{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T02:16:13Z","timestamp":1760235373540,"version":"build-2065373602"},"reference-count":21,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2021,8,23]],"date-time":"2021-08-23T00:00:00Z","timestamp":1629676800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000925","name":"National Health and Medical Research Council","doi-asserted-by":"publisher","award":["970859"],"award-info":[{"award-number":["970859"]}],"id":[{"id":"10.13039\/501100000925","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100003359","name":"Scoliosis Research Society","doi-asserted-by":"publisher","award":["30999"],"award-info":[{"award-number":["30999"]}],"id":[{"id":"10.13039\/100003359","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Genes"],"abstract":"<jats:p>Exogenous siRNAs are commonly used to regulate endogenous gene expression levels for gene function analysis, genotype\u2013phenotype association studies and for gene therapy. Exogenous siRNAs can target mRNAs within the cytosol as well as nascent RNA transcripts within the nucleus, thus complicating siRNA targeting specificity. To highlight challenges in achieving siRNA target specificity, we targeted an overlapping gene set that we found associated with a familial form of multiple synostosis syndrome type 4 (SYSN4). In the affected family, we found that a previously unknown non-coding gene TOSPEAK\/C8orf37AS1 was disrupted and the adjacent gene GDF6 was downregulated. Moreover, a conserved long-range enhancer for GDF6 was found located within TOSPEAK which in turn overlapped another gene which we named SMALLTALK\/C8orf37. In fibroblast cell lines, SMALLTALK is transcribed at much higher levels in the opposite (convergent) direction to TOSPEAK. siRNA targeting of SMALLTALK resulted in post transcriptional gene silencing (PTGS\/RNAi) of SMALLTALK that peaked at 72 h together with a rapid early increase in the level of both TOSPEAK and GDF6 that peaked and waned after 24 h. These findings indicated the following sequence of events: Firstly, the siRNA designed to target SMALLTALK mRNA for RNAi in the cytosol had also caused an early and transient transcriptional interference of SMALLTALK in the nucleus; Secondly, the resulting interference of SMALLTALK transcription increased the transcription of TOSPEAK; Thirdly, the increased transcription of TOSPEAK increased the transcription of GDF6. These findings have implications for the design and application of RNA and DNA targeting technologies including siRNA and CRISPR. For example, we used siRNA targeting of SMALLTALK to successfully restore GDF6 levels in the gene therapy of SYNS4 family fibroblasts in culture. To confidently apply gene targeting technologies, it is important to first determine the transcriptional interference effects of the targeting reagent and the targeted gene.<\/jats:p>","DOI":"10.3390\/genes12081290","type":"journal-article","created":{"date-parts":[[2021,8,23]],"date-time":"2021-08-23T10:24:17Z","timestamp":1629714257000},"page":"1290","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["siRNA Mediate RNA Interference Concordant with Early On-Target Transient Transcriptional Interference"],"prefix":"10.3390","volume":"12","author":[{"given":"Zhiming","family":"Fang","sequence":"first","affiliation":[{"name":"Ingham Institute, School of Psychiatry, University of NSW, Sydney, NSW 2170, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3477-0914","authenticated-orcid":false,"given":"Zhongming","family":"Zhao","sequence":"additional","affiliation":[{"name":"Center for Precision Health, School of Biomedical Informatics and School of Public Health, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6296-8306","authenticated-orcid":false,"given":"Valsamma","family":"Eapen","sequence":"additional","affiliation":[{"name":"Ingham Institute, School of Psychiatry, University of NSW, Sydney, NSW 2170, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Raymond A.","family":"Clarke","sequence":"additional","affiliation":[{"name":"Ingham Institute, School of Psychiatry, University of NSW, Sydney, NSW 2170, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,8,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1016\/j.ymgme.2003.08.011","article-title":"RNA interference and human disease","volume":"80","author":"Cheng","year":"2003","journal-title":"Mol. Genet. Metab."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"842","DOI":"10.1038\/nsmb.1444","article-title":"Antisense transcripts are targets for activating small RNAs","volume":"15","author":"Schwartz","year":"2008","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"494","DOI":"10.1038\/35078107","article-title":"Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells","volume":"411","author":"Elbashir","year":"2001","journal-title":"Nature"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1016\/B978-0-12-415795-8.00005-2","article-title":"Small RNA-induced transcriptional gene regulation in mammals mechanisms, therapeutic applications, and scope within the genome","volume":"102","author":"Green","year":"2011","journal-title":"Prog. Mol. Biol. Transl. Sci."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"122","DOI":"10.1021\/cb300490j","article-title":"Transcriptional Silencing by Single-Stranded RNAs Targeting a Noncoding RNA That Overlaps a Gene Promoter","volume":"8","author":"Matsui","year":"2013","journal-title":"ACS Chem. Biol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1038\/nchembio.400","article-title":"Transcriptional regulation by small RNAs at sequences downstream from 3\u2019 gene termini","volume":"6","author":"Yue","year":"2010","journal-title":"Nat. Chem. Biol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"413","DOI":"10.1038\/nature07756","article-title":"Small RNAs in transcriptional gene silencing and genome defence","volume":"457","author":"Moazed","year":"2009","journal-title":"Nature"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"100","DOI":"10.1038\/nrg3355","article-title":"RNA interference in the nucleus: Roles for small RNAs in transcription, epigenetics and beyond","volume":"14","author":"Castel","year":"2013","journal-title":"Nat. Rev. Genet."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1038\/nrg3606","article-title":"Long non-coding RNAs: New players in cell differentiation and development","volume":"15","author":"Fatica","year":"2014","journal-title":"Nat. Rev. Genet."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"8796","DOI":"10.1073\/pnas.132270899","article-title":"Transcriptional collision between convergent genes in budding yeast","volume":"99","author":"Prescott","year":"2002","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/j.mgene.2016.11.003","article-title":"CTNNA3 discordant regulation of nested LRRTM3, implications for autism spectrum disorder and Tourette syndrome","volume":"11","author":"Fang","year":"2017","journal-title":"Meta Gene"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1047","DOI":"10.1002\/dvdy.22251","article-title":"Identification of a distant cis-regulatory element controlling pharyngeal arch-specific expression of zebrafish gdf6a\/radar","volume":"239","author":"Reed","year":"2010","journal-title":"Dev. Dyn."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1125","DOI":"10.3390\/cancers2021125","article-title":"Markers for Detection of Prostate Cancer","volume":"2","author":"Clarke","year":"2010","journal-title":"Cancers"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"13377","DOI":"10.1038\/s41598-019-49802-w","article-title":"Overlapping protein-coding genes in human genome and their coincidental expression in tissues","volume":"9","author":"Chen","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"W273","DOI":"10.1093\/nar\/gkh458","article-title":"VISTA: Computational tools for comparative genomics","volume":"32","author":"Frazer","year":"2004","journal-title":"Nucleic Acids Res."},{"key":"ref_16","first-page":"1364","article-title":"Familial Klippel-Feil syndrome and paracentric inversion inv(8)(q22.2q23.3)","volume":"57","author":"Clarke","year":"1995","journal-title":"Am. J. Hum. Genet."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1017","DOI":"10.1002\/humu.20741","article-title":"Mutations in GDF6 are associated with vertebral segmentation defects in Klippel-Feil syndrome","volume":"29","author":"Tassabehji","year":"2008","journal-title":"Hum. Mutat."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"201","DOI":"10.1177\/000348949410300306","article-title":"Klippel-Feil syndrome associated with malformed larynx","volume":"103","author":"Clarke","year":"1994","journal-title":"Case report. Ann. Otol. Rhinol. Laryngol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2069","DOI":"10.1101\/gr.1306003","article-title":"A general approach for identifying distant regulatory elements applied to the Gdf6 gene","volume":"13","author":"Mortlock","year":"2003","journal-title":"Genome. Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"116","DOI":"10.1016\/S0012-1606(02)00022-2","article-title":"Multiple joint and skeletal patterning defects caused by single and double mutations in the mouse Gdf6 and Gdf5 genes","volume":"254","author":"Settle","year":"2003","journal-title":"Dev. Biol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Miles, J., Mitchell, J.A., Chakalova, L., Goyenechea, B., Osborne, C.S., O\u2019Neill, L., Tanimoto, K., Engel, J.D., and Fraser, P. (2007). Intergenic transcription, cell-cycle and the developmentally regulated epigenetic profile of the human beta-globin locus. PLoS ONE, 2.","DOI":"10.1371\/journal.pone.0000630"}],"container-title":["Genes"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4425\/12\/8\/1290\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:49:57Z","timestamp":1760165397000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4425\/12\/8\/1290"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,8,23]]},"references-count":21,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2021,8]]}},"alternative-id":["genes12081290"],"URL":"https:\/\/doi.org\/10.3390\/genes12081290","relation":{},"ISSN":["2073-4425"],"issn-type":[{"type":"electronic","value":"2073-4425"}],"subject":[],"published":{"date-parts":[[2021,8,23]]}}}