Journal: bioRxiv

Article Title: Epigenetic–splicing regulation of hTERT mediated by hTAPAS

doi: 10.64898/2026.05.08.723733

Figure Lengend Snippet: (A) BF images five out of the seven cell lines, including telomerase-negative fibroblasts (HFF-1, IMR90, BJ), telomerase-positive lines (HEK293T, NALM6, HG002 iPSCs), and the ALT-positive line VA13. (B) Quantification of hTAPAS and hTERT mRNA levels by qRT-PCR. (C) RT–PCR analysis of hTERT splice isoforms using primers spanning exons 2–3 and 5–9. An inverse relationship between hTAPAS and hTERT expression was observed in HFF-1 and BJ fibroblasts (low hTERT , detectable hTAPAS ) and in HEK293T and NALM6 cells (high hTERT , absent hTAPAS ), whereas intermediate patterns were detected in IMR90, VA13, and iPSCs, with iPSCs maintaining moderate hTERT expression despite high hTAPAS levels.

Article Snippet: Human embryonic kidney 293T (HEK293T; ATCC® CRL-3216TM), human embryonic lung fibroblast VA13 (WI-38 VA13 subline 2RA; ATCC® CCL-75.1TM), human foreskin fibroblast HFF-1 (ATCC® SCRC-1041TM), human lung fibroblast IMR-90 (ATCC® CCL-186TM), and human foreskin fibroblast BJ (ATCC® CRL-2522TM) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: bioRxiv

Article Title: Epigenetic–splicing regulation of hTERT mediated by hTAPAS

doi: 10.64898/2026.05.08.723733

Figure Lengend Snippet: Targeted DNA methylation profiling was performed using CRISPR–Cas9 enrichment followed by Nanopore sequencing across a ∼9 kb region spanning hTAPAS through hTERT intron 2 (Chr. 5: 1,196,006–1,205,206) and a ∼6.5 kb region covering introns 6–8 (Chr. 5: 1,174,035–1,180,535). The analysis included telomerase-negative fibroblasts (HFF-1, IMR90, BJ), telomerase-positive cell lines (HEK293T, NALM6, HG002 iPSCs), and the ALT-positive cell line VA13. DNA methylation levels at individual CpG sites are depicted across the indicated genomic regions, including hTAPAS , the THOR region, the core promoter, exon1, intron1, exon 2 and intron 2. Methylation for each individual CpG is shown as a percentage, with unmethylated CpGs depicted in red and methylated CpGs in blue. Methylation across intron 2 was consistently high (80–100%) in all cell lines, whereas regions encompassing hTAPAS , the THOR region, exon 2, and introns 6–8 displayed marked variability between cell types. CpGs within the hTAPAS region were highly methylated in telomerase-positive cells and in the ALT-positive VA13 line, but largely unmethylated in fibroblasts, with partial methylation observed in IMR90. The core hTERT promoter and exon 2–proximal regions remained mostly unmethylated in all cell lines except VA13, which exhibited substantial hypermethylation

Article Snippet: Human embryonic kidney 293T (HEK293T; ATCC® CRL-3216TM), human embryonic lung fibroblast VA13 (WI-38 VA13 subline 2RA; ATCC® CCL-75.1TM), human foreskin fibroblast HFF-1 (ATCC® SCRC-1041TM), human lung fibroblast IMR-90 (ATCC® CCL-186TM), and human foreskin fibroblast BJ (ATCC® CRL-2522TM) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: DNA Methylation Assay, CRISPR, Nanopore Sequencing, Methylation

Journal: bioRxiv

Article Title: Loss of MTPAP disrupts mitochondrial RNA processing causing upregulation of type I interferon signalling

doi: 10.64898/2026.05.04.722669

Figure Lengend Snippet: A-B: Western blot showing levels of MTPAP and mitochondrial complex I (CI) NDUFB8, II (CII) SDHB, III (CIII) CYTB, IV (CIV) COX2, V (CV) ATP5B proteins in lysates of fibroblasts from patient P3, P4 and P5 compared to fibroblasts from 3 healthy donor (controls) ( A ), and in lysates of BJ-5ta deleted for MTPAP by CRISPR/Cas9 using 3 different single guides (sg#1, #2, #3) compared to controls ( B ). Vinculin is used as a loading control. Image representative of 3 different experiments. C: Left, representative confocal images of the mitochondrial network stained with mitochondrial outer membrane protein TOMM40 antibody in patient fibroblasts (P3, P4 and P5) and one healthy donor fibroblast (control). Scale bar: 5 µm. Right, quantification of the mitochondrial footprint based on MiNA ImageJ analysis plugin in patient fibroblasts and the average of 3 healthy donor fibroblasts (control), represented as a superplot (each border free-point represents the measure of a cell, each colour is a different experiment). Mean ± SEM of 3 experiments; ns indicates non significance in a paired t-test performed on the average value for each experiment. D: As in C for MTPAP_KO (sg#2) and control cells. E: Left, representative spinning disk live images of TMRE signal in patient fibroblasts (P3, P4 and P5) compared to one healthy donor fibroblast (control). Scale bar: 5 µm. Right, quantification of TMRE signal in patient fibroblasts relative to the average of 3 healthy donor fibroblasts (control). Superplot, mean ± SEM; n=3 experiments (each colour is a different experiment); each border-free point represents the measure of a cell; * indicates p<0.05 in paired t-test performed on the average value for each experiment. F: As in E for MTPAP_KO (sg#2) and control cells. G-H: ATP production ( G ) and basal respiration ( H ) measured by Seahorse mito stress assay in MTPAP_KO (average of sg#1, sg#2 and sg#3 data for each experiment) versus control cells. mean ± SEM; n=3 experiments, each colour is a different experiment, * indicates p<0.05 in Mann-Whitney test. I-J: ISG score (median of fold changes of mRNA expression of 6 ISGs, see Methods) ( I ) and IFNB1 mRNA expression ( J ) in MTPAP_KO cells compared to controls measured by qPCR. Mean ± SEM; n=7 experiments; each colour corresponds to KO_MTPAP cells generated using a single guide RNA targeting MTPAP; ** p<0.01 in Kruskall-Wallis with Dunn’s post-hoc test.

Article Snippet: Primary fibroblasts (from three healthy donors (controls), and patients 2, 3, 4 and 5) and BJ-5ta fibroblasts (catalog no. CRL-4001; ATCC) were grown in DMEM supplemented with 10% (vol/vol) fetal bovine serum.

Techniques: Western Blot, CRISPR, Control, Staining, Membrane, MANN-WHITNEY, Expressing, Generated

Journal: bioRxiv

Article Title: Loss of MTPAP disrupts mitochondrial RNA processing causing upregulation of type I interferon signalling

doi: 10.64898/2026.05.04.722669

Figure Lengend Snippet: A-B: ISG score ( A ) and IFNB1 mRNA ( B ) in control and MTPAP_KO (sg#2) cells treated for 10 days with ddC. Mean ± SEM; n=4, each colour is a different experiment; * indicates p<0.05, ** p< 0.01, 2-way ANOVA with Holm-Sidak multiple comparison test. C-D: ISG score ( C ) and IFNB1 mRNA levels ( D ) in BJ-5ta cells double KO IRF3/MTPAP compared to controls. E-F : ISG score ( E ) and IFNB1 mRNA levels ( F ) in MAVS/MTPAP double KO cells compared to controls. G-H : ISG score ( G ) and IFNB1 mRNA levels ( H ) in MDA5/MTPAP double KO cells compared to controls. I-J : ISG score ( I ) and IFNB1 mRNA levels ( J ) in RIG-I/MTPAP double KO cells compared to controls. C-J: Mean ± SEM; n≥4, each colour is a different experiment, KO_MTPAP data are represented by the average of sg#1, sg#2 and sg#3 data; ns indicates non significance, * indicates p<0.05 in Wilcoxon test.

Article Snippet: Primary fibroblasts (from three healthy donors (controls), and patients 2, 3, 4 and 5) and BJ-5ta fibroblasts (catalog no. CRL-4001; ATCC) were grown in DMEM supplemented with 10% (vol/vol) fetal bovine serum.

Techniques: Control, Comparison

Journal: bioRxiv

Article Title: Loss of MTPAP disrupts mitochondrial RNA processing causing upregulation of type I interferon signalling

doi: 10.64898/2026.05.04.722669

Figure Lengend Snippet: A: (Left) Relative levels of mtDNA expressed as the averaged relative levels of MT-CO2 , MT-ND1 and mtDNA D-loop signals and ( Right ) of mtRNA expressed as the averaged relative expression levels of MT-ATP6/8 , MT-ND4 , MT-CO1 , MT-CYTB and MT-ND6 in lysates of control cells and of MTPAP_KO cells (sg#2) treated for 10 days with ddC. Mean ± SEM; n=3 experiments, each colour is a different experiment, ** indicates p<0.01, in two-way ANOVA with Holm-Sidak multiple comparison test. B-C-D-J-L-N: Western blot analysis showing protein levels of MTPAP and IRF3 ( B ), STING ( C ), cGAS ( D ), MAVS ( J ), MDA5 ( L ), RIG-I ( N ), in lysates of double KO cells (sg#2 for MTPAP_KO) compared to controls. Vinculin or cofilin are used as loading controls. Pictures representative of 3 different experiments. E: mRNA levels of IFNB1 measured by qPCR in cGAS/MTPAP and STING/MTPAP double KO BJ-5ta cells compared to controls 24 h after stimulation with transfected HT-DNA (sg#2 for MTPAP_KO). Mean ± SEM; n=3 experiments, each colour is a different experiment, **** indicates p< 0.0001 in two-way ANOVA with Holm-Sidak multiple comparison test. F-H: ISG score measured by qPCR in BJ-5ta cells double KO for STING/MTPAP ( F ) cGAS/MTPAP ( H ) cells compared to controls (sg#2 for MTPAP_KO). Mean ± SEM; n≥4 experiments, each colour is a different experiment, MTPAP_KO using sg#2; ns indicates non significance for Wilcoxon tests. G-I: IFNB1 mRNA expression measured by qPCR in double KO STING/MTPAP ( G ) cGAS/MTPAP ( I ) cells compared to controls (sg#2 for MTPAP_KO). N=4 experiments, each colour is a different experiment; ns indicates non significance for Wilcoxon tests. K-M: IFNB1 mRNA expression levels in MAVS_KO ( K ) and MDA5_KO ( M ) cells compared to BJ-5ta controls when transfected with poly(I:C). Mean ± SEM; n=3 experiments, ** indicates p<0.01, *** p< 0.001 in two-way ANOVA with Holm-Sidak multiple comparison test. O: IFNB1 mRNA expression levels in RIG-I_KO BJ-5ta cells compared to controls Infected with Sendai Virus. Mean ± SEM; n=5, ** indicates p<0.01 in two-way ANOVA with Holm-Sidak multiple comparison test.

Article Snippet: Primary fibroblasts (from three healthy donors (controls), and patients 2, 3, 4 and 5) and BJ-5ta fibroblasts (catalog no. CRL-4001; ATCC) were grown in DMEM supplemented with 10% (vol/vol) fetal bovine serum.

Techniques: Expressing, Control, Comparison, Western Blot, Transfection, Infection, Virus

Journal: bioRxiv

Article Title: Loss of MTPAP disrupts mitochondrial RNA processing causing upregulation of type I interferon signalling

doi: 10.64898/2026.05.04.722669

Figure Lengend Snippet: A: Representative confocal microscopy image of immunostaining with antibody to mitochondrial protein TOMM40 and J2 antibody to dsRNA in control and MTPAP_KO (sg#1) cells. Scale bar: 5 µm. B: Quantification of average pixel intensity of J2 dsRNA signal in mitochondria in control vs MTPAP_KO cells (average of sg#1, #2 and #3). Superplot, mean ± SEM; n=3 experiments; each borderless point represents the measure of a cell; each colour is a different experiment; * indicates p<0.05 in paired t-test performed on the mean value for each experiment. C: Percentage of dsRNA dots in mitochondria with an area over 1 µm 2 in control cells vs MTPAP_KO cells (average of sg#1, #2 and #3). Superplot, mean ± SEM; n=3 experiments; each borderless point represents the measure of a cell; each colour is a different experiment; * indicates p<0.05 in paired t-test performed on the mean value for each experiment. D: Representative confocal microscopy image of immunostaining with antibody to mitochondrial RNA granule protein GRSF1 and dsRNA (J2) in control and MTPAP_KO (sg#2) BJ-5ta cells. Scale bar: 5 µm. E: Representative image of the 3D volume reconstruction of STED images after immunostaining with antibody against mitochondrial protein TOMM40 and dsRNA (J2) in control and MTPAP_KO cells (sg#2). Yellow dots correspond to dsRNA particles detected outside of the mitochondrial network. Scale bar: 3 µm. F: Percentage of dsRNA dots detected outside of the mitochondrial network in control vs MTPAP_KO cells (sg#2). Mean ± SEM; n=3 experiments; each point represents the measure of a cell; each colour is a different experiment; **** indicates p<0.0001 in Mann-Whitney test. G: Quantification of the median volume of dsRNA particles detected inside or outside of the mitochondrial network in MTPAP_KO cells (sg#2). Mean ± SEM; n=3 experiments; each point represents the measure of a cell; each colour is a different experiment; *** indicates p<0.001 in Mann-Whitney test. H: Quantification of mtRNA levels measured by qPCR in cytosolic fractions of control and MTPAP_KO cells (sg#2). Mean ± SEM; n=8 experiments; each colour is a different experiment; ns indicates non significance, ** p< 0.01 in 2-way ANOVA with Holm-Sidak multiple comparison test. I-J: ISG score ( I ) and IFNB1 mRNA expression ( J ) measured by qPCR in MTPAP_KO cells (average of sg#1, sg#2 and sg#3 data for each experiment) treated for 24h with 300 µM DIDS compared to controls. Mean ± SEM; n=4 experiments, each colour is a different experiment; * indicates p<0.05, ** p<0.01, two-way ANOVA with Holm-Sidak multiple comparison test.

Article Snippet: Primary fibroblasts (from three healthy donors (controls), and patients 2, 3, 4 and 5) and BJ-5ta fibroblasts (catalog no. CRL-4001; ATCC) were grown in DMEM supplemented with 10% (vol/vol) fetal bovine serum.

Techniques: Confocal Microscopy, Immunostaining, Control, MANN-WHITNEY, Comparison, Expressing

Journal: bioRxiv

Article Title: Loss of MTPAP disrupts mitochondrial RNA processing causing upregulation of type I interferon signalling

doi: 10.64898/2026.05.04.722669

Figure Lengend Snippet: A: Representative confocal microscopy image of immunostained mitochondrial protein TOMM40 and dsRNA (J2) in control and in patient P3, P4 and P5 fibroblasts. Scale bar: 5µm. B: Quantification of average pixel intensity of immunostaining of dsRNA signal in mitochondria in control cells and in patients’ cells (P3, P4 and P5). Superplot, mean ± SEM; n=3 experiments; each borderless point represents the measure of a cell; each colour is a different experiment; ns indicates non significance, * p<0.05 in one-way ANOVA with Dunnett multiple comparison test test performed on the mean value for each experiment C: Percentage of cells with a J2 signal 1.5-fold above the average of 3 controls. Mean ± SEM; n=3 experiments, each colour is a different experiment. D: Representative confocal microscopy image of immunostained TOMM40 and dsRNA (J2) in control and MTPAP_KO BJ-5ta cells (sg#2) untreated (NT) or treated with actinomycin D (ActD) for up to 24h. Scale bar: 5 µm. E: Quantification of pixel intensity of immunostaining of dsRNA signal in mitochondria in control and MTPAP_KO cells (average of sg#1, #2, #3) untreated (NT) and treated with actinomycin D overtime. Mean ± SEM; n=6 experiments, **** indicates p<0.0001 in two-way ANOVA with Holm-Sidak multiple comparison test. F: Representative confocal microscopy image of immunostained TOMM40 and dsRNA (J2) in MTPAP_KO cells (sg#2) non treated (NT), treated in culture for 10 days with ddC, or untreated in culture but treated after fixation with dsRNA specific RNase III (see Methods). Scale bar: 5 µm. G: Representative confocal microscopy image of immunostained PNPT1 and dsRNA (J2) in control and MTPAP_KO cells (sg#3). Scale bar: 5µm. H-I: Quantification of the median sphericity ( H ) and median volume ( I ) of dsRNA particles in control and MTPAP_KO cells (sg#2). Mean ± SEM; n=3, each dot represents a different cell, each colour represents a different experiment, *** indicates p<0.001, **** p<0.0001 in Mann-Whitney test. J: Schematic representation of the experimental set-up to isolate the cytosolic fraction using digitonin and differential centrifugation. K: Western blot analysis of the different fractions generated to isolate cytosolic fractions. Vinculin is used as a cytosolic marker, Lamin A/C as a nuclear fraction marker, TIM44 and TOM40 as mitochondrial membrane markers and TFAM as mitochondrial matrix marker. Note the absence of all markers but vinculin in the cytosolic fraction.

Article Snippet: Primary fibroblasts (from three healthy donors (controls), and patients 2, 3, 4 and 5) and BJ-5ta fibroblasts (catalog no. CRL-4001; ATCC) were grown in DMEM supplemented with 10% (vol/vol) fetal bovine serum.

Techniques: Confocal Microscopy, Control, Immunostaining, Comparison, MANN-WHITNEY, Centrifugation, Western Blot, Generated, Marker, Membrane