tRNA Splicing

Abstract

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 (1-3). 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. tRNA Precursors in Eukarya and ArchaeaThe earliest studies of tRNA splicing were in the yeast Saccharomyces cerevisiae where tRNA introns were first discovered (4, 5). With the completion of the S. cerevisiae genome sequence it is now known that yeast contains 272 tRNA genes of which 59, encoding 10 different tRNAs, are interrupted by introns (6). The introns are 14 -60 nucleotides in length and interrupt the anticodon loop immediately 3Ј to the anticodon (7). Among the 10 different yeast pre-tRNAs there is no conservation of sequence at the splice junctions although the 3Ј-splice junction is invariably in a bulged loop (8). 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. 1A (9, 10).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 (11). 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) 1 motif (12). This structure, modeled in Fig. 1B from the related TAR RNA structure (13), 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 sys...

Transient ADP-ribosylation of a 2′-Phosphate Implicated in Its Removal from Ligated tRNA during Splicing in Yeast

“…2 Yeast Tpt1 was prepared by M. Steiger as a Tpt1-His 6 fusion protein and was purified from E. coli extracts by nickel column affinity chromatography. Yeast cyclic phosphodiesterase protein has been described (23).…”

“…Nonetheless, the amount of protein required to form the intermediate in each case conforms very closely to that expected from the kinetic parameters. 2 The fact that both RNA and NAD comprise the intermediate eliminates mechanism A as the catalytic pathway for the phosphotransfer reaction (Fig. 1).…”

“…To test this, we used p*ApApA P POCH3 as substrate because it has no vicinal hydroxyls on its riboses. 2 After forming the intermediate with AppN, we treated it with pyrophosphatase to release the AMP moiety (Fig. 9, lane c); this removed the vicinal hydroxyls on the ribose of AMP.…”

“…tRNA introns occur widely in Eukarya and Archaea (1,2). Splicing in these organisms is initiated by a highly conserved endonuclease that excises the intron (3)(4)(5), followed by joining of the two half-molecules by one of two different ligases (6 -13).…”

Achieving Specific RNA Cleavage Activity by an Inactive Splicing Endonuclease Subunit Through Engineered Oligomerization

“…The RNA splicing endonuclease, the subject of this study, excises phosphodiester bonds at two exon-intron junctions in precursor transfer and archaeal ribosomal RNAs. The eukaryotic enzyme, best characterized in yeast [14][15][16] , and the archaeal enzymes 15,17 are believed to utilize composite cleavage sites through subunit assembly. Therefore, the assembly of subunits or domains in RNases serves to control their activity and specificity.…”