Wheat Germ RNA Ligase Research Articles

Non-enzymatic excision of pre-tRNA introns?

We used human tRNA(Tyr) precursor as a substrate to study self-excision of a pre-tRNA intron. This RNA was synthesized in vitro in a HeLa cell extract. It contains a 5' leader, an intron of 20 nucleotides and a 3' trailer. Self-cleavage of pre-tRNA(Tyr) occurs in 100 mM NH4OAc at a pH ranging from 6 to 8.5 in the presence of spermine, MgCl2 and Triton X-100 under conditions very similar to enzymatic intron excision. The reaction is temperature-dependent, relatively fast as compared to the enzyme-catalysed reaction and leads to fragments which resist further degradation. The detailed structure of all major and minor cleavage products was established by fingerprint analyses. Non-enzymatic cleavage occurs predominantly at the 3' splice site and to a minor extent at the 5' splice site. Other minor cleavage sites are located within the intron and in the 3' trailer. Putative 5' and 3' tRNA halves resulting from pre-tRNA(Tyr) self-cleavage are substrates for wheat germ RNA ligase, suggesting that the cleavage reaction yields 2',3'-cyclic phosphate and 5'-hydroxyl termini. Pre-tRNA splicing endonuclease is believed to cleave both the 5' and the 3' splice site. However, on the basis of our results we propose that this enzyme may support the formation of a pre-tRNA tertiary structure favourable for autocatalytic intron excision and impair unspecific self-cleavage.

Purification and characterization of wheat germ 2',3'-cyclic nucleotide 3'-phosphodiesterase.

The 2',3'-cyclic nucleotide 3'-phosphodiesterase which hydrolyzes nucleoside 2',3'-cyclic phosphates (N greater than p) to nucleoside 2'-phosphates has been purified 16,000-fold to near homogeneity from wheat germ. The purified enzyme is a single polypeptide with a molecular weight of 23,000-24,000. It has a pH optimum of 7.0. The apparent Km values for A greater than p, G greater than p, C greater than p, and U greater than p are 13.1, 9.2, 25.2, and 25.3 mM, respectively. Vmax values for A greater than p, G greater than p, C greater than p, and U greater than p are 2090, 280, 2140, and 600 mumol/min/mg of purified protein, respectively. Wheat germ 2',3'-cyclic nucleotide 3'-phosphodiesterase does not hydrolyze 2',3'-cyclic esters in cyclic phosphate-terminated oligoribonucleotides or in nucleoside 5'-phosphate, 2',3'-cyclic phosphate (pN greater than p). This is in contrast to the 3'-phosphodiesterase activity associated with a wheat germ RNA ligase which hydrolyzes cyclic phosphate-terminated oligonucleotides and pN greater than p substrates much more efficiently than nucleoside 2',3'-cyclic phosphates. The enzyme characterized in this work appears to be the only known 2',3'-cyclic nucleotide 3'-phosphodiesterase specific for 2',3'-cyclic mononucleotides.

Purification of a RNA debranching activity from HeLa cells.

The splicing of messenger RNA precursors (pre-mRNA) of eukaryotic cells involves the formation of a branched RNA intermediate known as a RNA lariat. This structure is formed in the first step of the reaction when a cleavage at the 5' splice site generates the 5' exon and a RNA species containing the intron and 3' exon in which the phosphate moiety at the 5' end of the intron is forming a 2'-5' phosphodiester bond with the 2'-hydroxyl moiety of a specific adenine residue near the 3' end of the intron forming a RNA branch with the following structure: -pA2'-pX-3'-pZ-. We have purified a debranching activity approximately 700-fold from the cytosolic fraction of HeLa cells. This activity catalyzes the hydrolysis of the 2'-5' phosphodiester bond of branched RNA structures yielding a 5'-phosphate end and a 2'-hydroxyl group at the branch attachment site. The activity possessed a sedimentation coefficient of 3.5 S. The reaction catalyzed by the purified fraction requires a divalent cation and is optimal at pH 7.0. The purified activity can efficiently hydrolyze triester trinucleotide structures (pY2'-pX-3'-pZ-) prepared by digestion of RNA lariats with nuclease P1. In contrast, a 2' phosphate monoester product (-pG2'-p 3'-pC-), formed by the wheat germ RNA ligase, was not attacked.

Purification of wheat germ RNA ligase. II. Mechanism of action of wheat germ RNA ligase.

The mechanism of action of purified wheat germ RNA ligase has been examined. ATP was absolutely required for the ligation of substrates containing 5'-OH or 5'-P and 2',3'-cyclic P or 2'-P termini. Ligation of 1 mol of 5'-P-2',3'-cyclic P-terminated poly(A) was accompanied by the hydrolysis of 1 mol of ATP to 1 mol each of AMP and PPi. Purified RNA ligase catalyzed an ATP-PPi exchange reaction, specific for ATP and dATP, and formed a covalent enzyme-adenylate complex that was detected by autoradiography following incubation with [alpha-32P]ATP and separation of the products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A protein doublet with a molecular weight of approximately 110 kDa, the major product detected by silver staining, was labeled in these reactions. Isolated E-AMP complex was dissociated by the addition of ligatable poly(A), containing 5'-P-2',3'-cyclic P termini, to yield AMP and by the addition of PPi to yield ATP. The unique feature of the reactions leading to an exchange reaction between ATP and PPi and to the formation of an E-AMP complex was their marked stimulation (up to 400-fold) by the addition of RNA. This property distinguishes the wheat germ RNA ligase from other known RNA and DNA ligases which catalyze ATP-PPi exchange reactions and form E-AMP complexes in the absence of substrate. Thus, RNA appears to function in two capacities in the wheat germ system: as a cofactor, to stimulate the reaction of the enzyme with ATP, and as an authentic substrate for ligation.

Purification of wheat germ RNA ligase. I. Characterization of a ligase-associated 5'-hydroxyl polynucleotide kinase activity.

An RNA ligase that catalyzes the formation of a 2'-phosphomonoester-3',5'-phosphodiester bond in the presence of ATP and Mg2+ was purified approximately 6000-fold from raw wheat germ. A 5'-hydroxyl polynucleotide kinase activity copurified with RNA ligase through all chromatographic steps. Both activities cosedimented upon glycerol gradient centrifugation even in the presence of high salt and urea. RNA ligase and kinase activities sedimented as a single peak on glycerol gradients with a sedimentation coefficient of 6.2 S. The purified polynucleotide kinase activity required dithiothreitol and a divalent cation for activity and was inhibited by pyrophosphate and by ADP. The kinase phosphorylated a variety of 5'-hydroxyl-terminated polynucleotide chains including some that were substrates for the RNA ligase (e.g. 2',3'-cyclic phosphate-terminated poly(A)) and others that were not ligase substrates (e.g. DNA or RNA containing 3'-hydroxyl termini). RNA molecules containing either 5'-hydroxyl or 5'-phosphate and 2',3'-cyclic or 2'-phosphate termini were substrates for the purified RNA ligase activity. The rate of ligation of 5'-hydroxyl-terminated RNA chains was greater than that of 5'-phosphate-terminated molecules, suggesting that an interaction between the wheat germ kinase and ligase activities occurs during the course of ligation.

The enzymatic conversion of 3'-phosphate terminated RNA chains to 2',3'-cyclic phosphate derivatives.

The enzyme, RNA cyclase, has been purified from cell-free extracts of HeLa cells approximately 6000-fold. The enzyme catalyzes the conversion of 3'-phosphate ends of RNA chains to the 2',3'-cyclic phosphate derivative in the presence of ATP or adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) and Mg2+. The formation of 1 mol of 2',3'-cyclic phosphate ends is associated with the disappearance of 1 mol of 3'-phosphate termini and the hydrolysis of 1 mol of ATP gamma S to AMP and thiopyrophosphate. No other nucleotides could substitute for ATP or ATP gamma S in the reaction. The reaction catalyzed by RNA cyclase was not reversible and exchange reactions between [32P]pyrophosphate and ATP were not detected. However, an enzyme-AMP intermediate could be identified that was hydrolyzed by the addition of inorganic pyrophosphate or 3'-phosphate terminated RNA chains but not by 3'-OH terminated chains or inorganic phosphate. 3'-[32P](Up)10Gp* could be converted to a form that yielded, (Formula: see text) after degradation with nuclease P1, by the addition of wheat germ RNA ligase, 5'-hydroxylpolynucleotide kinase, RNA cyclase, and ATP. This indicates that the RNA cyclase had catalyzed the formation of the 2',3'-cyclic phosphate derivative, the kinase had phosphorylated the 5'-hydroxyl end of the RNA, and the wheat germ RNA ligase had catalyzed the formation of a 3',5'-phosphodiester linkage concomitant with the conversion of the 2',3'-cyclic end to a 2'-phosphate terminated residue.

Cell-free synthesis and processing of an infectious dimeric transcript of potato spindle tuber viroid RNA

A full-length cloned cDNA insert containing the sequence of potato spindle tuber viroid (PSTV) was dimerized and placed in the plasmid vector pSP65 adjacent to a promoter for bacteriophage SP6 RNA polymerase. In vitro transcription of this region yielded a single linear RNA chain 760 bases in length containing two copies of PSTV RNA with about 20 bases of vector sequence at each end. Bioassay on tomato plants revealed that this transcript has infectivity comparable to that of PSTV itself, yielding circular progeny RNAs indistinguishable from PSTV grown in vivo Comparative RNA fingerprinting analysis revealed that the viroid sequence in the dimeric transcript breeds true (compared to control viroid strains of different sequence grown in parallel) but loses the vector-specific sequences during growth in plants. Incubation of 32P-labeled dimeric PSTV RNA at neutral pH and 39° gave a 1–5% yield of three RNA segments, one comigrating with unit-length linear PSTV and two smaller fragments. Reaction of the unit-length cleavage product with wheat germ RNA ligase gave a high yield of circular molecules indistinguishable from PSTV circles arising in vivo, suggesting that the cleavage reaction yields 2′,3′ cyclic phosphate termini and could represent a part of the in vivo PSTV replication cycle.

Enzymatic mechanism of an RNA ligase from wheat germ.

We have characterized the mechanism of action of a wheat germ RNA ligase which has been partially purified on the basis of its ability to participate in in vitro splicing of yeast tRNA precursors (Gegenheimer, P., Gabius, H-J., Peebles, C.L., and Abelson, J. (1983) J. Biol. Chem. 258, 8365-8373). The preparation catalyzes the ligation of oligoribonucleotide substrates forming a 2'-phosphomonoester, 3',5'-phosphodiester linkage. The 5' terminus of an RNA substrate can have either a 5'-hydroxyl or a 5'-phosphate. The 5'-phosphate, which for a 5'-hydroxyl substrate can be introduced by a polynucleotide kinase activity in the preparation, is incorporated into the ligated junction. The 3' terminus can have either a 2',3'-cyclic phosphate or a 2'-phosphate. 2',3'-Cyclic phosphates can be converted into 2'-phosphates by a 2',3'-cyclic phosphate, 3'-phosphodiesterase activity in the preparation. The 2'-phosphate of the ligated product is derived from the phosphate at the 3' terminus of the substrate. Ligation proceeds with the adenylylation of the 5'-phosphorylated terminus to form an intermediate with a 5',5'-phosphoanhydride bond.