Schistosoma mansoni U6 gene promoter-driven short hairpin RNA induces RNA interference in human fibrosarcoma cells and schistosomules

Abstract

RNA interference (RNAi) mediated by short hairpin-RNA (shRNA) expressing plasmids can induce specific and long-term knockdown of specific mRNAs in eukaryotic cells. To develop a vector-based RNAi model for Schistosoma mansoni, the schistosome U6 gene promoter was employed to drive expression of shRNA targeting reporter firefly luciferase. An upstream region of a U6 gene predicted to contain the promoter was amplified from genomic DNA of S. mansoni. A shRNA construct driven by the predicted U6 promoter targeting luciferase was assembled and cloned into plasmid pXL-Bac II, the construct termed pXL-BacII_SmU6-shLuc. Luciferase expression in transgenic fibrosarcoma HT-1080 cells was significantly reduced 96 h following transduction with plasmid pXL-BacII_SmU6-shLuc, which encodes luciferase mRNA-specific shRNA. In a similar fashion, schistosomules of S. mansoni were transformed with the SmU6-shLuc or control constructs. Firefly luciferase mRNA was introduced into transformed schistosomules after which luciferase activity was analyzed. Significantly less activity was present in schistosomules transfected with pXL-BacII_SmU6-shLuc compared with controls. The findings revealed that the putative S. mansoni U6 gene promoter of 270 bp in length was active in human cells and schistosomes. Given that the U6 gene promoter drove expression of shRNA from an episome, the findings also indicate the potential of this putative RNA polymerase III dependent promoter as a component regulatory element in vector-based RNAi for functional genomics of schistosomes.

Fig. 1

Multiple sequence alignment of the U6 genes of Schistosoma mansoni, Homo sapiens and Mus musculus. The multiple alignment was assembled with ClustalW (Thompson et al., 1994) using BioEdit, version 7.0.5 (Hall, 1999) and the box shade feature of GeneDoc (Nicholas and Nicholas, 1997, Pittsburgh Supercomputing Center, Pittsburgh, PA 15213, USA), and the following sequences: S. mansoni (GenBank L25920 (coding region) and HQ540317 (upstream flanking region)); human, (GenBank X07425), mouse (GenBank X06980). Residue one (black, curved arrow) is the first nucleotide (nt) of the U6 RNA gene sequence. Characteristic motifs of the enhancer and core regions of the U6 gene promoter are identified or predicted (red colored rectangles). The enhancer distal sequence element includes an octamer motif (OCT) and SphI post-octamer homology (SPH) element and the core region comprises a proximal sequence element (PSE) and a TATA-like element. Consensus sequences are indicated under the PSE and OCT motifs. Note that the relative positions for the SPH and OCT elements are reversed in mouse U6.

Fig. 2

Episomal vector-based RNA interference (RNAi) of firefly luciferase activity in human fibrosarcoma HT1080 cells. A) Schematic illustration of the insert of plasmid of pXL-BacII_SmU6-shLuc, including the inverted terminal repeats (ITR) flanking the short hairpin (sh)RNA cassette, the Schistosoma mansoni U6 gene promoter, the sense strand (21 nucleotide (nt), residues 851–871 of the gene encoding firefly luciferase, the nine residue loop, the 21 nt anti-sense strand and the TTTTTT terminator residues. The predicted shRNA is shown below the plasmid construct. B) Luciferase activity in HT1080 cells expressed as relative light units (RLU)/s/10³ cells. HT1080 cells were transfected with 1 µg of small interfering RNA of 21 nt in length, specific for residues 851–871 of firefly luciferase (siLuc) ; 4 µg pXL-BacII_SmU6-shLuc (shLuc_4µg); 10 µg of pXL-BacII_SmU6-shLuc (shLuc_4µg); 4 µg plasmid pXL-BacII_SmU6-shScramLuc (shScrambled_4µg); 10 µg of control plasmid pXL-BacII_SmU6-shScramLuc (shScrambled_10µg); and no treatment control (mock). Bars are ± S.D. (n = 2). Significant differences between treated groups and the (mock) control are indicated: *, P ≤ 0.05; **, P ≤ 0.01.

Fig. 3

Episomal vector-based RNA interference (RNAi) of firefly luciferase activity in 1 day old schistosomules. Luciferase activity in schistosomules expressed as relative light units (RLU)/s/mg of protein. One day old schistosomules were electroporated in the absence of plasmid or RNAs (mock), 30 µg of firefly luciferase double stranded RNA (dsLuc), 10 µg of small interfering RNA of 21 nt in length, specific for residues 851–871 of firefly luciferase (siLuc), 20 µg of control plasmid pXL-BacII_SmU6-shScramLuc (ShScram) and 20 µg of plasmid pXL-BacII_SmU6-shLuc (shLuc). Bars are ± S.D. (n = 2). Significant differences between treated groups and the (mock) control are indicated: *, P ≤ 0.05; **, P ≤ 0.01.