Michelle Silhol

The 2′-5′ Oligoadenylate/RNase L/RNase L Inhibitor Pathway Regulates Both MyoD mRNA Stability and Muscle Cell Differentiation

ABSTRACT The 2′-5′ oligoadenylate (2-5A)/RNase L pathway is one of the enzymatic pathways induced by interferon. RNase L is a latent endoribonuclease which is activated by 2-5A and inhibited by a specific protein known as RLI (RNase L inhibitor). This system has an important role in regulating viral infection. Additionally, variations in RNase L activity have been observed during cell growth and differentiation but the significance of the 2-5A/RNase L/RLI pathway in these latter processes is not known. To determine the roles of RNase L and RLI in muscle differentiation, C2 mouse myoblasts were transfected with sense and antisense RLI cDNA constructs. Importantly, the overexpression of RLI in C2 cells was associated with diminished RNase L activity, an increased level of MyoD mRNA, and accelerated kinetics of muscle differentiation. Inversely, transfection of the RLI antisense construct was associated with increased RNase L activity, a diminished level of MyoD mRNA, and delayed differentiation. In agreement with these data, MyoD mRNA levels were also decreased in C2 cells transfected with an inducible RNase L construct. The effect of RNase L activity on MyoD mRNA levels was relatively specific because expression of several other mRNAs was not altered in C2 transfectants. Therefore, RNase L is directly involved in myoblast differentiation, probably through its role in regulating MyoD stability. This is the first identification of a potential mRNA target for RNase L.

The 2–5A/RNase L/RNase L Inhibitor (RNI) Pathway Regulates Mitochondrial mRNAs Stability in Interferon α-treated H9 Cells

Interferon α (IFNα) belongs to a cytokine family that exhibits antiviral properties, immuno-modulating effects, and antiproliferative activity on normal and neoplasic cells in vitro and in vivo. IFNα exerts antitumor action by inducing direct cytotoxicity against tumor cells. This toxicity is at least partly due to induction of apoptosis. Although the molecular basis of the inhibition of cell growth by IFNα is only partially understood, there is a direct correlation between the sensitivity of cells to the antiproliferative action of IFNα and the down-regulation of their mitochondrial mRNAs. Here, we studied the role of the 2–5A/RNase L system and its inhibitor RLI in this regulation of the mitochondrial mRNAs by IFNα. We found that a fraction of cellular RNase L and RLI is localized in the mitochondria. Thus, we down-regulated RNase L activity in human H9 cells by stably transfecting (i) RNase L antisense cDNA or (ii) RLI sense cDNA constructions. In contrast to control cells, no post-transcriptional down-regulation of mitochondrial mRNAs and no cell growth inhibition were observed after IFNα treatment in these transfectants. These results demonstrate that IFNα exerts its antiproliferative effect on H9 cells at least in part via the degradation of mitochondrial mRNAs by RNase L.

Regulation of mitochondrial mRNA stability by RNase L is translation-dependent and controls IFN α -induced apoptosis

Interferons (IFNs) inhibit the growth of many different cell types by altering the expression of specific genes. IFNs activities are partly mediated by the 2′-5′ oligoadenylates-RNase L RNA decay pathway. RNase L is an endoribonuclease requiring activation by 2′-5′ oligoadenylates to cleave single-stranded RNA. Here, we present evidence that degradation of mitochondrial mRNA by RNase L leads to cytochrome c release and caspase 3 activation during IFNα-induced apoptosis. We identify and characterize the mitochondrial translation initiation factor (IF2mt) as a new partner of RNase L. Moreover, we show that specific inhibition of mitochondrial translation with chloramphenicol inhibits the IFNα-induced degradation of mitochondrial mRNA by RNase L. Finally, we demonstrate that overexpression of IF2mt in human H9 cells stabilizes mitochondrial mRNA, inhibits apoptosis induced by IFNα and partially reverses IFNα-cell growth inhibition. On the basis of our results, we propose a model describing how RNase L regulates mitochondrial mRNA stability through its interaction with IF2mt.

New Insights Into the Membrane Translocating Process of the Tat Peptide Used for Cellular Delivery of Various Biological Molecules

A 14-mer peptide derived from the HIV Tat protein was shown to translocate efficiently through the plasma membrane [1], Basic amino acid residues in this highly cationic peptide are responsible for its internalization [2]. Non-permeant peptides, antisense oligonucleotides, proteins and even particulate material can be efficiently internalized when chemically conjugated or genetically fused to this Tat peptide. However, the mechanism of this uptake still remains to be elucidated.