Probing the Ubiquitination-Mediated Function of Epithelial Sodium Channel in A549 Cells.

BACKGROUND: The activity of the amiloride-sensitive epithelial sodium channel (ENaC) in the tight epithelia of the lung is regulated by proteolytic activation and ubiquitination. Pathophysiology of lung diseases is directly related to changes in one or both of these mechanisms. METHODS: In this study, we investigated the impact of ubiquitination and cathepsin-mediated proteolytic activation mechanisms on the functional regulation of ENaC in lung cancer A549 cells using the patch-clamp technique. RESULTS: Our findings suggest that inhibiting the proteasome (polyubiquitination) with MG132 improves ENaC activity, whereas altering the pH of the lysosome (monoubiquitination inhibition) with NH4Cl has no effect on ENaC activity. In A549 cells, inhibition of cathepsin B (CSTB) decreased the ENaC current, open probabilities (NPo and Po), and the number of active channels. CONCLUSION: These findings delineate novel modes of ENaC degradation and proteolytic activation of functional channels in A549 cells. Our findings indicate that both proteolytic activation and ubiquitination of ENaC significantly affect channel function and add new insights into the endogenous ENaC processing which might help to further understand the pathophysiology of the lung disease.

Short-lived AUF1 p42-binding mRNAs of RANKL and BCL6 have two distinct instability elements each.

Regulation of mRNA stability by RNA-protein interactions contributes significantly to quantitative aspects of gene expression. We have identified potential mRNA targets of the AU-rich element binding protein AUF1. Myc-tagged AUF1 p42 was induced in mouse NIH/3T3 cells and RNA-protein complexes isolated using anti-myc tag antibody beads. Bound mRNAs were analyzed with Affymetrix microarrays. We have identified 508 potential target mRNAs that were at least 3-fold enriched compared to control cells without myc-AUF1. 22.3% of the enriched mRNAs had an AU-rich cluster in the ARED Organism database, against 16.3% of non-enriched control mRNAs. The enrichment towards AU-rich elements was also visible by AREScore with an average value of 5.2 in the enriched mRNAs versus 4.2 in the control group. Yet, numerous mRNAs were enriched without a high ARE score. The enrichment of tetrameric and pentameric sequences suggests a broad AUF1 p42-binding spectrum at short U-rich sequences flanked by A or G. Still, some enriched mRNAs were highly unstable, as those of TNFSF11 (known as RANKL), KLF10, HES1, CCNT2, SMAD6, and BCL6. We have mapped some of the instability determinants. HES1 mRNA appeared to have a coding region determinant. Detailed analysis of the RANKL and BCL6 3'UTR revealed for both that full instability required two elements, which are conserved in evolution. In RANKL mRNA both elements are AU-rich and separated by 30 bases, while in BCL6 mRNA one is AU-rich and 60 bases from a non AU-rich element that potentially forms a stem-loop structure.

Properties of short double-stranded RNAs carrying randomized base pairs: toward better controls for RNAi experiments.

Short interfering RNAs (siRNAs) are mediators of RNA interference (RNAi), a commonly used technique for selective down-regulation of target gene expression. Using an equimolar mixture of A, G, C, and U phosphoramidites during solid-phase synthesis, we introduced degenerate positions into RNA guide and passenger strands so that, when annealed, a large pool of distinct siRNA duplexes with randomized base pairs at defined sites was created. We assessed the randomization efficiency by deep sequencing one of the RNAs. All possible individual sequences were present in the pool with generally an excellent distribution of bases. Melting temperature analyses suggested that pools of randomized guide and passenger strands RNAs with up to eight degenerate positions annealed so that mismatched base-pairing was minimized. Transfections of randomized siRNAs (rnd-siRNAs) into cells led to inhibition of luciferase reporters by a miRNA-like mechanism when the seed regions of rnd-siRNA guide strands were devoid of degenerate positions. Furthermore, the mRNA levels of a select set of genes associated with siRNA off-target effects were measured and indicated that rnd-siRNAs with degenerate positions in the seed likely show typical non-sequence-specific effects, but not miRNA-like off-target effects. In the wake of recent reports showing the preponderance of miRNA-like off-target effects of siRNAs, our findings are of value for the design of a novel class of easily prepared and universally applicable negative siRNA controls.

Multiple microRNAs derived from chemically synthesized precursors regulate thrombospondin 1 expression.

Thrombospondin 1 (THBS1) is a secreted protein with a variety of biological functions, including a potent anti-angiogenic activity and activation of latent transforming growth factor beta (TGF-ß). In many human cancers it is expressed at low levels, although mutations in the THBS1 gene have been rarely reported. Instead, the loss of THBS1 expression has been proposed to be due to transcriptional and post-transcriptional deregulations. In a systematic screen of predicted microRNA (miRNA) binding sites in the THBS1 3' untranslated region (UTR) we employed chemically synthesized pre-miRNAs-a new class of pre-miRNA mimics-to show that several miRNAs (let-7a, miR-18a, miR-29b, miR-194, and miR-221) can modulate THBS1 expression at the post-transcriptional level. Sequence-specific downregulation of THBS1 by let-7a, miR-18a or by a small interfering RNA induced TGF-ß1 and SMAD4 transcript levels. Ectopic expression of latent TGF-ß1 reduced THBS1 protein expression and was associated with increased expression of let-7a, let-7-b, and miR-18a in cells. These data suggest an inverse correlation of THBS1 and latent TGF-ß1 expression levels possibly involving miRNAs.

Synthetic pre-microRNAs reveal dual-strand activity of miR-34a on TNF-α.

Functional microRNAs (miRNAs) are produced from both arms of their precursors (pre-miRNAs). Their abundances vary in context-dependent fashion spatiotemporarily and there is mounting evidence of regulatory interplay between them. Here, we introduce chemically synthesized pre-miRNAs (syn-pre-miRNAs) as a general class of accessible, easily transfectable mimics of pre-miRNAs. These are RNA hairpins, identical in sequence to natural pre-miRNAs. They differ from commercially available miRNA mimics through their complete hairpin structure, including any regulatory elements in their terminal-loop regions and their potential to introduce both strands into RISC. They are distinguished from transcribed pre-miRNAs by their terminal 5' hydroxyl groups and their precisely defined terminal nucleotides. We demonstrate with several examples how they fully recapitulate the properties of pre-miRNAs, including their processing by Dicer into functionally active 5p; and 3p-derived mature miRNAs. We use syn-pre-miRNAs to show that miR-34a uses its 5p and 3p miRNAs in two pathways: apoptosis during TGF-ß signaling, where SIRT1 and SP4 are suppressed by miR-34a-5p and miR-34a-3p, respectively; and the lipopolysaccharide (LPS)-activation of primary human monocyte-derived macrophages, where TNF (TNFα) is suppressed by miR-34a-5p indirectly and miR-34a-3p directly. Our results add to growing evidence that the use of both arms of a miRNA may be a widely used mechanism. We further suggest that syn-pre-miRNAs are ideal and affordable tools to investigate these mechanisms.

Iron availability modulates aberrant splicing of ferrochelatase through the iron- and 2-oxoglutarate dependent dioxygenase Jmjd6 and U2AF(65.).

Erythropoietic protoporphyria (EPP) results from partial deficiency of ferrochelatase (FECH). Genetically, EPP patients differ from asymptomatic mutation carriers at the unmutated FECH allele, the expression of which is modulated by single nucleotide polymorphism IVS3-48C/T. The IVS3-48C genotype, which is present among patients, leads to correct splicing of 60% of the pre-mRNA and to alternative splicing of 40%, the latter mRNA-product being destroyed by nonsense-mediated decay. An IVS3-48T genotype generates 80% correct and 20% aberrant products. Our study demonstrated that under iron deficient conditions, the aberrant splice product was increased to 56% and 50% of total FECH mRNA in erythroleukemic K562 and lymphoblastoid cell lines, respectively, both being homozygous for IVS3-48T. Concomitantly, FECH protein was decreased. Iron deficiency had less effect on the FECH splice ratio in an IVS3-48C/C lymphoblastoid cell line. Effects similar to iron deficiency were generated by siRNA knockdown of either splicing factor U2AF(65) or Fe(II)- and 2-oxoglutarate-dependent dioxygenase Jumonji domain-containing protein 6 (Jmjd6), which interacts with U2AF(65) by lysyl-hydroxylation. Based on these results, we propose that the availability of iron, a co-factor of Jmjd6, modulates U2AF(65)-lysyl-hydroxylation. This in turn, influences the relative amounts of correct and aberrant FECH mRNA splice products and thus, regulates the FECH enzyme activity.

Properties of N(4)-methylated cytidines in miRNA mimics.

Experiments conducted with micro RNA (miRNA) mimics often result in subtle phenotypic changes and hence require careful controls. A commonly used type of control reagent in the antisense/RNA interference fields is the mismatched sequence. However, it is difficult to use mismatch controls for miRNAs, mainly because base permutation in the seed region may generate a new miRNA seed with its own associated target transcripts. We incorporated N(4)-methylcytidine and N(4),N(4)-dimethylcytidine into a series of RNAs using the convertible nucleoside approach and measured their effects on hybridization affinity with complementary RNAs, and on miRNA-mediated and small interfering RNA (SiRNA)-mediated silencing. We report here that incorporation of a single N(4),N(4)-dimethylcytidine into the seed region of miRNAs can be used as a new class of negative miRNA control which (1) does not constitute a new seed sequence; (2) is accepted by the RNA-induced silencing complex (RISC); (3) causes a significant loss of binding affinity to target RNAs; and (4) is synthesized conveniently into oligoribonucleotides.

Suppression of latent transforming growth factor (TGF)-beta1 restores growth inhibitory TGF-beta signaling through microRNAs.

Cancer cells secreting excess latent TGF-ß are often resistant to TGF-ß induced growth inhibition. We observed that RNAi against TGF-ß1 led to apoptotic death in such cell lines with features that were, paradoxically, reminiscent of TGF-ß signaling activity and that included transiently enhanced SMAD2 and AKT phosphorylation. A comprehensive search in Hela cells for potential microRNA drivers of this mechanism revealed that RNAi against TGF-ß1 led to induction of pro-apoptotic miR-34a and to a globally decreased oncomir expression. The reduced levels of the oncomirs miR-18a and miR-24 accounted for the observed derepression of two TGF-ß1 processing factors, thrombospondin-1, and furin, respectively. Our data suggest a novel mechanism in which latent TGF-ß1, thrombospondin 1, and furin form a microRNA-mediated regulatory feedback loop. For cells with high levels of latent TGF-ß, this provides a potentially widespread mechanism of escape from TGF-ß-mediated growth arrest at the earliest point in the signaling pathway, TGF-ß processing.

Destabilization of interleukin-6 mRNA requires a putative RNA stem-loop structure, an AU-rich element, and the RNA-binding protein AUF1.

Interleukin-6 mRNA is unstable and degraded with a half-life of 30 min. Instability determinants can entirely be attributed to the 3' untranslated region. By grafting segments of this region to stable green fluorescent protein mRNA and subsequent scanning mutagenesis, we have identified two conserved elements, which together account for most of the instability. The first corresponds to a short noncanonical AU-rich element. The other, 80 nucleotides further 5', comprises a sequence predicted to form a stem-loop structure. Neither element alone was sufficient to confer full instability, suggesting that they might cooperate. Overexpression of myc-tagged AUF1 p37 and p42 isoforms as well as suppression of endogenous AUF1 by RNA interference stabilized interleukin-6 mRNA. Both effects required the AU-rich instability element. Similarly, the proteasome inhibitor MG132 stabilized interleukin-6 mRNA probably through an increase of AUF1 levels. The mRNA coimmunoprecipitated specifically with myc-tagged AUF1 p37 and p42 in cell extracts but only when the AU-rich instability element was present. These results indicate that AUF1 binds to the AU-rich element in vivo and promotes IL-6 mRNA degradation.