Isolation of a U-insertion/deletion editing complex from Leishmania tarentolae mitochondria

A multiprotein, high molecular weight complex active in both U-insertion and U-deletion as judged by a pre-cleaved RNA editing assay was isolated from mitochondrial extracts of Leishmania tarentolae by the tandem affinity purification (TAP) procedure, using three different TAP-tagged proteins of the complex. This editing- or E-complex consists of at least three protein-containing components interacting via RNA: the RNA ligase-containing L-complex, a 3' TUTase (terminal uridylyltransferase) and two RNA-binding proteins, Ltp26 and Ltp28. Thirteen approximately stoichiometric components were identified by mass spectrometric analysis of the core L-complex: two RNA ligases; homologs of the four Trypanosoma brucei editing proteins; and seven novel polypeptides, among which were two with RNase III, one with an AP endo/exonuclease and one with nucleotidyltransferase motifs. Three proteins have no similarities beyond kinetoplastids.     

Characterization of two classes of ribonucleoprotein complexes possibly involved in RNA editing from Leishmania tarentolae mitochondria.

The molecular mechanism of RNA editing in trypanosomatid mitochondria is an unsolved problem. We show that two classes of ribonucleoprotein complexes exist in a mitochondrial extract from Leishmania tarentolae and appear to be involved in RNA editing. The 'G' class of RNP complexes consists of 170-300 A particles which contain guide RNAs and proteins, show little terminal uridylyl transferase (TUTase) activity and exhibit an in vitro RNA editing-like activity. The 'T' class consists of approximately six RNP complexes, the endogenous RNA of which can be self-labeled with [alpha-32P]UTP. The most abundant T complex, T-IV, is visualized by electron microscopy as 80-140 A particles. This complex exhibits TUTase activity in the native gel and contains guide RNAs. Both G and T complexes are possibly involved with RNA editing in vivo. These results are a starting point for the analysis of the biochemistry of RNA editing.     

Selective importation of RNA into isolated mitochondria from Leishmania tarentolae

All mitochondrial tRNAs in kinetoplastid protozoa are encoded in the nucleus and imported from the cytosol. Incubation of two in vitro-transcribed tRNAs, tRNA(Ile)(UAU) and tRNA(Gln)(CUG), with isolated mitochondria from Leishmania tarentolae, in the absence of any added cytosolic fraction, resulted in a protease-sensitive, ATP-dependent importation, as measured by nuclease protection. Evidence that nuclease protection represents importation was obtained by the finding that Bacillus subtilis pre-tRNA(Asp) was protected from nuclease digestion and was also cleaved by an intramitochondrial RNase P-like activity to produce the mature tRNA. The presence of a membrane potential is not required for in vitro importation. A variety of small synthetic RNAs were also found to be efficiently imported in vitro. The data suggest that there is a structural requirement for importation of RNAs greater than approximately 17 nt, and that smaller RNAs are apparently nonspecifically imported. The signals for importation of folded RNAs have not been determined, but the specificity of the process was illustrated by the higher saturation level of importation of the mainly mitochondria-localized tRNA(Ile) as compared to the level of importation of the mainly cytosol-localized tRNA(Gln). Furthermore, exchanging the D-arm between the tRNA(Ile) and the tRNA(Gln) resulted in a reversal of the in vitro importation behavior and this could also be interpreted in terms of tertiary structure specificity.     

Partially edited mRNAs for cytochrome b and subunit III of cytochrome oxidase from Leishmania tarentolae mitochondria: RNA editing intermediates

Partially edited mRNAs were selected by the polymerase chain reaction and sequenced. In the case of cytochrome b, 102 out of 106 clones displayed patterns of editing that were consistent with a strictly progressive 3' to 5' editing process, as predicted by the guide RNA model of RNA editing. In the case of cytochrome oxidase subunit III (COIII), 177 out of 304 clones displayed strictly progressive 3' to 5' patterns of editing. However, the remaining 127 COIII clones displayed unexpected patterns in which upstream editing preceded downstream editing, uridines were inserted at sites not normally edited, and purine residues were deleted. We suggest that many of these RNAs are produced by normal 3' to 5' editing of the COIII mRNA with incorrect guide RNA molecules.     

Isolation and characterization of a U-specific 3'-5'-exonuclease from mitochondria of Leishmania tarentolae

We have purified a 3'-5'-exoribonuclease from mitochondrial extract of Leishmania tarentolae over 4000-fold through six column fractionations. This enzyme digested RNA in a distributive manner, showed a high level of specificity for 3'-terminal Us, and was blocked by a terminal dU; there was slight exonucleolytic activity on a 3'-terminal A or C but no activity on a 3'-terminal G residue. The enzyme preferred single-stranded 3'-oligo(U) overhangs and did not digest duplex RNA. Two other 3'-5'-exoribonuclease activities were also detected in the mitochondrial extract, one of which was stimulated by a 3'-phosphate and the other of which degraded RNAs with a 3'-OH to mononucleotides in a processive manner. The properties of the distributive U-specific 3'-5'-exoribonuclease suggest an involvement in the U-deletion RNA editing reaction that occurs in the mitochondrion of these cells.     

Characterization of a protein fraction containing cytochromes b and c1 from mitochondria of Leishmania tarentolae

A soluble red band fraction was obtained from Leishmania tarentolae cells by sucrose gradient sedimentation of a Triton X-100 lysate. Spectral analysis indicated that cytochrome b was present in the red band: the reduced minus oxidized difference spectra revealed absorption maxima at 562,527, and 431 nm at room temperature and 562, 530, and 422 nm at 77K. In addition, a 28-kDa protein was identified in this fraction which retained heme-associated peroxidase activity even after denaturation on SDS-polyacrylamide gels. The amino acid composition of this protein showed a strong similarity to cytochrome c1 of both bovine and yeast.     

Association of two novel proteins, TbMP52 and TbMP48, with the Trypanosoma brucei RNA editing complex

RNA editing in kinetoplastid mitochondria inserts and deletes uridylates at multiple sites in pre-mRNAs as directed by guide RNAs. This occurs by a series of steps that are catalyzed by endoribonuclease, 3'-terminal uridylyl transferase, 3'-exouridylylase, and RNA ligase activities. A multiprotein complex that contains these activities and catalyzes deletion editing in vitro was enriched from Trypanosoma brucei mitochondria by sequential ion-exchange and gel filtration chromatography, followed by glycerol gradient sedimentation. The complex size is approximately 1,600 kDa, and the purified fraction contains 20 major polypeptides. A monoclonal antibody that was generated against the enriched complex reacts with an approximately 49-kDa protein and specifically immunoprecipitates in vitro deletion RNA editing activity. The protein recognized by the antibody was identified by mass spectrometry, and the corresponding gene, designated TbMP52, was cloned. Recombinant TbMP52 reacts with the monoclonal antibody. Another novel protein, TbMP48, which is similar to TbMP52, and its gene were also identified in the enriched complex. These results suggest that TbMP52 and TbMP48 are components of the RNA editing complex.     

The mouse poly(C)-binding protein exists in multiple isoforms and interacts with several RNA-binding proteins.

The murine poly(C)-binding protein (mCBP) was previously shown to belong to the group of K-homology (KH) proteins by virtue of its homology to hnRNP-K. We have isolated cDNA-splice variants of mCBP which differ by two variable regions of 93 bp and/or 39 +/- 3 bp respectively. Both variable regions are located between the second and third KH-domain of mCBP. The characterization of a partial genomic clone enabled us to propose a model for the generation of the second variable region by the use of a putative alternative splice signal. The mCBP mRNA is expressed ubiquitously and the protein is found predominantly in the nucleus with the exception of the nucleoli. We have identified five proteins which interact with mCBP in the yeast two hybrid system: mouse y-box protein 1 (msy-1), y-box-binding protein, hnRNP-L, filamin and splicing factor 9G8. The interaction between mCBP and splicing factor 9G8 was confirmed in vivo. These results suggest a function of mCBP in RNA metabolism.     

Resolution of the RNA editing gRNA-directed endonuclease from two other endonucleases of Trypanosoma brucei mitochondria.

RNA editing in kinetoplastids, the specific insertion and deletion of U residues, requires endonuclease cleavage of the pre-mRNA at each cycle of insertion/deletion. We have resolved three endoribonuclease activities from Trypanosoma brucei mitochondrial extracts that cleave CYb pre-mRNA specifically. One of these, which sediments at approximately 20S and is not affected substantially by DTT, has all the features of the editing endonuclease. It cleaves CYb pre-edited or partially edited mRNA only when annealed to the anchor region of a cognate guide RNA (gRNA), and it cleaves accurately just 5' of the duplex region. Its specificity is for the 5' end of extended duplex RNA regions, and this prevents cleavage of the gRNA or other positions in the mRNA. This gRNA-directed nuclease is evidently the same activity that functions in A6 pre-mRNA editing. However, it is distinct and separable from a previously observed DTT-requiring endonuclease that sediments similarly under certain conditions, but does not cleave precisely at the first editing site in either the presence or absence of a gRNA. The editing nuclease is also distinct from a DTT-inhibited endonuclease that cleaves numerous free pre-mRNAs at a common structure in the region of the first editing site.     

Identification of translocatable RNA-binding phloem proteins from melon, potential components of the long-distance RNA transport system

Phloem proteins (P-proteins) are an enigmatic group of proteins present in most angiosperm species. The best characterized P-proteins (PP1 and PP2) are synthesized in companion cells, transported into sieve elements via pore plasmodesmata and translocated through the plant. Characteristics such as long-distance translocation, RNA-binding activity and capacity of increasing plasmodesmata exclusion size suggest that certain phloem proteins could be involved in RNA transport within the plant, forming translocatable ribonucleoprotein complexes with endogenous or pathogenic RNAs. Long-distance movement of RNA through the phloem is a process known to occur, but both the mechanisms involved and the components constituting this potential information network remain unclear. Here, we demonstrate that several melon phloem proteins have a wide RNA-binding activity. Serological assays strongly suggest that one of these proteins is the melon phloem protein 2 (CmmPP2). Mass spectrometry analysis undoubtedly identifies another one as the recently characterized melon phloem lectin (CmmLec17). Grafting experiments demonstrate that the CmmLec17 is a translocatable phloem protein, able to move through intergeneric grafts from melon to pumpkin. Translocatability and RNA-binding activity was also demonstrated for an uncharacterized protein of approximately 14 kDa. In light of these results the possible involvement of these phloem proteins in the long-distance transport of melon RNAs is discussed.     

Site-specific DNA endonuclease and RNA maturase activities of two homologous intron-encoded proteins from yeast mitochondria

Two introns of the mitochondrial genome 777-3A of S. cerevisiae, bl4 in cob and al4 in coxl genes, contain ORFs that can be translated into two homologous proteins. We changed the UGA, AUA, and CUN codons of these ORFs to the universal genetic code, in order to study the functions of their translated products in E. coli and in yeast, by retargeting the nuclear encoded protein into mitochondria. The p27bl4 protein has been shown to be required for the splicing of both introns bl4 and al4. The homologous p28al4 protein is highly toxic to E. coli. It can specifically cleave double-stranded DNA at a sequence representing the junction of the two fused flanking exons. We present evidence that this system is a good model for studying the role of mitochondrial intron-encoded proteins in the rearrangement of genetic information at both the RNA (RNA splicing-bl4 maturase) and DNA levels (intron transposition-al4 transposase).     

MEF10 is required for RNA editing at nad2-842 in mitochondria of Arabidopsis thaliana and interacts with MORF8

A forwards genetic screen of a chemically mutated plant population identified mitochondrial RNA editing factor 10 (MEF10) in Arabidopsis thaliana. MEF10 is a trans-factor required specifically for the C to U editing of site nad2-842. The MEF10 protein is characterized by a stretch of pentatricopeptide repeats (PPR) and a C-terminal extension domain ending with the amino acids DYW. Editing is lost in mutant plants but is recovered by transgenic introduction of an intact MEF10 gene. The MEF10 protein interacts with multiple organellar RNA editing factor 8 (MORF8) but not with other mitochondrial MORF proteins in yeast two hybrid assays. These results support the model that specific combinations of MORF and MEF proteins are involved in RNA editing in plant mitochondria.     

Specific cleavage of kinetoplast minicircle DNA from Leishmania tarentolae by mung bean nuclease and identification of several additional minicircle sequence classes

Multiple sequence classes of kinetoplast minicircle DNA from Leishmania tarentolae were cleaved by mung bean nuclease in the presence of formamide, yielding unit length linear molecules which retained the anomalous electrophoretic mobility in acrylamide characteristic of minicircle DNA. No specific cleavage site sequence common to all minicircle sequence classes was apparent, although the main region of nuclease cleavage was localized approximately 350 bp from the unique SmaI restriction site of the conserved region found in all minicircle sequence classes. Covalent closure of the minicircle substrate was not a requirement for cleavage, as linearized network-derived or cloned minicircles were also cleaved by mung bean nuclease at similar locations. The partial sequences of several new minicircle sequence classes released from the network by mung bean nuclease are also reported.     

A tale of two paralogs: human Transformer2 proteins with differential RNA-binding affinities

The Transformer2 (Tra2) proteins in humans are homologues of the Drosophila Tra2 protein. One of the two RNA-binding paralogs, Tra2β, has been very well-studied over the past decade, but not much is known about Tra2α. It was very recently shown that the two proteins demonstrate the phenomenon of paralog compensation. Here, we provide a structural basis for this genetic backup circuit, using molecular modelling and dynamics studies. We show that the two proteins display similar binding specificities, but differential affinities to a short GAA-rich RNA stretch. Starting from the 6-nucleotide RNA in the solution structure, close to 4000 virtual mutations were modelled on RNA and the domain–RNA interactions were studied after energy minimisation to convergence. Separately, another known 13-nucleotide stretch was docked and the domain–RNA interactions were observed through a 100-ns dynamics trajectory. We have also demonstrated the ‘compensatory’ mechanism at the level of domains in one of the domain repeat-containing RNA-binding proteins.     

Identification of two novel proteins that interact with germ-cell-specific RNA-binding proteins DAZ and DAZL1

The human DAZ (deleted in azoospermia) gene family on the Y chromosome and an autosomal DAZ-like gene, DAZL1, encode RNA-binding proteins that are expressed exclusively in germ cells. Their role in spermatogenesis is supported by their homology with a Drosophila male infertility gene boule and sterility of Daz11 knock-out mice. While all mammals contain a DAZL1 homologue on their autosomes, DAZ homologues are present only on the Y chromosomes of great apes and Old World monkeys. The DAZ and DAZL1 proteins differ in the copy numbers of a DAZ repeat and the C-terminal sequences. We studied the interaction of DAZ and DAZL1 with other proteins as an approach to investigate functional similarity between these two proteins. Using DAZ as bait in a yeast two-hybrid system, we isolated two DAZAP (DAZ-associated protein) genes. DAZAP1 encodes a novel RNA-binding protein that is expressed most abundantly in the testis, and DAZAP2 encodes a ubiquitously expressed protein with no recognizable functional motif. DAZAP1 and DAZAP2 bind similarly to both DAZ and DAZL1 through the DAZ repeats. The DAZAP genes were mapped to chromosomal regions 19p13.3 and 2q33-q34, respectively, where no genetic diseases affecting spermatogenesis are known to map.