Littérature scientifique sur le sujet « Elav-like »

The neuron-specific Elav-like Hu RNA-binding proteins were described to play an important role in neuronal differentiation and plasticity by ensuring the post-transcriptional control of RNAs encoding for various proteins. Although Elav-like Hu proteins alterations were reported in diabetes or neuropathy, little is known about the regulation of neuron-specific Elav-like Hu RNA-binding proteins in sensory neurons of dorsal root ganglia (DRG) due to the diabetic condition. The goal of our study was to analyze the gene and protein expression of HuB, HuC, and HuD in DRG sensory neurons in diabetes. The diabetic condition was induced in CD-1 adult male mice with single-intraperitoneal injection of streptozotocin (STZ, 150 mg/kg), and 8-weeks (advanced diabetes) after induction was quantified the Elav-like proteins expression. Based on the glycemia values, we identified two types of responses to STZ, and mice were classified in STZ-resistant (diabetic resistant, glycemia < 260 mg/dL) and STZ-sensitive (diabetic, glycemia > 260 mg/dL). Body weight measurements indicated that 8-weeks after STZ-induction of diabetes, control mice have a higher increase in body weight compared to the diabetic and diabetic resistant mice. Moreover, after 8-weeks, diabetic mice (19.52 ± 3.52 s) have longer paw withdrawal latencies in the hot-plate test than diabetic resistant (11.36 ± 1.92 s) and control (11.03 ± 1.97 s) mice, that correlates with the installation of warm hypoalgesia due to the diabetic condition. Further on, we evidenced the decrease of Elav-like gene expression in DRG neurons of diabetic mice (Elavl2, 0.68 ± 0.05 fold; Elavl3, 0.65 ± 0.01 fold; Elavl4, 0.53 ± 0.07 fold) and diabetic resistant mice (Ealvl2, 0.56 ± 0.07 fold; Elavl3, 0.32 ± 0.09 fold) compared to control mice. Interestingly, Elav-like genes have a more accentuated downregulation in diabetic resistant than in diabetic mice, although hypoalgesia was evidenced only in diabetic mice. The Elav-like gene expression changes do not always correlate with the Hu protein expression changes. To detail, HuB is upregulated and HuD is downregulated in diabetic mice, while HuB, HuC, and HuD are downregulated in diabetic resistant mice compared to control mice. To resume, we demonstrated HuD downregulation and HuB upregulation in DRG sensory neurons induced by diabetes, which might be correlated with altered post-transcriptional control of RNAs involved in the regulation of thermal hypoalgesia condition caused by the advanced diabetic neuropathy.

Drosophila ELAV and human HuD are two neuronal RNA binding proteins that show remarkable sequence homology, yet differ in their respective documented roles in post-transcriptional regulation. ELAV regulates neural-specific alternative splicing of specific transcripts, and HuD stabilizes specific mRNAs that are otherwise unstable due to AU-rich elements(AREs) in their 3′ untranslated region (UTR). AREs are major determinants of transcript stability in mammalian cells. The role of each of these proteins was investigated and compared, by ectopically expressing them in Drosophila imaginal wing disc cells, which lack endogenous expression of either protein. The effect of the ectopic expression of ELAV and HuD was assessed on two sets of green fluorescent protein reporter transgenes,which were all driven with a broadly expressing promoter. Each set consisted of three reporter transgenes: (1) with an uninterrupted open reading frame(ORF); (2) with a constitutively spliced intron inserted into the ORF; and (3)with the intron nASI whose splicing is regulated in neurons by ELAV,inserted into the ORF. The two sets differed from each other only in their 3′UTR: Heat-shock-protein-70Ab (Hsp70Ab) trailer with ARE-like characteristics or Actin 5C (Act5C) trailer. Our results show that:(1) both ectopically expressed ELAV and HuD can enhance expression of transgenes with the Hsp70Ab 3′UTR, but not of transgenes with Act5C 3′UTR; (2) this enhancement is accompanied by an increase in mRNA level; (3) only ELAV can induce neural-specific splicing of nASI; and (4) although HuD is localized primarily to the cytoplasm,ELAV is localized to both the cytoplasm and the nucleus.

ELAV proteins are implicated in regulating the stability and translation of cytokine and growth regulatory mRNAs such as GM-CSF, IL-2, c-myc, c-fos and GLUT1 by binding to their AU-rich 3′UTRs. The tissue-specific ELAV protein HuB (aka. Hel-N1) is predominantly cytoplasmic and has been shown to stabilize GLUT1 and c-myc mRNAs and to increase their translation following ectopic expression in 3T3-L1 cells. We report that the most widely expressed mouse ELAV protein, mHuA, is predominately nuclear in cultured NIH-3T3 cells, but is localized in the cytoplasm during early G1 of the cell cycle. Therefore, much like the primarily cytoplasmic HuB, HuA becomes temporally localized in the cytoplasm where it can potentially regulate the stability or translation of bound mRNAs. Moreover, we report that stimulation of mouse spleen cells using either mitogenic or sub-mitogenic levels of anti-CD3/CD28 resulted in a dramatic increase in the level of HuA. Upregulation of HuA corresponds to previously documented increases in cytokine expression which are due to increased mRNA stability following T cell activation. Consistent with these findings, HuA was down regulated in quiescent cells and upregulated in 3T3 cells following serum stimulation. The increase of murine HuA during the cell cycle closely resembles that of cyclin B1 which peaks in G2/M. Together with our earlier studies, these data indicate that mammalian ELAV proteins function during cell growth and differentiation due in part to their effects on posttranscriptional stability and translation of multiple growth regulatory mRNAs. This supports the hypothesis that ELAV proteins can function as transacting factors which affect a default pathway of mRNA degradation involved in the expression of growth regulatory proteins.

3T3-L1 preadipocytes ectopically expressing the mammalian RNA-binding protein Hel-N1 expressed up to 10-fold more glucose transporter (GLUT1) protein and exhibited elevated rates of basal glucose uptake. Hel-N1 is a member of the ELAV-like family of proteins associated with the induction and maintenance of differentiation in various species. ELAV proteins are known to bind in vitro to short stretches of uridylates in the 3' untranslated regions (3'UTRs) of unstable mRNAs encoding growth-regulatory proteins involved in transcription and signal transduction. GLUT1 mRNA also contains a large 3'UTR with a U-rich region that binds specifically to Hel-N1 in vitro. Analysis of the altered GLUT1 expression at the translational and posttranscriptional levels suggested a mechanism involving both mRNA stabilization and accelerated formation of translation initiation complexes. These findings are consistent with the hypothesis that the Hel-N1 family of proteins modulate gene expression at the level of mRNA in the cytoplasm.

Post-transcriptional regulations (PTRs) have always been considered features of organisms with higher complexity. However recently, the interest toward the post- transcriptional mechanisms in prokaryotes increased. The bacterial proteome is much more complex compared to the genome size, suggesting a tight and articulate regulation of proteins production, extremely important for the bacterial adaptation to an always changing environment. Bacterial PTRs are responsible of modulation of mRNA stability and decay, translation initiation and elongation, modulation of the access of ribosome to the ribosome binding site and control of termination of the transcript. The main actors in the PTRs are small non-coding RNA (responsible of the inhibition of the transcription) and RNA binding proteins (RBPs), which modulate the translation and half-life of the mRNA. RBPs, are particularly of my interest since I wanted to find a possible orthologous of the eukaryotic Elav-like (Elavl) family of proteins in Acinetobacter baumannii. Elav-like proteins are present in all metazoans and are characterized by two highly conserved sequences: RNP-1 (a quite well conserved hexamer) and RNP-2 (a really well conserved octamer) that are responsible of binding to the mRNA. Each species has a different number of Elavl paralogous that is totally independent from the complexity of the organisms, suggesting a more ancient origin. In particular, I focused on the human paralog HuR (human antigen R). HuR is characterized by three RNA Recognition motif (RRM) -domains, is ubiquitously expressed and is mainly localized into the nucleus (where it is responsible of maturation of the mRNA), but under stress stimuli, can shuttle into the cytoplasm where protect the target mRNA from degradation, by binding AU/U rich sequences (ARE sequences). Its high concentration into the cytoplasm can lead to the overexpression of oncogenes and pro-tumorigenic factors. The choice of Acinetobacter baumannii comes from the increasing worldwide concern toward this pathogen that is becoming multidrug resistant. Indeed, in Italy, more the 50% of nosocomial infections are caused by A. baumannii. I found a putative protein (AB-Elavl), composed by a single RRM domain endowed with similar features of the eukaryotic RRM domain as the presence of a quite well conserved RNP-2 and a less conserved RNP-1. I expressed this protein with recombinant tools and confirmed the production of the protein in the host by western blot and mass spectrometry. I evaluated the binding activity of AB-Elavl testing the EC50 and the Kd with different biochemical assays (EMSA, AlphaScreen and HTRF- FRET) toward three different RNA sequences, in order to test the specificity. By X- RAY and NMR, I confirmed the folded structure that can be overlapped to the HuR’s one and the interaction with the probes tested, highlighting the presence of binding, but with different specificity. I also tested some small molecules developed for interfering in the binding of HuR with the target sequence and found a possible compound able to interact with AB-Elavl, by disrupting the binding with the target probe. All these results suggest an ancient origin of the metazoans’ Elavl family of proteins that probably share a common ancestor with AB-Elavl. More studies should be performed to better understand the role of AB-Elavl in A. baumannii as well as in other bacteria. In fact, I found the presence of other ARE sequence-binding proteins also in Pseudomonas aeruginosa. Interesting would be to check the presence of this protein in all the multidrug resistant ESKAPE bacteria.