Trypanosoma brucei, the etiologic agent of sleeping sickness, encodes a single intron-containing tRNA, tRNA super(Tyr), and splicing is essential for its viability. In Archaea and Eukarya, tRNA ...splicing requires a series of enzymatic steps that begin with intron cleavage by a tRNA-splicing endonuclease and culminates with joining the resulting tRNA exons by a splicing tRNA ligase. Here we explored the function of TbTrl1, the T. brucei homolog of the yeast Trl1 tRNA ligase. We used a combination of RNA interference and molecular biology approaches to show that down-regulation of TbTrl1 expression leads to accumulation of intron-containing tRNA super(Tyr) and a concomitant growth arrest at the G1 phase. These defects were efficiently rescued by expression of an "intronless" version of tRNA super(Tyr) in the same RNAi cell line. Taken together, these experiments highlight the crucial importance of the TbTrl1 for tRNA super(Tyr) maturation and viability, while revealing tRNA splicing as its only essential function.
Trypanosoma brucei
, the etiologic agent of sleeping sickness, encodes a single intron-containing tRNA, tRNA
Tyr
, and splicing is essential for its viability. In Archaea and Eukarya, tRNA splicing ...requires a series of enzymatic steps that begin with intron cleavage by a tRNA-splicing endonuclease and culminates with joining the resulting tRNA exons by a splicing tRNA ligase. Here we explored the function of TbTrl1, the
T. brucei
homolog of the yeast Trl1 tRNA ligase. We used a combination of RNA interference and molecular biology approaches to show that down-regulation of TbTrl1 expression leads to accumulation of intron-containing tRNA
Tyr
and a concomitant growth arrest at the G1 phase. These defects were efficiently rescued by expression of an “intronless” version of tRNA
Tyr
in the same RNAi cell line. Taken together, these experiments highlight the crucial importance of the TbTrl1 for tRNA
Tyr
maturation and viability, while revealing tRNA splicing as its only essential function.
All organisms encode transfer RNAs (tRNAs) that are synthesized as precursor molecules bearing extra sequences at their 5′ and 3′ ends; some tRNAs also contain introns, which are removed by splicing. ...Despite commonality in what the ultimate goal is (i.e., producing a mature tRNA), mechanistically, tRNA splicing differs between Bacteria and Archaea or Eukarya. The number and position of tRNA introns varies between organisms and even between different tRNAs within the same organism, suggesting a degree of plasticity in both the evolution and persistence of modern tRNA splicing systems. Here we will review recent findings that not only highlight nuances in splicing pathways but also provide potential reasons for the maintenance of introns in tRNA. Recently, connections between defects in the components of the tRNA splicing machinery and medically relevant phenotypes in humans have been reported. These differences will be discussed in terms of the importance of splicing for tRNA function and in a broader context on how tRNA splicing defects can often have unpredictable consequences. WIREs RNA 2015, 6:337–349. doi: 10.1002/wrna.1279
This article is categorized under:
RNA Processing > Splicing Mechanisms
RNA Processing > tRNA Processing
RNA in Disease and Development > RNA in Disease
Methanocaldococcus jannaschii prolyl-tRNA synthetase (ProRS) was previously reported to also catalyze the synthesis of cysteinyl-tRNA Cys (Cys-tRNA Cys ) to make up for the absence of the canonical ...cysteinyl-tRNA synthetase in this organism (Stathopoulos, C., Li, T., Longman,
R., Vothknecht, U. C., Becker, H., Ibba, M., and Söll, D. (2000) Science 287, 479â482; Lipman, R. S., Sowers, K. R., and Hou, Y. M. (2000) Biochemistry 39, 7792â7798). Here we show by acid urea gel electrophoresis that pure heterologously expressed recombinant M. jannaschii ProRS misaminoacylates M. jannaschii tRNA Pro with cysteine. The enzyme is unable to aminoacylate purified mature M. jannaschii tRNA Cys with cysteine in contrast to facile aminoacylation of the same tRNA with cysteine by Methanococcus maripaludis cysteinyl-tRNA synthetase. Although M. jannaschii ProRS catalyzes the synthesis of Cys-tRNA Pro readily, the enzyme is unable to edit this misaminoacylated tRNA. We discuss the implications of these results on the in vivo activity of the M. jannaschii ProRS and on the nature of the enzyme involved in the synthesis of Cys-tRNA Cys in M. jannaschii .
Rhodnius prolixusnot only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people ...worldwide. We sequenced the genome ofR. prolixus,generated assembled sequences covering 95% of the genome (∼702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immunedeficiency (IMD)-mediated immune responses were observed,R. prolixusputatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affectedTrypanosoma cruzi,the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms.R. prolixushas experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes fromWolbachiasp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences inR. prolixus,including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods.
Glycosphingolipids were extracted from hyphae of
Fusarium solani and from an unnamed
Fusarium species, and were purified by silica and Iatrobead column chromatography. Their structures were ...determined by compositional analysis, nuclear magnetic resonance spectroscopy, gas chromatography/mass spectrometry and by fast atom bombardment mass spectrometry of the native and peracetylated materials, which defined their sugar, long-chain base and fatty acid compositions. The locations of the double bonds in the bases were established by 2D NMR spectroscopy and by novel mass spectrometric approaches, including collisional activation of the protonated and lithium-cationized glycosphingolipids, and of the sphingadienene-derived fragment ion at
m/
z 276.
From these results we propose that the structures of the glycosphingolipids from
F. solani and
Fusarium sp. are
N-2′-hydroxyoctadecanoyl-1-
O-β-
d-glucopyranosyl-9-methyl-4,8-sphingadienine and
N-2′-hydroxyoctadecenoyl-1-
O-β-
d-glucopyranosyl-9-methyl-4,8-sphingadienine, respectively.
Monomethylamine methyltransferase of the archaeon Methanosarcina barkeri contains a rare amino acid, pyrrolysine, encoded by the termination codon UAG. Translation of this UAG requires the ...aminoacylation of the corresponding amber suppressor tRNAPyl. Previous studies reported that tRNAPyl could be aminoacylated by the synthetase-like protein PylS. We now show that tRNAPyl is efficiently aminoacylated in the presence of both the class I LysRS and class II LysRS of M. barkeri, but not by either enzyme acting alone or by PylS. In vitro studies show that both the class I and II LysRS enzymes must bind tRNAPyl in order for the aminoacylation reaction to proceed. Structural modeling and selective inhibition experiments indicate that the class I and II LysRSs form a ternary complex with tRNAPyl, with the aminoacylation activity residing in the class II enzyme.
Pyrrolysine (Pyl), the 22nd naturally encoded amino acid, gets acylated to its distinctive UAG suppressor tRNA... by the cognate pyrrolysyl-tRNA synthetase (PylRS). Here we determine the RNA elements ...required for recognition and aminoacylation of tRNA... in vivo by using the Pyl analog N-...-cyclopentyloxycarbonyl-L-lysine. Forty-two Methanosarcina barkeri tRNA... variants were tested in Escherichia coli for suppression of the lac amber A24 mutation; then relevant tRNA... mutants were selected to determine in vivo binding to M. barkeri PylRS in a yeast three-hybrid system and to measure in vitro tRNA... aminoacylation. tRNA... identity elements include the discriminator base, the first base pair of the acceptor stem, the T-stem base pair G51 :C63, and the anticodon flanking nucleotides U33 and A37. Transplantation of the tRNA... identity elements into the mitochondrial bovine tRNASer scaffold yielded chimeric tRNAs active both in vitro and in vivo. Because the anticodon is not important for PylRS recognition, a tRNA... variant could be constructed that efficiently suppressed the lac opal U4 mutation in E. coli. These data suggest that tRNA... (ProQuest-CSA LLC: ... denotes formulae/symbols omitted.)
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