The effect of intracellular iron concentration and nitrogen monoxide on NRAMP2 expression and non-transferrin-bound iron uptake

Recent studies have demonstrated that the protein product (natural resistance associated macrophage protein 2, Nramp2) encoded by the gene Nramp2 acts as an Fe transporter involved in the uptake of Fe from transferrin (Tf ) and low Mr Fe complexes. Interestingly, there are two splice variants of Nramp2, one with a putative ironresponsive element (IRE) in its 30 untranslated region (UTR) and another without. Due to the importance of Nramp2 in Fe transport, and the presence of an IRE in its 30-UTR, we have examined the effect of Fe-deprivation, Fe-loading, and nitrogen monoxide on the expression of Nramp2 mRNA. These results were compared to the expression of transferrin receptor (TfR) mRNAwhich also has IREs in its 30-UTR and is regulated by Fe and NO via the binding of iron-regulatory proteins (IRPs) to its IREs. Our experiments show that the IRE in Nramp2 mRNA does bind the IRPs in lysates from a mouse fibroblast cell line (LMTK2). Moreover, reverse transcription- PCR (RT-PCR) demonstrated that both the IRE and non-IRE-containing transcripts were present within these cells. However, there was no change in Nramp2 mRNA expression in LMTK2 cells after a 20-h incubation with either the Fe chelator, desferrioxamine (DFO), the Fe donor, ferric ammonium citrate (FAC), or the NO generator, S-nitroso-N-acetylpenicillamine (SNAP). In contrast, these agents caused a marked change in the RNA-binding activity of the IRPs and the expression of TfR mRNA. In addition, both FAC and DFO caused an appropriate change in [59Fe] uptake from [59Fe]Tf, viz., an increase in Fe uptake after exposure to DFO and a decrease after treatment with FAC. As Nramp2 can transport Fe from non-Tf-bound Fe, the effect of preincubation with DFO and FAC was also examined on Fe uptake from [59Fe]nitrilotriacetate and [59Fe]citrate. However, in contrast to the results found for [59Fe]Tf, incubation with DFO and FAC did not result in appropriate regulation of Fe uptake from [59Fe]nitrilotriacetate or [59Fe]citrate. These data demonstrate that non-Tf-bound Fe uptake was not under control of the IRP-IRE system in these cells. Collectively, the results indicate that in LMTK-fibroblasts Nramp2 mRNA expression was not regulated like TfR mRNA

All Small Nuclear RNAs (snRNAs) of the [U4/U6.U5] Tri-snRNP Localize to Nucleoli; Identification of the Nucleolar Localization Element of U6 snRNA

Previously, we showed that spliceosomal U6 small nuclear RNA (snRNA) transiently passes through the nucleolus. Herein, we report that all individual snRNAs of the [U4/U6.U5] tri-snRNP localize to nucleoli, demonstrated by fluorescence microscopy of nucleolar preparations after injection of fluorescein-labeled snRNA into Xenopus oocyte nuclei. Nucleolar localization of U6 is independent from [U4/U6] snRNP formation since sites of direct interaction of U6 snRNA with U4 snRNA are not nucleolar localization elements. Among all regions in U6, the only one required for nucleolar localization is its 3′ end, which associates with the La protein and subsequently during maturation of U6 is bound by Lsm proteins. This 3′-nucleolar localization element of U6 is both essential and sufficient for nucleolar localization and also required for localization to Cajal bodies. Conversion of the 3′ hydroxyl of U6 snRNA to a 3′ phosphate prevents association with the La protein but does not affect U6 localization to nucleoli or Cajal bodies