scholarly journals Assembly and Function of the RNA Editing Complex in Trypanosoma brucei Requires Band III Protein

2002 ◽  
Vol 22 (9) ◽  
pp. 3194-3203 ◽  
Author(s):  
Catherine E. Huang ◽  
Sean F. O'Hearn ◽  
Barbara Sollner-Webb
Keyword(s):  
Rna Editing ◽  
Guide Rna ◽  
Insertion And Deletion ◽  
Cell Extracts ◽  
Zinc Finger Motifs ◽  
Acid Protein ◽  
Mitochondrial Transcripts ◽  
Insertion Sites ◽  
And Function ◽  
Amino Acid Protein

ABSTRACT Trypanosome RNA editing, the posttranscriptional insertion and deletion of U residues in mitochondrial transcripts, is catalyzed by a protein complex containing seven distinct proteins. In this study, we cloned the gene for band III, a 555-amino-acid protein with two separate zinc finger motifs. We prepared antibodies that showed band III protein cofractionates with the previously characterized band IV protein throughout the purification of the editing complex and is not found free or in other protein associations; therefore, it is a true constituent of the editing complex. Double-stranded RNA interference efficiently depleted band III protein and demonstrated that band III expression is essential for growth of procyclic trypanosomes and for RNA editing. These depleted cell extracts were deficient specifically in guide RNA-directed endonuclease cleavage at both U deletion and U insertion sites and in the activity of the band IV ligase, but they retained the 3′-U-exonuclease and terminal-U-transferase activities as well as band V ligase of the editing complex. Loss of band III protein also resulted in almost complete loss of the band IV ligase protein and altered sedimentation of the band V ligase. These data indicate that band III is either the RNA editing endonuclease or a factor critical for cleavage activity in the editing complex. They also demonstrate that band III is required for proper assembly of the editing complex.

NHE3 in an ancestral vertebrate: primary sequence, distribution, localization, and function in gills

2005 ◽  
Vol 289 (5) ◽  
pp. R1520-R1534 ◽  
Author(s):  
Keith P. Choe ◽  
Akira Kato ◽  
Shigehisa Hirose ◽  
Consuelo Plata ◽  
Aleksandra Sindić ◽  
...  
Keyword(s):  
Complete Sequence ◽  
Coding Region ◽  
Degenerate Primers ◽  
Acid Protein ◽  
Rapid Amplification ◽  
And Function ◽  
Apical Membranes ◽  
Carboxyl Tail ◽  
Ancestral Vertebrate ◽  
Amino Acid Protein

In mammals, the Na+/H+ exchanger 3 (NHE3) is expressed with Na+/K+-ATPase in renal proximal tubules, where it secretes H+ and absorbs Na+ to maintain blood pH and volume. In elasmobranchs (sharks, skates, and stingrays), the gills are the dominant site of pH and osmoregulation. This study was conducted to determine whether epithelial NHE homologs exist in elasmobranchs and, if so, to localize their expression in gills and determine whether their expression is altered by environmental salinity or hypercapnia. Degenerate primers and RT-PCR were used to deduce partial sequences of mammalian NHE2 and NHE3 homologs from the gills of the euryhaline Atlantic stingray ( Dasyatis sabina). Real-time PCR was then used to demonstrate that mRNA expression of the NHE3 homolog increased when stingrays were transferred to low salinities but not during hypercapnia. Expression of the NHE2 homolog did not change with either treatment. Rapid amplification of cDNA was then used to deduce the complete sequence of a putative NHE3. The 2,744-base pair cDNA includes a coding region for a 2,511-amino acid protein that is 70% identical to human NHE3 (SLC9A3). Antisera generated against the carboxyl tail of the putative stingray NHE3 labeled the apical membranes of Na+/K+-ATPase-rich epithelial cells, and acclimation to freshwater caused a redistribution of labeling in the gills. This study provides the first NHE3 cloned from an elasmobranch and is the first to demonstrate an increase in gill NHE3 expression during acclimation to low salinities, suggesting that NHE3 can absorb Na+ from ion-poor environments.

scholarly journals Guide RNA-mRNA chimeras, which are potential RNA editing intermediates, are formed by endonuclease and RNA ligase in a trypanosome mitochondrial extract.

1995 ◽  
Vol 15 (6) ◽  
pp. 2933-2941 ◽  
Author(s):  
L N Rusché ◽  
K J Piller ◽  
B Sollner-Webb
Keyword(s):  
Rna Editing ◽  
Mitochondrial Rna ◽  
Site Specific ◽  
Rna Ligase ◽  
Guide Rna ◽  
Guide Rnas ◽  
Editing Domain ◽  
Mitochondrial Transcripts ◽  
Mitochondrial Extract

RNA editing in kinetoplast mitochondrial transcripts involves the insertion and/or deletion of uridine residues and is directed by guide RNAs (gRNAs). It is thought to occur through a chimeric intermediate in which the 3' oligo(U) tail of the gRNA is covalently joined to the 3' portion of the mRNA at the site being edited. Chimeras have been proposed to be formed by a transesterification reaction but could also be formed by the known mitochondrial site-specific nuclease and RNA ligase. To distinguish between these models, we studied chimera formation in vitro directed by a trypanosome mitochondrial extract. This reaction was found to occur in two steps. First, the mRNA is cleaved in the 3' portion of the editing domain, and then the 3' fragment derived from this cleavage is ligated to the gRNA. The isolated mRNA 3' cleavage product is a more efficient substrate for chimera formation than is the intact mRNA, inconsistent with a transesterification mechanism but supporting a nuclease-ligase mechanism. Also, when normal mRNA cleavage is inhibited by the presence of a phosphorothioate, normal chimera formation no longer occurs. Rather, this phosphorothioate induces both cleavage and chimera formation at a novel site within the editing domain. Finally, levels of chimera-forming activity correlate with levels of mitochondrial RNA ligase activity when reactions are conducted under conditions which inhibit the ligase, including the lack of ATP containing a cleavable alpha-beta bond. These data show that chimera formation in the mitochondrial extract occurs by a nuclease-ligase mechanism rather than by transesterification.

scholarly journals The Two RNA Ligases of the Trypanosoma brucei RNA Editing Complex: Cloning the Essential Band IV Gene and Identifying the Band V Gene

2001 ◽  
Vol 21 (4) ◽  
pp. 979-989 ◽  
Author(s):  
Laura N. Rusché ◽  
Catherine E. Huang ◽  
Kenneth J. Piller ◽  
Michael Hemann ◽  
Elizabeth Wirtz ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Mrna Levels ◽  
Mitochondrial Targeting ◽  
Terminal Sequence ◽  
Partial Amino Acid Sequence ◽  
Rna Ligase ◽  
E Coli ◽  
Insertion And Deletion ◽  
Mitochondrial Transcripts

ABSTRACT Kinetoplastid RNA editing is a posttranscriptional insertion and deletion of U residues in mitochondrial transcripts that involves RNA ligase. A complex of seven different polypeptides purified fromTrypanosoma brucei mitochondria that catalyzes accurate RNA editing contains RNA ligases of ∼57 kDa (band IV) and ∼50 kDa (band V). From a partial amino acid sequence, cDNA and genomic clones of band IV were isolated, making it the first cloned component of the minimal RNA editing complex. It is indeed an RNA ligase, for when expressed inEscherichia coli, the protein autoadenylylates and catalyzes RNA joining. Overexpression studies revealed that T. brucei can regulate of total band IV protein at the level of translation or protein stability, even upon massively increased mRNA levels. The protein's mitochondrial targeting was confirmed by its location, size when expressed in T. brucei and E. coli, and N-terminal sequence. Importantly, genetic knockout studies demonstrated that the gene for band IV is essential in procyclic trypanosomes. The band IV and band V RNA ligases of the RNA editing complex therefore serve different functions. We also identified the gene for band V RNA ligase, a protein much more homologous to band IV than to other known ligases.

scholarly journals Editing domains of Trypanosoma brucei mitochondrial RNAs identified by secondary structure.

1995 ◽  
Vol 15 (6) ◽  
pp. 2916-2924 ◽  
Author(s):  
K J Piller ◽  
C J Decker ◽  
L N Rusché ◽  
M E Harris ◽  
S L Hajduk ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Mung Bean Nuclease ◽  
Reaction Conditions ◽  
Rna Ligase ◽  
Guide Rna ◽  
Insertion And Deletion ◽  
Mitochondrial Transcripts ◽  
Cytochrome Oxidase Subunit Ii ◽  
Model Reactions

The posttranscriptional insertion and deletion of U residues in trypanosome mitochondrial transcripts called RNA editing initiates at the 3' end of precisely defined editing domains that can be identified independently of the cognate guide RNA. The regions where editing initiates in Trypanosoma brucei cytochrome b and cytochrome oxidase subunit II preedited mRNAs are specifically cleaved by a trypanosome mitochondrial endonuclease that acts like mung bean nuclease and therefore is single strand specific. The regions where editing initiates in virtually all examined preedited mRNAs are predicted to form loop structures, suggesting that editing domains could generally be recognized as prominent single-stranded loops. In contrast to preedited mRNA, edited mRNA can be either resistant or sensitive to cleavage by trypanosome mitochondrial endonuclease, depending on the reaction conditions. This selectivity appears dependent on the availability of extract RNAs, and in model reactions, edited mRNA becomes resistant to cleavage upon base pairing with its guide RNA. Natural partially edited mRNAs are also specifically cleaved with a sensitivity like preedited and unlike edited mRNAs, consistent with their being intermediates in editing. These results suggest that in vivo, the structure of editing domains could initially be recognized by the mitochondrial endonuclease, which could target its associated RNA ligase and terminal U transferase to begin cycles of enzymatic editing modifications.

scholarly journals Streptococcus parasanguis pepO Encodes an Endopeptidase with Structure and Activity Similar to Those of Enzymes That Modulate Peptide Receptor Signaling in Eukaryotic Cells

1999 ◽  
Vol 67 (10) ◽  
pp. 5206-5214 ◽  
Author(s):  
Eunice H. Froeliger ◽  
Joyce Oetjen ◽  
Jeffrey P. Bond ◽  
Paula Fives-Taylor
Keyword(s):  
Active Form ◽  
Neutral Endopeptidase ◽  
Opioid Peptide ◽  
Northern Hybridization ◽  
Reading Frame ◽  
Cell Extracts ◽  
Acid Protein ◽  
Potent Vasoconstrictor ◽  
Structure And Activity ◽  
Amino Acid Protein

ABSTRACT Studies in our laboratory have identified two fimbria-associated adhesins, FimA and Fap1, of Streptococcus parasanguisFW213. In this study, we isolated and sequenced DNA fragments linked tofimA to determine if they contained additional factors associated with adherence, virulence, or survival in the host. An open reading frame just upstream and divergently transcribed from thefimA operon was identified and named pepO. Northern hybridization indicated that pepO is transcribed as a monocistronic message. pepO encodes a predicted 631-amino-acid protein with a molecular mass of approximately 70.6 kDa. PepO contains the essential motif HEXXH, typical of many zinc-dependent metalloproteases and metallopeptidases. PepO has significant sequence identity to mammalian metallopeptidases, including endothelin-converting enzyme, which converts a potent vasoconstrictor into its active form, and neutral endopeptidase (NEP), which is involved in terminating the activity of opioid peptides. The opioid peptide metenkephalin is a natural substrate of NEP. Cell extracts of FW213 cleaved metenkephalin at the same site as does NEP, while an extract from an insertionally inactivated pepO mutant did not. These results indicate that FW213 pepO encodes an enzyme with activity similar to that of known mammalian endopeptidases. Phylogenetic analysis of PepO and its homologues suggests lateral genetic exchange between bacteria and eukaryotes.

scholarly journals In Trypanosoma brucei RNA Editing, Band II Enables Recognition Specifically at Each Step of the U Insertion Cycle

2005 ◽  
Vol 25 (7) ◽  
pp. 2785-2794 ◽  
Author(s):  
Julie A. Law ◽  
Catherine E. Huang ◽  
Sean F. O'Hearn ◽  
Barbara Sollner-Webb
Keyword(s):  
Rna Interference ◽  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Cell Lines ◽  
Protein Recognition ◽  
Essential Nature ◽  
Insertion And Deletion ◽  
Insertion Sites

ABSTRACT Trypanosome RNA editing is the posttranscriptional insertion and deletion of uridylate (U) residues, often to a massive extent, through cycles of cleavage, U addition or U removal, and ligation. These editing cycles are catalyzed by a complex that we purified to seven major proteins (bands I through VII). Here we analyze the role of band II using extracts of clonal band II RNA interference (RNAi) cell lines prepared by a rapid protocol that enables retention of activities that are lost during traditional extract preparation. By individually scoring each step of editing, we show that band II is critical for all steps of U insertion but is not important for any of the steps of U deletion or for their coordination into the U deletion cycle. This specificity supports the long- standing model that U-insertional and U-deletional activities are separated within the editing complex. Furthermore, by assaying the basic activities of the enzymes that catalyze the steps of U insertion, independent of their action in editing, we show that band II is not any of those enzymes. Rather, band II enables endonuclease action at authentic U insertion sites, terminal-uridylyl-transferase (TUTase) action at cleaved U insertion sites, and U-insertion-specific ligase (band V/IREL) action in the editing complex. Thus, band II facilitates each step of U insertion by providing proper RNA and/or protein recognition. We propose that band II (TbMP81) be called IRER, indicating its essential nature in U-insertional RNA editing recognition.

scholarly journals Dyskinetoplastic Trypanosoma brucei Contains Functional Editing Complexes

2003 ◽  
Vol 2 (3) ◽  
pp. 569-577 ◽  
Author(s):  
Gonzalo J. Domingo ◽  
Setareh S. Palazzo ◽  
Bingbing Wang ◽  
Brian Pannicucci ◽  
Reza Salavati ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Trypanosoma Evansi ◽  
Wild Type ◽  
Single Class ◽  
Catalytic Activities ◽  
Guide Rnas ◽  
Insertion And Deletion ◽  
And Function

ABSTRACT 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.

scholarly journals The structure of a highly conserved picocyanobacterial protein reveals a Tudor domain with a novel tRNA binding function

2019 ◽  
Author(s):  
Katherine M Bauer ◽  
Rose Dicovitsky ◽  
Maria Pellegrini ◽  
Olga Zhaxybayeva ◽  
Michael J Ragusa
Keyword(s):  
Amino Acid ◽  
Amino Acid Level ◽  
Computational Docking ◽  
Binding Function ◽  
Acid Protein ◽  
Tudor Domain ◽  
And Function ◽  
Positively Charged ◽  
Amino Acid Protein

ABSTRACTCyanobacteria of the Prochlorococcus and marine Synechococcus genera are the most abundant photosynthetic microbes in the ocean. Intriguingly, the genomes of these bacteria are very divergent even within each genus, both in gene content and at amino acid level of the encoded proteins. One striking exception to this is a 62 amino acid protein, termed Prochlorococcus/SynechococcusHyper Conserved Protein (PSHCP). PSHCP is not only found in all sequenced Prochlorococcus and marine Synechococcus genomes but it is also nearly 100% identical in its amino acid sequence across all sampled genomes. Such universal distribution and sequence conservation suggests an essential cellular role of the encoded protein in these bacteria. However, the function of PSHCP is unknown. We used Nuclear Magnetic Resonance (NMR) spectroscopy to determine its structure. We found that 52 of the 62 amino acids in PSHCP form a Tudor domain, while the remainder of the protein is disordered. NMR titration experiments revealed that PSHCP has only a weak affinity for DNA, but an 18.5 fold higher affinity for tRNA, hinting at an involvement of PSHCP in translation. Computational docking and mutagenesis studies identified a positively charged patch surrounding residue K30 that serves as the primary docking site for tRNA on PSHCP. These results provide the first insight into the structure and function of PSHCP and suggest a new function for Tudor domains in recognizing tRNA.

Cloning and expression of apyrase gene from Ancylostoma caninum in Escherechia coli

2014 ◽  
Vol 60 (1) ◽  
Author(s):  
Ying Qiao ◽  
Theerakamol Pengsakul
Keyword(s):  
Recombinant Protein ◽  
Molecular Size ◽  
Cloning And Expression ◽  
Host Immune System ◽  
Ancylostoma Caninum ◽  
Acid Protein ◽  
Partial Cdna ◽  
And Function ◽  
Phosphate Groups ◽  
Amino Acid Protein

AbstractApyrase encoding metal-ions activated plasma membrane protease is present in animal and plant tissues. This enzyme can hydrolyze ADP and ATP pyrophosphate bond, resulting in AMP and free phosphate groups, and plays an important role for insects and parasites to evade host immune system. However localization and function of apyrase in the canine hookworm, Ancylostoma caninum, remains unknown. To analyze apyrase gene in A. caninum (a eukaryotic parasitic hookworm), a pair of primers was designed according to the previous EST data. The full-length cDNA of apyrase gene was amplified from A. caninum by RT-PCR. The partial cDNA of apyrase encodes 249 amino acid protein was expressed in Escherechia coli. The recombinant protein was induced to express under proper conditions and the molecular size was as expected. The recombinant protein was purified. The transcripts of apyrase in different stages of A. caninum were analyzed by the Real-time PCR assay, and Immuno-localization assays were used to research the protein expression in different stages of A. caninum

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