4298 Nucleic Acids Research, 2006, Vol. 34, No. 15 Figure 3. pfuCbf5 modification of tRNA in the absence and presence of accessory proteins. [a-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 TY-loop (nt 54–65) was excised and purified from a 20% denaturing gel. This fragment was digested with nuclease P1, and Y 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 Y incorporated per mole of RNA substrate (taking into account the number of uridines in the region analyzed) is indicated below each lane. allele of the truB gene (16). It has been demonstrated that TruB is necessary (and sufﬁcient) for the formation of Y55 in vivo, affording us the opportunity to test if expression of either of the two archaeal enzymes would complement U55 modiﬁcation of E.coli tRNAs (23). The truB strains were transformed with plasmids containing the P.furiosus pus10 or cbf5 gene and the effect of the expression of each of these proteins on tRNA modiﬁcation was assayed by primer extension after CMCT treatment (22). The cells were incub- ated for 1 h at 50 C [a compromise between the high temper- ature optimum for enzymatic activity (see Figure 1E–F) and optimal E.coli growth temperature]. tRNA was isolated from these cells and Y residues within tRNACys and tRNAPhe were identiﬁed using the CMC/RT assay system (22). Similar res- ults were obtained with both tRNAs. As expected, Y55 was observed in the wild type (wt) strain but not in the truB (KO) strain (Figure 4). truB cells transformed with a plasmid containing the cbf5 gene showed no signiﬁcant modiﬁcation of U55 (data not shown), but because we were not able to detect expression of the Cbf5 protein in the E.coli, no conclu- sion can be drawn from the lack of activity. Nearly wild type levels of U55 modiﬁcation were observed in the truB strains transformed with a plasmid containing the pus10 gene (+Pus10). These results indicate that pfuPus10 can efﬁciently catalyze formation of tRNA Y55 in this heterologous system. DISCUSSION Identification of archaeal enzymes that modify U55 in tRNA Until the present work, no protein was attributed to the role of pseudouridylation of tRNA U55 in archaea. U55 modiﬁcation activity was demonstrated in a cell-free extract of P.furiosus, but no protein was implicated as the source of the activity (11). We have identiﬁed two archaeal proteins capable of site-speciﬁc modiﬁcation of U55: pfuCbf5, the only member of the TruB/Pus4/Cbf5 family (COG 0130) found in archaea, and pfuPus10 (previously PsuX, belonging to COG 1258), which was previously uncharacterized. The evidence reported here indicates that both of these archaeal enzymes are capable of speciﬁcally performing this modiﬁcation. It is possible that either or both of these proteins introduce the conserved Y55 in tRNA in P.furiosus. Pus10 (alias PsuX) is a previously uncharacterized enzyme. The data presented here clearly show that this enzyme can efﬁciently modify U55 both in vitro within a variety of substrates (wild type, 30CA mutant and miniS tRNAAsp), and in vivo within E.coli tRNAs. While Pus10 is not a member of the (TruB/Pus4/Cbf5) family of Y synthases that carry out tRNA U55 pseudouridylation in bacteria and eukaryotes, Pus10 orthologs are found in all sequenced archaeal genomes, consistent with the notion that this may be the conserved archaeal enzyme responsible for tRNA Y55 (15). Pus10 homologs are not present in bacteria (which employ TruB for Y55 synthesis), but do also appear to exist in certain higher eukaryotes (which employ Pus4 for Y55 synthesis) (15). Pus10 is renamed here as the 10th Y synthase acting on tRNA [for review on Pus1–9, see (24,25)]. The implication of Pus10 in tRNA Y55 formation (based on our ﬁndings) represents the ﬁrst evidence for func- tion of a protein outside the TruB/Pus4/Cbf5 family in intro- duction of this highly conserved modiﬁcation. The results of our studies indicate that pfuCbf5 also cata- lyzes speciﬁc tRNA Y55 formation in vitro. Cbf5 is the only enzyme in archaea that bears signiﬁcant sequence simil- arity to the known Y55 synthases in bacteria and eukaryotes. Therefore, it is the most logical choice for the relevant enzyme. However, a role for Cbf5 in RNA-guided rRNA modiﬁcation has been ﬁrmly established in archaea (12,13) as well as eukaryotes (14). Our ﬁnding that Cbf5 may also function in tRNA U55 modiﬁcation suggests an unexpected dual function for Cbf5 in archaea. There is some precedent for this concept in the dual speciﬁcity of Pus1p for tRNA and U2 snRNA in yeast (26). Pseudouridylation of rRNA by Cbf5 depends upon the accessory proteins Gar1, Nop10 and L7Ae (12,13). In this work we found that the activity of Cbf5 toward tRNA is