{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,13]],"date-time":"2026-01-13T06:10:48Z","timestamp":1768284648858,"version":"3.49.0"},"reference-count":51,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2022,4,1]],"date-time":"2022-04-01T00:00:00Z","timestamp":1648771200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004396","name":"Thailand Research Fund","doi-asserted-by":"publisher","award":["TRG5880047"],"award-info":[{"award-number":["TRG5880047"]}],"id":[{"id":"10.13039\/501100004396","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Research Development fund, Faculty of Medicine, Siriraj Hospital","award":["R0158333001"],"award-info":[{"award-number":["R0158333001"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Spinal muscular atrophy (SMA) is a severe, debilitating neuromuscular condition characterised by loss of motor neurons and progressive muscle wasting. SMA is caused by a loss of expression of SMN1 that encodes the survival motor neuron (SMN) protein necessary for the survival of motor neurons. Restoration of SMN expression through increased inclusion of SMN2 exon 7 is known to ameliorate symptoms in SMA patients. As a consequence, regulation of pre-mRNA splicing of SMN2 could provide a potential molecular therapy for SMA. In this study, we explored if splice switching antisense oligonucleotides could redirect the splicing repressor hnRNPA1 to the hnRNPA1b isoform and restore SMN expression in fibroblasts from a type I SMA patient. Antisense oligonucleotides (AOs) were designed to promote exon 7b retention in the mature mRNA and induce the hnRNPA1b isoform. RT-PCR and western blot analysis were used to assess and monitor the efficiency of different AO combinations. A combination of AOs targeting multiple silencing motifs in hnRNPA1 pre-mRNA led to robust hnRNPA1b induction, which, in turn, significantly increased expression of full-length SMN (FL-SMN) protein. A combination of PMOs targeting the same motifs also strongly induced hnRNPA1b isoform, but surprisingly SMN2 exon 5 skipping was detected, and the PMO cocktail did not lead to a significant increase in expression of FL-SMN protein. We further performed RNA sequencing to assess the genome-wide effects of hnRNPA1b induction. Some 3244 genes were differentially expressed between the hnRNPA1b-induced and untreated SMA fibroblasts, which are functionally enriched in cell cycle and chromosome segregation processes. RT-PCR analysis demonstrated that expression of the master regulator of these enrichment pathways, MYBL2 and FOXM1B, were reduced in response to PMO treatment. These findings suggested that induction of hnRNPA1b can promote SMN protein expression, but not at sufficient levels to be clinically relevant.<\/jats:p>","DOI":"10.3390\/ijms23073937","type":"journal-article","created":{"date-parts":[[2022,4,1]],"date-time":"2022-04-01T21:22:39Z","timestamp":1648848159000},"page":"3937","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Antisense Oligonucleotide Induction of the hnRNPA1b Isoform Affects Pre-mRNA Splicing of SMN2 in SMA Type I Fibroblasts"],"prefix":"10.3390","volume":"23","author":[{"given":"Jarichad","family":"Toosaranont","sequence":"first","affiliation":[{"name":"Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sukanya","family":"Ruschadaariyachat","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Warasinee","family":"Mujchariyakul","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10700, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8025-0557","authenticated-orcid":false,"given":"Jantarika Kumar","family":"Arora","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10700, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Varodom","family":"Charoensawan","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10700, Thailand"},{"name":"Integrative Computational BioScience (ICBS) Center, Mahidol University, Nakhon Pathom 73170, Thailand"},{"name":"Systems Biology of Diseases Research Unit, Faculty of Science, Mahidol University, Bangkok 10400, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8034-7757","authenticated-orcid":false,"given":"Bhoom","family":"Suktitipat","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand"},{"name":"Integrative Computational BioScience (ICBS) Center, Mahidol University, Nakhon Pathom 73170, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Thomas N.","family":"Palmer","sequence":"additional","affiliation":[{"name":"Perron Institute for Neurological and Translational Science, The University of Western Australia, Perth, WA 6009, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8632-641X","authenticated-orcid":false,"given":"Sue","family":"Fletcher","sequence":"additional","affiliation":[{"name":"Perron Institute for Neurological and Translational Science, The University of Western Australia, Perth, WA 6009, Australia"},{"name":"Centre for Molecular Medicine and Innovative Therapeutics, Health Futures Institute, Murdoch University, Perth, WA 6150, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Steve D.","family":"Wilton","sequence":"additional","affiliation":[{"name":"Perron Institute for Neurological and Translational Science, The University of Western Australia, Perth, WA 6009, Australia"},{"name":"Centre for Molecular Medicine and Innovative Therapeutics, Health Futures Institute, Murdoch University, Perth, WA 6150, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9311-7977","authenticated-orcid":false,"given":"Chalermchai","family":"Mitrpant","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand"},{"name":"Perron Institute for Neurological and Translational Science, The University of Western Australia, Perth, WA 6009, Australia"},{"name":"Centre for Molecular Medicine and Innovative Therapeutics, Health Futures Institute, Murdoch University, Perth, WA 6150, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,4,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1038\/ng0797-265","article-title":"Correlation between severity and SMN protein level in spinal muscular atrophy","volume":"3","author":"Lefebvre","year":"1997","journal-title":"Nat. Genet."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"408","DOI":"10.1016\/j.ajhg.2009.08.002","article-title":"A positive modifier of spinal muscular atrophy in the SMN2 gene","volume":"85","author":"Prior","year":"2009","journal-title":"Am. J. Hum. Genet."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1333","DOI":"10.1128\/MCB.26.4.1333-1346.2006","article-title":"Splicing of a critical exon of human Survival Motor Neuron is regulated by a unique silencer element located in the last intron","volume":"26","author":"Singh","year":"2006","journal-title":"Mol. Cell. Biol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1291","DOI":"10.1261\/rna.7580704","article-title":"In vivo selection reveals combinatorial controls that define a critical exon in the spinal muscular atrophy genes","volume":"10","author":"Singh","year":"2004","journal-title":"RNA"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"460","DOI":"10.1038\/ng1207","article-title":"A negative element in SMN2 exon 7 inhibits splicing in spinal muscular atrophy","volume":"4","author":"Kashima","year":"2003","journal-title":"Nat. Genet."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"3149","DOI":"10.1093\/hmg\/ddm276","article-title":"hnRNP A1 functions with specificity in repression of SMN2 exon 7 splicing","volume":"16","author":"Kashima","year":"2007","journal-title":"Hum. Mol. Genet."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1167","DOI":"10.1261\/rna.2154310","article-title":"An antisense microwalk reveals critical role of an intronic position linked to a unique long-distance interaction in pre-mRNA splicing","volume":"16","author":"Singh","year":"2010","journal-title":"RNA"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1625","DOI":"10.1093\/hmg\/ddr600","article-title":"A single administration of morpholino antisense oligomer rescues spinal muscular atrophy in mouse","volume":"21","author":"Porensky","year":"2012","journal-title":"Hum. Mol. Genet."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1089\/hum.2012.211","article-title":"A novel morpholino oligomer targeting ISS-N1 improves rescue of severe spinal muscular atrophy transgenic mice","volume":"24","author":"Zhou","year":"2013","journal-title":"Hum. Gene Ther."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Mitrpant, C., Porensky, P., Zhou, H., Price, L., Muntoni, F., Fletcher, S., Wilton, S.D., and Burghes, A.H. (2013). Improved antisense oligonucleotide design to suppress aberrant SMN2 gene transcript processing: Towards a treatment for spinal muscular atrophy. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0062114"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1089\/nat.2017.0693","article-title":"The effects of 2\u2032-O-Methoxyethyl Oligonucleotides on renal function in humans","volume":"28","author":"Crooke","year":"2018","journal-title":"Nucleic Acid Ther."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1089\/nat.2016.0650","article-title":"The effects of 2\u2019-O-Methoxyethyl containing antisense oligonucleotides on platelets in human clinical trials","volume":"27","author":"Crooke","year":"2017","journal-title":"Nucleic Acid Ther."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1177\/0192623314551840","article-title":"Antisense oligonucleotide therapies: The promise and the challenges from a toxicologic pathologist\u2019s perspective","volume":"43","author":"Frazier","year":"2015","journal-title":"Toxicol. Pathol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"5483","DOI":"10.1002\/j.1460-2075.1994.tb06883.x","article-title":"Function of conserved domains of hnRNP A1 and other hnRNP A\/B proteins","volume":"13","author":"Mayeda","year":"1994","journal-title":"EMBO J."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1776","DOI":"10.1128\/MCB.17.4.1776","article-title":"An intron element modulating 5\u2032 splice site selection in the hnRNP A1 pre-mRNA interacts with hnRNP A1","volume":"17","author":"Chabot","year":"1997","journal-title":"Mol. Cell. Biol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1939","DOI":"10.1093\/emboj\/18.7.1939","article-title":"Modulation of exon skipping by high-affinity hnRNP A1-binding sites and by intron elements that repress splice site utilization","volume":"18","author":"Blanchette","year":"1999","journal-title":"EMBO J."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"413","DOI":"10.1016\/j.nmd.2011.03.009","article-title":"Plastin 3 expression in discordant spinal muscular atrophy (SMA) siblings","volume":"21","author":"Bernal","year":"2011","journal-title":"Neuromuscul. Disord."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1038\/nbt.1515","article-title":"Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes","volume":"27","author":"Foust","year":"2009","journal-title":"Nat. Biotechnol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1038\/nbt.1610","article-title":"Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN","volume":"28","author":"Foust","year":"2010","journal-title":"Nat. Biotechnol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"377","DOI":"10.1038\/ng854","article-title":"Disruption of an SF2\/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1","volume":"30","author":"Cartegni","year":"2002","journal-title":"Nat. Genet."},{"key":"ref_21","first-page":"405","article-title":"A highly stable duplex structure sequesters the 5\u2019 splice site region of hnRNP A1 alternative exon 7B","volume":"3","author":"Blanchette","year":"1997","journal-title":"RNA"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"595","DOI":"10.1016\/S0140-6736(11)60756-3","article-title":"Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: An open-label, phase 2, dose-escalation study","volume":"378","author":"Cirak","year":"2011","journal-title":"Lancet"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"918","DOI":"10.1016\/S1474-4422(09)70211-X","article-title":"Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: A single-blind, placebo-controlled, dose-escalation, proof-of-concept study","volume":"8","author":"Kinali","year":"2009","journal-title":"Lancet Neurol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1002\/cpnc.21","article-title":"Using Morpholinos to Control Gene Expression","volume":"68","author":"Moulton","year":"2017","journal-title":"Curr. Protoc. Nucleic Acid Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"486","DOI":"10.1093\/hmg\/ddx418","article-title":"Downregulation of Survivin contributes to cell-cycle arrest during postnatal cardiac development in a severe spinal muscular atrophy mouse model","volume":"27","author":"Sheng","year":"2018","journal-title":"Hum. Mol. Genet."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"114","DOI":"10.1038\/cdd.2017.172","article-title":"Cell cycle arrest through indirect transcriptional repression by p53: I have a DREAM","volume":"25","author":"Engeland","year":"2018","journal-title":"Cell Death Differ."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"893","DOI":"10.1007\/s00018-010-0501-9","article-title":"B-MYB delays cell aging by repressing p16 (INK4alpha) transcription","volume":"68","author":"Huang","year":"2011","journal-title":"Cell Mol. Life Sci."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1384","DOI":"10.18632\/aging.100988","article-title":"FOXM1 regulates proliferation, senescence and oxidative stress in keratinocytes and cancer cells","volume":"8","author":"Smirnov","year":"2016","journal-title":"Aging"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"3779","DOI":"10.2147\/CMAR.S185438","article-title":"FOXM1 promotes the growth and metastasis of colorectal cancer via activation of beta-catenin signaling pathway","volume":"11","author":"Yang","year":"2019","journal-title":"Cancer Manag. Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"483","DOI":"10.3389\/fonc.2018.00483","article-title":"FoxM1 promotes cell proliferation, invasion, and stem cell properties in Nasopharyngeal Carcinoma","volume":"8","author":"Luo","year":"2018","journal-title":"Front. Oncol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"200","DOI":"10.1186\/1479-5876-10-200","article-title":"Overexpression of FoxM1 is associated with tumor progression in patients with clear cell renal cell carcinoma","volume":"10","author":"Xue","year":"2012","journal-title":"J. Transl. Med."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1063","DOI":"10.1111\/acel.12511","article-title":"hnRNP A1 antagonizes cellular senescence and senescence-associated secretory phenotype via regulation of SIRT1 mRNA stability","volume":"15","author":"Wang","year":"2016","journal-title":"Aging Cell"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"3568","DOI":"10.1093\/nar\/gkg616","article-title":"ESEfinder: A web resource to identify exonic splicing enhancers","volume":"31","author":"Cartegni","year":"2003","journal-title":"Nucleic Acids Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2490","DOI":"10.1093\/hmg\/ddl171","article-title":"An increased specificity score matrix for the prediction of SF2\/ASF-specific exonic splicing enhancers","volume":"15","author":"Smith","year":"2006","journal-title":"Hum. Mol. Genet."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1418","DOI":"10.1038\/mt.2009.49","article-title":"Rational design of antisense oligomers to induce dystrophin exon skipping. Molecular therapy","volume":"17","author":"Mitrpant","year":"2009","journal-title":"J. Am. Soc. Gene Ther."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"8665","DOI":"10.1093\/nar\/gkt579","article-title":"HnRNP A1 controls a splicing regulatory circuit promoting mesenchymal-to-epithelial transition","volume":"41","author":"Bonomi","year":"2013","journal-title":"Nucleic Acids Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/j.omtn.2018.01.011","article-title":"Antisense oligonucleotide-mediated terminal intron retention of the SMN2 transcript","volume":"11","author":"Flynn","year":"2018","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"881","DOI":"10.1016\/j.molcel.2005.10.026","article-title":"Cell motility is controlled by SF2\/ASF through alternative splicing of the Ron protooncogene","volume":"20","author":"Ghigna","year":"2005","journal-title":"Mol. Cell"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/S0378-1119(02)00800-4","article-title":"Characterization of the mouse hnRNP A2\/B1\/B0 gene and identification of processed pseudogenes","volume":"295","author":"Hatfield","year":"2002","journal-title":"Gene"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1038\/nsmb1209","article-title":"The gene encoding the splicing factor SF2\/ASF is a proto-oncogene","volume":"14","author":"Karni","year":"2007","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_41","first-page":"2343","article-title":"Interleukin-6 and the Interleukin-6 receptor in the human adrenal gland: Expression and effects on steroidogenesis1","volume":"82","author":"Bornstein","year":"1997","journal-title":"J. Clin. Endocrinol. Metab."},{"key":"ref_42","first-page":"1542","article-title":"MYBL2 is an independent prognostic marker that has tumor-promoting functions in colorectal cancer","volume":"5","author":"Ren","year":"2015","journal-title":"Am. J. Cancer Res."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"807","DOI":"10.1038\/onc.2016.249","article-title":"A novel FOXM1 isoform, FOXM1D, promotes epithelial-mesenchymal transition and metastasis through ROCKs activation in colorectal cancer","volume":"36","author":"Zhang","year":"2017","journal-title":"Oncogene"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"671","DOI":"10.1038\/nmeth.2089","article-title":"NIH Image to ImageJ: 25 years of image analysis","volume":"9","author":"Schneider","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_45","unstructured":"Andrew, S. (2022, February 01). FastQC: A Quality Control Tool for High Throughput Sequence Data. Available online: http:\/\/www.bioinformatics.babraham.ac.uk\/projects\/fastqc."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"907","DOI":"10.1038\/s41587-019-0201-4","article-title":"Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype","volume":"37","author":"Kim","year":"2019","journal-title":"Nat. Biotechnol."},{"key":"ref_47","unstructured":"Broad Institute (2021, January 15). Picard Tools. Available online: https:\/\/github.com\/broadinstitute\/picard."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"166","DOI":"10.1093\/bioinformatics\/btu638","article-title":"HTSeq\u2014A Python framework to work with high-throughput sequencing data","volume":"31","author":"Anders","year":"2015","journal-title":"Bioinformatics"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"550","DOI":"10.1186\/s13059-014-0550-8","article-title":"Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2","volume":"15","author":"Love","year":"2014","journal-title":"Genome Biol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"W193","DOI":"10.1093\/nar\/gkm226","article-title":"g:Profiler\u2014A web-based toolset for functional profiling of gene lists from large-scale experiments","volume":"3","author":"Reimand","year":"2007","journal-title":"Nucleic Acids Res."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"3251","DOI":"10.1093\/bioinformatics\/btm369","article-title":"GSEA-P: A desktop application for Gene Set Enrichment Analysis","volume":"23","author":"Subramanian","year":"2007","journal-title":"Bioinformatics"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/7\/3937\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T22:48:27Z","timestamp":1760136507000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/7\/3937"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,4,1]]},"references-count":51,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2022,4]]}},"alternative-id":["ijms23073937"],"URL":"https:\/\/doi.org\/10.3390\/ijms23073937","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,4,1]]}}}