tRNA Splicing

Introns interrupt the continuity of many eukaryal genes, and therefore their removal by splicing is a crucial step in gene expression. Interestingly, even within Eukarya there are at least four splicing mechanisms. mRNA splicing in the nucleus takes place in two phosphotransfer reactions on a complex and dynamic machine, the spliceosome. This reaction is related in mechanism to the two self-splicing mechanisms for Group 1 and Group 2 introns. In fact the Group 2 introns are spliced by an identical mechanism to mRNA splicing, although there is no general requirement for either proteins or co-factors. Thus it seems likely that the Group 2 and nuclear mRNA splicing reactions have diverged from a common ancestor. tRNA genes are also interrupted by introns, but here the splicing mechanism is quite different because it is catalyzed by three enzymes, all proteins and with an intrinsic requirement for ATP hydrolysis. tRNA splicing occurs in all three major lines of descent, the Bacteria, the Archaea, and the Eukarya. In bacteria the introns are self-splicing (1Biniszkiewicz D. Cesnaviciene E. Shub D.A. EMBO J. 1994; 13: 4629-4635Crossref PubMed Scopus (66) Google Scholar, 2Reinhold-Hurek B. Shub D.A. Nature. 1992; 357: 173-176Crossref PubMed Scopus (157) Google Scholar, 3Kuhsel M.G. Strickland R. Palmer J.D. Science. 1990; 250: 1570-1573Crossref PubMed Scopus (208) Google Scholar). Until recently it was thought that the mechanisms of tRNA splicing in Eukarya and Archaea were unrelated as well. In the past year, however, it has been found that the first enzyme in the tRNA splicing pathway, the tRNA endonuclease, has been conserved in evolution since the divergence of the Eukarya and the Archaea. Surprising insights have been obtained by comparison of the structures and mechanisms of tRNA endonuclease from these two divergent lines. The earliest studies of tRNA splicing were in the yeastSaccharomyces cerevisiae where tRNA introns were first discovered (4Goodman H.M. Olson M.V. Hall B.D. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5453-5457Crossref PubMed Scopus (218) Google Scholar, 5Valenzuela P. Venegas A. Weinberg F. Bishop R. Rutter W.J. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 190-194Crossref PubMed Scopus (158) Google Scholar). With the completion of the S. cerevisiaegenome sequence it is now known that yeast contains 272 tRNA genes of which 59, encoding 10 different tRNAs, are interrupted by introns (6Trotta C.R. Miao F. Arn E.A. Stevens S.W. Ho C.K. Rauhut R. Abelson J.N. Cell. 1997; 89: 849-858Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). The introns are 14–60 nucleotides in length and interrupt the anticodon loop immediately 3′ to the anticodon (7Ogden R.C. Lee M.C. Knapp G. Nucleic Acids Res. 1984; 12: 9367-9382Crossref PubMed Scopus (59) Google Scholar). Among the 10 different yeast pre-tRNAs there is no conservation of sequence at the splice junctions although the 3′-splice junction is invariably in a bulged loop (8Baldi M.I. Mattoccia E. Bufardeci E. Fabbri S. Tocchini-Valentini G.P. Science. 1992; 255: 1404-1408Crossref PubMed Scopus (59) Google Scholar). Early studies on the structure of yeast tRNA precursors showed that the conformation of the mature domain is retained suggesting the model of the tertiary structure of eukaryal pre-tRNA shown in Fig. 1 A (9Lee M.C. Knapp G. J. Biol. Chem. 1985; 260: 3108-3115Abstract Full Text PDF PubMed Google Scholar,10Swerdlow H. Guthrie C. J. Biol. Chem. 1984; 259: 5197-5207Abstract Full Text PDF PubMed Google Scholar). In the Archaea the introns are also small and often interrupt the anticodon loop, but they are found elsewhere as well, for example interrupting the dihydro U stem (11Thompson L.D. Brandon L.D. Nieuwlandt D.T. Daniels C.J. Can. J. Microbiol. 1989; 35: 36-42Crossref PubMed Scopus (31) Google Scholar). In several of the Archaea, tRNA genes have been found that contain two introns. The splice sites are found in an absolutely conserved structural motif consisting of two loops of three bases separated by a four-base pair helix, the bulge-helix-bulge (BHB) 1The abbreviations used are: BHB, bulge-helix-bulge; pre-tRNA, precursor tRNA; TAR, trans-activating response region.motif (12Thompson L.D. Daniels C.J. J. Biol. Chem. 1988; 263: 17951-17959Abstract Full Text PDF PubMed Google Scholar). This structure, modeled in Fig. 1 B from the related TAR RNA structure (13Li H. Trotta C.R. Abelson J.N. Science. 1998; 280: 279-284Crossref PubMed Scopus (120) Google Scholar), allows the archaeal splicing mechanism to be extended to introns in rRNA that also retain this motif. Thus, early on it was suggested that the eukaryal and archaeal splicing systems operate by a different mechanism. The early discovery by Hopper and co-workers (14Hopper A.K. Banks F. Evangelidis V. Cell. 1978; 14: 211-219Abstract Full Text PDF PubMed Scopus (139) Google Scholar) that pre-tRNAs accumulate in the yeast mutant rna1–1 provided a source of pre-tRNA substrates, which allowed the development of the first in vitro RNA splicing system (15Knapp G. Beckmann J.S. Johnson P.F. Fuhrman S.A. Abelson J.N. Cell. 1978; 14: 221-236Abstract Full Text PDF PubMed Scopus (190) Google Scholar, 16O'Farrell, P. Z., Cordell, B., Valenzuela, P., Rutter, W. J., and Goodman, H. M. (1978) Nature 438–445Google Scholar). Using this system the pathway of tRNA splicing was deduced (17Greer C.L. Peebles C.L. Gegenheimer P. Abelson J. Cell. 1983; 32: 537-546Abstract Full Text PDF PubMed Scopus (178) Google Scholar, 18Peebles C.L. Ogden R.C. Knapp G. Abelson J. Cell. 1979; 18: 27-35Abstract Full Text PDF PubMed Scopus (89) Google Scholar, 19Knapp G. Ogden R.C. Peebles C.L. Abelson J. Cell. 1979; 18: 37-45Abstract Full Text PDF PubMed Scopus (95) Google Scholar, 20Peebles C.L. Gegenheimer P. Abelson J. Cell. 1983; 32: 525-536Abstract Full Text PDF PubMed Scopus (154) Google Scholar). The tRNA splicing reaction in yeast occurs in three steps; each step is catalyzed by a distinct enzyme, which can function interchangeably on all of the substrates (Fig. 2). In the first step the pre-tRNA is cleaved at its two splice sites by an endonuclease. The products of the endonuclease reaction are the two tRNA half-molecules and the linear intron with 5′-OH and 3′-cyclic PO4 ends. The endonuclease has been purified to homogeneity (6Trotta C.R. Miao F. Arn E.A. Stevens S.W. Ho C.K. Rauhut R. Abelson J.N. Cell. 1997; 89: 849-858Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 21Rauhut R. Green P.R. Abelson J. J. Biol. Chem. 1990; 265: 18180-18184Abstract Full Text PDF PubMed Google Scholar). The enzyme behaves as an integral membrane protein, and since splicing takes place in the nucleus, it may be an inner nuclear envelope protein. The two tRNA half-molecules, in essence a nicked tRNA, are the substrate for the ensuing ligase reaction. This baroque reaction, catalyzed by the 90-kDa tRNA ligase (22Phizicky E.M. Schwartz R.C. Abelson J. J. Biol. Chem. 1986; 261: 2978-2986Abstract Full Text PDF PubMed Google Scholar, 23Westaway S.K. Phizicky E.M. Abelson J. J. Biol. Chem. 1988; 263: 3171-3176Abstract Full Text PDF PubMed Google Scholar), takes place in three steps. In the first step the cyclic PO4 is opened to give a 2′-PO4 and 3′-OH. In the second step the 5′-OH is phosphorylated with the γ-PO4 of GTP (24Westaway S.K. Belford H.G. Apostol B.L. Abelson J. Greer C.L. J. Biol. Chem. 1993; 268: 2435-2443Abstract Full Text PDF PubMed Google Scholar, 25Belford H.G. Westaway S.K. Abelson J. Greer C.L. J. Biol. Chem. 1993; 268: 2444-2450Abstract Full Text PDF PubMed Google Scholar). tRNA ligase is adenylated at an active site lysine (26Xu Q. Teplow D. Lee T.D. Abelson J. Biochemistry. 1990; 29: 6132-6138Crossref PubMed Scopus (49) Google Scholar), and then the AMP is transferred to the 5′-PO4 of the substrate. Formation of the 5′–3′-phosphodiester bond proceeds and AMP is released. The phosphate at the spliced junction is derived from the γ-phosphate of GTP, and the phosphate originally at the 5′-splice site remains at the spliced junction as a 2′-phosphate and must be removed to complete the splicing reaction. Phizicky and co-workers (27Culver G.M. McCraith S.M. Consaul S.A. Stanford D.R. Phizicky E.M. J. Biol. Chem. 1997; 272: 13203-13210Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) have characterized the enzymology of the removal of the 2′-PO4 from the spliced tRNA. A nicotinamide adenine dinucleotide (NAD)-dependent phosphotransferase catalyzes the transfer of the 2′-PO4 to NAD (28McCraith S.M. Phizicky E.M. Mol. Cell. Biol. 1990; 10: 1049-1055Crossref PubMed Scopus (42) Google Scholar, 29McCraith S.M. Phizicky E.M. J. Biol. Chem. 1991; 266: 11986-11992Abstract Full Text PDF PubMed Google Scholar). Surprisingly the structure of the transfer product is ADP-ribose 1′–2′-cyclic phosphate (30Culver G.M. McCraith S.M. Zillmann M. Kierzek R. Michaud N. Lareau R.D. Turner D.H. Phizicky E.M. Science. 1993; 261: 206-208Crossref PubMed Scopus (80) Google Scholar). The nicotinamide moiety is displaced, apparently supplying the energy for cyclization. It is tempting to speculate that this unique and hitherto unknown compound goes on to play some crucial regulatory role in the cell. The eukaryal endonuclease is solely responsible for the recognition of the splice sites contained in the pre-tRNA. Since only the mature domain in the pre-tRNA is conserved, it was postulated that endonuclease recognizes the splice sites by measuring the distance from the mature domain to the splice sites (31Reyes V.M. Abelson J. Cell. 1988; 55: 719-730Abstract Full Text PDF PubMed Scopus (104) Google Scholar). This hypothesis was confirmed by experiments in which insertion mutations in the pre-tRNA that changed the distance between the mature domain and the anticodon resulted in a predictable shift of the splice sites (31Reyes V.M. Abelson J. Cell. 1988; 55: 719-730Abstract Full Text PDF PubMed Scopus (104) Google Scholar). The intron is not completely passive in the recognition process. TheXenopus oocyte tRNA endonuclease has been shown to recognize a crucial element involving the intron (8Baldi M.I. Mattoccia E. Bufardeci E. Fabbri S. Tocchini-Valentini G.P. Science. 1992; 255: 1404-1408Crossref PubMed Scopus (59) Google Scholar). Yeast tRNA introns contain a conserved purine residue three nucleotides upstream of the 3′-splice site. This base must be able to pair with a pyrimidine at position 32 in the anticodon loop in order for the intron to be recognized by either yeast or Xenopus endonuclease (8Baldi M.I. Mattoccia E. Bufardeci E. Fabbri S. Tocchini-Valentini G.P. Science. 1992; 255: 1404-1408Crossref PubMed Scopus (59) Google Scholar, 32Di Nicola Negri E. Fabbri S. Bufardeci E. Baldi M.I. Gandini Attardi D. Mattoccia E. Tocchini-Valentini G.P. Cell. 1997; 89: 859-866Abstract Full Text Full Text PDF PubMed Google Scholar). These experiments suggested complexities in the structure of the pre-tRNA effecting the recognition by the eukaryal endonuclease, which had not been previously appreciated. It has also been demonstrated that there are different requirements for the recognition of the 5′- and 3′-splice sites (32Di Nicola Negri E. Fabbri S. Bufardeci E. Baldi M.I. Gandini Attardi D. Mattoccia E. Tocchini-Valentini G.P. Cell. 1997; 89: 859-866Abstract Full Text Full Text PDF PubMed Google Scholar). Recognition of archaeal tRNA splice sites by the archaeal tRNA endonuclease relies solely on the BHB motif (Fig. 1 B). Yeast pre-tRNAs are not substrates for the Haloferax volcaniiendonuclease (33Palmer J.R. Nieuwlandt D.T. Daniels C.J. J. Bacteriol. 1994; 176: 3820-3823Crossref PubMed Scopus (18) Google Scholar), and unlike the eukaryal endonuclease, the mature domain of the pre-tRNA is not required for intron excision by the archaeal enzyme (12Thompson L.D. Daniels C.J. J. Biol. Chem. 1988; 263: 17951-17959Abstract Full Text PDF PubMed Google Scholar). Despite the differences in both substrate and the mechanism for substrate recognition between the archaeal and eukaryal systems, as we shall discuss below, the endonuclease that catalyzes the first step in splicing has been conserved between Eukarya and Archaea. Different mechanisms for substrate recognition have evolved since the divergence from their common ancestor. The characterization of the yeast tRNA endonuclease was extremely difficult for two reasons. The enzyme was present at very low levels, approximately 150 molecules per cell, and it appeared to be an integral membrane protein. However, after many years of work the enzyme was successfully purified, and the genes for all four of its subunits were cloned (6Trotta C.R. Miao F. Arn E.A. Stevens S.W. Ho C.K. Rauhut R. Abelson J.N. Cell. 1997; 89: 849-858Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 21Rauhut R. Green P.R. Abelson J. J. Biol. Chem. 1990; 265: 18180-18184Abstract Full Text PDF PubMed Google Scholar). Of particular advantage in purifying the enzyme was the construction of a modified gene for the 44-kDa subunit,SEN2, containing His8 and Flag epitope affinity tags. The SEN2 gene had been found earlier in genetic screens (34Winey M. Culbertson M.R. Genetics. 1988; 118: 49-63Crossref PubMed Google Scholar, 35Ho C.K. Rauhut R. Vijayraghavan U. Abelson J. EMBO J. 1990; 9: 1245-1252Crossref PubMed Scopus (36) Google Scholar), and the sen2–3 allele was shown to specifically block 5′-splice site cleavage (35Ho C.K. Rauhut R. Vijayraghavan U. Abelson J. EMBO J. 1990; 9: 1245-1252Crossref PubMed Scopus (36) Google Scholar). The enzyme turned out to be an αβγδ heterotetramer whose subunits have molecular masses of 54 (SEN54), 44 (SEN2), 34 (SEN34), and 15 kDa (SEN15). Each of these genes proved to be essential for cell viability. The amino acid sequence of each of the four subunits contains a canonical nuclear localization sequence. Sen2 contains the only plausible transmembrane sequence, suggesting that it anchors the endonuclease complex to the nuclear membrane. Two subunits of endonuclease, Sen2 and Sen34, contain a homologous domain approximately 130 amino acids in length, suggesting that they perform a similar function. The excitement came when we learned that apparent homologs of this domain are encoded by the gene for the archaeal tRNA splicing endonuclease of H. volcanii cloned by Daniels and co-workers (36Kleman-Leyer K. Armbruster D.A. Daniels C.J. Cell. 1997; 89: 839-848Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) and in endonuclease homologs found in the sequenced genomes ofMethanococcus jannaschii, Methanobacterium thermoautotrophicum, and Archaeoglobus fulgidis. The homology between Sen2, Sen34, and the archaeal endonucleases immediately suggested a model in which the yeast endonuclease contains two distinct active sites, one for each splice site (6Trotta C.R. Miao F. Arn E.A. Stevens S.W. Ho C.K. Rauhut R. Abelson J.N. Cell. 1997; 89: 849-858Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 37Belfort M. Weiner A. Cell. 1997; 89: 1003-1006Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The fact that sen2–3 is defective in cleavage of the 5′-splice site suggested that Sen2 contains the active site for 5′-splice site cleavage and led to the prediction that Sen34 cleaves the 3′-splice site. This hypothesis was strongly supported by the observation that a Sen34 mutant enzyme is defective in 3′-splice site cleavage (6Trotta C.R. Miao F. Arn E.A. Stevens S.W. Ho C.K. Rauhut R. Abelson J.N. Cell. 1997; 89: 849-858Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Our biochemical experience with the endonuclease had suggested strong interactions between the subunits. To probe the of these interactions a was in which all of the four subunits were (6Trotta C.R. Miao F. Arn E.A. Stevens S.W. Ho C.K. Rauhut R. Abelson J.N. Cell. 1997; 89: 849-858Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). It turned out that strong interactions were only between Sen2 and and between Sen34 and These with to a model for the yeast endonuclease in which Sen2 contains the active site for 5′-splice site cleavage and Sen34 the active site for 3′-splice site cleavage is as no as to the mechanism although we that it be the of a very protein, and The tRNA splicing endonuclease of the H. shown to as a in (36Kleman-Leyer K. Armbruster D.A. Daniels C.J. Cell. 1997; 89: 839-848Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Since its the BHB has it seems very likely that the recognizes its substrate that each splice site is cleaved by a active site Thus we are led to a model of tRNA splicing in which the two splice sites are cleaved by each containing an active site. M. contains a gene that an endonuclease homologous to the H. volcanii enzyme but is the amino acids in the of M. that a structure of the archaeal enzyme on the mechanism of the but related eukaryal endonuclease and on a structural of the M. endonuclease, an structure to a of (13Li H. Trotta C.R. Abelson J.N. Science. 1998; 280: 279-284Crossref PubMed Scopus (120) Google Scholar). The M. endonuclease is an different from the H. volcanii enzyme (13Li H. Trotta C.R. Abelson J.N. Science. 1998; 280: 279-284Crossref PubMed Scopus (120) Google K. Armbruster D.A. Daniels C.J. Cell. 1997; 89: 839-848Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, J. EMBO J. 1997; PubMed Scopus Google Scholar). Fig. A that the M. endonuclease of two distinct the domain and the domain The domain of three and a of four The domain contains two a The is with but its interactions the in the the interactions between has turned out to be crucial to the structure and evolution of the of the tRNA splicing endonuclease Two of subunits and and to interactions between their (Fig. B). The of from one with the of from the to a the The two also and related by the on of the The with a at the These are for the and to an extremely which we has been conserved in evolution The is between the two The between the two is the insertion of loop from subunits and a in subunits and between the and of each The is between in loop and in the This the two to be to each by This subunits and and which not at and as we shall below, in an of subunits in which only one pair of active sites can recognize the substrate. These are also likely to have been conserved in evolution because they to a between the which in to the required of the two active The tRNA splicing endonucleases pre-tRNA 5′-OH and PO4 This is the in the The A mechanism has been and a first be that the endonuclease mechanism be similar J. Chem. 1994; PubMed Scopus Google Scholar, C. W. H. and Scholar). The reaction pathway for A is a reaction. A general base a from the of to an on the bond and the of a The general acid the to the cyclic PO4 In a second step a is from and the cyclic PO4 is to give the In is the general base in the first is the general and the is by In the endonuclease there is a conserved residue at position in the M. is strong that this is of the active site. A to in the at position in Sen34 was shown to 3′-splice site cleavage (6Trotta C.R. Miao F. Arn E.A. Stevens S.W. Ho C.K. Rauhut R. Abelson J.N. Cell. 1997; 89: 849-858Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Daniels J. has shown that the mutant in the H. volcanii enzyme cleavage as has J. EMBO J. 1997; PubMed Scopus Google Scholar) for a to mutant in the M. endonuclease. is found in a with conserved and on the of the and a which the phosphate to be cleaved is to (Fig. these three can be with the of A. In the is to of A and be the general be the general and the This to be a of because it is that the tRNA endonucleases and the not a common ancestor. two of the M. endonuclease active sites to recognize and the tRNA substrate. the of the subunits and to function as active subunits. The active sites on subunits and are at one of the that is shown to be from an This of the therefore the of the tRNA substrate. A model of the substrate derived from the TAR RNA structure with the active subunits and (Fig. The two the and active site and with the found in each site. The distance between of active sites in the is to with this model substrate. This is of the related in and which are that it is that in substrate for a it is of to a structure of the The H. volcanii and the M. recognize the substrate their active sites must be in This was difficult to out that the H. is a of the sequence of the endonuclease gene J. EMBO J. 1997; PubMed Scopus Google Scholar). The not contain the and it 2 of the active site It however, contain the structural of the in particular the sequence. a model of the H. volcanii enzyme, which is as a of two The structure of the is to contain a two an structural of the M. The H. volcanii enzyme only contains two active sites in the and these are to an identical to in the and subunits of The are to the conserved loop in the to in the and subunits in the M. The H. volcanii enzyme that only two of the active sites are but to these in one must retain of both the interactions and the interactions by The yeast endonuclease contains two active site Sen2 and The two subunits not to to the endonuclease gene however, out that both and contain a of sequence to the endonuclease their J. EMBO J. 1997; PubMed Scopus Google Scholar). of the structure of the M. endonuclease, this sequence conservation to contain the and interactions crucial to the of the to the M. endonuclease. have that the strong and interactions in experiments (13Li H. Trotta C.R. Abelson J.N. Science. 1998; 280: 279-284Crossref PubMed Scopus (120) Google Scholar) are by the These two are to to the heterotetramer the conserved 10 of and Thus it is likely that has been conserved since the divergence of the Eukarya and the Archaea is the endonuclease active site and the to two of in a and conserved for this pathway is supported by the of Tocchini-Valentini and co-workers S. P. Bufardeci E. Nicola Negri E. Baldi M.I. Gandini Attardi D. Mattoccia E. Tocchini-Valentini G.P. Science. 1998; 280: PubMed Scopus (49) Google Scholar), where it is demonstrated that both the eukaryal and archaeal endonucleases can a substrate containing the BHB motif. The eukaryal enzyme seems to with the mechanism for tRNA substrate recognition when the substrate. This to the that the of two active sites in endonuclease has been Thus, subunits and the active site of all tRNA splicing and subunits and position the two active sites in The eukaryal enzyme has evolved a distinct measuring mechanism for splice site recognition the of the and subunits the to recognize and the substrate.