Domain architecture of the DRpp29 protein and its interaction with the RNA subunit of Dictyostelium discoideum RNase P

Dictyostelium discoideum nuclear RNase P is a ribonucleoprotein complex that displays similarities with its counterparts from higher eukaryotes such as the human enzyme, but at the same time it retains distinctive characteristics. In the present study, we report the molecular cloning and interaction details of DRpp29 and RNase P RNA, two subunits of the RNase P holoenzyme from D. discoideum. Electrophoretic mobility shift assays exhibited that DRpp29 binds specifically to the RNase P RNA subunit, a feature that was further confirmed by the molecular modeling of the DRpp29 structure. Moreover, deletion mutants of DRpp29 were constructed in order to investigate the domains of DRpp29 that contribute to and/or are responsible for the direct interaction with the D. discoideum RNase P RNA. A eukaryotic specific, lysine- and arginine-rich region was revealed, which seems to facilitate the interaction between these two subunits. Furthermore, we tested the ability of wild-type and mutant DRpp29 to form active RNase P enzymatic particles with the Escherichia coli RNase P RNA.     

Heterodimerization of the human RNase P/MRP subunits Rpp20 and Rpp25 is a prerequisite for interaction with the P3 arm of RNase MRP RNA

Rpp20 and Rpp25 are two key subunits of the human endoribonucleases RNase P and MRP. Formation of an Rpp20–Rpp25 complex is critical for enzyme function and sub-cellular localization. We present the first detailed in vitro analysis of their conformational properties, and a biochemical and biophysical characterization of their mutual interaction and RNA recognition. This study specifically examines the role of the Rpp20/Rpp25 association in the formation of the ribonucleoprotein complex. The interaction of the individual subunits with the P3 arm of the RNase MRP RNA is revealed to be negligible whereas the 1:1 Rpp20:Rpp25 complex binds to the same target with an affinity of the order of nM. These results unambiguously demonstrate that Rpp20 and Rpp25 interact with the P3 RNA as a heterodimer, which is formed prior to RNA binding. This creates a platform for the design of future experiments aimed at a better understanding of the function and organization of RNase P and MRP. Finally, analyses of interactions with deletion mutant proteins constructed with successively shorter N- and C-terminal sequences indicate that the Alba-type core domain of both Rpp20 and Rpp25 contains most of the determinants for mutual association and P3 RNA recognition.     

Extensive deproteinization of Dictyostelium discoideum RNase P reveals a new catalytic activity.

Nuclear Dictyostelium discoideum RNase P was subjected to vigorous deproteinization procedures. After treatment with proteinase K followed by phenol extraction of samples containing D. discoideum RNase P activity, a new enzymatic activity was recovered. The proteinase K/phenol/SDS treated enzyme cleaves Schizossacharomyces pombe tRNAser (supS1), D. discoideum tRNASer and tRNALeu precursors several nucleotides upstream of the cleavage site of RNase P, liberating products with 5'-hydroxyl ends. This activity seems to be associated with one or two RNA molecules copurifying with D. discoideum RNase P activity as judged by its inhibition in the presence of micrococcal nuclease, which is in contrast to its resistance to proteinase K/phenol/SDS treatment.     

Heterodimerization regulates RNase MRP/RNase P association, localization, and expression of Rpp20 and Rpp25

Rpp20 and Rpp25 are subunits of the human RNase MRP and RNase P endoribonucleases belonging to the Alba superfamily of nucleic acid binding proteins. These proteins, which bind very strongly to each other, transiently associate with RNase MRP. Here, we show that the Rpp20-Rpp25 heterodimer is resistant to both high concentrations of salt and a nonionic detergent. The interaction of Rpp20 and Rpp25 with the P3 domain of the RNase MRP RNA appeared to be strongly enhanced by their heterodimerization. Coimmunoprecipitation experiments demonstrated that only a single copy of each of these proteins is associated with the RNase MRP and RNase P particles in HEp-2 cells. Both proteins accumulate in the nucleoli, which in case of Rpp20 is strongly dependent on its interaction with Rpp25. Finally, the results of overexpression and knock-down experiments indicate that their expression levels are codependent. Taken together, these data indicate that the Rpp20-Rpp25 heterodimerization regulates their RNA-binding activity, subcellular localization, and expression, which suggests that their interaction is also crucial for their role in RNase MRP/P function.     

The human homologue of Dictyostelium discoideum phg1A is expressed by human metastatic melanoma cells

Tumour cannibalism is a characteristic of malignancy and metastatic behaviour. This atypical phagocytic activity is a crucial survival option for tumours in conditions of low nutrient supply, and has some similarities to the phagocytic activity of unicellular microorganisms. In fact, Dictyostelium discoideum has been used widely as a model to study phagocytosis. Recently, phg1A has been described as a protein that is primarily involved in the phagocytic process of this microorganism. The closest human homologue to phg1A is transmembrane 9 superfamily protein member 4 (TM9SF4). Here, we report that TM9SF4 is highly expressed in human malignant melanoma cells deriving from metastatic lesions, whereas it is undetectable in healthy human tissues and cells. TM9SF4 is predominantly expressed in acidic vesicles of melanoma cells, in which it co‐localizes with the early endosome antigens Rab5 and early endosome antigen 1. TM9SF4 silencing induced marked inhibition of cannibal activity, which is consistent with a derangement of intracellular pH gradients, with alkalinization of acidic vesicles and acidification of the cell cytosol. We propose TM9SF4 as a new marker of malignancy, representing a potential new target for anti‐tumour strategies with a specific role in tumour cannibalism and in the establishment of a metastatic phenotype.     

NMR structure of an archaeal homologue of ribonuclease P protein Rpp29

A protein component of the Archaeoglobus fulgidus RNase P was expressed in Escherichia coli, purified, and structurally characterized using multidimensional NMR methods. The dominant structural feature of this 11 kDa protein is a sheet of six antiparallel beta-strands, wrapped around a core of conserved hydrophobic amino acids. Amide proton exchange and (15)N relaxation rate data provide evidence that the first 16 residues of the protein, located before the start of the first beta-strand, and the last 24 residues, located past the end of the last beta-strand, are relatively flexible; this contrasts with the relatively rigid and well-defined structure of the beta-sheet. Amino acid sequence comparisons among a diverse set of species indicate that the A. fulgidus protein is homologous to the human RNase P protein Rpp29, yeast RNase P protein Pop4, and a known archaeal RNase P protein from Methanobacter thermoautotrophicus; conserved hydrophobic residues indicate that the homologous protein in each of these species contains a similar beta-sheet structure. Conserved surface residues located in the loop connecting strands beta2 and beta3, the loop connecting strands beta4 and beta5, and in the flexible N- and C-terminal tails are most likely to have specific interactions with the RNA and other proteins of RNase P. The structural model of an RNase P protein component provided by the present work provides an essential step toward eventually understanding the overall architecture of this complex enzyme and the mechanism by which it performs its functions.     

Potential contact sites between the protein and RNA subunit in the Bacillus subtilis RNase P holoenzyme.

We have detected by nucleotide analog interference mapping (NAIM) AMPalphaS and IMPalphaS modifications in Bacillus subtilis RNase P RNA that interfere with binding of the homologous protein subunit. Interference as well as some enhancement effects were clustered in two main areas, in P10.1a/L10.1 and P12 of the specificity domain (cluster 1, domain I) and in P2, P3, P15.1, J18/2 and J19/4 of the catalytic domain (cluster 2a, domain II). Minor interferences in P1 and P19 and a strong and weak enhancement effect in P19 represent a third area located in domain II (cluster 2b). Our results suggest that P3, P2-J18/2 and J19/4 are key elements for anchoring of the protein to the catalytic domain close to the scissile phosphodiester in enzyme-substrate complexes. Sites of interference or enhancement in clusters 1 and 2a are located at distances between 65 and 130 A from each other in the current 3D model of a full-length RNase P RNA-substrate complex. Taking into account that the RNase P protein monomer can bridge a maximum distance of about 40 A, simultaneous direct contacts to the two aforementioned potential RNA-binding areas would be incompatible with our current understanding of bacterial RNase P RNA architecture. Our findings suggest that the current 3D model has to be rearranged in order to reduce the distance between clusters 1 and 2a. Alternatively, based on the recent finding that B. subtilis RNase P forms a tetramer consisting of two protein and two RNA subunits, cluster 1 may reflect one protein contact site in domain I, and cluster 2a a separate one in domain II.     

A RAS-encoded protein in Dictyostelium discoideum is acylated and membrane-associated

Dictyostelium dfscoideum synthesizes a 23000 M_r protein, p23^dd-ras, closely related to the mammalian oncogene-encoded protein p21^ras. To investigate the subcellular localization of P23^dd-ras, conditions were optimized to reduce protein degradation following cell breakage. Subcellular fractionation of D. discoideum showed that p23^dd-ras was associated predominantly with the membrane fraction during both vegetative growth and differentiation, in the absence of suitable protease inhibitors considerable amounts of a truncated form of p23^dd-ras were recovered in the cytosol fraction, suggesting that intact p23^dd-ras is attached to the membrane by a short terminal peptide sequence. Radio-isotope labelilng of D. discoideum with myristic acid or palmitic acid in the presence of excess un-labelled acetate resulted in radio-isotope incorporation into a select group of proteins including p23^dd-ras. No acyl label appeared in the truncated cytoplasmic form of p23^dd-ras when ceil breakage was performed In the absence of suitable protease inhibitors, indicating that the acyl group is associated with the short terminal peptide that is cleaved. These data suggest that p23^dd-ras like its mammailan counterpart, is acylated and associated with the plasma membrane. There was no evidence during a 30-minute pulse of methionine for a cytoplasmic precursor to the membrane-bound p23^dd-ras suggesting that the turnover of the presumptive precursor must be much more rapid in D. discoideum than for pro-p21^ras in mammalian cells.     

Identification of the regulatory subunit of a cAMP-dependent protein kinase in Dictyostelium discoideum

The occurrence of a cytosolic cAMP-binding protein of an approximate molecular weight of 41,000 daltons was monitored in vegetative and developing amoebae of $\text{Dictyostelium}\text{discoideum}$ by the use of the photoaffinity probe (³²P) 8N₃-cAMP. There was a large apparent increase in the amount of this binding protein during development; its molecular weight remained constant, if appropriate methods were employed for the disruption of the amoebae. Comigration during electrophoresis on two-dimensional gels identifies this cAMP-binding protein, photoaffinity-labeled in crude extracts, as the regulatory subunit of the cAMP-dependent protein kinase of $\text{D.}\text{discoideum}$.     

A syntaxin 7 homologue is present in Dictyostelium discoideum endosomes and controls their homotypic fusion

Endo-phagocytic activity is prominent in Dictyostelium discoideum and makes it a good model organism to study the molecular organization of membrane traffic in this pathway. We have identified a syntaxin 7 homologue (26% identity and 54% similarity to human syntaxin 7) in Dictyostelium cDNA and genomic data banks. In addition to the Habc and H3 helices and the C-terminal transmembrane domain characteristic of syntaxins, this protein contains a repetitive N-terminal extension of 68 amino acids. We first showed that Dictyostelium syntaxin 7 was able to form a complex with N-ethylmaleimide-sensitive fusion protein and alpha- and gamma-soluble N-ethylmaleimide-sensitive fusion protein attachment protein. Its intracellular localization was then studied by cell fractionation techniques and magnetic purification of the endocytic compartments. Most of D. discoideum syntaxin 7 is contained in endosomes. Finally, an in vitro endosome homotypic fusion assay (Laurent, O., Bruckert, F., Adessi, C., and Satre, M. (1998) J. Biol. Chem. 273, 793-799) was used to study a possible role for syntaxin 7 in this process. Purified anti-syntaxin 7 antibodies and a recombinant soluble fragment of syntaxin 7 both strongly inhibited fusion activity, indicating that this protein was necessary for endosome-endosome fusion. These results demonstrate the importance of this syntaxin 7 homologue in the early phases of Dictyostelium endo-phagocytic pathway.     

A cAMP-dependent protein kinase is present in differentiating Dictyostelium discoideum cells

We demonstrate the occurrence of a cAMP-dependent protein kinase in Dictyostelium discoideum cells at the terminal stage of differentiation. A cAMP-binding component was purified to homogeneity by affinity chromatography. This subunit inhibits the activity of purified catalytic subunit from beef heart protein kinase; the inhibition is reversed upon addition of cAMP. The protein is highly specific for cAMP and has a dissociation constant of 4 nM. The isolated regulatory subunit is a monomer of 39 K, with a sedimentation coefficient of 3.5S and a frictional coefficient of 1.24. The differences between this regulatory subunit and regulatory subunits of protein kinases from other sources are discussed.     

3D models of yeast RNase P/MRP proteins Rpp1p and Pop3p

Sensitive profile searches and fold recognition were used to predict the structures of two yeast RNase P/MRP proteins. Rpp1p, which is one of the subunits common to eukaryotes and archaea, is predicted to adopt the seven-stranded TIM-barrel fold found in PHP phosphoesterases. Pop3p, initially thought to be one of the RNase P/MRP subunits unique to yeast, has been assigned the L7Ae/L30e fold. This RNA-binding fold is also present in human RNase P subunit Rpp38, raising the possibility that Pop3p and Rpp38 are functional homologs.     

A functional RNase P protein subunit of bacterial origin in some eukaryotes

RNase P catalyzes 5'-maturation of tRNAs. While bacterial RNase P comprises an RNA catalyst and a protein cofactor, the eukaryotic (nuclear) variant contains an RNA and up to ten proteins, all unrelated to the bacterial protein. Unexpectedly, a nuclear-encoded bacterial RNase P protein (RPP) homolog is found in several prasinophyte algae including Ostreococcus tauri. We demonstrate that recombinant O. tauri RPP can functionally reconstitute with bacterial RNase P RNAs (RPRs) but not with O. tauri organellar RPRs, despite the latter's presumed bacterial origins. We also show that O. tauri PRORP, a homolog of Arabidopsis PRORP-1, displays tRNA 5'-processing activity in vitro. We discuss the implications of the striking diversity of RNase P in O. tauri, the smallest known free-living eukaryote.     

hPop5, a protein subunit of the human RNase MRP and RNase P endoribonucleases.

The RNase MRP and RNase P particles both function as endoribonucleases. RNase MRP has been implicated in the processing of precursor-rRNA, whereas RNase P has been shown to function in the processing of pre-tRNA. Both ribonucleoprotein particles have an RNA component that can be folded into a similar secondary structure and share several protein components. We have identified human, rat, mouse, cow, and Drosophila homologues of the Pop5p protein subunit of the yeast RNase MRP and RNase P complexes. The human Pop5 cDNA encodes a protein of 163 amino acids with a predicted molecular mass of 18.8 kDa. Polyclonal antibodies raised against recombinant hPop5 identified a 19-kDa polypeptide in HeLa cells and showed that hPop5 is associated with both RNase MRP and RNase P. Using affinity-purified anti-hPop5 antibodies, we demonstrated that the endogenous hPop5 protein is localized in the nucleus and accumulates in the nucleolus, which is consistent with its association with RNase MRP and RNase P. Catalytically active RNase P was partially purified from HeLa cells, and hPop5 was shown to be associated with it. Finally, the evolutionarily conserved acidic C-terminal tail of hPop5 appeared to be required neither for complex formation nor for RNase P activity.     

Identification of a homologue for annexin VII (synexin) in Dictyostelium discoideum

Immunological and biochemical data have been used to show that the slime mold Dictyostelium discoideum expresses a Ca2+/phospholipid-binding protein related to vertebrate annexins. The Dictyostelium protein (apparent molecular mass 46 kDa) is recognized by an antibody directed against an annexin consensus peptide and exhibits the properties characteristic for annexins, i.e. it interacts in a Ca2(+)-dependent manner with negatively charged phospholipids. Limited proteolysis converts the 46-kDa protein into a 32-kDa derivative which retains the Ca2+/phospholipid-binding properties of the 46-kDa polypeptide. Partial protein sequence data identify the Dictyostelium protein as the typical annexin and indicate that the 46-kDa protein is an annexin VII (synexin) homologue. The identification of an annexin in a simple eucaryote should lead to the introduction of genetic approaches to analyze the physiological role of the annexins.