Finding the Tail End: the Discovery of RNA Splicing PROFILE Melissa Suran, Science Writer

PROFILE

Finding the tail end: The discovery of RNA splicing PROFILE Melissa Suran, Science Writer

Major findings are sometimes hidden in small details. Molecular biologists at the time worked almost At least that was the case when, in a PNAS article, exclusively with bacterial systems, which are easy to molecular biologist Phillip Sharp and his research grow in a laboratory. Although Sharp primarily studied team described a little strand of RNA that led to an bacteria and published work about the Escherichia coli understanding of how proteins are synthesized in genome during his postdoctoral position at the cells (1). California Institute of Technology with biochemist During the 1970s, Sharp headed a laboratory in the Norman Davidson (2, 3), he started exploring tumor Center for Cancer Research at the Massachusetts biology and virology. When Sharp arrived at CSHL, he turned his attention toward DNA viruses known to in- Institute of Technology (MIT). Having been a post- fect animal cells. He was particularly curious about doctoral researcher for geneticist James Watson and gene expression—the conversion of DNA into instruc- then a staff member at Cold Spring Harbor Laboratory tions for creating proteins—in human cells and began (CSHL) prior to his appointment at MIT, Sharp was studying the transcriptional profile of a simian DNA drawn to studying genes and measuring chromosome virus called SV40. Found in both humans and mon- sizes. The field of genetics was relatively primitive; keys, SV40 can generate tumors. Sharp’s work with Watson and Francis Crick had discovered DNA’s struc- the virus stemmed from a collaboration with virolo- ture only 2 decades earlier. gist Joseph Sambrook. Meanwhile, an officemate, Ulf

Fig. 1. Members of the MIT Center for Cancer Research (Robert Weinberg, Second Row from Bottom, Far Left; Susan Berget, Third Row from Bottom, Third from Left; Claire Moore, Back Row, Fourth from Left; Philip Sharp, Back Row, Far Right). Image courtesy of Robert Weinberg.

Published under the PNAS license. See Classic Article “Spliced segments at the 5′ terminus of adenovirus 2 late mRNA” on page 3171 in issue 8 of volume 74.

www.pnas.org/cgi/doi/10.1073/pnas.1919416116 PNAS Latest Articles | 1of4 Downloaded by guest on September 26, 2021 Fig. 2. (A–C) Electron micrograph of a hexon mRNA hybridized to an adenovirus genome fragment. The arrows in the micrographs mark the single-stranded RNA tails at the ends of the RNA/DNA hybrid. The 5’ to 3’ orientation of the mRNA is indicated in C, and the displaced single-stranded viral DNA is shown as a thin line. Reproduced with permission from ref. 1.

Pettersson, worked on DNA replication of adenovirus, a member of the Whitehead Institute for Biomedical a common virus with a double-stranded DNA genome Research and professor of biology at MIT. that is known to cause tumors in rodents and a range In fact, Weinberg adds, not all groups at the center of illnesses in humans. focused directly on cancer as a disease. “The MIT “Ulf and I became friends,” Sharp says. “He was cancer center was built on the notion that curiosity- interested in adenovirus DNA replication; I was interested driven research is likely to yield great benefits.” in adenovirus transcriptional activities and the location And it did. During the 1970s and 1980s, MIT’s of genes.” Center for Cancer Research was a research powerhouse A few years earlier, microbiologists Daniel Nathans with notable scientists, including David Baltimore and and Hamilton Smith discovered enzymes called re- Susumu Tonegawa, who won Nobel Prizes for discov- striction endonucleases (4–6), which earned them a ering reverse transcriptase and the rearrangement of shared Nobel Prize with Werner Arber. Following their antibody genes, respectively. insights, Sharp developed a method to purify restriction Around 1975, Weinberg organized a weekly meeting for the center’s fifth floor laboratories to discuss their re- enzymes using gel electrophoresis and ethidium bro- search. Little did Sharp know that those meetings would mide staining. Eventually, Sharp, Pettersson, and others ultimately play an integral role in a landmark discovery. made restriction maps of the adenovirus 2 and 5 ge- In 1976, postdoctoral fellow Susan Berget began nomes as well as several other serotypes. These maps using electron microscopy to examine the relationship were used to identify the viral regions that contained between cytoplasmic RNA and the DNA structure of cancer-causing genes. adenovirus. Both Sharp and Berget worked closely “The transcriptional pattern was important for un- with Claire Moore, a technician who ran MIT’s electron derstanding how the virus created tumors,” Sharp microscopy facility for the cancer center. Through her says. “It also set the table for a long-term curiosity: work with Sharp, Moore became familiar with a tech- ” The significance of heterogeneous nuclear RNA. nique called R-loop analysis, which at the time was a ’ When Sharp was recruited by MIT s cancer center, new method to map RNA sequences on a genome. By he and postdoctoral fellow Jane Flint extended the creating optimal conditions using a combination of ’ transcriptional maps of adenovirus. One of Sharp s salt, formamide, and heat, Moore could make an colleagues, molecular biologist Robert Weinberg, RNA strand hybridize with its complementary DNA. ran an adjacent laboratory on the fifth floor and stud- “You would see a bit of string, and there would be ied SV40. Both adenovirus and SV40 can produce a bubble where the RNA had hybridized with the DNA multiple copies of their genomes in infected cells, and displaced the other strand,” says Moore, now a enabling analysis. professor of developmental, molecular, and chemical “The reason that Phil Sharp, and I, and others biology at Tufts University. “Phil thought that would worked on DNA tumor viruses, like adenovirus and be a great way to map the adenovirus because you SV40, was actually not because we were interested could precisely map the location of each gene.” in cancer per se, but because these viruses clone their Adenovirus replicates efficiently, so when Berget own genomes for us,” says Weinberg, who is currently infected a human cell line, the majority of the messenger

2of4 | www.pnas.org/cgi/doi/10.1073/pnas.1919416116 Suran Downloaded by guest on September 26, 2021 RNA (mRNA) molecules would be of viral origin; as its name suggests, mRNA serves as an intermediary tem- plate, transmitting information from DNA to proteins. Berget purified the most abundant viral mRNAs, which encode a capsid protein, and in collaboration with Moore, subjected the mRNAs to hybridization conditions in order to form R-loops with specific restriction endonuclease fragments of the adenovirus genome. The resulting R-loops were visualized using electron microscopy, and the length of the R-loops and double-stranded DNA were measured to de- termine the gene’s location. “My task was to prepare the samples,” Moore says. “There were a lot of tricks to getting a good spread.” With a viscous solution of formamide and a ramp constructed from a slide that ran into another solution, Moore had to drop a sample on the slide at just the right place so it would spread onto a film. Once Berget and Moore collected the film on a tiny grid coated with plastic, they processed it for electron microscopy. This film was subsequently inserted into the electron microscope and examined for interpretable R-loop structures that were then photographed. To get accu- rate measurements, the micrographs were photograph- ically printed, and a “little map measurer,” as Moore puts it, was traced along each strand. Throughout this process for dozens of micrographs, something seemed off. “I was getting nice, uniform R-loops, but there were these little strands of single-stranded RNA stick- ing out at the ends of the R-loops, and I didn’t know what to do with them,” Moore says. Other laboratories had previously found that adenovirus RNA in the nucleus was far longer than cytoplasmic mRNA. The team wondered if these long, viral RNAs were related to observations of cellular heterogeneous nuclear RNAs. Similar to adenovirus RNAs, these nuclear RNAs containing gene sequences were considerably longer than more stable mRNAs in Fig. 3. (A) Phillip Sharp receives the 1993 Nobel Prize in Physiology or Medicine the cell cytoplasm. from King Carl XVI Gustaf of Sweden. Image courtesy of Tobbe Gustavsson/ Berget, Moore, and Sharp concluded that one of Reportagebild/TT/Sipa USA. (B) Richard Roberts receives the 1993 Nobel Prize in the extensions must be a polyadenosine tail that is Physiology or Medicine from King Carl XVI Gustaf of Sweden. Image courtesy of AGIP/Rue des Archives/Granger, NYC. added posttranscriptionally to the end of mRNA and has nothing with which to hybridize. Assuming that the other tail was an artifact, possibly caused by DNA But 3 months and several experiments later, the rehybridizing and displacing RNA, the team con- extra tail was still a mystery. ducted a myriad of experiments. They eliminated “It was a puzzle until Sue presented the data at one the opposite DNA strand so nothing could compete of the floor meetings, and we got the idea that maybe with the end of the RNA. However, when the RNA it’s coming from a different region of the adenovirus,” hybridized to a single-stranded bit of DNA, the tail was Moore says. still present. Even trying different conditions of form- So the team tried hybridizing the RNA to a longer amide and salt proved unsuccessful. Nevertheless, piece of DNA. Sharp and his colleagues were determined to rule out “That’s when the little tail found its partner strands other explanations for the tail before proceeding. ” “Our first indication that there was something we and pulled the DNA into the loops, Moore says. didn’t understand was when we were doing the mi- Although Sharp was unsure what to anticipate, he “ ” croscopy of mapping the adenovirus mRNA on the recalls: When I saw those 3 loops, I knew what it was. genome, and we found this tail at the 5′ end of the It was the answer to the heterogeneous nuclear RNA,” Sharp says. “It didn’t behave as if it were part of RNA mystery. Berget, Moore, and Sharp had discov- the sequences immediately adjacent to the genome, ered RNA splicing. and we spent 3 months mostly trying to convince our- This stage in gene transcription, or the process by selves that it wasn’t an artifact.” which DNA information is transcribed into mRNA, is

Suran PNAS Latest Articles | 3of4 Downloaded by guest on September 26, 2021 similar to a message that needs to be decoded be- genes are needed to create various proteins via a pro- cause of a sentence with extra letters. Once those cess called alternative splicing. Similar to how rearrang- unnecessary letters that make the sentence seem like ing a group of letters can form different words, the gibberish are removed, a clear message becomes exons of a single gene can reorder to generate a mul- apparent. In the case of mRNA, the message conveys titude of proteins. directions to guide synthesis of proteins. Sharp compares discovering RNA splicing to find- The human genome has an estimated tens of ing the Rosetta Stone. His revelation earned him the thousands of genes that provide instructions for pro- 1993 Nobel Prize in Physiology or Medicine, which he ducing an even larger number of proteins, which are shared with Richard Roberts (8), a molecular biologist created via RNA splicing. When a gene is copied into who independently made the same discovery and RNA, it contains a long, jumbled message of nucleo- published the results in a 1977 issue of Cell (9). tide sequences called exons and introns. While exons While at CSHL, Roberts and his colleague Richard compose a message with specific instructions from a Gelinas focused their research on promoters, the particular gene, they are separated by introns that are areas of DNA where gene transcription into mRNA interspersed in the RNA, rendering the message in- begins. They specifically studied adenovirus to de- comprehensible. Introns must be spliced out so exons termine whether bacterial and eukaryotic promoters can form a coherent message called a mature mRNA. share the same sequence characteristics. This is the genetic information used to synthesize “Our experiments gave us results that didn’t make proteins in the cytoplasm. Thus, the concept of RNA sense if eukaryotic genes were the same as bacterial splicing explains why nuclear mRNA is longer than genes,” says Roberts, now the chief scientific officer at cytoplasmic RNA. New England Biolabs. “I came up with the idea for an The evidence for RNA splicing, which was pub- electron microscopy experiment that would shed light lished in PNAS in 1977, was groundbreaking (1). ’ “There were people who were tantalizingly close on all of the biochemistry we d been doing and de- to discovering splicing, but being close is not good vised the experiment on a Saturday morning; the ex- enough,” Weinberg says. “You either discover it, or periment was carried out by Louise Chow on a Tuesday you don’t. And Phil did.” morning, and by Tuesday afternoon, splicing was uni- ” Before knowledge of RNA splicing, the consensus versally accepted as being discovered. was that all organisms have the same gene structure as But Sharp believes it was just a matter of time before bacteria, which lack introns. French biochemist Jacques RNA splicing would become known to the world. “ ” Monod, who received the 1965 Nobel Prize in Physiol- Somebody would have made the discovery, he ogy or Medicine, famously asserted that “anything says. “We were fortunate to have made it first.” found to be true of E. coli must also be true of ele- Sharp, who is still a professor at MIT, remains in phants” (7). However, RNA splicing demonstrated that awe of what he accomplished with Berget and Moore eukaryotic cells, with their discontinuous genes, are far more than 40 years ago. “To have the privilege of more complex than bacterial cells. It also debunked the being part of discoveries that advance our under- dogma that one gene produces one mRNA, and all standing and our ability to help people is such an mRNAs from a gene produce one protein. Only a few extraordinary, extraordinary experience.”

1 S. M. Berget, C. Moore, P. A. Sharp, Spliced segments at the 5′ terminus of adenovirus 2 late mRNA. Proc. Natl. Acad. Sci. U.S.A. 74, 3171–3175 (1977). 2 P. A. Sharp, M. T. Hsu, E. Otsubo, N. Davidson, Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. I. Structure of F-prime factors. J. Mol. Biol. 71,471–497 (1972). 3 P. A. Sharp, S. N. Cohen, N. Davidson, Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. II. Structure of drug resistance (R) factors and F factors. J. Mol. Biol. 75,235–255 (1973). 4 K. Danna, D. Nathans, Specific cleavage of simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae. Proc. Natl. Acad. Sci. U.S.A. 68, 2913–2917 (1971). 5 H. O. Smith, K. W. Wilcox, A restriction enzyme from Hemophilus influenzae. I. Purification and general properties. J. Mol. Biol. 51, 379–391 (1970). 6 T. J. Kelly, Jr, H. O. Smith, A restriction enzyme from Hemophilus influenzae. II. J. Mol. Biol. 51, 393–409 (1970). 7 H. C. Friedmann, From “butyribacterium” to “E. coli”: An essay on unity in biochemistry. Perspect. Biol. Med. 47,47–66 (2004). 8 NobelPrize.org, The Nobel Prize in Physiology or Medicine 1993. https://www.nobelprize.org/prizes/medicine/1993/summary/. Accessed 25 September 2019. 9 L. T. Chow, R. E. Gelinas, T. R. Broker, R. J. Roberts, An amazing sequence arrangement at the 5′ ends of adenovirus 2 messenger RNA. Cell 12,1–8 (1977).

4of4 | www.pnas.org/cgi/doi/10.1073/pnas.1919416116 Suran Downloaded by guest on September 26, 2021