Abstract
MicroRNAs (miRNAs) are endogenously expressed single-stranded ∼21–23 nucleotide RNAs that inhibit gene expression post-transcriptionally by binding imperfectly to elements usually within the 3′untranslated region (3′UTR) of mRNAs. Small interfering RNAs (siRNAs) mediate site-specific cleavage by binding with perfect complementarity to RNA. Here, a cell-based miRNA reporter system was developed to screen for compounds from marine and plant extracts that inhibit miRNA or siRNA activity. The daphnane diterpenoid genkwanine M (GENK) isolated from the plant Wikstroemia polyantha induces an early inflammatory response and can moderately inhibit miR-122 activity in the liver Huh-7 cell line. GENK does not alter miR-122 levels nor does it directly inhibit siRNA activity in an in vitro cleavage assay. Finally, we demonstrate that GENK can inhibit HCV infection in Huh-7 cells. In summary, the development of the cell-based miRNA sensor system should prove useful in identifying compounds that affect miRNA/siRNA activity.
Highlights
Small interfering RNAs, which are produced by introducing foreign dsRNAs [19] or by endogenously transcribed double-stranded RNAs [20], bind to RNA sequences with perfect complementarity leading to specific cleavage and subsequent degradation of the target messenger RNAs (mRNAs)
We have identified a compound of the Daphne diterpenoid family - called genkwanine M (GENK) [54]- which affects diverse cellular functions including the inhibition of miRNA activity
We demonstrated that GENK inhibits miRNAmediated repression in a time-dependent manner, suggesting that tight control of miRNA activity is involved in the early inflammatory response
Summary
MicroRNAs (miRNAs) are small endogenous non-coding singlestranded RNAs (typically 21–23 nucleotide long) that act posttranscriptionally to repress translation [1,2,3,4,5] and/or induce mRNA decay [6,7,8]. miRNAs play significant roles in a number of cellular processes and their misregulation has been linked to many pathological states, including cancer [9,10], diabetes [11,12], neurodegenerative disease [13] and viral infections [14,15,16]. Small interfering RNAs (siRNAs), which are produced by introducing foreign dsRNAs [19] or by endogenously transcribed double-stranded RNAs (dsRNAs) [20], bind to RNA sequences with perfect complementarity leading to specific cleavage and subsequent degradation of the target mRNA. The ,70 nucleotide pre-miRNA is processed by another RNAse III enzyme, Dicer [28,29,30], and its binding partner, TRBP [31,32], into a mature 21–23 nucleotide dsRNA containing 2 nucleotide 59 overhangs. The miRNA-containing RISC complex binds to the target sequence within the 39UTR of the mRNA [38]. Several models have been proposed including miRNA-directed inhibition of the cap-binding complex, inhibition of translation elongation, and deadenylation stimulation leading to subsequent degradation of target mRNA. Besides the main miRNA biogenesis pathway described, a subset of miRNAs mature via alternate pathways including Drosha-independent and Dicer-independent pathways and can originate from tRNA precursors and introns [41,42]
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