DOI: 10.5061/dryad.gb5mkkx1z
title: An alternative cytoplasmic SFPQ isoform with reduced phase separation potential is upregulated in ALS

description: Splicing factor proline- and glutamine-rich (SFPQ) is an RNA-binding
 protein that broadly regulates RNA metabolism. Although its nuclear roles
 are well-studied, evidence of SFPQ’s cytoplasmic functionality is
 emerging. Altered expression and nuclear-to-cytoplasmic redistribution of
 SFPQ have been recognised in amyotrophic lateral sclerosis (ALS)
 pathology, yet the mechanistic basis of this remains undetermined. We
 identified altered SFPQ splicing in ALS, increasing the expression of an
 alternative mRNA isoform lacking a nuclear localisation sequence, which we
 termed ‘altSFPQ’. We find that altSFPQ mRNA contributes to SFPQ
 autoregulation and is highly unstable yet exhibits context-specific
 translation with cytoplasm-predominant localisation. Notably, reduced
 canonical SFPQ coincides with increased altSFPQ transcript expression in
 familial and sporadic ALS models, providing a mechanistic basis for SFPQ
 nuclear-to-cytoplasmic redistribution in ALS patients. Finally, we observe
 that the altSFPQ protein has reduced phase separation potential and
 differential protein binding compared to its canonical counterpart,
 providing insight into its mechanistic relevance to physiology and ALS
 pathogenesis.
description: # An alternative cytoplasmic SFPQ isoform with reduced phase separation
 potential is upregulated in ALS Dataset DOI:
 [10.5061/dryad.gb5mkkx1z](10.5061/dryad.gb5mkkx1z) ## Description of the
 data and file structure The attached documents contain raw data relating
 to the experiments undertaken within the associated manuscript (Neeves et
 al., 2025). ### File: Doc_1_Neeves_et_al_2025_raw_data_final.xlsx
 **Description:** tabulated Excel spreadsheet. Experimental types - qPCR,
 image analyses * qPCR - raw Ct values * High throughput immunofluorescence
 imaging (Opera phenix) - object data (number, area, immunofluorescence
 intensities) * Image analysis of LLPS droplet assays - object data
 (number, area, immunofluorescence intensities) The attached documents
 contain raw data relating to the experiments undertaken within the
 associated manuscript (Neeves et al., 2025). Document 1: tabulated Excel
 spreadsheet. Experimental types - qPCR, image analyses   * qPCR - raw Ct
 values * High throughput immunofluorescence imaging (Opera phenix) -
 object data (number, area, immunofluorescence intensities) * Image
 analysis of LLPS droplet assays - object data (number, area,
 immunofluorescence intensities) Document 1 individual tab notes: **Tab 1:
 1B_DRB** Description: assessing levels of target RNA(s) in HEK293T cells
 treated with 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB)
 transcription inhibitor; time (hours) indicates time after treatment at
 which cells were harvested Data type: qPCR, mean Ct (i.e., threshold
 cycle) values. *GAPDH* acts as a housekeeping gene to which other target
 RNAs are normalised. Comments: GADD45A was assessed on a separate plate,
 hence housekeeping gene *GAPDH* was rerun alongside it and used to
 normalise GADD45A values **Tab 2: 1C_S2E_SMG1i** Description: assessing
 levels of target RNA(s) in HEK293T cells treated with SMG1 small molecule
 inhibitor (or untreated, or DMSO vehicle treated) Data type: qPCR, mean Ct
 (i.e., threshold cycle) values. *GAPDH and POLR2B* act as housekeeping
 genes to which other target RNAs are normalised (using their geometric
 mean). **Tab 3: 1F_S2G_SFPQi** Description: assessing levels of target
 RNA(s) in human induced pluripotent stem cell (hiPSC)-derived motor
 neurons treated with SFPQ siRNA (or a control siRNA, or mock treated with
 vehicle/transfection reagents only). Data type: qPCR, mean Ct (i.e.,
 threshold cycle) values. *GAPDH* acts as a housekeeping gene to which
 other target RNAs are normalised. Alternatively, fig1F displays the ratio
 of altSFPQ to wtSFPQ RNA levels (i.e., altSFPQ / wtSFPQ). Comments:
 replicates reflect 3 separate experiments using the same iPSC line
 (control 3). SFPQ pre-mRNA (specifically primers target a constitutively
 spliced intronic region) was assessed on a separate plate; hence,
 housekeeping gene *GAPDH* was rerun alongside it and used to normalise
 SFPQ pre-mRNA values **Tab 4: 1G_S2I_S4A_SFPQ-OE** Description: assessing
 levels of target RNA(s) in HeLa cells transfected with recombinant SFPQ
 proteins (or a control empty vector plasmid, or mock treated with
 vehicle/transfection reagents only) Data type: qPCR, mean Ct (i.e.,
 threshold cycle) values. *GAPDH and POLR2B* act as housekeeping genes to
 which other target RNAs are normalised (using their geometric mean). **Tab
 5: 1H_S4B_SFPQ-OE** Description: assessing levels of target RNA(s) in
 NGN2-i3Neurons transduced with lentiviruses to express recombinant SFPQ
 proteins (or a control lentivirus, or untreated condition) Data type:
 qPCR, mean Ct (i.e., threshold cycle) values. *GAPDH* acts as a
 housekeeping gene to which other target RNAs are normalised. **Tab 6:
 S2A_Cq_panel** Description: assessing levels of target RNA(s) in (i) human
 induced pluripotent stem cells (hiPSC), (ii) hiPSC-derived DIV14 neural
 precursor cells (NPC), and (iii) hiPSC-derived DIV35 motor neurons (MN)
 biochemically separated into nuclear and cytoplasmic fractions Data type:
 qPCR, mean Ct (i.e., threshold cycle) values Comment: iPSC and NPC
 datasets constitute 4 different biological lines, one experimental
 replicate; MN dataset constitutes 4 different biological lines, two
 experimental replicates **Tab 7: S2B_a-amanitin** Description: assessing
 levels of target RNA(s) in HEK293T cells treated with alpha-amanitin
 transcription inhibitor; time (hours) indicates time after treatment at
 which cells were harvested Data type: qPCR, mean Ct (i.e., threshold
 cycle) values. *GAPDH* acts as a housekeeping gene to which other target
 RNAs are normalised. Comment: The dataset consists of just one single
 experimental replicate **Tab 8: S2C_CHX** Description: assessing levels of
 target RNA(s) in HEK293T cells treated with cycloheximide (CHX)
 translation inhibitor (or untreated, or DMSO vehicle treated) Data type:
 qPCR, mean Ct (i.e., threshold cycle) values. *GAPDH and POLR2B* act as
 housekeeping genes to which other target RNAs are normalised (using their
 geometric mean). **Tab 9: S2F_SMG1i** Description: assessing levels of
 target RNA(s) in hiPSC-derived motor neurons treated with SMG1 small
 molecule inhibitor (or untreated, or DMSO vehicle treated) Data type:
 qPCR, mean Ct (i.e., threshold cycle) values. *GAPDH and POLR2B* act as
 housekeeping genes to which other target RNAs are normalised (using their
 geometric mean). Comment: dataset consists of 3 different biological hiPSC
 lines (control lines 1, 3, and 4) **Tab 10: 2B_polysome** Description:
 assessing levels of target RNA(s) in polysome sucrose gradient fractions
 extracted from hiPSC-derived DIV7 neural precursors Data type: qPCR, mean
 Ct (i.e., threshold cycle) values. Data inthe paper is presented as a
 percentage per fraction relative to the total (i.e., the sum of the target
 RNA amount across all fractions) for each target Comment: dataset consists
 of 3 different biological hiPSC lines (control 3, ALS-VCP patient line 3,
 ALS-VCP patient line 4) **Tab 11: 2C_S3B_S3C_polysome** Description:
 assessing levels of target RNA(s) in polysome sucrose gradient fractions
 extracted from hiPSC-derived DIV14 neural precursors; lysates were then
 either treated with EDTA to disrupt polysomes/ribosomes or untreated Data
 type: qPCR, mean Ct (i.e., threshold cycle) values. Data inthe paper is
 presented as a percentage per fraction relative to the total (i.e., the
 sum of the target RNA amount across all fractions) for each target
 Comment: dataset consists of 3 different biological hiPSC lines (control
 1, control 3, ALS-VCP patient line 4) **Tab 12: 2F_S3H_ASO20**
 Description: assessing levels of target RNA(s) in HEK293T cells treated
 with *SFPQ* -targeting antisense oligonucleotide 20 (ASO20) (or
 non-targeting control ASO) Data type: qPCR, mean Ct (i.e., threshold
 cycle) values. *GAPDH* acts as a housekeeping gene to which other target
 RNAs are normalised. **Tab 13: S3A_polysome** Description: assessing
 levels of target RNA(s) in polysome sucrose gradient fractions extracted
 from HEK293T cells; lysates were then either treated with EDTA to disrupt
 polysomes/ribosomes or untreated Data type: qPCR, mean Ct (i.e., threshold
 cycle) values. Data in the paper is presented as a percentage per fraction
 relative to the total (i.e., the sum of the target RNA amount across all
 fractions) for each target. NA = non-applicable cells since no target RNA
 was identified in these fractions Comment: dataset consists of 1
 biological hiPSC line **Tab 14: S3G_ASO_screen** Description: assessing
 levels of target RNA(s) in HEK293T cells treated with 20 *SFPQ* -targeting
 antisense oligonucleotides (or mock, i.e., transfection reagents only)
 Data type: qPCR, mean Ct (i.e., threshold cycle) values. *wtSFPQ and*
 altSFPQ are normalised to* SFPQ constitutive *(i.e. region common to
 splice variants) to give ‘percentage splicing’. **Tab 15:
 S4D_NONO_HTP_IF** Description: high throughput image analysis of NONO
 (antibody) fluorescent intensity in HEK293T cells transfected with
 recombinant SFPQ expression plasmids (or an empty vector plasmid control
 condition); NONO intensity was measured in cell nuclei or perinuclear
 regions (defined as nuclear masks expanded by 10 um to generate a
 perinuclear ring region) to determine the nuclear:perinuclear distribution
 of the protein Data type: high throughput analysis of immunofluorescence
 performed on Opera Phenix; data represents summary values per well from 36
 fields of view imaged per well    * Replicate_bio: biological replicates
 (i.e., separate transfection / experimental setup) * Replicate_tech:
 technical replicates (i.e. duplicate wells per biological replicates) *
 Cell – Number of objects: total number of objects identified as cells per
 well - Cell - Intensity Nuclear region Alexa 568 (NONO) Mean - Mean per
 Well: average across well of mean intensity of AlexaFluor568-labelled NONO
 intensity (a.u.) in cell nuclei * Cell - Intensity Cytoplasmic Region
 Alexa 568 (NONO) Mean - Mean per Well: average across well of mean
 intensity of AlexaFluor568-labelled NONO intensity (a.u.) in cell
 perinuclear ring region - Cells positive for Alexa 488 (HA-tag) - Number
 of Objects: number of cell objects positive for AlexaFluor488 (HA-tag
 antibody; note that recombinant SFPQ proteins have an HA-tag) above a set
 threshold * % of Cells 488+ (i.e., transfected cells): percentage of cell
 objects positive for AlexaFluor488 (HA-tag antibody; note that recombinant
 SFPQ proteins have an HA-tag) above a set threshold, relative to the total
 number of cell objects identified per well - Ratio Alexa 568 (NONO) -
 Nucleus/Cytoplasm: value in ‘Cell - Intensity Nuclear region Alexa 568
 (NONO) Mean - Mean per Well’ divided by value in ‘Cell - Intensity
 Cytoplasmic Region Alexa 568 (NONO) Mean - Mean per Well’ * Number of
 Analyzed Fields: number of analysed fields per well Comments:
 Yellow-highlighted cells associated with one empty vector condition were
 not included in downstream analysis/data presentation, as was quantifiably
 determined as an outlier (based on 1.5 IQR rule). **Tab 16:
 S5F_S5G_WC_MN_stress** Description: assessing levels of target RNA(s) in
 hiPSC-derived motor neurons treated with sodium arsenite (oxidative
 stressor), sorbitol (osmotic stressor), or untreated Data type: qPCR, mean
 Ct (i.e., threshold cycle) values. *GAPDH* acts as a housekeeping gene to
 which other target RNAs are normalised. Comment: dataset consists of 3
 different biological hiPSC lines (controls 2, 3, and 5) **Tab 17:
 4E_4F_S5D_llps_droplet** Description: quantification of liquid-liquid
 phase separation (LLPS) droplet formation from homotypic eGFP-tagged
 wtSFPQ and altSFPQ proteins in vitro Data type: image analysis of LLPS
 droplet formation. Data represents summary values per image (one image per
 protein condition and per replicate) * Sum_GFP_Intensity_SolubleFraction:
 total GFP intensity in regions outside of droplets (a.u.) *
 Sum_GFP_Intensity_droplets: total GFP intensity within droplets (a.u.) *
 Median_Area: median area of droplets **Tab 18: 4IJ_S5HIJ_frac_MN_stress**
 Description: assessing levels of target RNA(s) in hiPSC-derived motor
 neurons treated with sodium arsenite (oxidative stressor), sorbitol
 (osmotic stressor), or untreated, and biochemically separated into nuclear
 and cytoplasmic fractions Data type: qPCR, mean Ct (i.e., threshold cycle)
 values. *NIT1 and NFX1* act as housekeeping genes (geometric mean) to
 which other target RNAs are normalised. Comment: dataset consists of 3
 different biological hiPSC lines (controls 2, 3, and 4) **Tab 19:
 5A_S7A_SFPQ_RIP** Description: RNA immunoprecipitation performed on N2A
 neuroblastoma cells expressing HA-tagged SFPQ variants (or transfected
 with empty vector plasmid). Data type: qPCR, mean Ct (i.e., threshold
 cycle) values. Each individual condition has an input and an
 RNA-immunoprecipitation sample for each replicate. Levels of target RNAs
 in RIP samples are normalised to levels in the associated input sample and
 expressed as ‘percentage of total input’. Comment: The dataset consists of
 3 different biological hiPSC lines (controls 2, 3, and 4). Note that
 different dilution factors need to be applied to RIP samples relative to
 input samples: * Replicates 1 & 2: dilution factor = 40 * Replicate 3:
 dilution factor = 0.4 **Tab 20: 6B_DIV14_SFPQ** Description: assessing
 levels of target RNA(s) in hiPSC-derived DIV14 neural precursor cells
 (NPC) biochemically separated into nuclear and cytoplasmic fractions Data
 type: qPCR, mean Ct (i.e., threshold cycle) values. *NIT1 and NFX1* act as
 housekeeping genes (geometric mean) to which other target RNAs are
 normalised (i.e., ‘transcript abundance’). ‘Splicing ratio’ is determined
 by dividing SFPQ splice variants (wtSFPQ and altSFPQ) by the constitutive
 SFPQ target region. Comment: dataset constitutes 8 different biological
 lines (controls 1, 3, 4, 5; VCP-ALS patient-derived lines 1, 2, 3, 4), and
 two experimental replicates ### File:
 Doc_2_BaseScope_SFPQ_HEK293T_SMG1i.xlsx **Description:** tabulated Excel
 spreadsheet.BaseScope RNA-FISH analysis of day 6 terminally differentiated
 hiPSC-derived motor neurons treated with sodium arsenite - summary data
 (object number and area) Document 2 notes: Target: * TV1 = wtSFPQ  * TV2 =
 altSFPQ RNA refers to BaseScope RNA puncta Conditions: * OX = sodium
 arsenite oxidative stress * UT = untreated Replicates reflect different
 biological hiPSC-derived day 6 (DIV24) motor neuron cell lines (control
 lines 1, 2, and 3) ### File: Doc_3_BaseScope_SFPQ_D6MN_OX.xlsx
 **Description:** tabulated Excel spreadsheet. BaseScope RNA-FISH analysis
 of HEK293T cells treated with SMG1 inhibitor - summary data (object number
 and area) Document 3 notes: Target: * TV1 = wtSFPQ  * TV2 = altSFPQ RNA
 refers to BaseScope RNA puncta Conditions: * SMG1i = SMG1 small molecule
 inhibitor * UT = untreated ### File:
 Doc_4_Neeves_et_al_SA_Western_blots_full.pdf **Description:** All full
 Western blots used within the study. ### File:
 Doc_5_Neeves_et_al_sedimentation_gels.pdf **Description:** All full gel
 images used for quantification of sedimentation assays within the study.
 ### File: Doc_6_SFPQ_cotransfection_imaging.xlsx **Description:**
 tabulated Excel spreadsheet. Image analysis of SFPQ cotransfection
 immunofluorescence assay- nuclear and perinuclear (generated via expansion
 of nuclear margins per cell) per object data (object number, area,
 integrated intensity, median intensity) Document 6 notes: Replicate: N1 –
 3 (note upper case N) Field: n1 – 7 (note lower case N) Condition: * wt =
 eGFP-wtSFPQ expressing cells * alt = eGFP-altSFPQ expressing cells * cotf
 = eGFP-wtSFPQ + mAPPLE-altSFPQ cotransfected cells Each row reflects an
 individual object Each tab represents an analysis of each of the 3
 conditions for each replicate. Data contained within represents the
 following for each individual object (i.e., cell), in both nuclear and
 perinuclear cell regions: * Area * Integrated intensity of GFP *
 Integrated intensity of mAPPLE * Median intensity of GFP * Median
 intensity of mAPPLE For more details, please refer to the methods sections
 within the associated article. ## Code/software All qPCR data were
 analysed in Excel and Prism. Image analysis was performed through a
 combination of ImageJ and CellProfiler processing, except Opera Phenix
 high-content imaging (Harmony high-content imaging and analysis software
 used). Western blot & gel densitometry analyses were performed in
 ImageJ or ImageStudioLite. For more details, please refer to the methods
 sections within the associated article. ## Access information NA ## Human
 subjects data Informed consent was obtained from healthy control donors
 and patients in this study. Experimental protocols were enacted according
 to regulations and guidelines outlined by UCLH’s National Hospital for
 Neurology and Neurosurgery and UCL’s Institute of Neurology joint research
 ethics committee (09/0272).