Insulin receptor alternative splicing is regulated by insulin signaling and modulates beta cell survival Pushkar Malakar,4, Lital Chartarifsky,4, Ayat Hija, Gil Leibowitz 3, Benjamin Glaser 3, Yuval Dor, and Rotem Karni * Figures SS5, Figure Legends, Table S List of primers used in the study
Normalized level of Exon Inclusion Normalized level of Total INSR Relative Inclusion of Exon / Total INSR Normalized level of Exon Inclusion Normalized level of Total INSR Relative Inclusion of Exon / Total INSR Normalized level of Exon Inclusion Normalized level of Total INSR Relative Inclusion of Exon / Total INSR A 7 6 5 4 3 U6: + + +.4..8.6.4. U6: + + + 6 5 4 3 U6: + + + B 5 4.5 4 3.5 3.5.5.5 Human Islets U6: + + +.6.4..8.6.4. Human Islets U6: + + + 5 Human Islets 5 5 U6: + + + C 3.6.5.5.5.5.4..8.6.4..5.5 DMSO GKA5 DMSO GKA5 DMSO GKA5 Malakar et al. Figure S
Relative ERK activity A Insulin perk/ Total ERK/ pakt Total AKT βcatenin B..8.6.4. phosphoerk/total ERK ** Vehicle UO6 Malakar et al. Figure S
A U6: + + + B.5.5 MBNL * CUGBP MBNL SRSF3.5 βcatenin U6: + + + C.4..8.6.4. U6: CUGBP + + + D.6.4..8.6.4. U6: SRSF3 + + + Malakar et al. Figure S3
A..8.6.4 SRSF 4 Hrs.4..8.6.4 SRSF 48 Hrs...5 5 Glucose Concentrations (mm).5 5 Glucose Concentrations (mm) B Glucose (mm) C. MBNL.5 5 MBNL.8 * CELF.6 * SRSF3 SRSF.4 βcatenin. 48 Hrs.5 5 Glucose Concentrations (mm) Malakar et al. Figure S4
A..8.6.4. SRSF sirna: Luciferase SRSF B 3.5 Inclusion of Exon * DMSO GKA5.5.5 sirna: Luciferase SRSF C.4. Total INSR DMSO GKA5.8.6.4. sirna: Luciferase SRSF Malakar et al. Figure S5
Figure Legends: Figure S: Inclusion of INSR exon by insulin and GCK activation through the MAPK ERK pathway. A: cells were starved for 4 hours in.% serum and then stimulated with nm insulin in the presence or absence of µm U6. Total RNA was extracted and subjected to QRTPCR using primers from exon and exon, for inclusion and exon and exon, for total INSR. Actin was used for normalization. B: Human pancreatic islets were starved for 4 hours in.% serum and then stimulated with nm insulin in the presence or absence of µm U6. RNA was extracted and the level of exon inclusion of and total INSR was measured using QRT PCR. Actin was used for normalization. C: cells were grown in regular medium for 4 hours and then the medium was replaced with DMEM medium without glucose containing DMSO with or without 5 µm GKA5 for 48 hours. RNA was isolated and QRTPCR performed to measure the exon inclusion, total INSR and the relative inclusion of exon as compared to total INSR. Figure S: Inhibition of ERK activity by UO6. A: cells were starved for 4 hours in.% serum and then stimulated with nm insulin in presence or absence of µm U6. Protein lysates were prepared and analyzed by western blot for ERK activity. βcatenin was used as a loading control. B: Quantification of ERK activity in cells described in (A) as measured by phosphoerk/totalerk levels Error Bars, SD; n=3,**p<.. Figure S3: Analysis of RNA binding proteins for their involvement in INSR splicing. A: cells were starved for 4 hours in.% serum and then stimulated with nm insulin in the presence or absence of µm U6. Western blot of lysates. βcatenin was used as a loading control BD: Quantification of the expression levels of MBNL (B), CUGBP(C) and SRSF3 (D). Error Bars, SD; n=3, *P<.5. Figure S4: Analysis of RNA binding proteins for involvement in glucose induced toxicity leading to exclusion of INSR exon. A: cells were exposed to DMEM medium with or without.5mm or 5mM glucose for 4 and 48 hours. After stipulated period of time, RNA was isolated and subjected to QRTPCR to
detect the expression of SRSF. Tubulin was used for normalization. B: Western blot of lysates of cells incubated with DMEM medium without or with.5mm or 5mM glucose for 48 hours. βcatenin was used for normalization. C: Quantification of MBNL expression from Western blot shown in (B). Error Bars, SD; n=3, *P<.5. Figure S5: SRSF is important for the inclusion of INSR exon through GCK activity. A: cells transfected with sirna targeted against SRSF were grown for 4 hours and then the medium was replaced with DMEM medium without glucose containing DMSO with or without 5µM GKA5 for 48 hours. Cells were lysed, RNA and protein were isolated. Knockdown of SRSF was measured by QRTPCR. Actin was used for normalization. B: QRTPCR to detect exon of INSR with (red) or without (blue) GCK activation by GKA5 in cells with either control sirna or SRSF sirna. Actin was used for normalization. C: Quantification of total INSR transcript levels (exons ) measured by QRTPCR Actin was used for normalization. Error Bars, SD; n=3,*p<..
Primers h = human, m = mouse Semiquantitative PCR INSR E Forward AGATCCTGAAGGAGCTGGAGGA he Reverse GGTCGAGGAAGTGTTGGG me Reverse GAGGAGACGTTGGGGAAATCTG GAPDH Forward ATCAAGAAGGTGGTGAAGCAG Reverse CTTACTCCTTGGAGGCCATGT Actin Forward GTCCCTGTATGCCTCTGGTC Reverse CGCTCGGTCAGGATCTTCAT Tubulin Forward AATGTCGGCCACCTTCATTG Reverse CTCAGCCTCAGTGAACTCCA Real Time PCR msrsf E Forward GAGTGGTTGTCTCTGGACTG E3 Reverse TCTTCTTTCCGTACAAACTCCA hsrsf EE Forward GAGTTCGAGGACCCGCGAGACG E Reverse GAGCTCCGCCACCTCCAC Tubulin Forward AATGTCGGCCACCTTCATTG Reverse CTCAGCCTCAGTGAACTCCA minsr Inc EE Forward AGGAGCTGGAGGAGTCTTCA Inc EE Reverse CTACTGTCCTCGGCACCATT
minsr Total INSR EE Forward GCTGTGCCATTGCTGGTG Total INSR EE Reverse CAGCTCATGTAGCCTGGTCA hinsr Inc EE Forward CCTGAAGGAGCTGGAGGAGT Inc EE Reverse TAGGGTCCTCGGCACCAGT hinsr Total INSR EE Forward CTGTACCCCGGAGAGGTGT Total INSR EE Reverse GGGCCTCGTTTTGAACATC Actin Forward GTCCCTGTATGCCTCTGGTC Reverse CGCTCGGTCAGGATCTTCAT Tubulin Forward AATGTCGGCCACCTTCATTG Reverse CTCAGCCTCAGTGAACTCCA hactin Forward GGCACCCAGCACAATGAAGA Reverse AGGATGGAGCCGGCGATC sirnas Luciferase. AntiLuc sirna from Dharmacon. Catalog number (D5). Target sequence GAUUAUGUCCGGUUAUGUA SRSF#. From Sigma. Product Number NM_7866. SiRNA ID: SASI_Hs_336. Target Sequence: Sense CAUGUCUGAAGAUAGAUGA[dT][dT] Antisense UCAUCUAUCUUCAGACAUG[dT][dT] SRSF#. From Sigma. Product Number NM_7866. SiRNA ID: SASI_Hs_336. Target Sequence: Sense CAUCUACGUGGGUAACUUA[dT][dT] Antisense UAAGUUACCCACGUAGAUG[dT][dT]