Accurate and efficient insertional RNA editing in isolated Physarum mitochondria.

RNA editing is a process whereby nucleotide insertion, deletion, or base substitution results in the production of an RNA whose sequence differs from that of its template. The mitochondrial RNAs of Physarum polycephalum are processed specifically at multiple sites by both mono- and dinucleotide insertions, as well as apparent cytidine (C) to uridine (U) changes. The precise mechanism and timing of these processing events are currently unknown. We describe here the development of an isolated mitochondrial system in which exogenously supplied nucleotides can be incorporated into RNAs under defined conditions. The results of S1 nuclease protection, nearest neighbor and RNase T1 fingerprint analyses indicate that the vast majority of these newly synthesized mitochondrial RNAs have been accurately and efficiently processed by both mono- and dinucleotide insertions. This work provides a direct demonstration of faithful nucleotide insertion in a mitochondrial editing system. In contrast, the newly synthesized RNAs are not processed by C to U changes in the isolated mitochondria, suggesting that the base changes observed in Physarum are unlikely to occur via a deletion/insertion mechanism.     

Trypanosoma brucei U insertion and U deletion activities co-purify with an enzymatic editing complex but are differentially optimized.

RNA editing, the processing that generates functional mRNAs in trypanosome mitochondria, involves cycles of protein catalyzed reactions that specifically insert or delete U residues. We recently reported purification from Trypanosoma brucei mitochondria of a complex showing seven major polypeptides which exhibits the enzymatic activities inferred in editing and that a pool of fractions of the complex catalyzed U deletion, the minor form of RNA editing in vivo . We now show that U insertion activity, the major form of RNA editing in vivo , chromatographically co-purifies with both U deletion activity and the protein complex. Furthermore, these editing activities co-sediment at approximately 20 S. U insertion does not require a larger, less characterized complex, as has been suggested and could have implied that the editing machinery would not function in a processive manner. We also show that U insertion is optimized at rather different and more exacting reaction conditions than U deletion. By markedly reducing ATP and carrier RNA and increasing UTP and carrier protein relative to standard editing conditions, U insertion activity of the purified fraction is enhanced approximately 100-fold.     

PPR蛋白参与RNA编辑机制的研究进展

RNA编辑是一种发生在转录后核苷酸特异位点的加工修饰现象,包括核苷酸的插入、删除和改变.高等植物中RNA编辑主要发生在线粒体与叶绿体中,具有重要的生物学功能,其机制仍在探索中.而PPR蛋白作为RNA编辑的反式作用因子,成为近几年来分子生物学的研究热点.该文就PPR蛋白、RNA编辑及PPR蛋白参与RNA编辑的机制等进行了综述.     

The mitochondrial RNA ligase from Leishmania tarentolae can join RNA molecules bridged by a complementary RNA.

A biochemical characterization was performed with a partially purified RNA ligase from isolated mitochondria of Leishmania tarentolae. This ligase has a K(m) of 25 +/- 0.75 nM and a V(max) of 1.0 x 10(-4) +/- 2.4 x 10(-4) nmol/min when ligating a nicked double-stranded RNA substrate. Ligation was negatively affected by a gap between the donor and acceptor nucleotides. The catalytic efficiency of the circularization of a single-stranded substrate was 5-fold less than that of the ligation of a nicked substrate. These properties of the mitochondrial RNA ligase are consistent with an expected in vivo role in the process of uridine insertion/deletion RNA editing, in which the mRNA cleavage fragments are bridged by a cognate guide RNA.     

Compositionally and functionally distinct editosomes in Trypanosoma brucei

Uridylate insertion/deletion RNA editing in Trypanosoma brucei mitochondria is catalyzed by a multiprotein complex, the approximately 20S editosome. Editosomes purified via three related tagged RNase III proteins, KREN1 (KREPB1/TbMP90), KREPB2 (TbMP67), and KREN2 (KREPB3/TbMP61), had very similar but nonidentical protein compositions, and only the tagged member of these three RNase III proteins was identified in each respective complex. Three new editosome proteins were also identified in these complexes. Each tagged complex catalyzed both precleaved insertion and deletion editing in vitro. However, KREN1 complexes cleaved deletion but not insertion editing sites in vitro, and, conversely, KREN2 complexes cleaved insertion but not deletion editing sites. These specific nuclease activities were abolished by mutations in the putative RNase III catalytic domain of the respective proteins. Thus editosomes appear to be heterogeneous in composition with KREN1 complexes catalyzing cleavage of deletion sites and KREN2 complexes cleaving insertion sites while both can catalyze the U addition, U removal, and ligation steps of editing.     

Computer methods for locating kinetoplastid cryptogenes.

RNA editing in the mitochondria of kinetoplastid protoza involves the insertion and/or deletion of precise numbers of uridine residues at precise locations in the numbers of uridine residues at precise locations in the transcribed RNA of certain genes. These genes are known as cryptogenes. In this paper we study computational algorithms to search for unknown cryptogenes and for the associated templates for insertion of uridines, gRNA sequences. The pairwise similarity search algorithm of Smith and Waterman (1) is modified to study this problem. The algorithm searches for unknown gRNAs given the cryptogene sequence. The method is tested on 4 known cryptogenes from L.tarentolae which are known to have 7 associated gRNAs. The statistical distribution of the longest gRNA when comparing random sequences is derived. Finally we develop an algorithm to search for cryptogenes using amino acid sequences from related proteins.     

Uridine insertion/deletion RNA editing in trypanosomatid mitochondria: In search of the editosome

The RNA ligase-containing or L-complex is the core complex involved in uridine insertion/deletion RNA editing in trypanosome mitochondria. Blue native gels of glycerol gradient-separated fractions of mitochondrial lysate from cells transfected with the TAP-tagged editing protein, LC-8 (TbMP44/KREPB5), show a ∼1 MDa L-complex band and, in addition, two minor higher molecular weight REL1-containing complexes: one (L*a) co-sedimenting with the L-complex and running in the gel at around 1.2 MDa; the other (L*b) showing a continuous increase in molecular weight from 1 MDa to particles sedimenting over 70S. The L*b-complexes appear to be mainly composed of L-complex components, since polypeptide profiles of L- and L*b-complex gradient fractions were similar in composition and L*b-complex bands often degraded to L-complex bands after manipulation or freeze–thaw cycles. The L*a-complex may be artifactual since this gel shift can be produced by various experimental manipulations. However, the nature of the change and any cellular role remain to be determined. The L*b-complexes from both lysate and TAP pull-down were sensitive to RNase A digestion, suggesting that RNA is involved with the stability of the L*b-complexes. The MRP1/2 RNA binding complex is localized mainly in the L*b-complexes in substoichiometric amounts and this association is RNase sensitive. We suggest that the L*b-complexes may provide a scaffold for dynamic interaction with other editing factors during the editing process to form the active holoenzyme or “editosome.”     

Endonuclease associations with three distinct editosomes in Trypanosoma brucei

Three distinct editosomes, typified by mutually exclusive KREN1, KREN2, or KREN3 endonucleases, are essential for mitochondrial RNA editing in Trypanosoma brucei. The three editosomes differ in substrate endoribonucleolytic cleavage specificity, which may reflect the vast number of editing sites that need insertion or deletion of uridine nucleotides (Us). Each editosome requires the single RNase III domain in each endonuclease for catalysis. Studies reported here show that the editing endonucleases do not form homodimeric domains, and may therefore function as intermolecular heterodimers, perhaps with KREPB4 and/or KREPB5. Editosomes isolated via TAP tag fused to KREPB6, KREPB7, or KREPB8 have a common set of 12 proteins. In addition, KREN3 is only found in KREPB6 editosomes, KREN2 is only found in KREPB7 editosomes, and KREN1 is only found in KREPB8 editosomes. These are the same associations previously found in editosomes isolated via the TAP-tagged endonucleases KREN1, KREN2, or KREN3. Furthermore, TAP-tagged KREPB6, KREPB7, and KREPB8 complexes isolated from cells in which expression of their respective endonuclease were knocked down were disrupted and lacked the heterotrimeric insertion subcomplex (KRET2, KREPA1, and KREL2). These results and published data suggest that KREPB6, KREPB7, and KREPB8 associate with the deletion subcomplex, whereas the KREN1, KREN2, and KREN3 endonucleases associate with the insertion subcomplex.     

Editosome accessory factors KREPB9 and KREPB10 in Trypanosoma brucei

Multiprotein complexes, called editosomes, catalyze the uridine insertion and deletion RNA editing that forms translatable mitochondrial mRNAs in kinetoplastid parasites. We have identified here two new U1-like zinc finger proteins that associate with editosomes and have shown that they are related to KREPB6, KREPB7, and KREPB8, and thus we have named them Kinetoplastid RNA Editing Proteins, KREPB9 and KREPB10. They are conserved and syntenic in trypanosomatids although KREPB10 is absent in Trypanosoma vivax and both are absent in Leishmania. Tandem affinity purification (TAP)-tagged KREPB9 and KREPB10 incorporate into ~20S editosomes and/or subcomplexes thereof and preferentially associate with deletion subcomplexes, as do KREPB6, KREPB7, and KREPB8. KREPB10 also associates with editosomes that are isolated via a chimeric endonuclease, KREN1 in KREPB8 RNA interference (RNAi) cells, or MEAT1. The purified complexes have precleaved editing activities and endonuclease cleavage activity that appears to leave a 5' OH on the 3' product. RNAi knockdowns did not affect growth but resulted in relative reductions of both edited and unedited mitochondrial mRNAs. The similarity of KREPB9 and KREPB10 to KREPB6, KREPB7, and KREPB8 suggests they may be accessory factors that affect editing endonuclease activity and as a consequence may affect mitochondrial mRNA stability. KREPB9 and KREPB10, along with KREPB6, KREPB7, and KREPB8, may enable the endonucleases to discriminate among and accurately cleave hundreds of different editing sites and may be involved in the control of differential editing during the life cycle of T. brucei.     

Purification of a functional enzymatic editing complex from Trypanosoma brucei mitochondria.

Kinetoplastid mitochondrial RNA editing, the insertion and deletion of U residues, is catalyzed by sequential cleavage, U addition or removal, and ligation reactions and is directed by complementary guide RNAs. We have purified a approximately 20S enzymatic complex from Trypanosoma brucei mitochondria that catalyzes a complete editing reaction in vitro. This complex possesses all four activities predicted to catalyze RNA editing: gRNA-directed endonuclease, terminal uridylyl transferase, 3' U-specific exonuclease, and RNA ligase. However, it does not contain other putative editing complex components: gRNA-independent endonuclease, RNA helicase, endogenous gRNAs or pre-mRNAs, or a 25 kDa gRNA-binding protein. The complex is composed of eight major polypeptides, three of which represent RNA ligase. These findings identify polypeptides representing catalytic editing factors, reveal the nature of this approximately 20S editing complex, and suggest a new model of editosome assembly.