A potential role for RNA interference in controlling the activity of the human LINE-1 retrotransposon - PubMed (original) (raw)
A potential role for RNA interference in controlling the activity of the human LINE-1 retrotransposon
Harris S Soifer et al. Nucleic Acids Res. 2005.
Abstract
Long interspersed nuclear elements (LINE-1 or L1) comprise 17% of the human genome, although only 80-100 L1s are considered retrotransposition-competent (RC-L1). Despite their small number, RC-L1s are still potential hazards to genome integrity through insertional mutagenesis, unequal recombination and chromosome rearrangements. In this study, we provide several lines of evidence that the LINE-1 retrotransposon is susceptible to RNA interference (RNAi). First, double-stranded RNA (dsRNA) generated in vitro from an L1 template is converted into functional short interfering RNA (siRNA) by DICER, the RNase III enzyme that initiates RNAi in human cells. Second, pooled siRNA from in vitro cleavage of L1 dsRNA, as well as synthetic L1 siRNA, targeting the 5'-UTR leads to sequence-specific mRNA degradation of an L1 fusion transcript. Finally, both synthetic and pooled siRNA suppressed retrotransposition from a highly active RC-L1 clone in cell culture assay. Our report is the first to demonstrate that a human transposable element is subjected to RNAi.
Figures
Figure 1
L1 dsRNA is a substrate for recombinant human DICER. (A) Schematic representation of a retrotransposition-competent LINE-1 (RC-L1) illustrating the 5′-UTR with internal promoter, ORF1 product p40, inter-ORF region (bold black line), ORF2 domains EN (endonuclease) and RT (reverse transcriptase), and 3′-UTR. Arrowheads above the diagram indicate the positions of the primers (see Table 1) used to generate cDNA templates for in vitro transcription (B) About 1 μg of each L1 dsRNA was incubated in the presence (+) or absence (−) of recombinant human DICER for 18 h and resolved on a 2.5% agarose gel. Open arrowhead, long dsRNA; closed arrowhead, L1 siRNA, measuring ∼21–23 nt.
Figure 2
L1 siRNA specifically inhibits the expression of a hybrid luciferase transcript driven by the L1 5′-UTR internal promoter. HCT116 cells were co-transfected with the indicated firefly (FF) luciferase expression vector and increasing amounts of L1 siRNA or control siRNA. Relative FF luciferase activity in the absence of siRNA was set at 1.0. The amount of each specific siRNA is indicated below the graphs. (A) Luciferase assays performed with in vitro ‘diced’ siRNA. Diced LacZ siRNA and synthetic FF luciferase siRNA served as negative and positive controls, respectively. (B) Luciferase assays performed with synthetic siRNA targeting nucleotides 749–769 of the L1 5′-UTR. Irrelevant synthetic siRNA targeting the HIV Rev transcript was included as the negative control.
Figure 3
L1 siRNA inhibits luciferase expression through RNAi-mediated degradation of the hybrid transcript. (A) RNA harvested from HCT116 cells co-transfected with pHSS101/RP and the indicated siRNA was subjected to semi-quantitative RT–PCR analysis with primers set to amplify the FF luciferase transgene and a separate set of primers to amplify the GAPDH gene as an internal loading control. FF luciferase expression was normalized to GAPDH levels. Relative luciferase expression in the absence of siRNA was set at 1.0. (B) Graphical representation of the semi-quantitative RT–PCR data. Columns represent mean FF luciferase expression (±standard deviations) of three independent PCR experiments.
Figure 4
L1 siRNA can limit the activity of the RC-L1 clone, L1RP, in a transient retrotransposition assay. HCT116 or HeLa cells were transfected with pCEP4-L1RPneo alone or co-transfected with different siRNA. The retrotransposition activity was determined using a transient retrotransposition assay following G418 selection. The relative retrotransposition frequency of pCEP4-L1RPneo in the absence of siRNA was set at 100%. The negative control pCEP4-L1.3neo-JM111, an RC-L1 clone with two missense mutations in ORF1 rendering it inactive, showed no retrotransposition activity. (A) Results of the retrotransposition assay in HCT116 cells. Control siRNA used for transfection included 250 ng synthetic HIV Rev and 50 ng diced LacZ siRNA. Diced 5′-UTR siRNA (50 and 250 ng), diced ORF1 siRNA (25, 50 and 250 ng) and 250 ng 5′-UTR #749 synthetic siRNA were assayed separately. (B) Results of the retrotransposition assay in HeLa cells. Synthetic HIV Rev siRNA (250 ng) was included as a control. Diced 5′-UTR siRNA (50 and 250 ng) was also assayed. (C). The results of the retrotransposition assay achieved in HCT116 cells. The wild-type pCEP4-L1RPneo (or the negative control pCEP4-L1.3neo-JM111) was transfected alone or with the indicated amount of siRNA. G418 colonies were fixed and stained 14 days post-selection with 4% Giemsa for visualization.
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