cis-acting sequences and trans-acting factors in the localization of mRNA for mitochondrial ribosomal proteins

mRNA localization is a conserved post-transcriptional process crucial for a variety of systems. Although several mechanisms have been identified, emerging evidence suggests that most transcripts reach the protein functional site by moving along cytoskeleton elements. We demonstrated previously that mRNA for mitochondrial ribosomal proteins are asymmetrically distributed in the cytoplasm, and that localization in the proximity of mitochondria is mediated by the 3′-UTR. Here we show by biochemical analysis that these mRNA transcripts are associated with the cytoskeleton through the microtubule network. Cytoskeleton association is functional for their intracellular localization near the mitochondrion, and the 3′-UTR is involved in this cytoskeleton-dependent localization. To identify the minimal elements required for localization, we generated DNA constructs containing, downstream from the GFP gene, deletion mutants of mitochondrial ribosomal protein S12 3′-UTR, and expressed them in HeLa cells. RT-PCR analysis showed that the localization signals responsible for mRNA localization are located in the first 154 nucleotides. RNA pulldown assays, mass spectrometry, and RNP immunoprecipitation assay experiments, demonstrated that mitochondrial ribosomal protein S12 3′-UTR interacts specifically with TRAP1 (tumor necrosis factor receptor-associated protein1), hnRNPM4 (heterogeneous nuclear ribonucleoprotein M4), Hsp70 and Hsp60 (heat shock proteins 70 and 60), and α-tubulin in vitro and in vivo

Alternative splicing and nonsense-mediated mRNA decay regulate mammalian ribosomal gene expression

Messenger RNAs containing premature stop codons are generally targeted for degradation through nonsense-mediated mRNA decay (NMD). This mechanism degrades aberrant transcripts derived from mutant genes containing nonsense or frameshift mutations. Wild-type genes also give rise to transcripts targeted by NMD. For example, some wild-type genes give rise to alternatively spliced transcripts that are targeted for decay by NMD. In Caenorhabditis elegans, the ribosomal protein (rp) L12 gene generates a nonsense codon-bearing alternatively spliced transcript that is induced in an autoregulatory manner by the rpL12 protein. By pharmacologically blocking the NMD pathway, we identified alternatively spliced mRNA transcripts derived from the human rpL3 and rpL12 genes that are natural targets of NMD. The deduced protein sequence of these alternatively spliced transcripts suggests that they are unlikely to encode functional ribosomal proteins. Overexpression of rpL3 increased the level of the alternatively spliced rpL3 mRNA and decreased the normally expressed rpL3. This indicates that rpL3 regulates its own production by a negative feedback loop and suggests the possibility that NMD participates in this regulatory loop by degrading the non-functional alternatively spliced transcript

Extended interactions with prothrombinase enforce affinity and specificity for its macromolecular substrate.

The specific action of serine proteinases on protein substrates is a hallmark of blood coagulation and numerous other physiological processes. Enzymic recognition of substrate sequences preceding the scissile bond is considered to contribute dominantly to specificity and function. We have investigated the contribution of active site docking by unique substrate residues preceding the scissile bond to the function of prothrombinase. Mutagenesis of the authentic P(1)-P(3) sequence in prethrombin 2/fragment 1.2 yielded substrate variants that could be converted to thrombin by prothrombinase. Proteolytic activation was also observed with a substrate variant containing the P(1)-P(3) sequence found in a coagulation zymogen not known to be activated by prothrombinase. Lower rates of activation of the variants derived from a decrease in maximum catalytic rate but not in substrate affinity. Replacement of the P(1) residue with Gln yielded an uncleavable derivative that retained the affinity of the wild type substrate for prothrombinase but did not engage the active site of the enzyme. Thus, active site docking of the substrate contributes to catalytic efficiency, but it is does not determine substrate affinity nor does it fully explain the specificity of prothrombinase. Therefore, extended interactions between prothrombinase and substrate regions removed from the cleavage site drive substrate affinity and enforce the substrate specificity of this enzyme complex

Autoregulatory circuit of human rpL3 expression requires hnRNP H1, NPM and KHSRP

Alternative pre-mRNA splicing (AS) is a major mechanism that allows proteomic variability in eukaryotic cells. However, many AS events result in mRNAs containing a premature termination codon, which are degraded by nonsense-mediated mRNA decay (NMD) pathway. We have previously demonstrated that human rpL3 autoregulates its expression through the association of AS with NMD. In fact, overexpression of rpL3 promotes downregulation of canonical splicing and upregulation of alternative splicing that produces an NMD-targeted mRNA isoform. The result of these events is a decreased production of rpL3. We have also identified heterogeneous nuclear ribonucleoprotein (hnRNP) H1 as a splicing factor involved in the regulation of rpL3 alternative splicing and identified its regulatory cis-elements within intron 3 transcript. Here, we report that NPM and KHSRP are two newly identified proteins involved in the regulation of rpL3 gene expression via AS-NMD. We demonstrate that hnRNP H1, KHSRP and NPM can be found associated, and present also in ribonucleoproteins (RNPs) including rpL3 and intron 3 RNA in vivo, and describe protein-protein and RNA-protein interactions. Moreover, our data provide an insight on the crucial role of hnRNP H1 in the regulation of the alternative splicing of the rpL3 gene

Thrombin mutants with altered enzymatic activity have an impaired mitogenic effect on mouse fibroblasts and are inefficient modulators of stellation of rat cortical astrocytes

AbstractWe produced recombinant human thrombin mutants to investigate the correlation between the thrombin enzyme and mitogenic activity. Single amino acid substitutions were introduced in the catalytic triad (H43N, D99N, S205A, S205T), in the oxy-anion binding site (G203A) and in the anion binding exosite-1 region (R73E). Proteins were produced as prethrombin-2 mutants secreted in the culture medium of DXB11-derived cell lines. All mutants were activated by ecarin to the corresponding thrombin mutants; the enzymatic activity was assayed on a chromogenic substrate and on the procoagulant substrate fibrinogen. Mutations S205A and G203A completely abolished the enzyme activity. Mutations H43N, D99N and S205T dramatically impaired the enzyme activity toward both substrates. The R73E mutation dissociated the amidolytic activity and the clotting activity of the protein. The ability of thrombin mutants to induce proliferation was investigated in NIH3T3 mouse fibroblasts and rat cortical astrocytes. The ability of the thrombin mutants to revert astrocyte stellation was also studied. The mitogenic activity and the effect on the astrocyte stellation of the thrombin mutants correlated with their enzymatic activity. Furthermore the receptor occupancy by the inactive S205A mutant prevented the thrombin effects providing strong evidence that a proteolytically activated receptor is involved in cellular responses to thrombin

Proteasi nexina-1: produzione e caratterizzazione funzionale della proteina ricombinante, e studi strutturali mediante "molecular modelling".

Protease Nexin-1 (PN-1) is a member of the serpin superfamily which mainly inhibits Thrombin; this inhibition is enhanced by the interaction with heparin. This protein is involved in embryogenesis of the nervous system and of the sexual organs and probably exherts a role in a variety of pathologies such as Alzheimer disease and scleroderma. Even though this protein can be obtained by the media of different cell lines and different systems to obtain recombinant PN-1 have been developed, the yields are in general rather low. For this reason in our lab we have tried to develop a method to produce recombinant PN-1 and obtain higher yields compared to the ones previously reported to be able to perform a structural study of this protein. Two different expression systems have been used: hamster BHK cells and E. coli. Both systems have given satisfactory yields but the one in E. coli,because of slightly higher yields of expressed PN-1 and because of the easier preparation resulted as best. At the end of the process of purification the total yield was of 0.2-0.15 mg/L of bacterial colture. Recombinant PN-1 has been characterized for its inhibitory and cellular activities. Recombinant PN-1 correctly inhibits Thrombin: this interaction is, as for the native protein, enhanced by heparin. Furthermore Thrombin mutants, previously produced in our lab, correctly recognise and cleave PN-1 only if still enzimatically active. We have also characterized the binding of PN-1 to heparin determining the equilibrium interaction constant (Kd=31nM). The cellular activity of PN-1 has been tested by analysing its ability to cause stellation in neural cells. We used Nb2A mouse nuroblastoma cells, and a recombinant PN-1 concentration of 50 nM has been able to cause complete stellation. We have also employed the technique of molecular modelling to perform a structural analysis of PN-1 using two serpins, PAI-1 and ATIII as models. First of all we localised the heparin binding site on the structure helix D and the site responsable for Thrombin-PN-1 complexes clearance on the loop connecting helices A and B. We also showed that presumably the interaction with heparin does not cause, for PN-1, the same structural activation of ATIII. These results show how it has been possible to develop a method to produce a recombinant PN-1 and that this protein is active both as an inhibitor and on cells. This protein will be used to confirm and widen the structural data obtained by molecular modelling. The recombinant PN-1 so obtained could also be used to invetigate the phisiological and pathological activities that have been connected to this serpin in the past few years

Post-transcriptional regulatory strategies and extraribosomal functions of human rpL3

rpL3 gene produces in addition to canonical mRNA isoform normally translated in protein, an alternative isoform, containing a PTC (premature termination codon) degraded by NMD (Nonsense mediated mRNA decay). Moreover, overexpression of rpL3 causes an increase of alternative isoform level, unproductive, which results in rpL3 decrease. This negative feedback loop triggered by the accumulation of ribosomal protein, is a strategy that finely regulates the amount of ribosomal protein to an appropriate level, especially relevant to any extra-ribosomal functions of rpL3. hnRNP H1, NPM and KHSRP are involved with different roles in the post-transcriptional regulation of rpL3 gene and we hypothesize a model of regulation that provides a sequence of interactions between these proteins and rpL3 transcript. This post-transcriptional regulation, that provides the association of alternative splicing and NMD, can be very important to finely modulate the rpL3 amount available to extra-ribosomal functions

FIK7, un nuovo "partner" proteico per il fattore di trascrizione neurospecifico mKLF7.

mKLF 7 é un fattore di trascrizione murino appartenente alla famiglia "Kruppel like", proteine con sequenza omologa al prodotto proteico del gene "Kruppel" di Drosophila. Esperimenti di ibridazione in situ hanno mostrato che mKLF 7 presenta un caratteristico profilo di espressione durante l'embriogenesi, che segue quello del gene neurospecifico Neuro D. In particolare, il profilo di espressione di mKLF 7 sembra coinvolgere questo fattore di trascrizione nella regolazione della differenziazione dei nuroblasti e della funzionalità dei neuroni mediante il mantenimento di un fenotipo post-mitotico. In questo progetto di ricerca l'obiettivo era quello di isolare un nuovo/i "partner" proteico per mKLF 7 che fosse coinvolto nella regolazione del suo ruolo di fattore di trascrizione neurospecico. A tale scopo é stata analizzata una libreria di espressione preparata da mRNA neuronale mediante il sistema del doppio ibrido nel lievito. Da questa analisi é stato isolato l'amminoterminale di una nuova proteina. Un frammento più esteso dell'amminoterminale é stato isolato mediante l'analisi di una libreria fagica di cDNA embrionale, mentre mediante l'analisi di una banca dati EST murina é stato isolata la restante porzione carbossiterminale. Per la presenza all'amminoterminale di un dominio proteico altamente conservato chiamato F-box, questa nuova proteina é stata chiamata FIK7, proteina F-box Interagente con mKLF 7. Le proteine F-box sono una famiglia di recettori solubili che reclutano substrati fosforilati al complesso proteico Ubiquitina-Ligasi. In questo lavoro é stato caratterizzato il profilo di espessione di FIK7 mediante esperimenti di Northern Blot ed inbridazione in situ. FIK7 é una proteina particolarmlente espressa in linee cellulari poco differenziate ed altamente proliferanti come le cellule delle cripte a livello intestinale e gli spermatogoni a livello dei tubuli seminiferi. Inoltre, esperimenti di "pull down" e co-immunoprecipitazione nelle cellule, realizzati con diversi mutanti di delezione dell'amminoterminale di FIK7 hanno dimostrato che i dominii proteici compresi tra gli aminoacidi 1-99 e 150-350 sono direttamente coinvolti nell'interazione con mKLF 7. I dati biochimici di interazione proteica con mKLF 7 insieme con il classico profilo di espressione di FIK7 suggeriscono fortemente la possibilità di un ruolo per questa proteina F-box di coregolatore negativo per mKLF 7. Questa ipotesi é avvalorata dalla presenza nella sequenza proteica di mKLF 7 di un dominio P.E.S.T., un segnale proteolitico condizionale che implica il coinvolgimento della via degradativa ubiquitino-dipendente. Questa via, quindi, potrebbe risultare uno dei sistemi di regolazione più importanti della attività trascrizionale di mKLF 7, che garantirebbe l'uscita delle cellule dalla fase G1 e/o il mantenimento di un fenotipo indifferenziato sia durante lo sviluppo del SNC e del SNP che durante la vita adulta