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.

RNA recognition motifs (RRMs) are a ubiquitous class of proteins which bind RNA in a sequence-specific fashion, often with high affinity. The mechanisms through which this single protein domain recognizes diverse RNA sequences is not fully understood. High-resolution three-dimensional structures are particularly important in understanding the structural features required for RNA recognition and binding. This work presents the structure of an expanded RRM domain from the Drosophila melanogaster Bruno protein. The Bruno protein is involved in establishing proper body patterning during development. This is accomplished through the translational repression of several mRNAs, in particular, the oskar mRNA. Previous work has identified an expanded RRM domain within the Bruno protein. This RRM requires an additional forty amino acids prior to the start of the canonical RRM domain for high affinity RNA binding. The protein was found to contain a canonical RRM domain comprised of four anti-parallel [beta] strands and two [alpha] helices. The RRM is preceded by a ten amino acid loop that interacts with [alpha]₁ and [beta]₂, while the remaining amino acids are flexible in solution. Interestingly, the deletion of these residues does not alter the fold or stability of the RRM domain. Thus, these additional residues must be involved in RNA binding, as they are not required for structure. From these studies, the Bruno RRM represents a new example of protein features required for recognition and high affinity binding of RNA. / text

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