Polyamine depletion increases cytoplasmic levels of RNA-binding protein HuR leading to stabilization of nucleophosmin and p53 mRNAs

Polyamines are essential for maintaining normal intestinal epithelial integrity, an effect that relies, at least in part, on their ability to keep low levels of nucleophosmin (NPM) and p53 mRNAs. The RNA-binding protein HuR associates with the p53 mRNA, as reported previously, and with the NPM mRNA, computationally predicted to be a target of HuR. Here, we show that HuR binds the NPM and p53 3'-untranslated regions and stabilizes these mRNAs in polyamine-depleted intestinal epithelial cells. Depletion of cellular polyamines by inhibiting ornithine decarboxylase with alpha-difluoromethylornithine dramatically enhanced the cytoplasmic abundance of HuR, whereas ectopic ornithine decarboxylase overexpression decreased cytoplasmic HuR; neither intervention changed whole-cell HuR levels. HuR was found to specifically bind the 3'-untranslated regions of NPN and p53 mRNAs. HuR silencing rendered the NPM and p53 mRNAs unstable and prevented increases in NPM and p53 mRNA and protein in polyamine-deficient cells. These results indicate that polyamines modulate cytoplasmic HuR levels in intestinal epithelial cells, in turn controlling the stability of the NPM and p53 mRNAs and influencing NPM and p53 protein levels.     

Expression of procyclin mRNAs during cyclical transmission of Trypanosoma brucei

Trypanosoma brucei, the parasite causing human sleeping sickness, relies on the tsetse fly for its transmission. In the insect, EP and GPEET procyclins are the major surface glycoproteins of procyclic (midgut) forms of the parasite, with GPEET predominating in the early procyclic form and two isoforms of EP in the late procyclic form. EP procyclins were previously detected on salivary gland trypanosomes, presumably epimastigotes, by immunoelectron microscopy. However, no procyclins could be detected by mass spectrometry when parasites were isolated from infected glands. We have used qualitative and quantitative RT-PCR to analyse the procyclin mRNAs expressed by trypanosomes in the tsetse midgut and salivary glands at different time points after infection. The coding regions of the three EP isoforms (EP1, EP2 and EP3) are extremely similar, but their 3' untranslated regions contain unique sequences that make it possible to assign the cDNAs amplified by this technique. With the exception of EP2, we found that the spectrum of procyclin mRNAs expressed in the midgut mirrors the protein repertoire of early and established procyclic forms. Surprisingly, procyclin mRNAs, including that of GPEET, are present at relatively high levels in salivary gland trypanosomes, although the proteins are rarely detected by immunofluorescence. Additional experiments using transgenic trypanosomes expressing reporter genes or mutant forms of procyclin point to a mechanism of translational or post-translational control, involving the procyclin coding regions, in salivary gland trypanosomes. It is widely accepted that T. brucei always has a coat of either variant surface glycoprotein or procyclin. It has been known for many years that the epimastigote form does not have a variant surface glycoprotein coat. The finding that this life cycle stage is usually negative for procyclin as well is new, and means that the paradigm will need to be revised.     

Cloning and recombinant expression of the La RNA-binding protein from Trypanosoma brucei

We report the isolation, cloning and recombinant expression of a Trypanosoma brucei homolog of the La RNA-binding protein. Based on peptide sequence information we have isolated a cDNA clone which encodes a protein of 335 amino acids with a predicted molecular weight of 37.7 kDa. The amino acid sequence fits the domain structure of known La proteins and contains a putative ATP-binding site located in the COOH-terminal domain. The cDNA was expressed as a glutathione S-transferase fusion protein in Escherichia coli, and the recombinant protein displayed RNA-binding activity in an electrophoretic mobility shift assay.     

RBP16 is a multifunctional gene regulatory protein involved in editing and stabilization of specific mitochondrial mRNAs in Trypanosoma brucei

RBP16 is a Trypanosoma brucei mitochondrial RNA-binding protein that associates with guide RNAs (gRNAs), mRNAs, and ribosomal RNAs. Based on its inclusion in the multifunctional Y-box protein family and its ability to bind multiple RNA classes, we hypothesized that RBP16 plays a role in diverse aspects of mitochondrial gene regulation. To gain insight into RBP16 function, we generated cells expressing less than 10% of wild-type RBP16 levels by tetracycline-regulated RNA interference (RNAi). Poisoned primer extension analyses revealed that edited, but not unedited, CYb mRNA is reduced by approximately 98% in tetracycline-induced RBP16 RNAi cells, suggesting that RBP16 is critical for CYb RNA editing. The down-regulation of CYb editing in RBP16 RNAi transfectants apparently entails a defect in gRNA utilization, as gCYb[560] abundance is similar in uninduced and induced cells. We observed a surprising degree of specificity regarding the ability of RBP16 to modulate editing, as editing of mRNAs other than CYb is not significantly affected upon RBP16 disruption. However, the abundance of the never edited mitochondrial RNAs COI and ND4 is reduced by 70%-80% in RBP16 RNAi transfectants, indicating an additional role for RBP16 in the stabilization of these mRNAs. Analysis of RNAs bound to RBP16 immunoprecipitated from wild-type cells reveals that RBP16 is associated with multiple gRNA sequence classes in vivo, including those whose abundance and usage appear unaffected by RBP16 disruption. Overall, our results indicate that RBP16 is an accessory factor that regulates the editing and stability of specific populations of mitochondrial mRNAs.     

The role of the AU-rich elements of mRNAs in controlling translation

Adenosine- and uridine-rich elements (AREs) located in 3'-untranslated regions are the best-known determinants of RNA instability. These elements have also been shown to control translation in certain mRNAs, including mRNAs for prominent pro-inflammatory and tumor growth-related proteins, and physiological anti-inflammatory processes that target ARE-controlled translation of mRNAs coding for pro-inflammatory proteins have been described. A major research effort is now being made to understand the mechanisms by which the translation of these mRNAs is controlled and the signalling pathways involved. This review focuses on the role of ARE-containing gene translation in inflammation, and the disease models that have improved our understanding of ARE-mediated translational control.     

Impaired gametogenesis in mice that overexpress the RNA-binding protein HuR

A series of experiments, using cell culture models or in vitro assays, has shown that the RNA-binding protein HuR increases the half-life of some messenger RNAs that contain adenylate/uridylate-rich decay elements. However, its function in an integrated system has not yet been investigated. Here, using a mouse model, we report that misregulation of HuR, due to expression of an HuR transgene, prevents the production of fully functional gametes. This work provides the first evidence for a physiological function of HuR, and highlights its involvement in spermatogenesis.     

Overexpression of HuR, a nuclear-cytoplasmic shuttling protein, increases the in vivo stability of ARE-containing mRNAs.

The messenger RNAs of many proto-oncogenes, cytokines and lymphokines are targeted for rapid degradation through AU-rich elements (AREs) located in their 3'' untranslated regions (UTRs). HuR, a ubiquitously expressed member of the Elav family of RNA binding proteins, exhibits specific affinities for ARE-containing RNA sequences in vitro which correlate with their in vivo decay rates, thereby implicating HuR in the ARE-mediated degradation pathway. We have transiently transfected HuR into mouse L929 cells and observed that overexpression of HuR enhances the stability of beta-globin reporter mRNAs containing either class I or class II AREs. The increase in mRNA stability parallels the level of HuR overexpression, establishing an in vivo role for HuR in mRNA decay. Furthermore, overexpression of HuR deletion mutants lacking RNA recognition motif 3 (RRM 3) does not exert a stabilizing effect, indicating that RRM 3 is important for HuR function. We have also developed polyclonal anti-HuR antibodies. Immunofluorescent staining of HeLa and L929 cells using affinity-purified anti-HuR antibody shows that both endogenous and overexpressed HuR proteins are localized in the nucleus. By forming HeLa-L929 cell heterokaryons, we demonstrate that HuR shuttles between the nucleus and cytoplasm. Thus, HuR may initially bind to ARE-containing mRNAs in the nucleus and provide protection during and after their export to the cytoplasmic compartment.