Structure of a large segment of the genome of simian virus 40 that does not encode known proteins.

The nucleotide sequence of the region of DNA of simian virus 40 extending from 0.595 to 0.790 map unit has been derived. The sequence includes the DNA complementary to the 5' end of early mRNA and to the 5' end of some of the forms of late RNA. Because there are termination codons in all three phases in early and late RNA, there is a sequence of almost 800 nucleotides of simian virus 40 DNA that probably does not code for known viral proteins. The sequence spans the 5' end of the early mRNA at 0.67 map unit and overlaps a species of late RNA whose 5' end is at 0.65 map unit and whose 3' end is at 0.77 map unit. This RNA is retained on oligo(dT)-cellulose columns in high salt concentrations. Analysis of the sequence of late strand RNA suggests that this RNA is not covalently linked to the mRNA that encodes structural proteins. There is another species of late RNA of simian virus 40 whose 5' end is at 0.775 map unit. The nucleotide sequence of this region of simian virus 40 DNA contains several examples of repeated sequences, most of which are located in DNA that does not encode known peptides. These may be analogous to the reiterated sequences that have been described in animal cell DNA.     

Comparison of the 5′ nuclease activities of Taq DNA polymerase and its isolated nuclease domain

Many eubacterial DNA polymerases are bifunctional molecules having both polymerization (P) and 5' nuclease (N) activities, which are contained in separable domains. We previously showed that the DNA polymerase I of Thermus aquaticus (TaqNP) endonucleolytically cleaves DNA substrates, releasing unpaired 5' arms of bifurcated duplexes. Here, we compare the substrate specificities of TaqNP and the isolated 5' nuclease domain of this enzyme, TaqN. Both enzymes are significantly activated by primer oligonucleotides that are hybridized to the 3' arm of the bifurcation; optimal stimulation requires overlap of the 3' terminal nucleotide of the primer with the terminal base pair of the duplex, but the terminal nucleotide need not hybridize to the complementary strand in the substrate. In the presence of Mn2+ ions, TaqN can cleave both RNA and circular DNA at structural bifurcations. Certain anti-TaqNP mAbs block cleavage by one or both enzymes, whereas others can stimulate cleavage of nonoptimal substrates.     

Enhancer-dependent interaction between 5'' and 3'' splice sites in trans.

Splice-site selection and alternative splicing of nuclear pre-mRNAs can be controlled by splicing enhancers that act by promoting the activity of upstream splice sites. Here we show that RNA molecules containing a 3' splice site and enhancer sequence are efficiently spliced in trans to RNA molecules containing normally cis-spliced 5' splice sites or to normally trans-spliced spliced leader RNAs from lower eukaryotes. In addition, we show that this reaction is stimulated by (Ser + Arg)-rich splicing factors that are known to promote protein-protein interactions in the cis-splicing reaction. Thus, splicing enhancers facilitate the assembly of protein complexes on RNAs containing a 3' splice site, and this complex is sufficiently stable to functionally interact with 5' splice sites located on separate RNAs. This trans-splicing is mediated by interactions between (Ser + Arg)-rich splicing factors bound to the enhancer and general splicing factors bound to the 5' and 3' splice sites. These same interactions are likely to play a crucial role in alternative splicing and splice-site selection in cis.     

Mapping of transcription sites of simian virus 40-specific late 16S and 19S mRNA by electron microscopy.

Simian virus 40 (SV40) late 19S and 16S mRNAs were annealed to complementary regions of partially melted viral double-stranded SV40(LHpa II) DNA or SV40(LBam HI) DNA. The RNA-DNA hybrid regions within the DNA molecules were visualized as loops [with SV40(LBam HI) DNA] in the electron microscope. The data confirm the previous localizations of the 3' and 5' ends of 16S SV40 mRNA and of the 3' end of late 19S SV40 mRNA. The 5' end of the major stable SV40 late 19S mRNA has been positioned at 0.755 map unit. Thus, the sequences of viral DNA from 0.655 to 0.755 map unit, including the replication origin, are not converted into major stable species of late viral mRNA.     

Detection and mapping of spliced RNA from a human hepatoma cell line transfected with the hepatitis B virus genome.

HepG2 cells, known to support the replication and virion formation of hepatitis B virus (HBV), were transfected with a cosmid constructed to contain 12 tandem head-to-tail repeats of the HBV genome for effective HBV genome expression. We detected previously identified RNAs of 3.3, 2.3, and 2.0 kilobases (kb) that code for core antigen, large surface antigen, and middle/major surface antigen, respectively. We also detected four additional RNAs of 2.1, 1.7, 1.1, and 0.7 kb [the lengths exclude the poly(A) tail]. S1 mapping and nucleotide sequencing data showed that the 2.1-kb RNA is a spliced RNA whose 5' and 3' ends are identical to those of the 3.3-kb RNA. The results suggest that the 2.1-kb RNA codes for an altered core antigen lacking the last amino acid, cysteine, and that expression of the 3.3-kb pregenomic RNA is regulated, at least in part, by splicing. The map positions of the 1.7- and 1.1-kb RNAs suggest that they code for the carboxyl-terminal portions of the putative polymerase, whereas the 0.7-kb RNA codes for the X protein.     

In vitro splicing of influenza viral NS1 mRNA and NS1-beta-globin chimeras: possible mechanisms for the control of viral mRNA splicing.

In influenza virus-infected cells, the splicing of the viral NS1 mRNA catalyzed by host nuclear enzymes is controlled so that the steady-state amount of the spliced NS2 mRNA is only 5-10% of that of the unspliced NS1 mRNA. Here we examine the splicing of NS1 mRNA in vitro, using nuclear extracts from HeLa cells. We show that in addition to its consensus 5' and 3' splice sites, NS1 mRNA has an intron branch-point adenosine residue that was functional in lariat formation. Nonetheless, this RNA was not detectably spliced in vitro under conditions in which a human beta-globin precursor was efficiently spliced. Using chimeric RNA precursors containing both NS1 and beta-globin sequences, we show that the NS1 5' splice site was effectively utilized by the beta-globin branch-point sequence and 3' splice site to form a spliced RNA, whereas the NS1 3' splice site did not function in detectable splicing in vitro, even in the presence of the beta-globin branch-point sequence or in the presence of both the branch-point sequence and 5' exon and splice site from beta-globin. With the chimeric precursors that were not detectably spliced, as with NS1 mRNA itself, a low level of a lariat structure containing only intron and not 3' exon sequences was formed. The inability of the consensus 3' splice site of NS1 mRNA to function effectively in in vitro splicing suggests that this site is structurally inaccessible to components of the splicing machinery. Based on these results, we propose two mechanisms whereby NS1 mRNA splicing in infected cells is controlled via the accessibility of its 3' splice site.     

tRNAHis guanylyltransferase catalyzes a 3'-5' polymerization reaction that is distinct from G-1 addition

Yeast tRNA(His) guanylyltransferase, Thg1, is an essential protein that adds a single guanine to the 5' end (G(-1)) of tRNA(His). This G(-1) residue is required for aminoacylation of tRNA(His) by histidyl-tRNA synthetase, both in vitro and in vivo. The guanine nucleotide addition reaction catalyzed by Thg1 extends the polynucleotide chain in the reverse (3'-5') direction of other known polymerases, albeit by one nucleotide. Here, we show that alteration of the 3' end of the Thg1 substrate tRNA(His) unleashes an unexpected reverse polymerase activity of wild-type Thg1, resulting in the 3'-5' addition of multiple nucleotides to the tRNA, with efficiency comparable to the G(-1) addition reaction. The addition of G(-1) forms a mismatched G.A base pair at the 5' end of tRNA(His), and, with monophosphorylated tRNA substrates, it is absolutely specific for tRNA(His). By contrast, reverse polymerization forms multiple G.C or C.G base pairs, and, with preactivated tRNA species, it can initiate at positions other than -1 and is not specific for tRNA(His). Thus, wild-type Thg1 catalyzes a templated polymerization reaction acting in the reverse direction of that of canonical DNA and RNA polymerases. Surprisingly, Thg1 can also readily use dNTPs for nucleotide addition. These results suggest that 3'-5' polymerization represents either an uncharacterized role for Thg1 in RNA or DNA repair or metabolism, or it may be a remnant of an earlier catalytic strategy used in nature.     

Masking the 5' terminal nucleotides of the hepatitis C virus genome by an unconventional microRNA-target RNA complex

Hepatitis C virus subverts liver-specific microRNA, miR-122, to upregulate viral RNA abundance in both infected cultured cells and in the liver of infected chimpanzees. These findings have identified miR-122 as an attractive antiviral target. Thus, it is imperative to know whether a distinct functional complex exists between miR-122 and the viral RNA versus its normal cellular target mRNAs. Toward this goal, effects on viral RNA abundance of mutated miR-122 duplex molecules, bound at each of the two target sites in the viral genome, were compared to effects on microRNA- or siRNA-mediated regulation of reporter target mRNAs. It was found that miR-122 formed an unusual microRNA complex with the viral RNA that is distinct from miR-122 complexes with reporter mRNAs. Notably, miR-122 forms an oligomeric complex in which one miR-122 molecule binds to the 5' terminus of the hepatitis C virus (HCV) RNA with 3' overhanging nucleotides, masking the 5' terminal sequences of the HCV genome. Furthermore, specific internal nucleotides as well as the 3' terminal nucleotides in miR-122 were absolutely required for maintaining HCV RNA abundance but not for microRNA function. Both miR-122 molecules utilize similar internal nucleotides to interact with the viral genome, creating a bulge and tail in the miR-122 molecules, revealing tandemly oriented oligomeric RNA complexes. These findings suggest that miR-122 protects the 5' terminal viral sequences from nucleolytic degradation or from inducing innate immune responses to the RNA terminus. Finally, this remarkable microRNA-mRNA complex could be targeted with compounds that inactivate miR-122 or interfere with this unique RNA structure.     

Structure of Moloney murine leukemia viral DNA: nucleotide sequence of the 5'' long terminal repeat and adjacent cellular sequences.

Some unintegrated and all integrated forms of murine leukemia viral DNA contain long terminal repeats (LTRs). The entire nucleotide sequence of the LTR and adjacent cellular sequences at the 5' end of a cloned integrated proviral DNA obtained from BALB/Mo mouse has been determined. It was compared to the nucleotide sequence of the LTR at the 3' end. The results indicate: (i) a direct 517-nucleotide repeat at the 5' and 3' termini; (ii) 145 nucleotides out of 517 nucleotides represent sequences between the 5'-CAP nucleotide and 3' end of the primer tRNA (strong-stop DNA); (iii) an 11-nucleotide inverted repeat is present at the ends of the 5'-LTR and a total of 17 out of 21 nucleotides at the termini are inverted repeats; (iv) sequences CAATAAAAG (at positions -24 to -31) and CAATAAAC (at positions +46 to +53) resembling the hypothetical DNA-dependent RNA polymerase II promoter site can be identified in the 5'-LTR; (v) the sequence GAAA appears to be repeated on both sides of the junction of viral and cellular sequences; and (vi) in analogy with the bacterial transposons, the presence of an inverted repeat sequence at the termini of 5'-LTR suggests that M-MLV also has the integration properties of a transposon.