Alternative 3'-end processing of U5 snRNA by RNase III

The cellular components required to form the 3' ends of small nuclear RNAs are unknown. U5 snRNA from Saccharomyces cerevisiae is found in two forms that differ in length at their 3' ends (U5L and U5S). When added to a yeast cell free extract, synthetic pre-U5 RNA bearing downstream genomic sequences is processed efficiently and accurately to generate both mature forms of U5. The two forms of U5 are produced in vitro by alternative 3'-end processing. A temperature-sensitive mutation in the RNT1 gene encoding RNase III blocks accumulation of U5L in vivo. In vitro, alternative cleavage of the U5 precursor by RNase III determines the choice between the two multistep pathways that lead to U5L and U5S, one of which (U5L) is strictly dependent on RNase III. These results identify RNase III as a trans-acting factor involved in 3'-end formation of snRNA and show how RNase III might regulate alternative RNA processing pathways.     

alpha-Oligodeoxyribonucleotide N3'-->P5' phosphoramidates: synthesis and duplex formation

The synthesis and hybridization properties of novel nucleic acid analogs, alpha-anomeric oligodeoxyribonucleotide N3'-->P5' phosphoramidates, are described. The alpha-3'-aminonucleoside building blocks used for oligonucleotide synthesis were synthesized from 3'-azido-3'-deoxythymidine or 3'-azido-2',3'-dideoxyuridine via acid catalyzed anomerization or transglycosylation reactions. The base-protected alpha-5'-O-DMT-3'-aminonucleosides were assembled into dimers and oligonucleotides on a solid support using the oxidative phosphorylation method.1H NMR analysis of the alpha-N3'-->P5' phosphoramidate dimer structures indicates significant differences in the sugar puckering of these compounds relative to the beta-N3'-->P5' phosphoramidates and to the alpha-phosphodiester counterparts. Additionally, the ability of the alpha-oligonucleotide N3'-->P5' phosphoramidates to form duplexes was studied using thermal denaturation experiments. Thus the N3'-->P5' phosphoramidate decamer containing only alpha-thymidine residues did not bind to poly(A) and exhibited lower duplex thermal stability with poly(dA) than that for the corresponding beta-anomeric phosphoramidate counterpart. A mixed base decamer alpha-CTTCTTCCTT formed duplexes with the RNA and DNA complementary strands only in a parallel orientation. Melting temperatures of these complexes were significantly lower, by 34-47 or 15-25 degrees C, than for the duplexes formed by the isosequential beta-phosphoramidates in antiparallel and parallel orientations respectively. In contrast, the alpha-decaadenylic N3'-->P5' phosphoramidate formed duplexes with both RNA and DNA complementary strands with a stability similar to that of the corresponding beta-anomeric phosphoramidate. Moreover, the self-complementary oligonucleotide alpha-ATATATATAT did not form an alpha:alpha homoduplex. These results demonstrate the effects of 3'-aminonucleoside anomeric configuration on sugar puckering and consequently on stability of the duplexes.     

Exonuclease IX of Escherichia coli removes 3' phosphoglycolate end groups from DNA

The bacteria Escherichia coli contains several exonucleases acting on both double- and single-stranded DNA, and in both a 5'--> 3' and a 3' --> 5' direction. These enzymes are involved in replicative, repair and recombination functions. A new exonuclease recently identified in E. coli, termed exonuclease IX, acts preferentially on single-stranded DNA as a 3'--> 5' exonuclease and also functions as a 3' phosphodiesterase on DNA containing 3' incised apurinic/apyrimidinic (AP) sites to remove the product trans-4-hydroxy-2-pentenal-5-phosphate. We now demonstrate that the enzyme is also able to remove 3' phosphoglycolate end groups from DNA. This activity may have an important role in DNA base excision repair in E. coli.     

Rotavirus RNA replication requires a single-stranded 3' end for efficient minus-strand synthesis

The segmented double-stranded (ds) RNA genome of the rotaviruses is replicated asymmetrically, with viral mRNA serving as the template for the synthesis of minus-strand RNA. Previous studies with cell-free replication systems have shown that the highly conserved termini of rotavirus gene 8 and 9 mRNAs contain cis-acting signals that promote the synthesis of dsRNA. Based on the location of the cis-acting signals and computer modeling of their secondary structure, the ends of the gene 8 or 9 mRNAs are proposed to interact in cis to form a modified panhandle structure that promotes the synthesis of dsRNA. In this structure, the last 11 to 12 nucleotides of the RNA, including the cis-acting signal that is essential for RNA replication, extend as a single-stranded tail from the panhandled region, and the 5' untranslated region folds to form a stem-loop motif. To understand the importance of the predicted secondary structure in minus-strand synthesis, mutations were introduced into viral RNAs which affected the 3' tail and the 5' stem-loop. Analysis of the RNAs with a cell-free replication system showed that, in contrast to mutations which altered the structure of the 5' stem-loop, mutations which caused complete or near-complete complementarity between the 5' end and the 3' tail significantly inhibited (>/=10-fold) minus-strand synthesis. Likewise, incubation of wild-type RNAs with oligonucleotides which were complementary to the 3' tail inhibited replication. Despite their replication-defective phenotype, mutant RNAs with complementary 5' and 3' termini were shown to competitively interfere with the replication of wild-type mRNA and to bind the viral RNA polymerase VP1 as efficiently as wild-type RNA. These results indicate that the single-strand nature of the 3' end of rotavirus mRNA is essential for efficient dsRNA synthesis and that the specific binding of the RNA polymerase to the mRNA template is required but not sufficient for the synthesis of minus-strand RNA.     

Effects of Xenopus laevis mitochondrial single-stranded DNA-binding protein on primer-template binding and 3'-->5' exonuclease activity of DNA polymerase gamma

Mitochondrial DNA (mtDNA) is replicated by DNA polymerase gamma by a strand displacement mechanism involving mitochondrial single-stranded DNA-binding protein (mtSSB). mtSSB stimulates the overall rate of DNA synthesis on singly-primed M13 DNA mainly by stimulating the processivity of DNA synthesis rather than by stimulating primer recognition. We used electrophoretic mobility shift methods to study the effects of mtSSB on primer-template recognition by DNA pol gamma. Preliminary experiments showed that single mtSSB tetramers bind tightly to oligo(dT) single strands containing 32 to 48 residues. An oligonucleotide primer-template was designed with an 18-mer primer annealed to the 3'-portion of a 71-mer template containing 40 dT residues at its 5'-end as a binding site for mtSSB. DNA pol gamma bound to this primer-template either in the absence or presence of mtSSB in complexes that remained intact and enzymatically active following native gel electrophoresis. Association of mtSSB with the 5'-dT40-tail in the 18:71-mer primer-template reduced the binding of DNA polymerase gamma and the efficiency of primer extension. Binding of mtSSB to single-stranded DNA was also observed to block the action of the 3'-->5' exonuclease of DNA polymerase gamma. The size of fragments protected from 3'-->5' exonuclease trimming increases with increasing ionic strength in a manner consistent with the known salt dependence of the binding site size of Escherichia coli SSB.     

A 5'-3' exonuclease activity involved in forming the 3' products of histone pre-mRNA processing in vitro

Histone RNA 3' processing in vitro produces one or more 5' cleavage products corresponding to the mature histone mRNA 3' end, and a group of 3' cleavage products whose 5' ends are mostly located several nucleotides downstream of the mRNA 3' end. The formation of these 3' products is coupled to the formation of 5' products and dependent on the U7 snRNP and a heat-labile processing factor. These short 3' products therefore are a true and general feature of the processing reaction. Identical 3' products are also formed from a model RNA containing all spacer nucleotides downstream of the mature mRNA 3' end, but no sequences from the mature mRNA. Again, this reaction is dependent on both the U7 snRNP and a heat-labile factor. Unlike the processing with a full-length histone pre-mRNA, this reaction produces only 3' but no 5' fragments. In addition, product formation is inhibited by addition of cap structures at the model RNA 5' end, indicating that product formation occurs by 5'-3' exonucleolytic degradation. This degradation of a model 3' product by a 5'-3' exonuclease suggests a mechanism for the release of the U7 snRNP after processing by shortening the cut-off histone spacer sequences base paired to U7 RNA.     

Structure-specific DNA binding by bacteriophage T5 5'-->3' exonuclease

Phage T5 exonuclease is a 5'-->3'exodeoxyribonuclease that also exhibits endonucleolytic activity on flap structures (branched duplex DNA containing a free single-stranded 5'-end). Oligonucleotides were used to construct duplexes with either blunt ends, 5'-overhangs, 3'-overhangs, a flap or a forked end (pseudo-Y). The binding of T5 exonuclease to various structures was investigated using native electrophoretic mobility shift assays (EMSA) in the absence of the essential divalent metal cofactor. Binding of T5 exonuclease to either blunt-ended duplexes or single-stranded oligonucleotides could not be detected by EMSA. However, duplexes with 5'-overhangs, flaps and pseudo-Y structures showed decreased mobility with added T5 exonuclease. On binding to DNA the wild-type enzyme was rendered partially resistant to proteolysis, yielding a biologically active 31.5 kDa fragment. However, the protein-DNA complex remained susceptible to inactivation by p-hydroxymercuribenzoate (PHMB, a cysteine-specific modifying agent), suggesting that neither cysteine is intimately associated with substrate binding. Replacement of both cysteine residues of the molecule with serine did not greatly alter the catalytic or binding characteristics of the protein but did render it highly resistant to inhibition by PHMB.     

A human U2 RNA mutant stalled in 3' end processing is impaired in nuclear import

The biosynthesis of U1, U2, U4 and U5 spliceosomal small nuclear RNAs (snRNAs) involves the nuclear export of precursor molecules extended at their 3' ends, followed by a cytoplasmic phase during which the pre-snRNAs assemble into ribonucleoprotein particles and undergo hypermethylation of their 5' caps and 3' end processing prior to nuclear import. Previous studies have demonstrated that the assembly of pre-snRNAs into ribonucleoprotein particles containing the Sm core proteins is essential for nuclear import in mammalian cells but that 5' cap hypermethylation is not. In the present investigation we have asked whether or not 3' end processing is required for nuclear import of U2 RNA. We designed human pre-U2 RNAs that carried modified 3' tails, and identified one that was stalled (or greatly slowed) in 3' end processing, leading to its accumulation in the cytoplasm of human cells. Nonetheless, this 3' processing arrested pre-U2 RNA molecule was found to undergo cytoplasmic assembly into Sm protein-containing complexes to the same extent as normal pre-U2 RNA. The Sm protein-associated, unprocessed mutant pre-U2 RNA was not observed in the nuclear fraction. Using an assay based on suppression of a genetically blocked SV40 pre-mRNA splicing pathway, we found that the 3' processing deficient U2 RNA was significantly reduced in its ability to rescue splicing, consistent with its impaired nuclear import.     

Decoying the cap- mRNA degradation system by a double-stranded RNA virus and poly(A)- mRNA surveillance by a yeast antiviral system

The major coat protein of the L-A double-stranded RNA virus of Saccharomyces cerevisiae covalently binds m7 GMP from 5' capped mRNAs in vitro. We show that this cap binding also occurs in vivo and that, while this activity is required for expression of viral information (killer toxin mRNA level and toxin production) in a wild-type strain, this requirement is suppressed by deletion of SKI1/XRN1/SEP1. We propose that the virus creates decapped cellular mRNAs to decoy the 5'-->3' exoribonuclease specific for cap- RNA encoded by XRN1. The SKI2 antiviral gene represses the copy numbers of the L-A and L-BC viruses and the 20S RNA replicon, apparently by specifically blocking translation of viral RNA. We show that SKI2, SKI3, and SKI8 inhibit translation of electroporated luciferase and beta-glucuronidase mRNAs in vivo, but only if they lack the 3' poly(A) structure. Thus, L-A decoys the SKI1/XRN1/SEP1 exonuclease directed at 5' uncapped ends, but translation of the L-A poly(A)- mRNA is repressed by Ski2,3,8p. The SKI2-SKI3-SKI8 system is more effective against cap+ poly(A)- mRNA, suggesting a (nonessential) role in blocking translation of fragmented cellular mRNAs.