Formation of the conserved pseudouridine at position 55 in archaeal tRNA - PubMed (original) (raw)

Formation of the conserved pseudouridine at position 55 in archaeal tRNA

Martine Roovers et al. Nucleic Acids Res. 2006.

Abstract

Pseudouridine (Psi) located at position 55 in tRNA is a nearly universally conserved RNA modification found in all three domains of life. This modification is catalyzed by TruB in bacteria and by Pus4 in eukaryotes, but so far the Psi55 synthase has not been identified in archaea. In this work, we report the ability of two distinct pseudouridine synthases from the hyperthermophilic archaeon Pyrococcus furiosus to specifically modify U55 in tRNA in vitro. These enzymes are (pfu)Cbf5, a protein known to play a role in RNA-guided modification of rRNA, and (pfu)PsuX, a previously uncharacterized enzyme that is not a member of the TruB/Pus4/Cbf5 family of pseudouridine synthases. (pfu)PsuX is hereafter renamed (pfu)Pus10. Both enzymes specifically modify tRNA U55 in vitro but exhibit differences in substrate recognition. In addition, we find that in a heterologous in vivo system, (pfu)Pus10 efficiently complements an Escherichia coli strain deficient in the bacterial Psi55 synthase TruB. These results indicate that it is probable that (pfu)Cbf5 or (pfu)Pus10 (or both) is responsible for the introduction of pseudouridine at U55 in tRNAs in archaea. While we cannot unequivocally assign the function from our results, both possibilities represent unexpected functions of these proteins as discussed herein.

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Figures

Figure 1

Figure 1

Ψ formation by pfuCbf5 and pfuPus10 in pfutRNAAsp. (A and B) Increasing molar amounts of pfuCbf5 and pfuPus10 were incubated at 70°C for 60 min. (C and D) 0.12 nmol (5 µg) of pfuCbf5 and 0.10 nmol (5 µg) of pfuPus10 were incubated with T7-transcribed pfutRNAAsp for increasing time intervals at 70°C. (E and F) 0.12 nmol (5 µg) of pfuCbf5 and 0.10 nmol (5 µg) of pfuPus10 were incubated at increasing temperatures for 60 min. After incubation, the tRNA was recovered, hydrolyzed by nuclease P1 and the resulting nucleotides were separated by 2D-TLC chromatography (see Materials and Methods). Mol of Ψ produced per mol of tRNA was measured after counting the radioactivity in the spots corresponding to UMP and ΨMP on the chromatogram and taking into account the nucleotide composition of the tRNA substrate. Precision is estimated to be ∼10%. The results shown are representative of two similar experiments.

Figure 2

Figure 2

Analysis of pfuCbf5 and pfuPus10 modification of various tRNA substrates. (A) Cloverleaf structure of tRNAAsp of P.furiosus used as the wild-type tRNA substrate in this work. The universal numbering system for nucleotides in tRNA corresponds to that of (4). U55 is indicated. C75 and A76 (missing in −3′CA substrates) are indicated by a dashed box. The portion of the tRNA sequence included in the miniS substrate is indicated by the plain box. (B) The expected patterns of nuclease P1 and RNase T2 cleavage in the U55 region of an [α-32P]UTP- and [α-32P]CTP-labeled tRNA, respectively. Nuclease P1 generates 5′-phosphate-nucleosides while RNase T2 generates 3′phosphate-nucleosides. (C) 2D-TLC analysis of pfuCbf5 and pfuPus10 modification of various tRNA substrates. WT indicates wild-type pfutRNAAsp (panels 1–4, 9–12); U55C indicates U55 replacement mutant (panels 5,6,13,14); miniS indicates mini-substrate (panels 7,15) and −3′CA indicates 3′ terminal CA deletion (panels 8,16). See text for more details. UTP/CTP/ATP and GTP refer to the (32P)-labeled nucleotide incorporated at transcription. Incubation was for 1 h at 70°C in the presence of 0.12 nmol (5 µg) of pfuCbf5 (panels 1–8) and 0.01 nmol (0.5 µg) of pfuPus10 (panels 9–16). After incubation, the RNA was digested by nuclease P1 or RNase T2 (as indicated in each panel) and the resulting nucleotides were analyzed by 2D-TLC on cellulose plates and autoradiography. Circles in dotted lines show the migration of the canonical nucleotides used as UV markers.

Figure 3

Figure 3

pfuCbf5 modification of tRNA in the absence and presence of accessory proteins. [α-32P]UTP-labeled wild-type (wt) or U55 mutant (U55C) tRNA (left and middle panels) was incubated with no protein (-), pfuCbf5 alone (C), or pfuCbf5 plus P.furiosus accessory proteins Gar1, Nop10, and L7Ae (C+). After incubation, the tRNA was digested with RNase T1 and the fragment corresponding to the TΨ-loop (nt 54–65) was excised and purified from a 20% denaturing gel. This fragment was digested with nuclease P1, and Ψ and U were separated and analyzed by TLC and phosphoimaging. In the right panel, site-specifically radiolabeled target rRNA was incubated with the same combinations of proteins in the presence of Pf9 guide rRNA. The resulting RNA was digested with nuclease P1 and analyzed as for tRNA. The number of moles of Ψ incorporated per mole of RNA substrate (taking into account the number of uridines in the region analyzed) is indicated below each lane.

Figure 4

Figure 4

Detection of Ψ in E.coli tRNACys and tRNAPhe. (A) CMC/RT analysis of tRNACys of E.coli. tRNA was isolated from wild-type (wt), truB (KO) or truB transformed with a plasmid containing the pfu_pus10_ gene (+Pus10) E.coli and CMC modified. Primer extension was performed as described in Materials and Methods with a primer complementary to ecotRNACys, nucleotides 61–76. The arrow indicates a strong stop at Ψ55. (B) CMC/RT analysis of ecotRNAPhe as described above using a primer complementary to nucleotides 61–76 of ecotRNAPhe.

Figure 5

Figure 5

Domain organization of Cbf5 and Pus10. Schematic representation of predicted domains of Cbf5, TruB, Pus4 and Pus10 proteins. Conserved catalytic, PUA, Zn and THUMP domains are indicated. See text for discussion.

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