Biochemical characterization of a U6 small nuclear RNA-specific terminal uridylyltransferase.

The HeLa cell terminal uridylyltransferase (TUTase) that specifically modifies the 3'-end of mammalian U6 small nuclear RNA (snRNA) was characterized with respect to ionic dependence and substrate requirements. Optimal enzyme activity was obtained at moderate ionic strength (60 mm KCl) and depended on the presence of 5 mm MgCl2. In vitro synthesized U6 snRNA without a 3'-terminal UMP residue was not accepted as substrate. In contrast, U6 snRNA molecules containing one, two or three 3'-terminal UMP residues were filled up efficiently, generating the 3'-terminal structure with four UMP residues observed in newly transcribed cellular U6 snRNA. In this reaction, the addition of more than one UMP nucleotide depended on higher UTP concentrations. The analysis of internally mutated U6 snRNA revealed that the fill-in reaction by the U6-TUTase was not controlled by opposite-strand nucleotides, excluding an RNA-dependent RNA polymerase mechanism. Furthermore, electrophoretic mobility-shift analyses showed that the U6-TUTase was able to form stable complexes with the U6 snRNA in vitro. On the basis of these findings, a protocol was developed for affinity purification of the enzyme. In agreement with indirect labeling results, PAGE of a largely purified enzyme revealed an apparent molecular mass of 115 kDa for the U6-TUTase.     

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.     

Mammalian U6 small nuclear RNA undergoes 3'' end modifications within the spliceosome.

Mammalian U6 small nuclear RNA (snRNA) is heterogeneous with respect to the number of 3' terminal U residues. The major form terminates with five U residues and a 2',3' cyclic phosphate. Because of the presence in HeLa cell nuclear extracts of a terminal uridylyl transferase, a minor form of U6 snRNA is elongated, producing multiple species containing up to 12 U residues. In this study we have used glycerol gradients to demonstrate that these U6 snRNA forms are assembled into U6 ribonucleoprotein (RNP), U4/U6 snRNPs, and U4/U5/U6 tri-snRNP complexes. Furthermore, glycerol gradients combined with affinity selection of biotinylated pre-mRNAs led us to show that elongated forms of U6 snRNAs enter the spliceosome and that some of these become shortened with time to a single species having the same characteristics as the major form of U6 snRNA present in mammalian nuclear extracts. We propose that this elongation-shortening process is related to the function of U6 snRNA in mammalian pre-mRNA splicing.     

Terminal uridylyl transferase of Vigna unguiculata: purification and characterization of an enzyme catalyzing the addition of a single UMP residue to the 3''-end of an RNA primer.

An enzyme which catalyzes the addition of a single UMP residue from UTP to the 3'-end of an RNA primer and which is referred to as terminal uridylyl transferase (TUT) has been extensively purified from the membrane fraction of vigna unguiculata leaves. The purification procedure involved (i) solubilization by cation depletion (ii) DEAE-Sepharose CL-6B column chromatography (iii) affinity chromatography of poly(U)-Sepharose 4B and (iv) glycerol gradient centrifugation. The molecular weight of the native enzyme was approximately 50,000 as determined by velocity sedimentation. Under conditions that were optimal for UMP-incorporation (5 mM Mg2+, low salt, 30 degrees C) TUT displayed a marked specificity for UTP as substrate, was unable to incorporate deoxyribonucleoside triphosphates and required a single-stranded oligo- or polyribonucleotide as primer. When oligoA20, tRNAasp of E. coli or alfalfa mosaic virus RNA 4 were used as primers at various substrate to primer ratio's, the vast majority of the product appeared to consist of primer molecules elongated with a single UMP residue as shown by polyacrylamide gelelectrophoresis and nearest neighbour analysis. We believe TUT to be a novel enzyme which has not been reported before and which may be a feasible tool in RNA sequencing as it enables the specific 3'-terminal labeling of RNA molecules.     

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.     

Lin28-mediated control of let-7 microRNA expression by alternative TUTases Zcchc11 (TUT4) and Zcchc6 (TUT7)

The pluripotency factor Lin28 recruits a 3' terminal uridylyl transferase (TUTase) to selectively block let-7 microRNA biogenesis in undifferentiated cells. Zcchc11 (TUTase4/TUT4) was previously identified as an enzyme responsible for Lin28-mediated pre-let-7 uridylation and control of let-7 expression. Here we investigate the protein and RNA determinants for this interaction. Biochemical dissection and reconstitution assays reveal the TUTase domains necessary and sufficient for Lin28-enhanced pre-let-7 uridylation. A single C2H2-type zinc finger domain of Zcchc11 was found to be responsible for the functional interaction with Lin28. We identify Zcchc6 (TUTase7) as an alternative TUTase that functions with Lin28 in vitro, and accordingly, we find Zcchc11 and Zcchc6 redundantly control let-7 biogenesis in embryonic stem cells. Our study indicates that Lin28 uses two different TUTases to control let-7 expression and has important implications for stem cell biology as well as cancer.     

Characterization of two classes of ribonucleoprotein complexes possibly involved in RNA editing from Leishmania tarentolae mitochondria.

The molecular mechanism of RNA editing in trypanosomatid mitochondria is an unsolved problem. We show that two classes of ribonucleoprotein complexes exist in a mitochondrial extract from Leishmania tarentolae and appear to be involved in RNA editing. The 'G' class of RNP complexes consists of 170-300 A particles which contain guide RNAs and proteins, show little terminal uridylyl transferase (TUTase) activity and exhibit an in vitro RNA editing-like activity. The 'T' class consists of approximately six RNP complexes, the endogenous RNA of which can be self-labeled with [alpha-32P]UTP. The most abundant T complex, T-IV, is visualized by electron microscopy as 80-140 A particles. This complex exhibits TUTase activity in the native gel and contains guide RNAs. Both G and T complexes are possibly involved with RNA editing in vivo. These results are a starting point for the analysis of the biochemistry of RNA editing.     

UTP-bound and Apo structures of a minimal RNA uridylyltransferase

3'-Uridylylation of RNA is emerging as a phylogenetically widespread phenomenon involved in processing events as diverse as uridine insertion/deletion RNA editing in mitochondria of trypanosomes and small nuclear RNA (snRNA) maturation in humans. This reaction is catalyzed by terminal uridylyltransferases (TUTases), which are template-independent RNA nucleotidyltransferases that specifically recognize UTP and belong to a large enzyme superfamily typified by DNA polymerase beta. Multiple TUTases, recently identified in trypanosomes, as well as a U6 snRNA-specific TUTase enzyme in humans, are highly divergent at the protein sequence level. However, they all possess conserved catalytic and UTP recognition domains, often accompanied by various auxiliary modules present at the termini or between conserved domains. Here we report identification, structural and biochemical analyses of a novel trypanosomal TUTase, TbTUT4, which represents a minimal catalytically active RNA uridylyltransferase. The TbTUT4 consists of only two domains that define the catalytic center at the bottom of the nucleoside triphosphate and RNA substrate binding cleft. The 2.0 Angstroms crystal structure reveals two significantly different conformations of this TUTase: one molecule is in a relatively open apo conformation, whereas the other displays a more compact TUTase-UTP complex. A single nucleoside triphosphate is bound in the active site by a complex network of interactions between amino acid residues, a magnesium ion and highly ordered water molecules with the UTP's base, ribose and phosphate moieties. The structure-guided mutagenesis and cross-linking studies define the amino acids essential for catalysis, uracil base recognition, ribose binding and phosphate coordination by uridylyltransferases. In addition, the cluster of positively charged residues involved in RNA binding is identified. We also report a 2.4 Angstroms crystal structure of TbTUT4 with the bound 2' deoxyribonucleoside, which provides the structural basis of the enzyme's preference toward ribonucleotides.     

RNA-specific ribonucleotidyl transferases

RNA-specific nucleotidyl transferases (rNTrs) are a diverse family of template-independent polymerases that add ribonucleotides to the 3'-ends of RNA molecules. All rNTrs share a related active-site architecture first described for DNA polymerase beta and a catalytic mechanism conserved among DNA and RNA polymerases. The best known examples are the nuclear poly(A) polymerases involved in the 3'-end processing of eukaryotic messenger RNA precursors and the ubiquitous CCA-adding enzymes that complete the 3'-ends of tRNA molecules. In recent years, a growing number of new enzymes have been added to the list that now includes the "noncanonical" poly(A) polymerases involved in RNA quality control or in the readenylation of dormant messenger RNAs in the cytoplasm. Other members of the group are terminal uridylyl transferases adding single or multiple UMP residues in RNA-editing reactions or upon the maturation of small RNAs and poly(U) polymerases, the substrates of which are still not known. 2'-5'Oligo(A) synthetases differ from the other rNTrs by synthesizing oligonucleotides with 2'-5'-phosphodiester bonds de novo.     

Trypanosoma brucei guide RNA poly(U) tail formation is stabilized by cognate mRNA

Guide RNAs (gRNAs) are small RNAs that provide specificity for uridine addition and deletion during mRNA editing in trypanosomes. Terminal uridylyl transferase (TUTase) adds uridines to pre-mRNAs during RNA editing and adds a poly(U) tail to the 3' end of gRNAs. The poly(U) tail may stabilize the association of gRNAs with cognate mRNA during editing. Both TUTase and gRNAs associate with two ribonucleoprotein complexes, I (19S) and II (35S to 40S). Complex II is believed to be the fully assembled active editing complex, since it contains pre-edited mRNA and enzymes thought necessary for editing. Purification of TUTase from mitochondrial extracts resulted in the identification of two chromatographically distinct TUTase activities. Stable single-uridine addition to different substrate RNAs is performed by the 19S complex, despite the presence of a uridine-specific 3' exonuclease within this complex. Multiple uridines are added to substrate RNAs by a 10S particle that may be an unstable subunit of complex I lacking the uridine-specific 3' exonuclease. Multiple uridines could be stably added onto gRNAs by complex I when the cognate mRNA is present. We propose a model in which the purine-rich region of the cognate mRNA protects the uridine tail from a uridine exonuclease activity that is present within the complex. To test this model, we have mutated the purine-rich region of the pre-mRNA to abolish base-pairing interaction with the poly(U) tail of the gRNA. This RNA fails to protect the uridine tail of the gRNA from exoribonucleolytic trimming and is consistent with a role for the purine-rich region of the mRNA in gRNA maturation.     

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.     

The highly conserved U small nuclear RNA 3''-end formation signal is quite tolerant to mutation.

Formation of the 3' end of U1 and U2 small nuclear RNA (snRNA) precursors is directed by a conserved sequence called the 3' box located 9 to 28 nucleotides downstream of all metazoan U1 to U4 snRNA genes sequenced so far. Deletion of part or all of the 3' box from human U1 and U2 genes drastically reduces 3'-end formation. To define the essential nucleotides within this box that direct 3'-end formation, we constructed a set of point mutations in the conserved residues of the human U1 3' box. The ability of the various mutations to direct 3'-end formation was tested by microinjection into Xenopus oocytes and transfection into HeLa cells. We found that the point mutations had diverse effects on 3'-end formation, ranging from no effect at all to severe inhibition; however, no single or double point mutation we tested completely eliminated 3'-end formation. We also showed that a rat U3 3' flank can effectively substitute for the human U1 3' flank, indicating that the 3' boxes of the different U snRNA genes are functionally equivalent.