Localized expression of small RNA inhibitors in human cells

Abstract

Several types of small RNAs have been proposed as gene expression repressors with great potential for use in gene therapy. RNA polymerase III (pol III) provides an ideal means of expressing small RNAs in cells because its normal products are small, highly structured RNAs that are found in a variety of subcellular compartments. We have designed cassettes that use human pol III promoters for the high-level expression of small RNAs in the cytoplasm, nucleoplasm, and nucleolus. The levels and subcellular destinations of the transcripts are compared for transcripts expressed using the U6 small nuclear RNA (snRNA), 5S ribosomal RNA (rRNA), and the 7SL RNA component of the signal recognition particle. The most effective location for a particular inhibitory RNA is not necessarily predictable; thus these cassettes allow testing of the same RNA insert in multiple subcellular locations. Several small interfering RNA (siRNA) inserts were tested for efficacy. An siRNA insert that reduces lamin expression when transcribed from the U6 snRNA promoter in the nucleus has no effect on lamin expression when transcribed from 5S rRNA and 7SL RNA-based cassettes and found in the nucleolus and cytoplasm. To test further the generality of U6-driven siRNA inhibitors, siRNAs targeting HIV were tested by co-transfection with provirus in cell culture. Although the degree of HIV-1 inhibition varied among inserts, results show that the U6 cassette provides a means of expressing an siRNA-like inhibitor of HIV gene expression.

MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells

“…1 online). Fluorescence in situ hybridization with a probe against the U6 sponge RNAs primarily labeled the nucleus, as in previous work 13 (data not shown). How an inhibitor localized primarily to the nucleus can function against microRNA localized primarily in the cytoplasm is not clear.…”

“…1c). We subcloned tandemly arrayed microRNA binding sites from the GFP sponge constructs into a modified U6 small nuclear RNA promoter-terminator vector, which produces short (<300 nt) RNAs with structurally stabilized 5′ and 3′ ends 13 . As they lack an open reading frame, these U6 sponges are substrates for microRNA binding, but not for translation or translational repression.…”

Gene therapy progress and prospects: Novel gene therapy approaches for AIDS

“…U6-driven siRNAs targeting HIV significantly inhibited HIV-1 gene expression when cotransfected with provirus. 37 Other studies specifically targeted Rev 38 or Tat and Rev. 39 Although clearly efficient for short-lived inhibition, RNAi might be less efficient in the long run, mainly due to point mutations at HIV-1 target sites, leading to resistance.…”

Utility of Epstein–Barr virus-encoded small RNA promoters for driving the expression of fusion transcripts harboring short hairpin RNAs

“…In this regard, some shRNAs expressed from the U6 promoter have been found mainly in the nucleus. 22,50 The subcellular localization of RNAs expressed from Pol III promoters depends mainly on the expression cassette. 50 Interestingly, while RNAi mediated by mRNA degradation occurs in the cytoplasm, siRNAs can also localize to the nucleus and induce specific RNA degradation there.…”