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.     

Donor activation in the T4 RNA ligase reaction

T4 RNA ligase catalyzes the adenylation of donor oligonucleotide substrates. These activated intermediates react with an acceptor oligonucleotide which results in phosphodiester bond formation and the concomitant release of AMP. Adenylation of the four common nucleoside 3',5'-bisphosphates as catalyzed by T4 RNA ligase in the absence of an acceptor oligonucleotide has been examined. The extents of product formation indicate that pCp is the best substrate in the reaction and pGp is the poorest. Kinetic parameters for the joining reaction between the preadenylated nucleoside 3',5'-bisphosphates, A(5')pp(5')Cp or A(5')pp(5')Gp, and a good acceptor substrate (ApApA) or a poor acceptor substrate (UpUpU) have been determined. The apparent Km values for both preadenylated donors in the joining reaction are similar, and the reaction velocity is much faster than observed in the overall joining reaction. The nonnucleotide adenylated substrate P1-(5'-adenosyl) P2-(o-nitrobenzyl) diphosphate also exhibits a similar apparent Km but reacts with a velocity 80-fold slower than the adenylated nucleoside 3',5'-bisphosphates. By use of preadenylated donors, oligonucleotide substrates can be elongated more efficiently than occurs with the nucleoside 3',5'-bisphosphates.     

Synthesis of RNA having cap structure.

RNA consisting 43 nucleotides bearing cap structure was synthesized (Figure). In the first place, 9 mer of a leader sequence with the cap structure (F-1) was synthesized by the phosphotriester method and followed by the capping reaction. Next, 32 mer of a cistron was divided into two fragments and each was synthesized by the phosphoramidite method. The 3'-end nucleotide of the RNA, a modified guanosine 5'-phosphate, was introduced to F-3 by use of P1-2',3'-O-methoxymethylene guanosine-5'-yl P2-adenosine-5'-yl diphosphate (A5' ppGmM) with T4 RNA ligase. The chemically synthesized RNA fragments were ligated with T4 RNA ligase to afford the desired RNA.     

Absence of detectable RNA ligase activity in eukaryotic cells.

Reports of the existence of eukaryotic RNA ligases may be incorrect. Evidence for this activity has been based upon the conversion of [5′-32p]-terminated oligoribonucleotides to an alkaline phosphatase resistant form and upon the detection of radioactive ribonucleoside monophosphates after alkaline hydrolysis of the reaction products. Although we have in part confirmed these observations, we find the labeled ribonucleoside monophosphate to be the 5′-isomer, and not the expected 2′ (3′)-isomer. In addition, roughly equivalent amounts of ribonucleoside monophosphate were observed whether or not alkaline hydrolysis was performed. We conclude that the existence of RNA ligase activity in eukaryotic cells is suspect.     

Preparation of 2-azidoadenosine 3',5'-[5'-32P]bisphosphate for incorporation into transfer RNA. Photoaffinity labeling of Escherichia coli ribosomes

2-Azidoadenosine was synthesized from 2-chloroadenosine by sequential reaction with hydrazine and nitrous acid and then bisphosphorylated with pyrophosphoryl chloride to form 2-azidoadenosine 3',5'-bisphosphate. The bisphosphate was labeled in the 5'-position using the exchange reaction catalyzed by T4 polynucleotide kinase in the presence of [gamma-32P]ATP. Polynucleotide kinase from a T4 mutant which lacks 3'-phosphatase activity (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) was required to facilitate this reaction. 2-Azidoadenosine 3',5'-[5'-32P]bisphosphate can serve as an efficient donor in the T4 RNA ligase reaction and can replace the 3'-terminal adenosine of yeast tRNAPhe with little effect on the amino acid acceptor activity of the tRNA. In addition, we show that the modified tRNAPhe derivative can be photochemically cross-linked to the Escherichia coli ribosome.     

Isolating and sequencing the infrequent 3'-ends of a specific mRNA

Procedures are described for identification of very infrequent in vivo 3'-ends of RNA. After purification by filter hybridization, the 3'-ends were labeled with [5'-32P] cytosine-3'-P in the RNA ligase reaction. Significantly fewer counts were incorporated in the ligase reaction than in the polynucleotide kinase reaction to label 5'-ends. The incorporation was increased by increasing the RNA concentration 5-10 fold by using only one round of filter hybridization. Non-specific RNA binding could be eliminated by RNase A treatment of the filter if a great excess of denatured heterologous DNA was immobilized along with the DNA probe. Significant amounts of DNA were released when eluting the hybrid RNA from such filters. DNA inhibited the ligase reaction, while its DNase products were even more inhibitory. Treatment of the DNase products with alkaline phosphatase completely eliminated the inhibition. We detected no spurious 5'- or 3'-ends generated in the hybrid RNA by RNase A activity used to reduce the non-specific RNA. Also, RNase T1 could be used in place of RNase A to eliminate non-specific RNA binding, but about 25 times more RNase T1 (microgram/microgram) was needed. We used partial alkali digestion to sequence 3'-ends. A major (one hit) and minor (two hit) set of products were produced which could be distinguished from each other by alkaline phosphatase treatment and homochromatography of the products.     

Equimolar addition of oligoribonucleotides with T4 RNA ligase.

T4 induced RNA ligase will join equimolar concentrations of two oligoribonucleotides, (Ap)3C and p(Up) 5, to form a single product, (Ap)3Cp(Up) 5, in high yield. The presence of the 3' phosphate on p(Up)5 prevents the oligomer from adding to itself. The pH optimum of the reaction is about 7.5, but less of the undesirable adenylated intermediate, App(Up) 5, forms at pH 8.2. The reaction rate is a linear function of oligomer concentration from 3 micronM to 0.6 mM. The data suggest that T4 RNA ligase will be a useful enzyme for the synthesis of oligomers of defined sequence.     

The incorporation of cytidine 5′-monophosphate and other ribonucleotides by an enzyme preparation from rat-liver microsomes

1. An enzyme preparation from rat-liver microsomes incorporated all four ribonucleotides from the corresponding triphosphates into ribosomal RNA. The reaction was Mn(2+)-dependent, but UMP incorporation also occurred in the presence of Mg(2+). 2. The incorporation of any one ribonucleotide was inhibited by the presence of the other three ribonucleoside triphosphates and by denatured DNA. 3. The product of the reaction consisted of short chains of homopolymer attached to the primer ribosomal RNA. 4. ;Soluble' RNA, synthetic polyribonucleotides, and oligoribonucleotides were also effective primers for CMP incorporation. 5. When phosphodiesterase-treated ;soluble' RNA was the primer, CMP was incorporated into positions usually occupied by the normal terminal trinucleotide sequence of intact ;soluble' RNA, but the enzyme did not synthesize a specific terminal sequence consisting of a defined number of CMP residues.     

Molecular design of a eukaryotic messenger RNA and its chemical synthesis.

A designed mRNA consisting of 42 ribonucleotides having the cap structure was synthesized. The capped leader sequence of the brome mosaic virus (BMV) mRNA 4, m7G5'pppGUAUUAAUA (F-1), was synthesized by the phosphotriester method and followed by the capping reaction. A 32-mer consisting of an initiation codon (AUG), the coding region corresponding to a bacterial pheromone cAD1 and two stop codons, was constructed by the 18-mer (F-2) and 14-mer (F-3), which were synthesized by the phosphoramidite method. 2'-,3'-O-Methoxymethylene-guanosine 5'-phosphate was condensed with F-3 using P1-2',3'-O-methoxymethyleneguanosine-5'-yl P2-adenosine-5'-yl pyrophosphate (9) with T4 RNA ligase. The chemically synthesized RNA fragments were ligated successively with T4 RNa ligase to afford the whole RNA molecule.     

Affinity modification of the 40S subparticle from human placenta with derivatives of pAUG and pAUGU3]

Affinity labeling of 40S subunits from human placenta with 4-(N-2-chloroethyl-N-methylamino)benzylmethyl-[32P]phosphoamide s of oligoribonucleotides pAUG and pAUGU3 was studied. Covalent attachment of these derivatives to 40S subunits within the complexes with 40S subunits, formed in the presence of Met-tRNAf.eIF-2.GTP, was detected. Both rRNA and ribosomal proteins were modified. Fragments of 18S rRNA, containing sites of the reagent attachment were identified: 1058-1164 for pAUG derivative and 976-1057--for pAUG and pAUGU3 ones. The data obtained allowed to conclude that the presence of the neighbouring codon at the A-site, regardless of the presence of the tRNA in it, affects significantly the arrangement of the trinucleotide template in the codon-anticodon interaction region. The large subunit does not cause significant alterations in the structural organization of the codon-anticodon interaction region.     

Synthesis of 2′-O-Photocaged Ribonucleoside Phosphoramidites

The chemical synthesis and incorporation of the phosphoramidite derivatives of 2?′-O-photocaged ribonucleosides (A, C, G and U) with o-nitrobenzyl, α-methyl-o-nitrobenzyl or 4,5-dimethoxy-2-nitrobenzyl group into oligoribonucleotides are described. The efficiency of UV irradiated uncaging of these 2′-O-photocaged oligoribonucleotides was found in the order of α-methyl-o-nitrobenzyl < 4,5-dimethoxy-2-nitrobenzyl < 2′-O-o-nitrobenzyl.     

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 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.     

Thermodynamic stability of RNA structures formed by CNG trinucleotide repeats. Implication for prediction of RNA structure

Trinucleotide repeat expansion diseases (TREDs) are correlated with elongation of CNG DNA and RNA repeats to pathological level. This paper shows, for the first time, complete data concerning thermodynamic stabilities of RNA with CNG trinucleotide repeats. Our studies include the stability of oligoribonucleotides composed of two to seven of CAG, CCG, CGG, and CUG repeats. The thermodynamic parameters of helix propagation correlated with the presence of multiple N-N mismatches within CNG RNA duplexes were also determined. Moreover, the total stability of CNG RNA hairpins, as well as the contribution of trinucleotide repeats placed only in the stem or loop regions, was evaluated. The improved thermodynamic parameters allow to predict much more accurately the thermodynamic stabilities and structures of CNG RNAs.     

Optimization of conditions for labeling the 3' OH end of tRNA using T4 RNA ligase

For several years most primary structure studies of ribonucleic acids have used the [32P] in vitro post-labeling techniques. We adapted our methods from the literature, and simplified them to make them accessible to any laboratory. These procedures are especially useful for preparation and purification of post labeling enzymes: T4 polynucleotide kinase, T4 RNA ligase and of gamma [32P] ATP. We developed a test tube method for 5' [32P] pCp preparation followed by tRNA labeling with T4 RNA ligase. The parameters for optimal labeling were determined. Labeling of 3.10(6) to 5.10(6) Cerenkov CPM per microgram tRNA are currently obtained.     

Primer RNA for DNA synthesis on single-stranded DNA template in a cell free system from Drosophila melanogaster embryos

A cytoplasmic extract of Drosophila melanogaster early embryos supported DNA synthesis which was dependent on an added single stranded DNA template, phi X174 viral DNA. The product DNA made during early reaction was about 100 to 600 nucleotides in length and complementary to the added template. After alkali treatment, 70 to 80 per cent of the product DNA chains exposed 5'-hydroxyl ends, suggesting covalent linkage of primer RNA at their 5'-ends. Post-labeling of 5'-ends of the product DNA with polynucleotide kinase and [gamma-32P]ATP revealed that oligoribonucleotides, mainly hexa- and heptanucleotides, were covalently linked to the 5'-ends of the majority of the DNA chains. The nucleotide sequence of the linked RNA was mainly 5'(p)ppApA(prN)4-5, where tri- (or di-) phosphate terminus was detected by the acceptor activity for the cap structure with guanylyltransferase and [alpha-32P]GTP. The structure of this primer RNA was comparable to that of the octaribonucleotide primer isolated from the nuclei of Drosophila early embryos.     

Joining of synthetic ribotrinucleotides with defined sequences catalyzed by T4 RNA ligase

RNA ligase catalyzed the joining of pC-C-Ap with C-A-A in the synthesis of C-A-A-C-C-Ap, which has the sequence of the Escherichia coli tRNAfMet 3'-end. pC-C-A was also shown to be joined to C-A-A without any undesired self-polymerization. Joining of pC-C-A to various synthetic ribotriplets, such as C-C-A, A-A-A, C-C-C, U-U-U, U-A-G, C-C-G and U-U-C, was performed as well as joining to the partially substituted trimers with a photolabile o-nitrobenzyl group, C-Anbzl-A and C-C-Anbzl. The yields were C-A-A-C-C-A (69%), C-C-A-C-C-A (38%), A-A-A-C-C-A (66%), C-C-C-C-C-A (71%), U-U-U-C-C-A (50%), U-A-G-C-C-A (23%), C-C-G-C-C-A (43%) and U-U-C-C-C-A (46%). C-Anbzl-A was a slightly poorer acceptor than C-A-A and C-C-Anbzl did not serve as an acceptor. Recognition of acceptor molecules by RNA ligase is discussed in terms of affinity of oligonucleotides for the enzyme.     

Solid phase synthesis of oligoribonucleotides using the o-nitrobenzyl group for 2''-hydroxyl protection and H-phosphonate chemistry.

Oligoribonucleotides with chain length of 7, 11, 15, 17, 24 and 34 were synthesized on long chain alkylamine controlled pore glass beads (LCA-CPG) using o-nitrobenzyl protection of 2'-hydroxyls via a H-phosphonate approach either manually or by using an automatic synthesizer. The oligoribonucleotides were obtained in yields of 0.6 0.6-20%, based on initial nucleoside bound to the LCA-CPG support.     

Erratum

RNA polynucleotide kinase has been shown to transfer [γ³²P] from ATP to 5-OH termini of endogenous nuclear RNA. The products of this reaction have been isolated in RNA larger than 125 after in vitro incubation of mouse L cell nuclei. About 20%–30% of these 5′-OH kinase products are polyadenylated. A sizeable fraction of the [γ³²P] label from ATP is also found in internal phosphodiester bonds after 30-minute nuclear incubation in vitro. The possibility of substantial [³²P] recycling via the α position of nucleoside triphosphate was ruled out because: (1) 2mM nucleoside triphosphates in the incubation medium, (2) limited nearestneighbor distribution 3′ and 5′ to the phosphodiester bond compared with that from [α³²P] UTP, (3) different nearest-neighbor distribution for RNA molecules > 12S and 12-3S, (4) relative insensitivity of the [γ³²P] incorporation to α-amanitin as compared with total RNA synthesis, (5) internal [³²P] appearance in RNA > 12S in less than five minutes of incubation, and (6) < 0.03% to 0.6% of the total [³²P] in the α position of nucleoside triphosphates after 30 minutes of incubation. The [γ³²P] incorporation was dependent on high ATP concentration and was insensitive to competition by inorganic phosphate. These results are consistent with the levels of 5′ RNA polynucleotide kinase activity in L cell nuclei and suggest the presence of an RNA ligase that can utilize the termini generated by the 5′-OH RNA kinase in a ligation reaction.     

Heterogeneity of the 3'' end of minus-strand RNA in the poliovirus replicative form.

The 3' terminus of the strand (minus strand) complementary to poliovirion RNA (plus strand) has been examined to see whether this sequence extends to the 5'-nucleotide terminus of the plus strand, or whether minus-strand synthesis terminates prematurely, perhaps due to the presence of a nonreplicated nucleotide primer for initiation of plus-strand synthesis. The 3' terminus was labeled with 32P using [5'-32P]pCp and RNA ligase, and complete RNase digests were performed with RNases A, T1, and U2. 32P-oligonucleotides were analyzed for size by polyacrylamide-urea gel electrophoresis. The major oligonucleotide products formed were consistent with the minus strand containing 3' ends complementary and flush with the 5' end of the plus strand. However, a variable proportion of the isolated minus strands from different preparations were heterogeneous in length and appeared to differ from each other by the presence of one, two, or three 3'-terminal A residues.