RNA-editing terminal uridylyl transferase 1: identification of functional domains by mutational analysis

The catalytic, RNA-binding and oligomerization domains of the RNA-editing terminal uridylyl transferase 1 (RET1) from Leishmania tarentolae mitochondria were characterized by mutational analysis. Significant N- and C-terminal portions of the protein were found to be dispensable for UTP polymerization in vitro. Changes of conserved amino acids in the active site demonstrated a general similarity of sugar-phosphate moiety recognition of the incoming ribonucleotide triphosphate by RET1 and eukaryotic poly(A) polymerases. Overlapping RNA-binding and oligomerization regions were mapped to the C-terminal region, which is conserved only among trypanosomatid RET1 enzymes. In the absence of an RNA primer, RET1 can use UTP itself to initiate nucleotide transfer and produce poly(U) molecules of several hundred nucleotides. An N-terminal zinc finger motif is essential for enzyme activity; deletion of this motif or chelation of zinc inhibits activity.     

Identification, cloning, and functional analysis of the human U6 snRNA-specific terminal uridylyl transferase

Mammalian cells contain a highly specific terminal uridylyl transferase (TUTase) that exclusively accepts U6 snRNA as substrate. This enzyme, termed U6-TUTase, was purified from HeLa cell extracts and analyzed by microsequencing. All sequenced peptides matched a unique human cDNA coding for a previously unknown protein. Domain structure analysis revealed that the U6-TUTase also belongs to the well-characterized poly(A) polymerase protein superfamily. However, by amino acid sequence as well as RNA-binding motifs, human U6-TUTase is highly divergent from both the poly(A) polymerases and from the TUTases identified within the editing complexes of trypanosomes. After cloning, the recombinant U6-TUTase was expressed in HeLa cells. Analysis of its catalytical activity confirmed the identity of the cloned protein as U6-TUTase, exhibiting the same exclusive substrate specificity for U6 snRNA as the endogenous enzyme. That unique selectivity even excluded as substrate U6atac RNA, the functional homolog of the minor spliceosome. Finally, RNAi knockdown experiments revealed that U6-TUTase is essential for cell proliferation. Surprisingly, large amounts of the recombinant enzyme were found to accumulate within nucleoli.     

Purification and characterization of human erythrocyte uridylyl transferase

A new method for the purification of human erythrocyte uridylyl transferase (UDPglucose: alpha-D-galactose-1-phosphate uridylyltransferase EC 2.7.7.12) is described. It consists of a hydrophobic purification step associated with hydroxyapatite chromatography and provided for the first time a purification of more than 45 000-fold with a high activity (15 I.U/mg) and a yield of 32%. We show that the enzyme is a dimer and has a molecular weight of 88 000. It can be resolved into three bands by isoelectric focusing with an apparent pI between 5.0 and 5.4. It could be shown by steady-state initial rate measurements that the interconversion of the two substrates of human transferase (Gal-1-P and UDP-glucose) follows ping-pong bi-bi kinetics, with Km values of 0.2 and 0.065 mM, respectively.     

TbMP57 is a 3' terminal uridylyl transferase (TUTase) of the Trypanosoma brucei editosome

RNA editing produces mature trypanosome mitochondrial mRNAs by uridylate (U) insertion and deletion. In insertion editing, Us are added to the pre-mRNA by a 3' terminal uridylyl transferase (TUTase) activity. We report the identification of a TUTase activity that copurifies with in vitro editing and is catalyzed by the integral editosome protein TbMP57. TbMP57 catalyzes the addition of primarily a single U to single-stranded (ss) RNA and adds the number of Us specified by a guide RNA to insertion editing-like substrates. TbMP57 is distinct from a previously identified TUTase that adds many Us to ssRNA and which we find is neither a stable editosome component nor does it add Us to editing-like substrates. Recombinant TbMP57 specifically interacts with the editosome protein TbMP81, and this interaction enhances the TUTase activity. These results suggest that TbMP57 catalyzes U addition to pre-mRNA during editing.     

A highly specific terminal uridylyl transferase modifies the 3'-end of U6 small nuclear RNA.

HeLa cell extracts contain significant amounts of terminal uridylyl transferase (TUTase) activity. In a template-independent reaction with labeled UTP, these enzymes are capable of modifying a broad spectrum of cellular RNA molecules in vitro . However, fractionation of cell extracts by gel filtration clearly separated two independent activities. In addition to a non-specific enzyme, an additional terminal uridylyl transferase has been identified that is highly specific for cellular and in vitro synthesized U6 small nuclear RNA (snRNA) molecules. This novel TUTase enzyme was also able to select as an efficient substrate U6 snRNA species from higher eucaryotes. In contrast, no labeling was detectable with purified fission yeast RNA. Using synthetic RNAs containing different amounts of transcribed 3'-end UMP residues, high resolution gel electrophoresis revealed that U6 snRNA species with three terminal U nucleotides served as the optimal substrate for the transferase reaction. The 3'-end modification of the optimal synthetic substrate was identical to that observed with endogenous U6 snRNA isolated from HeLa cells. Therefore, we conclude that the specific addition of UMP residues to 3'-recessed U6 snRNA molecules reflects a recycling process, ensuring the functional regeneration for pre-mRNA splicing of this snRNA.     

Specific and non-specific mammalian RNA terminal uridylyl transferases

Uridylylation of various types of RNA molecules is a wide-spread phenomenon in molecular biology and is catalyzed by enzymes mediating the transfer of UMP residues to the 3'-ends of preexisting RNA. In most cases, however, the biological significance of these modifications remains elusive. As an exception, the RNA terminal uridylyl transferases (TUTases) of the mRNA editing complex within mitochondria of Trypanosomatidae have been characterized in great detail. Current knowledge on those editing enzymes has been summarized recently by R. Aphasizhev [Cell. Mol. Life Sci. 62 (2005) 2194-203] and, therefore, will not be included here. Rather, this review will focus on cellular non-editing TUTases, characterized by distinct modes of catalytic activity and substrate specificity. Putative biological functions of this rapidly growing number of RNA modifying enzymes are discussed.     

Trypanosome mitochondrial 3' terminal uridylyl transferase (TUTase): the key enzyme in U-insertion/deletion RNA editing

A 3' terminal RNA uridylyltransferase was purified from mitochondria of Leishmania tarentolae and the gene cloned and expressed from this species and from Trypanosoma brucei. The enzyme is specific for 3' U-addition in the presence of Mg(2+). TUTase is present in vivo in at least two stable configurations: one contains a approximately 500 kDa TUTase oligomer and the other a approximately 700 kDa TUTase complex. Anti-TUTase antiserum specifically coprecipitates a small portion of the p45 and p50 RNA ligases and approximately 40% of the guide RNAs. Inhibition of TUTase expression in procyclic T. brucei by RNAi downregulates RNA editing and appears to affect parasite viability.     

Microheterogeneity of human galactose-1-Phosphate uridylyl transferase. Isoelectrofocusing results

Using thin-layer acrylamide gel isoelectrofocusing, several bands of galactose-1-Phosphate uridylyl transferase were found in various human tissues. Liver transferase, as well as that of some other tissues, was resolved into several bands with pHi between 5.30 and 5.80; red cell enzyme was resolved into five bands with pHi between 5.0 and 5.45. The comparison of erythrocytes with their precursors, reticulocytes and erythroblasts, showed a striking difference: the pHi of the erythroblast enzyme was between 5.55 and 5.90 and that of reticulocytes between 5.30 and 5.50. It is possible that molecular aging is the cause of the anodisation of the erythrocyte transferase and the microheterogeneity of the enzyme observed in other tissues.     

Molecular biology of terminal transferase

Terminal transferase is an unusual deoxynucleotide polymerizing enzyme found only in prelymphocytes. The protein was purified to homogeneity from calf thymus glands in 1971 as a 32 kDa protein with a two peptide structure. Subsequent biochemical and immunological analyses of terminal transferase protein in crude extracts from a number of animal species showed a single peptide with a molecular weight of about 58,000. The two peptide structure found earlier was caused by proteolysis. Homogeneous 58 kDa terminal transferase has now been produced from human lymphoblastoid cells and calf thymus glands by immunoaffinity chromatography. In vitro phosphorylation studies showed that the terminal transferase protein contains one phosphorylation site near one end of the polypeptide chain, and the phosphorylation of the enzyme has been confirmed by in vivo labeling experiments. Unambiguous demonstration of the molecular weight of the human terminal transferase was obtained by translation of the cloned human terminal transferase DNA sequence to a 58,308 Da protein. The translated amino acid sequence also provided a possible phosphorylation site near the amino-terminus of the protein. Preliminary analysis of the genomic structure shows a simple intron/exon pattern with the total human terminal transferase gene spanning at least 65 Kb.     

Synthesis of compositionally unique DNA by terminal deoxynucleotidyl transferase

Studies on the composition and characterization of DNA product(s) synthesized by calf thymus terminal deoxynucleotidyl transferase were performed using homopolymeric single-stranded, calf thymus double-stranded, and native DNA resident in calf thymus chromatin preparations as priming DNA species. Synthesis was carried out using equimolar concentrations of all four deoxynucleoside triphosphates as substrates and Mg²⁺ or Mn²⁺ as an effective divalent cation. Irrespective of the nature of the priming DNA or the divalent cation, the DNA product contained 60–70% dGMP residues, 10–15% each of the two pyrimidine residues, and 5–10% dAMP residues. The product synthesized using chromatin DNA as initiator was predominantly single-stranded and its synthesis was resistant to actinomycin D. The predilection of terminal deoxynucleotidyl transfease to synthesize dGMP-rich products on natural or homopolymeric DNA primers suggests that such products may represent biologically important recognition signal sequences.     

In vitro recombination and terminal elongation of RNA by Q beta replicase.

SV-11 is a short-chain [115 nucleotides (nt)] RNA species that is replicated by Q beta replicase. It is reproducibly selected when MNV-11, another 87 nt RNA species, is extensively amplified by Q beta replicase at high ionic strength and long incubation times. Comparing the sequences of the two species reveals that SV-11 contains an inverse duplication of the high-melting domain of MNV-11. SV-11 is thus a recombinant between the plus and minus strands of MNV-11 resulting in a nearly palindromic sequence. During chain elongation in replication, the chain folds consecutively to a metastable secondary structure of the RNA, which can rearrange spontaneously to a more stable hairpin-form RNA. While the metastable form is an excellent template for Q beta replicase, the stable RNA is unable to serve as template. When initiation of a new chain is suppressed by replacing GTP in the replication mixture by ITP, Q beta replicase adds nucleotides to the 3' terminus of RNA. The replicase uses parts of the RNA sequence, preferentially the 3' terminal part for copying, thereby creating an interior duplication. This reaction is about five orders of magnitude slower than normal template-instructed synthesis. The reaction also adds nucleotides to the 3' terminus of some RNA molecules that are unable to serve as templates for Q beta replicase.     

Polypeptide structure of human terminal transferase

The polypeptide structure of terminal transferase purified from human lymphoblasts was examined with an immunoblot procedure using rabbit anti-calf thymus terminal transferase antibodies. Two doublets of bands of M_r 58-56,000 and M_r 44-42,000 are the major immunoreactive polypeptides. Only the M_r 44-42,000 polypeptides can be efficiently renatured $\text{in}\text{situ}$ after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Controlled degradation with trypsin produces fully active enzyme containing the α and β polypeptides typical of the low molecular weight terminal transferase, suggesting that the different forms of purified terminal transferase may arise by proteolysis of the M_r 58,000 polypeptide.