Optimizations of SiRNA Design for the Activation of Gene Transcription by Targeting the TATA-Box Motif

Abstract

Small interfering RNAs (siRNAs) are widely used to repress gene expression by targeting mRNAs. Some reports reveal that siRNAs can also activate or inhibit gene expression through targeting the gene promoters. Our group has found that microRNAs (miRNAs) could activate gene transcription via interaction with the TATA-box motif in gene promoters. To investigate whether siRNA targeting the same region could upregulate the promoter activity, we test the activating efficiency of siRNAs targeting the TATA-box motif of 16 genes and perform a systematic analysis to identify the common features of the functional siRNAs for effective activation of gene promoters. Further, we try various modifications to improve the activating efficiency of siRNAs and find that it is quite useful to design the promoter-targeting activating siRNA by following several rules such as (a) complementary to the TATA-box-centered region; (b) UA usage at the first two bases of the antisense strand; (c) twenty-three nucleotides (nts) in length; (d) 2′-O-Methyl (2′-OMe) modification at the 3′ terminus of the antisense strand; (e) avoiding mismatches at the 3′ end of the antisense strand. The optimized activating siRNAs potently enhance the expression of interleukin-2 (IL-2) gene in human and mouse primary CD4+ T cells with a long-time effect. Taken together, our study provides a guideline for rational design the promoter-targeting siRNA to sequence-specifically enhance gene expression.

Alterations of DNA methylation and mRNA levels of CYP1A1, GSTP1, and GSTM1 in human bronchial epithelial cells induced by benzo[a]pyrene

“…Usually, acute exposure to environmental pollutant is more likely to cause changes in gene transcription levels rather than in DNA methylation levels; hence, we speculate that the decreased DNA methylation may be a negative feedback effect in response to over-inhibition of gene transcription to prevent further reductions in gene level (Marik et al, 2010). Besides DNA methylation, there are other epigenetic modifications which were involved in the gene transcriptional regulation such as intracellular circular RNA, microRNA, siRNA and histone modification, and chromatin remodeling (Borel et al, 2012;Fan et al, 2014;Li et al, 2015;Monga et al, 2011;Zhu et al, 2020), and those other modifications were not mentioned in this study.…”

Small activating RNA binds to the genomic target site in a seed-region-dependent manner

“…It has been suggested that 200- to 1200-bp upstream of the TSS is the optimal targeting area ( 5 ) and actually, most of the known active saRNA target sites are located upstream of and no more than 1 kb from the TSS. Particularly, it has been proposed that saRNAs targeting sequences around the TSS or the TATA-box-centered region might have higher activation efficiency ( 6 , 44 ).…”

The TATA-box motif and its impact on transcriptional gene regulation by miRNAs

“…Recently, Fan et al designed and evaluated the potential of TATA-box-targeting small interfering RNAs (siRNAs) in the regulation of gene expression. They also identified four characteristics that contribute to the high efficiency of TATA-box-targeting activating siRNAs on IL-2 promoter activation: UA at the 3′ end of the antisense strand (23 nucleotides in length), which targets the center of TATA-box (~34 bp upstream the TSS), as well as 2′-OMe modified bases at the 3′ terminus of the antisense strand (31). In other recent studies, the same group revealed that a novel HIV-1-encoded miRNA, miR-H3, could target the TATA-box motif within HIV-1 5′ long terminal repeat (LTR) and enhance viral replication (32).…”