Genevieve Degols

Intracellular distribution of microinjected antisense oligonucleotides.

Antisense oligomers constitute an attractive class of specific tools for genetic analysis and for potential therapeutic applications. Targets with different cellular locations have been described, such as mRNA translation initiation sites, pre-mRNA splicing sites, or the genes themselves. However the mechanism(s) of action and the intracellular distribution of antisense oligomers remain poorly understood. Antisense oligomers conjugated with various fluorochromes or with BrdUrd were microinjected into the cytoplasm of somatic cells, and their cellular distribution was monitored by fluorescence microscopy in fixed and nonfixed cells. A fast translocation in the nuclei and a concentration on nuclear structures were observed whatever probe was used. Nuclear transport occurs by diffusion since it is not affected by depletion of the intracellular ATP pool, temperature, or excess unlabeled oligomer. Accumulation of the oligomers in the nuclei essentially takes place on a set of proteins preferentially extracted between 0.2 M and 0.4 M NaCl as revealed by crosslinking of photosensitive oligomers. The relationship between nuclear location of antisense oligomers and their mechanism of action remains to be ascertained and could be of major interest in the design of more efficient antisense molecules.

The exonuclease ISG20 mainly localizes in the nucleolus and the Cajal (Coiled) bodies and is associated with nuclear SMN protein-containing complexes.

We have previously shown that ISG20, an interferon (IFN)‐induced gene, encodes a 3′ to 5′ exoribonuclease member of the DEDD superfamily of exonucleases. ISG20 specifically degrades single‐stranded RNA. In this report, using immunofluorescence analysis, we demonstrate that in addition to a diffuse cytoplasmic and nucleoplasmic localization, the endogenous ISG20 protein was present in the nucleus both in the nucleolus and in the Cajal bodies (CBs). In addition, we show that the ectopic expression of the CBs signature protein, coilin, fused to the red fluorescent protein (coilin‐dsRed) increased the number of nuclear dots containing both ISG20 and coilin‐dsRed. Using electron microcopy analysis, ISG20 appeared principally concentrated in the dense fibrillar component of the nucleolus, the major site for rRNA processing. We also present evidences that ISG20 was associated with survival of motor neuron (SMN)‐containing macromolecular nuclear complexes required for the biogenesis of various small nuclear ribonucleoproteins. Finally, we demonstrate that ISG20 was associated with U1 and U2 snRNAs, and U3 snoRNA. The accumulation of ISG20 in the CBs after IFN treatment strongly suggests its involvement in a new route for IFN‐mediated inhibition of protein synthesis by modulating snRNA and rRNA maturation. J. Cell. Biochem. 98: 1320–1333, 2006.

The exonuclease ISG20 is directly induced by synthetic dsRNA via NF-κB and IRF1 activation

Many interferon (IFN)-stimulated genes are also induced by double-stranded RNA (dsRNA), a component closely associated with the IFN system in the context of virus–host interactions. Recently, we demonstrated that the IFN-induced 3′ → 5′ exonuclease ISG20 possesses antiviral activities against RNA viruses. Here we show that ISG20 induction by synthetic dsRNA (pIpC) is stronger and faster than its induction by IFN. Two families of transcription factors are implicated in the transcriptional activation of ISG20 by dsRNA. Initially, the NF-κB factors p50 and p65 bind and activate the κB element of the Isg20 promoter. This is followed by IRF1 binding to the ISRE. As pIpC often induces protein movements in the cells, we questioned whether it could influence ISG20 localization. Interestingly and contrary to IFN, dsRNA induces a nuclear matrix enrichment of the ISG20 protein. dsRNA induction of ISG20 via NF-κB and its antiviral activity led us to suggest that ISG20 could participate in the cellular response to virus infection.