{"title":"The Journey","authors":"D. Rosenthal, S. Moore","doi":"10.4324/9781351169882-2","DOIUrl":null,"url":null,"abstract":"Twenty years of the journal RNA, 20 years of RNA research— the journal has witnessed and facilitated an explosive growth of the RNA field, a relatively young field in many ways. Having studied alternative splicing for more than 20 years, it is a great time for me to pause and reflect on what we have learned and still need to learn on this particular topic. There was heightened excitement when the first issue of RNA was making its way to Sue Berget’s laboratory at Baylor College of Medicine where I was conducting part of my postdoctoral research. Only, the excitement was not caused by the anticipated arrival of the journal, although the arrival of this new journal was indeed discussed repeatedly in the laboratory, but by another bigger than usual cleanup of a radioactive material spill in our room specifically reserved for generating and using in vitro RNA splicing substrates. Sue’s was one of the hardcore laboratories that used biochemical means to study the mechanism of splicing, which drewme to her laboratory. When I was a graduate student studying splicing in plants, I was always envious of people who conducted in vitro biochemical splicing analysis, as it was not feasible to use such an approach in plant systems due to the difficulty of preparing splicing-competent nuclear extracts from plant cells. In Sue’s laboratory, a standing order of the radioisotope P-UTP guaranteed that one vial of 1mCi of P-UTP was delivered to the laboratory every week, which could result in 20 in vitro transcribed RNA substrates. As such, radioactive RNA substrates were generated almost every day, which unavoidably led to the occasional bigger than usual decontamination buzz. Another buzzing activity that usually involved the whole laboratory, students, postdoctoral fellows, and technicians alike, occurred on the nuclear extract making days, which happened approximately once a month. On those days, 100 L (sometimes twice as much) worth of HeLa cell pellets would arrive at our laboratory from a company in Minnesota. Everybody worked hard as a team on those days for a whole day following Sue while she was shouting out orders. At the end of the day, wewould have 100mL of pure nuclear extracts at 10–15 mg/mL, which, if tested splicing-competent, would last us a couple of months. Those were some of my most vividly memorable good old days in Sue’s laboratory. When the first issue of RNA did arrive, we pored over the articles. Being in a splicing laboratory, we obviously read very carefully the article from Paula Grabowski’s laboratory that used in vitro splicing analysis to reveal a role of exon enhancers in promoting U2AF binding at the polypyrimidine tract. Looking back, this article represents the state of the approach at the time for mechanistic studies of splicing. The core questions asked by the investigators in the splicing field were what and how the sequence elements located on pre-mRNA and RNA-binding proteins (RBPs) that act in trans regulate splicing. To answer these questions, typically, one generated in vitro transcribed splicing substrate containing wild type or mutated sequence elements and carried out splicing assays in a test tube in which a potential splicing regulator was added or depleted from the nuclear extracts. In parallel, experiments were carried out using splicing reporters and protein expression vectors through cell transfection techniques. While it was relatively easy to identify sequence elements through deletion/mutation analysis, the bottleneck was identifying the trans-acting protein factors that recognize and interact with the sequence elements. The heroic classical biochemical purification approaches, i.e., with investigators spending many hours in cold rooms, led to the identification of a number of splicing factors. These elegant studies, combined with the power of yeast genetics, have built our knowledge foundation of splicing, a process carried out by the spliceosome that is one of the most complex macromolecular machines in eukaryotic cells. Studies of alternative splicing using in vitro biochemical approaches met with more difficulties due to an inherent nature of alternative exons, being surrounded by 3′ and 5′ splicing signals that can deviate significantly from the consensus sequences, which are recognized and bound by spliceosomal components. The presence of these sub-optimal splicing signals makes the already inefficient in vitro splicing system almost not suitable to study alternative splicing events. One had to become a true artist in making “super duper” splicing-competent nuclear extract and extremely “hot” and clean","PeriodicalId":211110,"journal":{"name":"The Psychology of Retirement","volume":"47 1","pages":"0"},"PeriodicalIF":0.0000,"publicationDate":"2018-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"The Psychology of Retirement","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.4324/9781351169882-2","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
Abstract
Twenty years of the journal RNA, 20 years of RNA research— the journal has witnessed and facilitated an explosive growth of the RNA field, a relatively young field in many ways. Having studied alternative splicing for more than 20 years, it is a great time for me to pause and reflect on what we have learned and still need to learn on this particular topic. There was heightened excitement when the first issue of RNA was making its way to Sue Berget’s laboratory at Baylor College of Medicine where I was conducting part of my postdoctoral research. Only, the excitement was not caused by the anticipated arrival of the journal, although the arrival of this new journal was indeed discussed repeatedly in the laboratory, but by another bigger than usual cleanup of a radioactive material spill in our room specifically reserved for generating and using in vitro RNA splicing substrates. Sue’s was one of the hardcore laboratories that used biochemical means to study the mechanism of splicing, which drewme to her laboratory. When I was a graduate student studying splicing in plants, I was always envious of people who conducted in vitro biochemical splicing analysis, as it was not feasible to use such an approach in plant systems due to the difficulty of preparing splicing-competent nuclear extracts from plant cells. In Sue’s laboratory, a standing order of the radioisotope P-UTP guaranteed that one vial of 1mCi of P-UTP was delivered to the laboratory every week, which could result in 20 in vitro transcribed RNA substrates. As such, radioactive RNA substrates were generated almost every day, which unavoidably led to the occasional bigger than usual decontamination buzz. Another buzzing activity that usually involved the whole laboratory, students, postdoctoral fellows, and technicians alike, occurred on the nuclear extract making days, which happened approximately once a month. On those days, 100 L (sometimes twice as much) worth of HeLa cell pellets would arrive at our laboratory from a company in Minnesota. Everybody worked hard as a team on those days for a whole day following Sue while she was shouting out orders. At the end of the day, wewould have 100mL of pure nuclear extracts at 10–15 mg/mL, which, if tested splicing-competent, would last us a couple of months. Those were some of my most vividly memorable good old days in Sue’s laboratory. When the first issue of RNA did arrive, we pored over the articles. Being in a splicing laboratory, we obviously read very carefully the article from Paula Grabowski’s laboratory that used in vitro splicing analysis to reveal a role of exon enhancers in promoting U2AF binding at the polypyrimidine tract. Looking back, this article represents the state of the approach at the time for mechanistic studies of splicing. The core questions asked by the investigators in the splicing field were what and how the sequence elements located on pre-mRNA and RNA-binding proteins (RBPs) that act in trans regulate splicing. To answer these questions, typically, one generated in vitro transcribed splicing substrate containing wild type or mutated sequence elements and carried out splicing assays in a test tube in which a potential splicing regulator was added or depleted from the nuclear extracts. In parallel, experiments were carried out using splicing reporters and protein expression vectors through cell transfection techniques. While it was relatively easy to identify sequence elements through deletion/mutation analysis, the bottleneck was identifying the trans-acting protein factors that recognize and interact with the sequence elements. The heroic classical biochemical purification approaches, i.e., with investigators spending many hours in cold rooms, led to the identification of a number of splicing factors. These elegant studies, combined with the power of yeast genetics, have built our knowledge foundation of splicing, a process carried out by the spliceosome that is one of the most complex macromolecular machines in eukaryotic cells. Studies of alternative splicing using in vitro biochemical approaches met with more difficulties due to an inherent nature of alternative exons, being surrounded by 3′ and 5′ splicing signals that can deviate significantly from the consensus sequences, which are recognized and bound by spliceosomal components. The presence of these sub-optimal splicing signals makes the already inefficient in vitro splicing system almost not suitable to study alternative splicing events. One had to become a true artist in making “super duper” splicing-competent nuclear extract and extremely “hot” and clean