Biomedical subjects

Reza Salavati

Publications and source records attributed to Reza Salavati.

17 recordsLinked to original sources

PubMed2006

Taking U out, with two nucleases?

BACKGROUND: REX1 and REX2 are protein components of the RNA editing complex (the editosome) and function as exouridylylases. The exact roles of REX1 and REX2 in the editosome are unclear and the consequences of the presence of two related proteins are not fully understood. Here, a variety of computational studies were performed to enhance understanding of the structure and function of REX proteins in Trypanosoma and Leishmania species. RESULTS: Sequence analysis and homology modeling of the Endonuclease/Exonuclease/Phosphatase (EEP) domain at the C-terminus of REX1 and REX2 highlights a common active site shared by all EEP domains. Phylogenetic analysis indicates that REX proteins contain a distinct subfamily of EEP domains. Inspection of three-dimensional models of the EEP domain in Trypanosoma brucei REX1 and REX2, and Leishmania major REX1 suggests variations of previously characterized key residues likely to be important in catalysis and determining substrate specificity. CONCLUSION: We have identified features of the REX EEP domain that distinguish it from other family members and hence subfamily specific determinants of catalysis and substrate binding. The results provide specific guidance for experimental investigations about the role(s) of REX proteins in RNA editing.

Binding Sites↗

PubMed2006

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.

Amino Acid Sequence↗

PubMed2006

KREPA4, an RNA binding protein essential for editosome integrity and survival of Trypanosoma brucei.

The 20S editosome, a multiprotein complex, catalyzes the editing of most mitochondrial mRNAs in trypanosomatids by uridylate insertion and deletion. RNAi mediated inactivation of expression of KREPA4 (previously TbMP24), a component of the 20S editosome, in procyclic form Trypanosoma brucei resulted in inhibition of cell growth, loss of RNA editing, and disappearance of 20S editosomes. Levels of MRP1 and REAP-1 proteins, which may have roles in editing but are not editosome components, were unaffected. Tagged KREPA4 protein is incorporated into 20S editosomes in vivo with no preference for either insertion or deletion subcomplexes. Consistent with its S1-like motif, recombinant KREPA4 protein binds synthetic gRNA with a preference for the 3' oligo (U) tail. These data suggest that KREPA4 is an RNA binding protein that may be specific for the gRNA Utail and also is important for 20S editosome stability.

Amino Acid Motifs↗

PubMed2004

High resolution crystal structure of a key editosome enzyme from Trypanosoma brucei: RNA editing ligase 1.

Trypanosomatids are causative agents of several devastating tropical diseases such as African sleeping sickness, Chagas' disease and leishmaniasis. There are no effective vaccines available to date for treatment of these protozoan diseases, while current drugs have limited efficacy, significant toxicity and suffer from increasing resistance. Trypanosomatids have several remarkable and unique metabolic and structural features that are of great interest for developing new anti-protozoan therapeutics. One such feature is "RNA editing", an essential process in these pathogenic protozoa. Transcripts for key trypanosomatid mitochondrial proteins undergo extensive post-transcriptional RNA editing by specifically inserting or deleting uridylates from pre-mature mRNA in order to create mature mRNAs that encode functional proteins. The RNA editing process is carried out in a approximately 1.6 MDa multi-protein complex, the editosome. In Trypanosoma brucei, one of the editosome's core enzymes, the RNA editing ligase 1 (TbREL1), has been shown to be essential for survival of both insect and bloodstream forms of the parasite. We report here the crystal structure of the catalytic domain of TbREL1 at 1.2 A resolution, in complex with ATP and magnesium. The magnesium ion interacts with the beta and gamma-phosphate groups and is almost perfectly octahedrally coordinated by six phosphate and water oxygen atoms. ATP makes extensive direct and indirect interactions with the ligase via essentially all its atoms while extending its base into a deep pocket. In addition, the ATP makes numerous interactions with residues that are conserved in the editing ligases only. Further away from the active site, TbREL1 contains a unique loop containing several hydrophobic residues that are highly conserved among trypanosomatid RNA editing ligases which may play a role in protein-protein interactions in the editosome. The distinct characteristics of the adenine-binding pocket, and the absence of any close homolog in the human genome, bode well for the design of selective inhibitors that will block the essential RNA ligase function in a number of major protozoan pathogens.

Adenosine Triphosphate↗

PubMed2004

In vitro assays for kinetoplastid U insertion-deletion editing and associated activities.

This chapter describes biochemical assays that have been used in analyzing RNA editing in kinetoplastid mitochondria and to characterize the general mechanism of editing by the editosome. Studies using these assays have shown that the characteristics of each activity contribute to editing site selection, U addition and removal, and RNA ligation resulting in accurately edited mRNAs.

Animals↗

PubMed2003

Comparative analysis of editosome proteins in trypanosomatids.

Detailed comparisons of 16 editosome proteins from Trypanosoma brucei, Trypanosoma cruzi and Leishmania major identified protein motifs associated with catalysis and protein or nucleic acid interactions that suggest their functions in RNA editing. Five related proteins with RNase III-like motifs also contain a U1-like zinc finger and either dsRBM or Pumilio motifs. These proteins may provide the endoribonuclease function in editing. Two other related proteins, at least one of which is associated with U-specific 3' exonuclease activity, contain two putative nuclease motifs. Thus, editosomes contain a plethora of nucleases or proteins presumably derived from nucleases. Five additional related proteins, three of which have zinc fingers, each contain a motif associated with an OB fold; the TUTases have C-terminal folds reminiscent of RNA binding motifs, thus indicating the presence of numerous nucleic acid and/or protein binding domains, as do the two RNA ligases and a RNA helicase, which provide for additional catalytic steps in editing. These data indicate that trypanosomatid RNA editing is orchestrated by a variety of domains for catalysis, molecular interaction and structure. These domains are generally conserved within other protein families, but some are found in novel combinations in the editosome proteins.

Algorithms↗

PubMed2003

Leishmania major chromosome 3 contains two long convergent polycistronic gene clusters separated by a tRNA gene.

Leishmania parasites (order Kinetoplastida, family Trypanosomatidae) cause a spectrum of human diseases ranging from asymptomatic to lethal. The approximately 33.6 Mb genome is distributed among 36 chromosome pairs that range in size from approximately 0.3 to 2.8 Mb. The complete nucleotide sequence of Leishmania major Friedlin chromosome 1 revealed 79 protein-coding genes organized into two divergent polycistronic gene clusters with the mRNAs transcribed towards the telomeres. We report here the complete nucleotide sequence of chromosome 3 (384 518 bp) and an analysis revealing 95 putative protein-coding ORFs. The ORFs are primarily organized into two large convergent polycistronic gene clusters (i.e. transcribed from the telomeres). In addition, a single gene at the left end is transcribed divergently towards the telomere, and a tRNA gene separates the two convergent gene clusters. Numerous genes have been identified, including those for metabolic enzymes, kinases, transporters, ribosomal proteins, spliceosome components, helicases, an RNA-binding protein and a DNA primase subunit.

Animals↗

PubMed2003

Kinetoplastid RNA editing ligases: complex association, characterization, and substrate requirements.

RNA editing processes kinetoplastid mitochondrial transcripts post-transcriptionally by inserting and deleting uridylates (Us) to produce functional mRNAs. The activities of the RNA ligases in the multienzyme complex (the editosome) that catalyzes editing and of the recombinant proteins were characterized and found to be similar. Ligation of two RNA fragments was enhanced when bridged by a complementary RNA or DNA, which left no gaps or overhangs. An acceptor nucleotide preference of G>U>C>A was observed in the absence of exogenous ATP but U was preferred upon addition of ATP and ligase activity was increased. The substrate specificity and catalytic characteristics indicate that RNA ligase activity contributes to the accuracy of RNA editing.

Animals↗

PubMed2003

TbMP57 is a 3' terminal uridylyl transferase (TUTase) of the Trypanosoma brucei editosome.

RNA editing produces mature trypanosome mitochondrial mRNAs by uridylate (U) insertion and deletion. In insertion editing, Us are added to the pre-mRNA by a 3' terminal uridylyl transferase (TUTase) activity. We report the identification of a TUTase activity that copurifies with in vitro editing and is catalyzed by the integral editosome protein TbMP57. TbMP57 catalyzes the addition of primarily a single U to single-stranded (ss) RNA and adds the number of Us specified by a guide RNA to insertion editing-like substrates. TbMP57 is distinct from a previously identified TUTase that adds many Us to ssRNA and which we find is neither a stable editosome component nor does it add Us to editing-like substrates. Recombinant TbMP57 specifically interacts with the editosome protein TbMP81, and this interaction enhances the TUTase activity. These results suggest that TbMP57 catalyzes U addition to pre-mRNA during editing.

Animals↗

PubMed2003

Separate insertion and deletion subcomplexes of the Trypanosoma brucei RNA editing complex.

The Trypanosoma brucei editosome catalyzes the maturation of mitochondrial mRNAs through the insertion and deletion of uridylates and contains at least 16 stably associated proteins. We examined physical and functional associations among these proteins using three different approaches: purification of complexes via tagged editing ligases TbREL1 and TbREL2, comprehensive yeast two-hybrid analysis, and coimmunoprecipitation of recombinant proteins. A purified TbREL1 subcomplex catalyzed precleaved deletion editing in vitro, while a purified TbREL2 subcomplex catalyzed precleaved insertion editing in vitro. The TbREL1 subcomplex contained three to four proteins, including a putative exonuclease, and appeared to be coordinated by the zinc finger protein TbMP63. The TbREL2 subcomplex had a different composition, contained the TbMP57 terminal uridylyl transferase, and appeared to be coordinated by the TbMP81 zinc finger protein. This study provides insight into the molecular architecture of the editosome and supports the existence of separate subcomplexes for deletion and insertion editing.

Animals↗

PubMed2003

Dyskinetoplastic Trypanosoma brucei contains functional editing complexes.

Mitochondrial pre-mRNAs undergo posttranscriptional RNA editing as directed by small guide RNAs (gRNAs) to produce functional mRNAs in kinetoplastid protozoa. The pre-mRNAs and gRNAs are encoded in the maxicircle and minicircle components, respectively, of the kinetoplastid mitochondrial DNA (kDNA), and editing is catalyzed by a multienzyme protein complex. Trypanosoma evansi AnTat3/3, which lacks maxicircles but retains a single class of minicircles, and a dyskinetoplastic mutant of Trypanosoma brucei EATRO164, which is devoid of kDNA, were both shown to retain genes and proteins for the editing complex. The proteins are present in complexes that immunoprecipitate and sediment indistinguishably from wild-type complexes. The complexes catalyze precleaved insertion and deletion editing as well as full-round deletion editing in vitro. Thus, mutants which lack the natural substrates for RNA editing and all or most gRNAs retain editing complexes that contain the four primary catalytic activities of editing and function in editing, at least in vitro. Therefore neither pre-mRNA nor gRNA is required to form functional RNA-editing complexes.

Animals↗

PubMed2003

TbMP44 is essential for RNA editing and structural integrity of the editosome in Trypanosoma brucei.

RNA editing produces mature mitochondrial mRNAs in trypanosomatids by the insertion and deletion of uridylates. It is catalyzed by a multiprotein complex, the editosome. We identified TbMP44 among the components of enriched editosomes by a combination of mass spectrometry and DNA sequence database analysis. Inactivation of an ectopic TbMP44 allele in cells in which the endogenous alleles were disrupted abolished RNA editing, inhibited cell growth, and was eventually lethal to bloodstream form trypanosomes. Loss of TbMP44 mRNA was followed initially by a reduction in the editosome sedimentation coefficient and then by the absence of other editosome proteins despite the presence of the mRNA. Reactivation of TbMP44 gene expression resulted in the resumption of cell growth and the reappearance of editosomes. These data indicate that TbMP44 is a component of the editosome that is essential for editing and critical for the structural integrity of the editosome.

Alleles↗

PubMed2003

Identification of novel components of Trypanosoma brucei editosomes.

The editosome is a multiprotein complex that catalyzes the insertion and deletion of uridylates that occurs during RNA editing in trypanosomatids. We report the identification of nine novel editosome proteins in Trypanosoma brucei. They were identified by mass spectrometric analysis of functional editosomes that were purified by serial ion exchange/gel permeation chromatography, immunoaffinity chromatography specific to the TbMP63 editosome protein, or tandem affinity purification based on a tagged RNA editing ligase. The newly identified proteins have ribonuclease and/or RNA binding motifs suggesting nuclease function for at least some of these. Five of the proteins are interrelated, as are two others, and one is related to four previously identified editosome proteins. The implications of these findings are discussed.

Amino Acid Sequence↗

PubMed2002

TbMP81 is required for RNA editing in Trypanosoma brucei.

Most mitochondrial mRNAs are edited in Trypano soma brucei by a series of steps that are catalyzed by a multienzyme complex that is in its initial stages of characterization. RNA interference (RNAi)-mediated repression of the expression of TbMP81, a zinc finger protein component of the complex, inhibited growth of bloodstream and insect forms, and blocked in vivo RNA editing. This repression preferentially inhibited insertion editing compared with deletion editing in vitro. It resulted in reduced specific endoribonucleolytic cleavage and a greater reduction of U addition and associated RNA ligation activities than U removal and associated RNA ligation activities. The repressed cells retained 20S editing complexes with several demonstrable proteins and adenylatable TbMP52 RNA ligase, but adenlyatable TbMP48 was not detected. Elimination of TbMP48 by RNAi repression did not inhibit cell growth or in vivo editing in either bloodstream or procyclic forms. These results indicate that TbMP81 is required for RNA editing and suggest that the editing complex is functionally partitioned.

Animals↗

PubMed2002

Endoribonuclease activities of Trypanosoma brucei mitochondria.

RNA editing in kinetoplastids is a type of post-transcriptional processing that changes mitochondrial mRNA sequences by the addition or deletion of uridines. Multiple enzymatic activities, such as endoribonuclease and RNA ligase, are associated with this process and exist in a multienzyme complex. Endonuclease activities from Trypanosoma brucei mitochondrial extracts were fractionated by sequential ion exchange and gel filtration chromatography. The RNA editing specific endonuclease activity co-fractionated with in vitro editing while another endonuclease activity with a different substrate specificity, and the majority of mtRNase P activity fractionated away from the editing activity. The pH, salt, temperature, and Mg(2+) optima of all three endonucleases were determined. All three activities are sensitive to high temperature and protease digestion. In addition, treatment with micrococcal nuclease resulted in partial disruption of the editing complex and decreased pre-cleaved in vitro insertion editing activity, suggesting that both RNA(s) and protein(s) are necessary in the intact functional complex.

Animals↗

PubMed2002

A hammerhead ribozyme substrate and reporter for in vitro kinetoplastid RNA editing.

Current in vitro assays for RNA editing in kinetoplastids directly examine the products generated by incubation of pre-mRNA substrate with guide RNA (gRNA) and mitochondrial (mt) extract. RNA editing substrates that are modeled on hammerhead ribozymes were designed with catalytic cores that contained or lacked additional uridylates (Us). They proved to be sensitive reporters of editing activity when used for in vitro assays. A deletion editing substrate that is based on A6 pre-mRNA had no ribozyme activity, but its incubation with gRNA and mt extract resulted in its deletion editing and production of a catalytically active ribozyme. Hammerhead ribozymes are thus sensitive tools to assay in vitro RNA editing.

Animals↗

PubMed2002

Role of uridylate-specific exoribonuclease activity in Trypanosoma brucei RNA editing.

Editing of mitochondrial mRNAs in kinetoplastid protozoa occurs by a series of enzymatic steps that insert and delete uridylates (U's) as specified by guide RNAs (gRNAs). The characteristics of the 3' exonuclease activity that removes the U's following cleavage during deletion editing were determined by using an in vitro precleaved deletion assay that is based on ATPase subunit 6 pre-mRNA and gA6[14] gRNA. The exonuclease in partially purified editing complexes is specific for U's. The specificity occurs in the absence of gRNA, but its activity is enhanced by the presence of gRNA. The 3' pre-mRNA fragment enhances the specificity, but not the efficiency, of U removal. The activity is sensitive to the 5' phosphate of the 3' fragment, which is not required for U removal. The ability of the 3' U's to base pair with purines in the gRNA protects them from removal, suggesting that the U-specific 3' exonuclease (exoUase) is specific for U's which are not base paired. ExoUase is stereospecific and cannot remove (Rp)alpha-thio-U. The specificity of the exoUase activity thus contributes to the precision of RNA editing.

Animals↗