Mapping of late adenovirus genes by cell-free translation of RNA selected by hybridization to specific DNA fragments.

The Problem: Short-Lived Heterogeneous Nuclear RNA During the 1960s and 1970s, there was a major conundrum in eukaryotic molecular biology regarding the synthesis of mRNA in animal cells. Pulse and pulsechase radio-labeling experiments with tritiated uridine had shown that a large fraction of nuclear RNA was degraded very rapidly after synthesis, whereas only a small fraction of the initially synthesized RNA was exported from the nucleus to the cytoplasm where it functions as far more stable mRNAs (1, 2). This shortlived nuclear RNA ranged in lengths up to several tens of kilobases, much longer than most cytoplasmic mRNAs, and was referred to as heterogeneous nuclear RNA (hnRNA) (3, 4). Pulse-chase labeling yielded results consistent with hnRNA functioning as a precursor of mRNA. hnRNA is rapidly labeled, whereas label appeared in mRNAs more slowly. Also, as for mRNA, a large fraction of hnRNA was shown to be polyadenylated at its 3′ end like mRNA, consistent with the model that RNA sequences near the 3′ end of hnRNAs are retained in shorter mRNAs exported to the cytoplasm (5, 6). Also, like mRNAs, hnRNAs were found to have the 7-methyl guanine 5′-5′ phosphotriester "cap" structure at their 5′ ends, like mRNAs. Remarkably, the methyl groups in hnRNA cap structures appeared to be conserved as RNA was exported to the cytoplasm, even though, on average, hnRNA molecules in the nucleus are at least four times longer than mRNAs in the cytoplasm (7, 8). However, it was not possible to prove rigorously from the flow of radiolabel in pulse-chase labeling experiments that hnRNAs are precursors to mRNAs. There were two principal reasons for this. First, the pool of ribonucleoside triphosphates in mammalian cells is so large that it takes more than 30 min to saturate the pools when labeled nucleosides are added to the media and equally long to dilute the intracellular pool of labeled nucleoside triphosphates by addition of a large excess of unlabeled nucleoside to the culture medium during the chase period. Consequently, a clean chase could not be achieved, and newly synthesized hnRNAs continued to be labeled as labeled mRNAs first appeared in the cytoplasm. Also, the small fraction of pulse-labeled nuclear RNA converted to more stable mRNAs, only 5-10%, added to the difficulty of proving a precursor product relationship from pulse-chase labeling experiments.The synthesis and processing of ribosomal RNAs was much easier to analyze than processing of hnRNAs because about 50% of all nuclear RNA synthesis in growing mammalian cells in culture is synthesis of the single ∼13.7-kb pre-rRNA precursor. Moreover, this full-length pre-rRNA precursor accumulates to significant levels before it is processed into stable mature rRNAs of 18S (1,870 bases), 5.8S (156 bases), and 28S (5,034 bases), so that the fraction of pre-RNA processed into stable cytoplasmic rRNAs is ∼50%, much more than the 5-10% of pulse-labeled hnRNA processed into mRNAs. This fortuitous situation for the study of prerRNA by pulse-chase labeling made it possible to ...

Section: The Experimentsmentioning

confidence: 99%

Discovery of RNA splicing and genes in pieces

Berk

2016

“…Persistent early RNAs (and polypeptides) have been distinguished from late RNAs (and polypeptides) by the greater sensitivity of late gene expression to inhibition ofviral DNA synthesis (11)(12)(13)(14)(15)(16)26). We now report the mapping of persistent early and late polypeptides to specific fragments ofviral DNA by hybrid selection and in vitro translation (27). The translation products can be correlated with the persistent early and late RNAs encoded by these fragments (26) (23).…”

mentioning

confidence: 99%

Mapping of polypeptides encoded by the Epstein-Barr virus genome in productive infection.

Hummel

Kieff²

1982

Over 30 viral-specified polypeptides are translated in vitro from RNA of cells productively infected with Epstein-Barr virus (EBV). The polypeptides map to sites in EBV DNA by hybrid selection. Almost all of the polypeptides are reactive with EBV immune human serum. Several of the polypeptides are part of the early antigen complex. Two others are likely to be major structural components of the virus. Genes encoding persistent early and late polypeptides are intermixed through most of the EBV genome. (26,28,29).Selection of DNA Fragment-Specific mRNA. Fifty micrograms of plasmid, phage, or viral DNA was bound to a 1-cm diazobenzyloxymethyl paper circle (30). Cytoplasmic polyadenylylated RNA (1 mg/ml) was hybridized in a single reaction vessel to several different paper circles for 4 hr at 500C (31). Unbound RNA was removed by washing the circles individually five times for 5 min at 50'C in 5 ml of 50% recrystallized formamide/0.5% NaDodSO4/40 mM Pipes, pH 6.5/15 mM NaCl. The selected RNA was eluted (31) and ethanol-precipitated with 20 ,g of tRNA as a carrier. The size of unbound and bound RNAs was monitored on RNA blots (26) and was not affected by hybridization and elution.In Vitro Translation, Immunoprecipitation, and Gel Electrophoresis. Half of the RNA that was eluted from a filter was used for a 25-,ul in vitro translation reaction with reticulocyte lysate (New England Nuclear) and [3S]methionine (32). After preabsorption with EBV-negative serum and staphylococcal protein A-Sepharose (Pharmacia), EBV antigens were immunoprecipitated from the in vitro translation in buffer without NaDodSO4 (33) by using 1 ,ul of human serum with high titers to EBV antigens [VCA, 1: 40,960; EA, 1: 10,240 The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

“…Sharp, data not shown). Furthermore, this RNA has been mapped by the R-loop technique (see below) to a region of the genome known to code for hexon (12) and is complementary to the r strand of the viral DNA (11). This mRNA species, therefore, will be referred to in the following sections as hexon mRNA.…”

mentioning

confidence: 99%

Spliced segments at the 5′ terminus of adenovirus 2 late mRNA

Berget

Moore

Sharp

1977

1,106

588

An mRNA fraction coding for hexon polypeptide, the major virion structural protein, was purified by gel electrophoresis from extracts of adenovirus 2-infected cells late in the lytic cycle. The mRNA sequences in this fraction were mapped between 51.7 and 61.3 units on the genome by visualizing RNA-DNA hybrids in the electron microscope. When hybrids of hexon mRNA and single-stranded restriction endonuclease cleavage fragments of viral DNA were visualized in the electron microscope, branched forms were observed in which 160 nucleotides of RNA from the 5! terminus were not hydrogen bonded to the single-stranded DNA. DNA se uences complementary to the RNA sequences in each 5' tail were found by electron microscopy to be located at 17,20, and 27 units on the same strand as that coding for the body of the hexon mRNA. Thus, four segments of vira RNA may be joined together during the synthesis of mature hexon mRNA. A model is presented for adenovirus late mRNA synthesis that involves multiple splicing during maturation of a larger precursor nuclear RNA.Most eukaryotic mRNAs bear modifications at both termini; their 3' termini have a tract of poly(A) that ranges in length from 30 to 200 bases (1-4), while their 5' termini are typically capped with a methylated guanine joined through a 5'-5' pyrophosphate linkage to a second nucleotide methylated at its 2' position (5, 6). Both types of modifications of eukaryotic mRNA are known to occur after transcription.All adenovirus mRNAs are thought to contain poly(A) tracts at their 3' termini (7) and be capped with a methylated guanine (8, 9). Specific restriction endonuclease cleavage fragments of adenovirus 2 (Ad2) DNA have permitted the mapping of regions of the genome expressed as mRNA and viral proteins during different stages of the lytic cycle (10-12). Little is known about the molecular mechanisms of viral mRNA synthesis. An important aspect of late mRNA synthesis is thought to be the processing and selection of viral mRNAs from the nucleus (18,14). We have purified a late Ad2 hexon mRNA and found evidence providing some insight into the mechanism of synthesis of this mRNA. MATERIALS AND METHODSIsolation of Ad2 DNA and RNA. Polyribosomal RNA was prepared from Ad2-infected cells 32 hr after infection as described by Flint and Sharp (14,15) and selected by chromatography on poly(U)-Sephadex (16).R-Loop Mapping. The R-loop hybridization mixture was essentially that of Thomas et al. (17) (20). The sample was incubated at 57-60°for 2-3 hr. RESULTSAdenovirus late mRNAs begin to appear on polyribosomes about 13 hr after infection and continue to accumulate in the cell throughout the lytic cycle (21). Thus, to fractionate the most abundant late mRNAs, polyribosomes were prepared from cells 32 hr after infection with Ad2 and poly(A)-containing mRNA was selected by chromatography on poly(U)Sephadex columns. These mRNAs were then resolved into different molecular weight fractions by electrophoresis in 2.4-4.0% linear gradient polyacrylamide gels containing a uniform conc...