G C A T T A C G G C A T genes Article siRNA Mediate RNA Interference Concordant with Early On-Target Transient Transcriptional Interference Zhiming Fang 1, Zhongming Zhao 2 , Valsamma Eapen 1 and Raymond A. Clarke 1,* 1 Ingham Institute, School of Psychiatry, University of NSW, Sydney, NSW 2170, Australia; [email protected] (Z.F.); [email protected] (V.E.) 2 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; [email protected] * Correspondence: [email protected] Abstract: Exogenous siRNAs are commonly used to regulate endogenous gene expression levels for gene function analysis, genotype–phenotype 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 multi- ple 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 downregu- lated. 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 Citation: Fang, Z.; Zhao, Z.; Eapen, V.; Clarke, R.A. siRNA Mediate RNA caused an early and transient transcriptional interference of SMALLTALK in the nucleus; Secondly, Interference Concordant with Early the resulting interference of SMALLTALK transcription increased the transcription of TOSPEAK; On-Target Transient Transcriptional Thirdly, the increased transcription of TOSPEAK increased the transcription of GDF6. These findings Interference. Genes 2021, 12, 1290. have implications for the design and application of RNA and DNA targeting technologies including https://doi.org/10.3390/genes12081290 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 Academic Editor: Michele Cioffi targeting technologies, it is important to first determine the transcriptional interference effects of the targeting reagent and the targeted gene. Received: 22 July 2021 Accepted: 11 August 2021 Keywords: transcriptional interference; siRNA; lncRNA; multiple synostosis syndrome type 4 Published: 23 August 2021 (SYNS4); sets of interacting transcription units (SITRUS) Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Exogenous siRNAs are used routinely in many biotechnology and research applica- tions to regulate endogenous gene expression levels. The most common is the use of siRNA for post transcriptional gene silencing (PTGS) via the knockdown of mRNA expression levels [1] which is also referred to as RNA interference (RNAi). Exogenous siRNA can also Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. target nascent RNA transcripts in the nucleus with the potential to cause near permanent This article is an open access article transcriptional silencing (PTS) or upregulation (PTU) of the associated gene through epi- distributed under the terms and genetic modification of the DNA [2–7]. The mechanisms of siRNA mediated PTGS and conditions of the Creative Commons PTS/PTU are well documented and involve siRNA targeting within the cytosol and the Attribution (CC BY) license (https:// nucleus, respectively [1–7]. creativecommons.org/licenses/by/ PTGS/RNAi represents the most widely documented siRNA approach for the tran- 4.0/). sient reduction (knockdown) of target mRNA levels in the cytosol [1]. To achieve PTGS, Genes 2021, 12, 1290. https://doi.org/10.3390/genes12081290 https://www.mdpi.com/journal/genes Genes 2021, 12, 1290 2 of 15 an exogenous siRNA is designed to be complementary to a mature mRNA (exon derived) sequence. In contrast, for PTS/PTU, the siRNA are designed complementary to the im- mediate gene promoter or 30 flanking region of the target gene, respectively [2–7]. During PTS/PTU, the exogenous siRNA enters the cytosol before relocating to the nucleus where it binds the nascent RNA transcript derived from the target gene causing epigenetic mod- ification of the DNA of the target gene [5,8]. When successful, the resulting epigenetic modifications of the DNA in PTS/PTU can cause near permanent changes to the transcrip- tion of the target gene [8]. From these applications, it is obvious that siRNA target RNA transcripts within both the cytosol and the nucleus where the region of the RNA targeted determines the nature of the interference. We, therefore, questioned whether an siRNA designed for PTGS of a target mRNA in the cytosol could target and interfere with the transcription of the nascent RNA precursor of the target in the nucleus and whether it would be possible to differentiate between these two distinct on-target interference effects. To differentiate between siRNA targeting effects on the mRNA and its nascent pre- cursor, we determined to target one gene that overlaps another gene with the following specifications: Firstly, the transcription of the two overlapping genes must converge from opposite directions on the DNA so as to initiate RNA polymerase collision mediated tran- scriptional interference [9–11]. Secondly, the target gene must be expressed at higher levels than the gene it overlaps [11]. The rationale for this approach was that the more highly expressed of the two convergently transcribed genes has been shown to repress the tran- scription of the lower expressed gene [11] presumably through increased incidence of RNA polymerase collision events [10]. In this way, if the siRNA were to alter the transcription of the target gene (after binding its nascent RNA transcript) as reported elsewhere [11] this could in turn effect the transcription and expression of the overlapping gene [11] which could in turn be monitored using comparative rtPCR [11]. The Model: In this study, we discovered and were the first to characterize the long noncoding gene which we named TOSPEAK. We found that TOSPEAK was disrupted in a family with SYSN4 with multiple joint fusions, malformation of laryngeal cartilages and severe speech impairment. TOSPEAK/C8orf37AS1 was found to overlap a nested long-range enhancer ECR5 that regulates expression of the adjacent bone morphogenetic protein gene growth differentiation factor 6 (GDF6)[12]. This was of great interest as GDF6 expression was previously found reduced in affected members of the SYSN4 family [13] thus raising the question as to whether the SYNS4 skeletal phenotype was a function of the GDF6 or TOSPEAK genotype. Moreover, there was another interesting feature of TOSPEAK that warranted further investigation, namely that TOSPEAK physically overlaps another more highly expressed gene, which we named SMALLTALK/C8orf37. To examine the expression of this overlapping gene set, we used siRNA to target SMALLTALK in primary fibroblast cell cultures. As expected, the siRNA targeting of SMALLALK resulted in a prolonged PTGS/RNAi mediated decrease in the mRNA level of SMALLTALK. In addition, we detected a transient increase in the mRNA levels of both TOSPEAK and GDF6. Together, our findings indicate a role for both TOSPEAK and GDF6 in the joint, bone and cartilage malformations of the SYNS4 family [13]. Given that over 20% of human protein coding genes physically overlap [14] and that many others share promoters, and those like GDF6 that have regulatory elements nested within adjacent genes, the findings of this study have important implications for genotype–phenotype correlation studies and gene targeting strategies and for the design of gene therapies including those that use exogenous siRNA. 2. Materials and Methods RNA isolation and cDNA synthesis: Total RNA was extracted from tissues (fresh skin biopsies and primary fibroblast cell lines derived there from, and from fresh white blood cells) using Trizol following the manufacture’s protocol (ThermoFisher Scientific Australia, Sydney, Australia). RNA was treated with DNase I (NEB Biolabs, Ipswich, Australia), ethanol precipitated, resuspended in DEPC-treated water and quality tested Genes 2021, 12, 1290 3 of 15 using spectrophotometry (A260/280 ratio was ~1.7) and gel electrophoresis. Then, 1 ug of total RNA extracted was reverse transcribed using 250 ng of random hexamers (Promega Pty Ltd., Sydney, Australia) in a standard 20 µL reaction including 4 µL of first strand buffer (Invitrogen Pty Ltd.), 2 µL of 0.1M DDT, 1 µL of 10 mM dNTP, 1 µL RNase inhibitor (2500 U) and 1 µL of reverse transcriptase (10,000 U) (Invitrogen Pty Ltd.). After annealing of the hexamers for 10 min at 72 ◦C, cDNA synthesis was performed for 42 ◦C for 90 min followed by an enzyme inactivation step at 70 ◦C for 15 min. All cDNA products were diluted