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Discovery of RNA Splicing and Genes in Pieces PERSPECTIVE Arnold J

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Discovery of RNA Splicing and Genes in Pieces PERSPECTIVE Arnold J PERSPECTIVE Discovery of RNA splicing and genes in pieces PERSPECTIVE Arnold J. Berka,1 Edited by Inder M. Verma, The Salk Institute for Biological Studies, La Jolla, CA, and approved December 29, 2015 (received for review December 18, 2015) The Problem: Short-Lived Heterogeneous of unlabeled nucleoside to the culture medium during Nuclear RNA the chase period. Consequently, a clean chase could During the 1960s and 1970s, there was a major co- not be achieved, and newly synthesized hnRNAs nundrum in eukaryotic molecular biology regarding continued to be labeled as labeled mRNAs first the synthesis of mRNA in animal cells. Pulse and pulse- appeared in the cytoplasm. Also, the small fraction of chase radio-labeling experiments with tritiated uridine pulse-labeled nuclear RNA converted to more stable had shown that a large fraction of nuclear RNA was mRNAs, only 5–10%, added to the difficulty of proving degraded very rapidly after synthesis, whereas only a a precursor product relationship from pulse-chase small fraction of the initially synthesized RNA was labeling experiments. exported from the nucleus to the cytoplasm where it The synthesis and processing of ribosomal RNAs functions as far more stable mRNAs (1, 2). This short- was much easier to analyze than processing of lived nuclear RNA ranged in lengths up to several tens hnRNAs because about 50% of all nuclear RNA syn- of kilobases, much longer than most cytoplasmic thesis in growing mammalian cells in culture is syn- mRNAs, and was referred to as heterogeneous nuclear thesis of the single ∼13.7-kb pre-rRNA precursor. RNA (hnRNA) (3, 4). Pulse-chase labeling yielded re- Moreover, this full-length pre-rRNA precursor accu- sults consistent with hnRNA functioning as a precursor mulates to significant levels before it is processed into of mRNA. hnRNA is rapidly labeled, whereas label stable mature rRNAs of 18S (1,870 bases), 5.8S (156 appeared in mRNAs more slowly. Also, as for mRNA, a bases), and 28S (5,034 bases), so that the fraction of large fraction of hnRNA was shown to be poly- pre-RNA processed into stable cytoplasmic rRNAs is adenylated at its 3′ end like mRNA, consistent with the ∼50%, much more than the 5–10% of pulse-labeled model that RNA sequences near the 3′ end of hnRNAs hnRNA processed into mRNAs. This fortuitous situa- are retained in shorter mRNAs exported to the cyto- tion for the study of prerRNA by pulse-chase labeling plasm (5, 6). Also, like mRNAs, hnRNAs were found to made it possible to show that the rRNAs were pro- have the 7-methyl guanine 5′–5′ phosphotriester cessed by the cleavage of the pre-RNA precursor into “cap” structure at their 5′ ends, like mRNAs. Re- the stable rRNAs that were transported to the cyto- markably, the methyl groups in hnRNA cap struc- plasm in nearly fully mature ribosomal subunits. So- tures appeared to be conserved as RNA was exported called “transcribed spacer” sequences between the to the cytoplasm, even though, on average, hnRNA stable rRNAs in the pre-rRNA precursor were rapidly molecules in the nucleus are at least four times longer degraded after they were generated by cleavage of than mRNAs in the cytoplasm (7, 8). However, it was the pre-rRNA. not possible to prove rigorously from the flow of ra- Because of this precedent for the processing of a diolabel in pulse-chase labeling experiments that large pre-rRNA precursor into the mature rRNAs by hnRNAs are precursors to mRNAs. There were two simple cleavage of the pre-rRNA precursor, the pre- principal reasons for this. First, the pool of ribonucle- dominant hypothesis for the synthesis of mRNAs from oside triphosphates in mammalian cells is so large that longer hnRNAs was that they were similarly cleaved it takes more than 30 min to saturate the pools when out of larger hnRNA molecules. However, what would labeled nucleosides are added to the media and specify where each postulated pre-mRNA precursor is equally long to dilute the intracellular pool of labeled cleaved to generate mRNAs much more stable than nucleoside triphosphates by addition of a large excess the precursor hnRNAs? Other significant questions aMolecular Biology Institute, Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095 Author contributions: A.J.B. wrote the paper. The author declares no conflict of interest. This article is a PNAS Direct Submission. This article is part of the special series of PNAS 100th Anniversary articles to commemorate exceptional research published in PNAS over the last century. See the companion article, “Spliced segments at the 5′ terminus of adenovirus 2 late mRNA,” on page 3171 in issue 8 of volume 74. 1Email: [email protected]. www.pnas.org/cgi/doi/10.1073/pnas.1525084113 PNAS | January 26, 2016 | vol. 113 | no. 4 | 801–805 Downloaded by guest on September 28, 2021 were raised by the observation of unstable hnRNA. For example, level. It was hoped that studies of Ad2 gene expression would be do the many unstable hnRNA sequences removed from hnRNAs similarly revealing about mechanisms controlling gene expression during their processing have specific functions? and mRNA synthesis in animal cells. Importantly, Ad2 had also To address these questions and other aspects of mRNA syn- been shown to express short-lived, high-molecular-weight nuclear thesis and processing, many researchers turned to the study of RNAs containing the sequences of shorter, more stable viral DNA viruses that infect animal cells in culture. Detailed analysis mRNAs (9). of gene expression by bacteriophage (bacterial viruses) had re- vealed (and continues to reveal) insights into the mechanisms of The Experiment gene expression and its control in bacteria. In the days before One of the first goals in the analysis of Ad2 mRNA synthesis was to molecular cloning of cellular genes (around the late 1970s), it was map the regions of the viral genome encoding the abundantly very difficult to analyze the expression of individual cellular genes expressed viral mRNAs for virion structural proteins expressed late (aside from pre-rRNA) because a large number of cellular genes in infection. This was of interest because in bacteria, DNA se- (>10,000) are all expressed at the same time and because it was quences near the transcription start sites for bacterial mRNAs not yet possible to obtain DNA clones of individual mammalian function as promoters where bacterial RNA polymerase binds genes. DNA viruses express a much smaller number of genes, and DNA and initiates transcription. Other bacterial DNA-binding the purified template DNAs encoding them could be isolated proteins that bind sequences within a few tens of base pairs from from purified virion particles. A worldwide group of researchers promoters regulate the frequency of transcription initiation. It was chose human adenovirus 2 (Ad2) and the closely related human ′ adenovirus 5 as model systems for studying mRNA synthesis in anticipated that DNA sequences near the 5 ends of regions animal cells. These viruses had the advantage that they have a encoding the abundant late viral mRNAs might also harbor pro- genome of ∼36 kb and can be readily grown in large amounts in moter and other regulatory sequences in Ad2 DNA. In the 1970s, cultured cells so that milligram amounts of the viral genome could methods had been developed for observing regions of RNA base be isolated from purified virions. Also, at 36 kb, the Ad2 genome paired to a longer DNA molecule by electron microscopy (EM) of was similar in size and therefore potential biological complexity to the hybrid molecules. The resolution of the method allowed the genomes of bacteriophages T7 and λ. T7 and λ had provided mapping of the ends of the RNA-DNA hybrid region to within an intriguing examples of gene control for analysis at a molecular accuracy of about 100 bp on the viral genome. Fig. 1. Single-stranded RNA tails observed at both 5′ and 3′ ends of R-loops of Ad2 hexon mRNA and the Ad2 HindIII A fragment. (Upper) R-loop formation near the temperature of DNA melting in 70% formamide, 0.4 M NaCl. Arrowheads indicate 3′ ends of strands. Adapted from ref. 10. (Lower) R-loop in Ad2 HindIII A restriction fragment formed with purified hexon mRNA. (A) EM of R-loop showing all of the Hind III A DNA fragment with an R-loop at the lower end. (B) Higher magnification of the R-loop. In A and B, the upper arrow points to a single-stranded region at the 3′ end of the RNA, and the bottom arrow points to a single-stranded region at the 5′ end. In these formamide spreads, single-stranded DNA appears thinner and has a more irregular path than double-stranded DNA or RNA-DNA hybrid, which appear similar. (C) Interpretation of EM in B. The hexon mRNA is represented in red and each strand of the Hind III A DNA in black. Regions where the red RNA is parallel to the black DNA strand represent base paired regions of RNA-DNA hybrid. Regions where the two black DNA strands are parallel represent DNA-DNA duplex. A, B,andC adapted from ref. 12. 802 | www.pnas.org/cgi/doi/10.1073/pnas.1525084113 Berk Downloaded by guest on September 28, 2021 When I arrived to begin my postdoc in Phil Sharp’s laboratory RNA to a DNA fragment above the melting temperature of the in the summer of 1976, Sue Berget, another postdoc who had DNA in 80% formamide and 0.4 M NaCl, but below the 9 °C higher started in the laboratory about a year earlier, was working on melting temperature of the RNA-DNA hybrid, allowed formation of mapping the Ad2 genome location encoding the most abun- RNA-DNA hybrid in the absence of hybridization of the comple- dantly expressed Ad2 mRNA, the mRNA for the major virion mentary DNA strands.
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