methylated SNRPB and SNRPD1 were mixed with 100 μL glutathione agarose beads (Thermo) in 50 mM Tris buffer, pH 7.8, containing 150 mM NaCl, 1 mM EDTA, 5% glycerol and 5 mM β-ME. Tubes were incubated for 30 min at room temperature under rotary conditions. At the end of incubation, the beads were centrifuged at 800 × g for 1 min and the supernatants were removed. The beads were similarly washed 3X in the same buffer to remove traces of unbound SNRPs.

Approximately 800 μg SMN was added to the beads in the same buffer in a total volume of 400 μL. The tubes were incubated at 4°C for 1h under rotary conditions. The beads were washed 5X with binding buffer containing 250 mM NaCl and the bound proteins were eluted with 100 μL binding buffer containing 10 mM glutathione. Glutathione agarose incubated with SMN alone served as the control.

Chromatin associated poly(A)-RNA protein enrichment and immunoblotting

Poly(A)-RNA isolation was performed with modifications to a previously described protocol.⁷⁰ A549 cells were grown in the presence of 0.01% DMSO, 1 μM GSK591 (Cayman), or 1 μM MS023 (Cayman) for two days. Cells were washed with 4°C PBS and irradiated on ice with 100 mJ cm-2 in a UV Stratalinker 1800. Cells were centrifuged at 500 × g for 10 minutes at 4 °C. Chromatin was isolated with nuclear lysis buffer (NLB; 10 mM Tris-HCl pH 7.5 at 4°C, 0.1% NP-40, 400 mM KCl, 1 mM DTT) supplemented with 40 U/mL RNaseOUT (Thermo), protease inhibitor (Thermo), and phosphatase inhibitor (Thermo). The chromatin pellet was resuspended in NLB and sonicated for five seconds at 20% amplitude with a probe-tip sonicator using a 1/8’’ tip. The sonicate was centrifuged at 10,000 × g for 10 minutes and the soluble material transferred to a low-adhesion RNase-free microcentrifuge tube. An aliquot from each sample was saved to serve as the unenriched control. The samples were split into two separate tubes, one of which received 10 μg of competitor 25-nt poly(A) RNA. Magnetic oligo-(dT) beads (NEB) were equilibrated in NLB and added to the enrichments. The samples were vortexed at room temperature for 10 minutes. The beads were then captured on a magnetic column, and the supernatant transferred to fresh tube for additional rounds of depletion. The beads were washed once with buffer A (10 mM Tris pH 7.5, 600 mM KCl, 1 mM EDTA, 0.1% Triton X-100), followed by buffer B (10 mM Tris pH 7.5, 600 mM KCl, 1 mM EDTA) and lastly buffer C (10 mM Tris pH 7.5, 200 mM KCl, 1 mM EDTA). The RNA was eluted by incubating the beads in 10 μL of 10 mM Tris pH 7.5 at 80°C for two minutes, capture of magnetic beads using a magnetic column, and quickly transferring the supernatant to a new tube. Protein from each condition and fraction was then immunoblotted with antibodies as indicated.

Cellular Fractionation for Proteomics

This cellular fractionation was done as previously described.⁹ Briefly, cells were harvested via trypsinization post treatment and washed with PBS. All subsequent steps were performed at 4°C. Cells were suspended in 300 μL of Hypotonic Lysis Buffer (10 mM Tris-HCl pH 8 at 4°C, 0.1% NP-40, 1 mM KCl, 1.5 mM MgCl₂, supplemented with protease inhibitor, phosphatase inhibitor, and 1μM of appropriate PRMT inhibitor) and rotated at for 30 min. Nuclei were spun down at 10,000 × g for 10 min and the cytoplasm was collected. Nuclei were washed with Hypotonic Lysis Buffer. Nuclei were then resuspended in 300 μL of Nuclear Lysis Buffer (10 mM Tris-HCl pH 8 at 4°C, 0.1% NP-40, 400 mM KCl, 1 mM DTT, supplemented with protease inhibitor, phosphatase inhibitor, and 1μM of appropriate PRMT inhibitor) and rotated for 30 min. Chromatin was spun down at 10,000 × g for 10 min and nucleoplasm was collected. The chromatin pellet was washed with additional Nuclear Lysis Buffer. Chromatin was resolubilized with prove-tip sonication at 20% amplitude for 5s. Total proteomic mass spectrometry was then performed on the cellular fractions.

Total and fractionated proteomic mass spectrometry

Cells were homogenized with a probe sonicator in a buffer containing 5% SDS, 5 mM DTT and 50 mM ammonium bicarbonate (pH = 8), and left on the bench for about 1 hour for disulfide bond reduction. Samples were then alkylated with 20 mM iodoacetamide in the dark for 30 minutes. Afterward, phosphoric acid was added to the sample at a final concentration of 1.2%. Samples were diluted in six volumes of binding buffer (90% methanol and 10 mM ammonium bicarbonate, pH 8.0). After gentle mixing, the protein solution was loaded to an S-trap filter (Protifi) and spun at 500 × g for 30 sec. The sample was washed twice with binding buffer. Finally, 1 μg of sequencing grade trypsin (Promega), diluted in 50 mM ammonium bicarbonate, was added into the S-trap filter and samples were digested at 37°C for 18 h. Peptides were eluted in three steps: (i) 40 μl of 50 mM ammonium bicarbonate, (ii) 40 μl of 0.1% TFA and (iii) 40 μl of 60% acetonitrile and 0.1% TFA. The peptide solution was pooled, spun at 1,000 × g for 30 sec and dried in a vacuum centrifuge. Peptides were then desalted as performed for the histone peptides.

Samples were resuspended in 10 μl of 0.1% TFA and loaded onto a Dionex RSLC Ultimate 300 (Thermo Scientific), coupled online with an Orbitrap Fusion Lumos (Thermo Scientific). Chromatographic separation was performed with a two-column system, consisting of a C-18 trap cartridge (300 μm ID, 5 mm length) and a picofrit analytical column (75 μm ID, 25 cm length) packed in-house with reversed-phase Repro-Sil Pur C18-AQ 3 μm resin. Peptides were separated using a 90 min gradient from 4–30% buffer B (buffer A: 0.1% formic acid, buffer B: 80% acetonitrile + 0.1% formic acid) at a flow rate of 300 nl/min. The mass spectrometer was set to acquire spectra in a data-dependent acquisition (DDA) mode. Briefly, the full MS scan was set to 300–1200 m/z in the orbitrap with a resolution of 120,000 (at 200 m/z) and an AGC target of 5x10⁵. MS/MS was performed in the ion trap using the top speed mode (2 secs), an AGC target of 1x10⁴ and an HCD collision energy of 35.

Proteome raw files were searched using Proteome Discoverer software (v2.5, Thermo Scientific) using SEQUEST search engine and the SwissProt human database (updated March 2024). The search for total proteome included variable modification of N-terminal acetylation, and fixed modification of carbamidomethyl cysteine. Trypsin was specified as the digestive enzyme with up to 2 missed cleavages allowed. Mass tolerance was set to 10 pm for precursor ions and 0.2 Da for product ions. Peptide and protein false discovery rate was set to 1%. For all proteomic studies, DEP2 (Differential Enrichment analysis of Proteomics data 2)⁷¹ was used for data analysis with a maximum of one missing value and either GSimp or k-nearest number data imputation and False Discovery Rate control with Strimmer’s q-value. Heatmaps were generated from the log₂ of the centered intensity measurements for the top differentially abundant proteins across all measurements. ClusterProfiler was used for protein ontology analysis with default background gene environments.⁶²

Lentiviral inducible protein overexpression

SNRPB constructs were derived from plasmids created by Maron et al. and cloned into a Gateway Lentiviral system (Invitrogen).⁹ SmB WT, RtoA, and RtoK ORFs were individually PCR amplified and cloned into pDONR221 (Invitrogen) via a Gateway BP Clonase II Enzyme Mix (Invitrogen) using the manufacture’s protocol. Entry clones were then cloned into the destination vector, pLIX_403, via a Gateway LR Clonase II Enzyme Mix (Invitrogen). pLIX_403 was a gift from David Root (Addgene plasmid # 41395; http://n2t.net/addgene:41395; RRID:Addgene_41395). Lentiviral particles containing these constructs were generated and transduced into A549 cells as described above. Transduced cells were selected with 2μg/mL Puromycin (Cayman) and maintained with 1μg/mL. Maximal protein induction was found after treating cells with Doxycycline at 1 μg/mL after three days (data not shown).

RNase treatment of Nuclei

Cells were pre-treated with 1 μM GSK591 for seven days. Cells were collected with trypsin, as described above. 5x10⁶ cells were lysed in 1 mL hypotonic lysis buffer (10 mM Tris pH 8 at 4°C, 1 mM KCl, 1.5 mM MgCl₂, 0.1% NP-40, protease inhibitor cocktail, am 1 mM DTT) with end over end rotation for 30 min. Nuclei were pelleted at 10,000 × g for 5 min. Nuclei were washed in 400 μL hypotonic lysis buffer and split in half. To one sample, 2 μg RNase A and 250 U RNase T1 were added; both samples were incubated at 37°C for 1 hour with intermittent mixing. Following incubation and at 4°C, nuclei were recovered by centrifugation at 10,000 × g for 5 min. Nuclei were lysed with nuclear extraction buffer (see cellular fractionation for proteomics above) and chromatin was pelleted at 10,000 × g for 5 min. Nucleoplasm was collected as the supernatant. Chromatin was resuspended in 150 μL nuclear lysis buffer and solubilized with a tip sonicator. Nucleoplasm and chromatin fractions were then analyzed by immunoblotting.

Subcellular RNA fractionation

This protocol followed previously described procedures with minor alterations.^9,14,51,52 Briefly, following appropriate experimental treatments, cells were collected via a brief trypsinization followed by a PBS wash. Cells were then lysed in a hypotonic lysis buffer containing 10 mM Tris, 1 mM KCl, 1.5 mM MgCl2, 0.1% NP40, 0.5 mM DTT, and 0.5 mM EDTA, as well as 1x protease inhibitor and 40 U/ml RNaseOUT. Cells were rotated at 4°C for 15 minutes to promote lysis. The lysate was overlaid onto a 24% sucrose cushion, a density gradient solution consisting of 10 mM Tris, 15 mM KCl, 1 mM EDTA, 24% Sucrose (w/v), 0.5 mM DTT, 1x protease inhibitor, and 40 U/ml RNaseOUT, and centrifuged at 10,000 × g for 10 min. Nuclei were washed in a PBS/1mM EDTA solution, and recollected by centrifuging at 10,000 × g for 2 min. Nuclei were then resuspended in glycerol buffer, composed of 20 mM Tris, 75 mM NaCl, 0.5 mM EDTA, 50% Glycerol (v/v), 0.85 mM DTT, 40 U/ml RNaseOUT, and 1x protease inhibitor, and subsequently lysed using equal volume of nuclei lysis buffer, which contained 20 mM HEPES, 1 mM DTT, 7.5 mM MgCl2, 0.2 mM EDTA, 300 mM NaCl, 1% NP40, 1M Urea, 1x protease inhibitor, and 40 U/ml RNaseOUT. Following another PBS/EDTA wash, all three fractions were dissolved into TRIzol (Thermo Fisher Scientific), followed by subsequent isolation of RNA, DNA, and Protein as described in the manufacturer’s manual. RNA and DNA were subsequently quantified using the Qubit RNA BR Assay Kit and dsDNA BR Assay Kit, respectively (Invitrogen).

Polyadenylated RNA quantification

Following Trizol purification, equal amounts (ng) of RNA were reverse transcribed using the Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (Invitrogen) and oligo-dT primers. Residual RNA was removed via a RNase H digestion. cDNA was then quantified via the ssDNA Qubit Assay Kit (Invitrogen).

Fractionated RNA-sequencing

The fractionation method used for the sequencing experiment is identical to the above subcellular RNA fractionation. Cells were counted in duplicate to obtain 5 million cells per condition and per biological replicate (collected on separate days). A batch of TRIzol was used with a spike in of total D. melanogaster S2 cells such that there are 250k (5% relative starting cell number) S2 cells per 750 μL of TRIzol. TRIzol was then aliquoted at 750 μL/tube and stored at −80°C until needed.

Statistical analysis

All immunoblots were performed in at least two independent biological replicates. Statistical analysis was performed either using GraphPad Prism (v10.2.3) or R (v4.4.0). Statistical details for each experiment, including the test used, exact value of n, and definition of n, are provided in the figure legends. Unless otherwise indicated, n refers to biological replicates. Data are represented as mean ± standard error of the mean (SEM) unless noted. Comparisons between two groups were performed using two-tailed unpaired Student’s t tests. For multiple group comparisons, one-way ANOVA with appropriate post hoc testing was used. Wilcoxon Rank rank-sum test was applied to compare medians between groups, and Kolmogorov-Smirnov test was used to compare distributions. GeneOverlap⁶³ with Fisher’s Exact Test was used to determine odds ratios of retained intron overlaps and transcript overlaps between different RNA-seq data sets. Significance thresholds are defined in the figure legends (*p < 0.05, **p < 0.01, ***p < 0.001).