Strand assignment of polyadenylated nuclear RNAs synthesized early in infection with adenovirus 2

Craig, Elizabeth A.; Sayavedra, Marta S.; Raskas, Heschel J.

doi:10.1016/0042-6822(77)90480-9

Cited by 14 publications

(7 citation statements)

References 17 publications

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“…Several lines of evidence suggest that cytoplasmic Ad2 mRNAs are derived from larger nuclear precursors that lose part of their sequences during processing (6,(25)(26)(27)(28)(29)(30)(31). If processing occurs by removal of sequences not only at the 5' and 3' ends of the molecule but also internally, the resulting mRNA would be expected to generate displacement loops when hybridized with the template strand of DNA.…”

Section: Discussionmentioning

confidence: 99%

Loop structures in hybrids of early RNA and the separated strands of adenovirus DNA.

Kitchingman¹,

Lai²,

Westphal³

1977

Proc. Natl. Acad. Sci. U.S.A.

100

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Separated strands of adenovirus DNA were annealed with ear1y cytoplasmic RNA and visualized in the electron microscope. DNA-RNA duplex regions within the DNA filaments could be recognized by their heavy contour. This contour was often interrupted at distinct locations by loops of displaced, sin-tranded DNA. Loops have been observed and mapped in al four early regions of the genome. The structures appear to signal hitherto unknown mechanisms of eukaryotic gene expression. The synthesis of mRNA in eukaryotic cells consists of a number of distinct but poorly understood steps. After transcription from the template DNA, the RNA transcript appears to undergo a series of nucleolytic cleavages, modification of the 5' end to form the "cap" structure, and addition of poly(A) to the 3' end (1, 2). We have been studying the RNAs synthesized after infection of KB cells by human adenovious type 2 (Ad2) in an attempt to understand these processes. The Ad2 system provides an excellent model in which to study these processes in that its DNA is far less complex than that of a eukaryotic cell and has few gene products but appears to share the same mechanism for RNA transcription and processing.The viral gene products can be placed into two separate classes: those produced early during infection (that is, before the onset of viral DNA synthesis); and those produced after its onset (late RNAs). We have mapped early and late cytoplasmic RNAs (3-5) as well as late nuclear RNAs (6) by using the R-loop electron microscope technique (7,8). In addition, use of quantitative electron microscopy has enabled us to determine the relative abundance of individual populations of cytoplasmic Ad2 RNA (9, 10). Hybridization of RNA with separated strands of Ad2 DNA revealed structures reminiscent of insertions previously observed in Drosophila melanogaster rDNA'RNA hybrids (7,11,12 RESULTSEarly cytoplasmic Ad2 RNA hybridizes to four regions of the genome (14, 15) which we (10) located at map positions 1.1-10.6, 61.6-68.1, 76.7-83.7, and 91.5-96.9. The rightward DNA strand contains regions 1 and 3; the leftward strand contains regions 2 and 4. Fig. 1A depicts a rightward Ad2 DNA molecule that carries RNA in region 1 (left end) and in region 3 (right of center). Arrows point to small loops of single-stranded nucleic acid that protrude, at two locations, from the DNA-RNA duplex structures. We have also observed loops in regions 2 and 4 on the leftward strand and a second loop occurring at a different site in region 1. Fig. 1 B and C shows enlargements of the two loops found in region 1. These RNAs have been mapped at positions 1.2-5.5 (region la) and 4.4-11.3 (region lb) with the loops mapping between points 2.9 and 4.0 and 5.9 and 9.7, respectively. The 3' terminus of region la RNA (5.5) overlaps with the 5' terminus of region lb (4.4) RNA. Both of these loops occur infrequently, with RNAs not generating a loop and mapping between positions 1.1 and 10.6 being the predominant RNAs found in this region.Two single-stranded DNA displacement loop...

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Section: Discussionmentioning

confidence: 99%

Loop structures in hybrids of early RNA and the separated strands of adenovirus DNA.

Kitchingman¹,

Lai²,

Westphal³

1977

Proc. Natl. Acad. Sci. U.S.A.

100

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“…Adenovirus serotype 2 (Ad2) (class C) mRNA's have been mapped by a variety of techniques, including hybridization to separated strands of DNA restriction fragments (12,29), fractionation by gel electrophoresis followed by hybridization to restriction fragments (5,9,11,13,23,26), and electron microscopic RNA loop analysis (8,24,25). Surprisingly, its mRNA's have a very complex sequence organization.…”

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confidence: 99%

“…39.1 ± 0.8 (10) 42.9 ± 0.8 (8) 45.5 ± 1.0 (9) 49.1 ± 0.9 (12) 51.4 ± 0.8 (15) 65.0 ± 0.6 (5) 68.2 ± 1.0…”

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confidence: 99%

Comparison of late mRNA splicing among class B and class C adenoviruses

Kilpatrick¹,

Gelinas²,

Broker³

et al. 1979

J Virol

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Adenovirus class B (Ad3 and Ad7) and class C (Ad1, Ad5, and Ad6) late r-strand mRNA's were found to have segmented 5' leaders. These leaders were very similar among serotype within a class but differed in sequence from the leaders on late mRNA's of a different class. However, the leader components of class B viruses mapped at essentially the same map coordinates as those of class C viruses. The 5' coordinates of the main bodies of class B messages to which the tripartite leaders are attached as well as the map positions of several of their early mRNA's were very similar to those of Ad2 transcripts. Infrequent examples of late r-strand polysomal RNAs of Ad3 and Ad7 had, in addition to the three common leader segments, a fourth leader segment derived from RNA encoded at various sites between the second and third leaders. The extra components formed several distinct groups. These molecules are presumably intermediates in the splicing processes that generate mature messages.

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“…This model can also explain why d1712, in which the 951-*2157 and 951-*2880 slices are enhanced early, makes almost exclusively mRNAs with E3B 3'-ends early (8). Arguing against this model are the observations that polyadenylation precedes splicing in adenovirus late pre-mRNA (26) and in at least some early E3 pre-mRNA (14,31). However, polyadenylation is not obligatory for splicing (39) and it is possible that in many pre-mRNAs splicing precedes polyadenylation.…”

Section: Discussionmentioning

confidence: 99%

“…We are interested in adenovirus region E3 (12,13,35) as a model to understand gene organization and regulation, and in particular differential RNA processing, in a "complex" transcription unit (14,25). E3 is located at genomic map position 76 to 86 and is expressed off r strand.…”

mentioning

confidence: 99%

Genetic analysis of mRNA synthesis in adenovirus region E3 at different stages of productive infection by RNA-processing mutants

Bhat¹,

Wold²

1986

J Virol

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Region E3 of adenovirus encodes about nine overlapping mRNAs (a to i) with different spliced structures and with two major RNA 3' end sites termed E3A and E3B. Synthesis of E3 mRNAs was examined by the nuclease-gel procedure at early and late stages of infection by wild-type virus (rec700) and by several E3 deletion mutants. Our results, together with those obtained by electron microscopy (L. T. Chow, T. R. Broker, and J. B. Lewis, J. Mol. Biol. 134:265-303, 1979), suggest that E3 may be differentially regulated at early and late stages at both the promoter and RNA-processing levels. Early after infection, the E3 promoter is used to make mainly mRNAs a and h. Late after infection, the E3 promoter appears to be shut off and the major late promoter is used to make mainly mRNAs d and e. The late L4 mRNA 3' end site is not used early even though early E3 pre-mRNAs transcribe through the L4 RNA 3' end site. The nucleotide 768-951 exon, which is the y leader on many L5 mRNAs, is very abundant late. (Nucleotide + 1 is the major E3 transcription initiation site.) Early after infection, the 951 5' splice site is enhanced 5to 10-fold in the d1712 (A1691 to 2122) group of mutants; late after infection, these mutants resemble the wild type. We speculate that the activity of the 951 5' splice site is regulated at early and late stages; it is suppressed early to permit synthesis of mRNA a, and it is activated late to permit synthesis of mRNAs d, e, and L5. With d1719 (A2173 to 2237), the 951-*2157 splice is enhanced both early and late; we suggest that this deletion enhances the 2157 3' splice site.

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Strand assignment of polyadenylated nuclear RNAs synthesized early in infection with adenovirus 2

Cited by 14 publications

References 17 publications

Loop structures in hybrids of early RNA and the separated strands of adenovirus DNA.

Loop structures in hybrids of early RNA and the separated strands of adenovirus DNA.

Comparison of late mRNA splicing among class B and class C adenoviruses

Genetic analysis of mRNA synthesis in adenovirus region E3 at different stages of productive infection by RNA-processing mutants

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