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siRNA Mediate RNA Interference Concordant with Early On-Target Transient Transcriptional Interference

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 siRN...

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Autores principales: Fang, Zhiming, Zhao, Zhongming, Eapen, Valsamma, Clarke, Raymond A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8393430/
https://www.ncbi.nlm.nih.gov/pubmed/34440463
http://dx.doi.org/10.3390/genes12081290
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author Fang, Zhiming
Zhao, Zhongming
Eapen, Valsamma
Clarke, Raymond A.
author_facet Fang, Zhiming
Zhao, Zhongming
Eapen, Valsamma
Clarke, Raymond A.
author_sort Fang, Zhiming
collection PubMed
description 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 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.
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spelling pubmed-83934302021-08-28 siRNA Mediate RNA Interference Concordant with Early On-Target Transient Transcriptional Interference Fang, Zhiming Zhao, Zhongming Eapen, Valsamma Clarke, Raymond A. Genes (Basel) Article 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 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. MDPI 2021-08-23 /pmc/articles/PMC8393430/ /pubmed/34440463 http://dx.doi.org/10.3390/genes12081290 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Fang, Zhiming
Zhao, Zhongming
Eapen, Valsamma
Clarke, Raymond A.
siRNA Mediate RNA Interference Concordant with Early On-Target Transient Transcriptional Interference
title siRNA Mediate RNA Interference Concordant with Early On-Target Transient Transcriptional Interference
title_full siRNA Mediate RNA Interference Concordant with Early On-Target Transient Transcriptional Interference
title_fullStr siRNA Mediate RNA Interference Concordant with Early On-Target Transient Transcriptional Interference
title_full_unstemmed siRNA Mediate RNA Interference Concordant with Early On-Target Transient Transcriptional Interference
title_short siRNA Mediate RNA Interference Concordant with Early On-Target Transient Transcriptional Interference
title_sort sirna mediate rna interference concordant with early on-target transient transcriptional interference
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8393430/
https://www.ncbi.nlm.nih.gov/pubmed/34440463
http://dx.doi.org/10.3390/genes12081290
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