Altered utilization of splice sites and 5'' termini in late RNAs produced by leader region mutants of simian virus 40.

We compared the 5' termini and splices of the late 16S and 19S RNAs synthesized by wild-type simian virus 40 and five mutants containing deletions in their late leader region. All mutants produced more unspliced 19S RNA than did wild-type virus, and in two mutants, unspliced 19S RNA constituted more than 60% of the total 19S species. The other three mutants each utilized predominantly a different one of the three spliced species of 19S mRNA. All mutants also produced decreased quantities of 16S mRNA, indicating that they may be defective for splicing both late RNAs. None of the 5' termini of the 16S and 19S RNAs made by the five mutants predominated as in those made by the wild type. Some of the mutant 5' termini were the same as those used by the wild type, whereas others were different. Although present, the major 5'-end positions used by the wild type were frequently not used as major sites by the mutants. In addition, mutants with very similar deletion endpoints synthesized RNAs with different 5' ends. Thus, downstream mutations have a pronounced effect on the location of 5' ends of the late RNAs, and there is no obvious involvement of a measuring function in the placement of 5' ends. For all mutants and wild-type virus, the 5' termini used for 16S and 19S RNAs showed major differences, with some degree of correlation found between the 5' ends and the internal splices of specific mRNA species. A model for the regulation of simian virus 40 late gene expression is presented to explain these findings.     

Detailed characterization of the mRNA mapping in the HindIII fragment K region of the herpes simplex virus type 1 genome.

We have isolated as recombinant DNA clones, in the plasmid pBR322, regions of the herpesvirus type 1 genome spanning the region between 0.53 and 0.6 on the prototypical arrangement. This 11,000-base-pair region corresponds to 10% of the large unique region and encodes five major and several minor mRNA species abundant at different times after infection, which range in length from 7 to 1 kilobase. In this report, we have used RNA transfer blots and S1 nuclease digestion of hybrids between viral DNA and polyribosomal RNA to precisely localize (+/- 0.1 kilobase) these mRNA's. Comparison of neutral and alkaline gels of S1 nuclease-digested hybrids indicates no internal introns in the coding sequences of these mRNA's, although noncontiguous leader sequences near (ca. 0.1 kilobase) the 5' ends of any or all mRNA's could not be excluded. The 5' ends of several late mRNA's that are encoded opposite DNA strands map very close to one another, and the 3' ends of a major late and a major early mRNA, which are partially colinear, terminate in the same region. In vitro translation of the viral mRNA's isolated by hybridization with DNA bound to cellulose and fractionation of mRNA species on denaturing agarose gels allowed us to assign specific polypeptide products to each of the mRNA's characterized. Among other results, it was demonstrated unequivocally that two major late mRNA's, which partially overlap, encode the same polypeptide.     

Antibodies elicited by N2,N2-7-trimethylguanosine react with small nuclear RNAs and ribonucleoproteins

The 5' ends of U1, U2, U3, U4, and U5 small nuclear RNAs (snRNA) are capped by a structure which contains N2,N2-7-trimethylguanosine (m2,2,7 G). m2,2,7 G was used as hapten to raise antibodies in rabbits, and these antibodies were linked to Sepharose. When deproteinized RNA was passed through this antibody column, these snRNA species were retained by the column. Conversely, 4 S, 5 S, 5.8 S, U6, and 7 S RNA, whose 5' termini do not contain m2,2,7 G, were not recognized. After a nuclear extract was loaded on the column, U1 RNA and some U2 RNA were retained. Therefore, the 5' ends of at least U1 RNA are accessible when this RNA species is in small nuclear ribonucleoprotein particle (snRNP) form. This is of interest, since it has been proposed that the 5' terminus sequence of U1 RNA may hybridize with splice junctions in heterogeneous nuclear ribonucleoprotein particles (hnRNP) during mRNA splicing. The retention of m2,2,7 G-containing RNA species by these antibodies is not due to association of snRNAs or snRNPs with heterogeneous nuclear RNA (hnRNA) or hnRNP (and antibody recognition of 7-monomethylguanosine residues in hnRNA), since the reaction still occurs after removal of hnRNA or hnRNP by sucrose gradient centrifugation.     

Novel RNA family structure of hepatitis B virus expressed in human cells, using a helper-free adenovirus vector.

Ad5-HBL is a type 5 adenovirus bearing the large BglII fragment (2.8 kilobases; 87% of the total genome) of hepatitis B virus (HBV), subtype adr. Eight HBV RNAs expressed in HeLa cells infected with Ad5-HBL were mapped by the nuclease S1 technique. Three major RNAs spanning 2.4, 2.0, and 0.7 kilobases of the HBV sequences cover the coding regions of "presurface" plus surface antigen, surface antigen alone, and "X" protein, respectively. The 5' segment of an RNA which could code for core antigen (HBcAg) was also detected. All major HBV RNAs initiate from mutually exclusive 5' ends, terminate at the unique 3' end within the HBcAg coding region (except readthrough species), and have no spliced deletion, forming a novel RNA family structure. No TATA box-like sequences were found near the 5' end of these RNAs, except in the case of the 2.4-kilobase RNA. About two thirds of total HBV RNA does not terminate at the mapped 3'-end position, suggesting the termination signal is functionally inefficient. Since the potential 5' end of HBcAg mRNA was mapped at the same position as the minus-strand nick of HBV DNA previously reported, we propose a model that requires inefficient poly(A) addition to produce an RNA which serves both as HBcAg mRNA and as the putative RNA template of minus-strand DNA synthesis in the HBV life cycle.     

Heterogneeous 5'-terminal structures occur on vesicular stomatitis virus mRNAs.

Four alternative structures occur at the 5' ends of vesicular stomatitis virus mRNAs synthesized in infected cells and are separated conveniently by a technique described here. Sixty-five to seventy per cent of the mRNA molecules have the 5' end structure m7G5'ppp5'(m)AmpAp and about 20% have a more highly modified structure m7G5'ppp5'(m)AmpmAmpCp. The base of the first adenosine in each sequence is methylated in about one-half of the ends of each type and kinetic experiments suggest that the latter sequence is derived from the former by further methylations. The remaining 10 to 15% of the 5' ends are pppAp and pppGp in approximately equimolar yields. This heterogeneity with respect to 5' end structure is found within each of the vesicular stomatitis virus mRNA species examined. The mRNA molecules with 5'-triphosphate ends accumulate throughout the infection but are not found on ribosomes, suggesting that they lack a structure(s) required for ribosome recognition. In contrast to mRNA, virion RNA has a single 5' end structure, pppAp.     

Detection and characterization of degradative intermediates of avian apo very low density lipoprotein II mRNA present in estrogen-treated birds and following destabilization by hormone withdrawal

The apo very low density lipoprotein II (apoVLDLII) gene is dormant in embryos, chicks, and roosters but can be activated by estrogen. ApoVLDLII mRNA is relatively stable in estrogen-treated birds. However, its stability decreases 4-5-fold following withdrawal of hormone. We have characterized degradative intermediates of apoVLDLII mRNA detected in liver total RNA from estrogen-treated birds and searched for alterations in the pattern of intermediates that occur upon hormone-withdrawal. Primer extension and S1 nuclease analyses have demonstrated that these intermediates consist of fragments of the molecule with intact 5' ends but which lack various 3' regions. Estrogen withdrawal results in a decrease in the steady state levels of several of these intermediates and the detection of two new species. The end points of the major fragments present in RNA from both estrogen-treated and withdrawn birds all map in, or within four nucleotides of, the tetranucleotide, GAUG. The two fragments detected only in RNA from withdrawn birds have 3' ends that immediately precede the sequence, CAGU. Based on secondary structures predicted by a global folding program, the end points also appear to be preferentially located in, or at the base of, internal "bulge-loops".