Nobel Laureate Assignment: Craig Mello. From an early age, Craig Mello was an explorer. He spent much of his

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1 1 Brett Habermehl December 4, 2014 BIO Nobel Laureate Assignment: Craig Mello From an early age, Craig Mello was an explorer. He spent much of his childhood exploring nature and playing in the creek in his backyard. He would turn over stones looking for small animals and other interesting things. To this day, he still has this same wants and needs for exploration by continuing to turn over stones, hoping to find something new. This early interest in exploration and the world around him lead Craig Mello to winning the Noble Prize for their discovery of RNA interference (RNAi) gene silencing by double-stranded RNA (shared with Andrew Fire) in 2006 (NobelPrize.org, 2007). Mello was born in New Haven, Connecticut on October 18, 1960 to a paleontologist father and an artist mother. Not long after his birth, the family moved to Fairfax, Virginia, where his father started working for the Smithsonian Museum of Natural History. Mello would often go on trips with his family in the mountainous regions of Colorado, Wyoming, and more frequently Virginia where he would hike, search for fossils, and explore (Marquis Who s Who, 2007). He attributes his ability to learn and debate to discussions with his family around the dinner table. From an early age, he understood that science is grounded on dialogue. It was from these discussions that prompted his decision to become a scientist (Mello, 2007). Mello received his undergraduate Bachelor of Science degree in Biochemistry from Brown University in After Brown, Mello attended University of Colorado-Boulder for graduate school. While there, he was first introduced to C.

2 2 elegans in the laboratory of Dr. David Hirsh. He was also introduced to the practice of molecular biology by Dan Stinchcomb (Marquis Who s Who, 2007). His research included identifying and understanding essential functional DNA elements that direct the replication and partitioning of chromosomes (replication origins and centromeres) and using them to produce stable artificial chromosomes for worm molecular genetics. During that time, work in yeast had identified such elements, but had not been done for C. elegans (Mello, 2007). After his first year at Boulder, Dr. David Hirsh left academia for an industry position, so Mello chose to move to Harvard University. Dan Stinchcomb was starting up a lab at Harvard, so Mello was able to continue his research there (Marquis Who s Who, 2007). During his time at Harvard, Mello learned how important it is to focus on ideas from every conceivable angle. In his research, by focusing on identifying worm centromere activities using yeast as a model system, he ended up learning on the yeast centromere and not the worm centromere. To study the yeast centromere, one should be working with yeast sequences. In order to study the worm centromere, Mello recognized that he should be injecting DNA into the worm. He realized his flaw, which changed his experiments and further enhanced his research (Mello, 2007). After graduating from Harvard, Mello started working at the Fred Hutchison Cancer Research Center in Seattle, Washington, where he started better understanding genetics. He was able to identify genes that act as regulators of early development in C. elegans. It actually turned out that some of the genes he studied during this time were connected to RNAi-related mechanisms in ways that are

3 3 currently being researched. Not long after, Mello learned of an antisense RNA injection technique that was able to silence target genes. He started using this technique to identify the functions of specific genes, especially since the genomesequencing project for C. elegans had revealed homologs for developmental function to genes Mello had earlier discovered. This technique significantly accelerated Mello s studies. However, even though this accelerated Mello s studies, the technique did not always deliver properly (Mello, 2007). In 1995 Mello joined the University of Massachusetts Medical School as a professor of molecular medicine and as a researcher. It was at the Fed Hutchison Cancer Research center where Mello met Andrew Fire. Due to both of them working on developing techniques for DNA transformation in worms, they often worked together through much of the 1990s. Between the two of them, they made DNA transformation a routine procedure for worms (Mello, 1995). This led to their longtime collaboration and combined research on RNA interference and later led to both of them winning the Nobel Prize. Mello and Fire received the Nobel Prize in Physiology or Medicine in 2006 for their discovery of RNA interference gene silencing by double stranded RNA (NobelPrize.org, 2007). They discovered a new fundamental RNA trigger mechanism that controls the flow of genetic information. In their paper, they reported a discovery that dsrna introduced into the cell can suppress gene expression in a potent sequence specific manner. The dsrna is able to produce interference to endogenous genes more so than either single strand individually (as shown by using sense mrna and antisense RNA). The process was shown to be an

4 4 active response and systemic, due to the small amounts of dsrna needed to affect a cell and that RNA injected anywhere in the body, or even by ingestion, can get into all the tissues (which was actually discovered due to a graduate student accidently injecting the RNA into areas other than the germline) (Fire et al., 1998), (Mello, 2007). The dsrna is cut and one of the RNA strands then base pairs with a complementary messenger RNA and elicits degradation of the message and the corresponding protein cannot be synthesized. RNAi has thus silenced the gene (Mello, 2014). Mello and Fire s paper lead to an explosion of research in similar fields and topics all over the world and it was discovered that RNAi would be of exceptional significance. RNAi machinery cannot only handle dsrna that enters the cell, but also dsrna that is generated within the cell. The development of an organism and the proper function of its cells and tissues are dependent on an intake RNAi machinery. Further research has led to find normal function of RNAi might defend cells against damaging effects of transposons and other foreign elements. Infection by RNA viruses can blocked by RNAi and foreign elements in the genome can be kept silent (Siomi and Siomi, 2009). The discovery of RNAi has also provided us with a powerful new tool to study the functions of genes. It is very apparent that it has become a valuable tool in molecular biology because synthetic dsrna introduced into cells can selectively and robustly induce suppression of specific genes of interest in a variety of model organisms. It can be used for large-scale screens that can shut down each gene in a specific cell, which helps to identify components necessary for a cellular process

5 5 involved with that cell (Mello, 2014). RNAi improved upon earlier techniques (such as the antisense RNA technique mentioned before) in that it was able to better target specific cells and that it is a very potent mechanism since only a few dsrna molecules are needed for effective interference. Also, the mechanism is able to cause interference in cells and tissues far removed from the site of introduction. RNAi was described as a workhorse technique for basic research due to the fact that it is very easy to get it to work (Bender, 2014). The discovery of RNAi also gave promise for its use in medicine. RNAi can prevent proteins that are triggering an illness from ever being translated, which avoids the need to attack the disease further downstream. RNAi also has the ability to target proteins that cannot be reached by some antibodies due to their intracellular location and unresponsiveness to drugs. Even though there was much excitement surrounding RNAi as a treatment in the early years after it s discovery, there was little success found due to technical difficulties. However, in recent years, RNAi treatment has been showing some promise in addressing liver disease, viral infections, and certain types of cancer (Bender, 2014). Since winning the Nobel Prize, Mello still is involved in much research dealing with RNAi. He has been investigating and characterizing genes that mediate RNAi. He also investigates how embryonic cells differentiate and communicate during development (Howard Hughes Medical Institute, 2009). Mello has also received a number of honors. Mello and Fire were named to the National Academy of Sciences in 2005 and were awarded the Ludwig Darmstaedter Prize in 2006, which is one of the most internationally renowned awards in the field of medicine.

6 6 He also founded RXi Pharmaceuticals (now called Galena Biopharma) in 2006 and has been its Chairman of the Scientific Advisory Board since 2007 (Bloomberg BusinessWeek, 2014). Primary Paper Analysis Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., Mello, C.C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 19, This paper was chosen for the primary paper analysis and was instrumental for Craig Mello and Andrew Fire being awarded the Nobel Prize because of its announcement of a discovery of RNA interference, a fundamental mechanism for controlling the flow of genetic information. The paper describes the mechanism that interferes with mrna on a specific gene through a process. It was known before this time that RNA played a key role in gene silencing (as shown by sense and antisense single stranded RNA), but it wasn t until the release of this paper that an explanation was provided. RNAi became an important molecular technique for silencing genes and shows promise as a medical application. In the paper, the main question that was being explored was the difference between native RNA and RNA molecules being inserted into the cell for interference. Both sense and antisense single stranded RNAs and double stranded RNAs were prepared to test whether dsrna might contribute to interference. C. elegans, a nematode often used in genetics studies, were being used as the model organism.

7 7 The unc-22 gene was chosen for targeting due to it encoding a large amount, but nonessential myofilament proteins. The phenotype of loss unc-22 produces a severe twitch in the next generation of the affected sample. It was shown that antisense and sense RNAs each alone had shown slight inference activity, but a sense-antisense mixture caused a very high affect in interference with endogenous gene activity. Mello and Fire postulated that dsrna silences genes at a much higher level due to the fact that when sense and antisense RNA meet they form dsrna. To test this postulate, gel electrophoresis was used to show that the injection was primarily double stranded. After this process, the dsrna was found to retain potent interfering activity. Antisense and sense RNA were injected again with a long interval between the injections. This resulted in a great decrease in interfering activity demonstrating that single stranded RNA may degrade without the opposite strand. Three other genes that have distinct phenotypes, unc-54, fem-1, and hlh-1, were also targeted using dsrna. Figure 1 shows the intron-exon structure of each gene with the labeled segment that was tested for RNA interference. With one exception, each gene showed the interfering phenotypes, while single stranded RNA showed no interference. The exception occurred in the segment unc-54c, which did not show the expected mutant phenotype, and could be attributed to the fact that this segment is a conserved myosin-motor domain and might not interfere with other highly related myosin heavy-chain genes. Table 1 shows the effects of the 26 dsrna segments that were injected and all of them showed limitations in the phenotype for the null mutant, except for the unc54c segment.

8 8 To assess the effects of dsrna interference at the cell level, a transgenic line from two different green fluorescent protein (GRP) reporter proteins in muscle was used. The dsrna showed decreases in the fraction of fluorescent cells. Figure 2 shows the GRP pattern results for a young larva and an adult for control RNA, dsgfpg RNA and ds-laczl RNA. The control appears to have no change from the parent strand. In the ds-grpg RNA, the reporter proteins were not seen in the affected cells in the progeny, where in the adult the vulva muscles are expressed in active GRP. In the laczl RNA, the nuclear targeted GFP-LacZ are not seen for almost all cells and the adult lacks almost muscles, except for the vulva muscles. Mello and Fire state they do not know the mechanisms between the dsrna interference, but they provide some observations that can aid in possible mechanisms. First, they found the dsrna that corresponds to intron and promoter sequences did not produce detectable interference. A second observation is that injection of dsrna made a dramatic decrease or elimination of endogenous mrna transcripts. Figure 3 shows the effects of antisense and double stranded mex-3 RNA using in situ hybridization in embryos. It is apparent that the antisense mex-3rna interferes with the mex-3 mrna, but the expression appears to only be slightly less than the wild type. For the mex-3 dsrna, there is no mex-3 mrna identified. Third, dsrna interference was able to cross cellular boundaries in that injections in the head or tail showed specific and robust interference in the progeny. It was even seen in the germline cells. Table 2 shows the effects of the site of injection for each dsrna in the adult and the progeny.

9 9 One of the main conclusions that Mello and Fire come to is the use of dsrna injection adds to the tools that can be used in studying gene function, especially in C. elegans and other similar organisms. There is a potential for use in vertebrates and plants. The new tool now allows for the possibility of analyzing many interesting coding regions for which a specific function has not defined. Even though RNA inference is potent and specific, there are some restraints that must be observed when performing experiments. One restraint is a sequence that shares many several closely related genes might inhibit with other members of the gene family (as shown in the exception to the results above). Another restraint is if there is a low level of expression in the genes or if there is small number of cells, there is a chance for the effects of RNA interference not to occur. Mello and Fire also conclude that by whatever mechanism RNA interference occurs, it is most likely a biological process and that genetic interference by dsrna could be used by the organism for physiological gene silencing due to the fact dsrna can work at a distance from the site of injection and its ability to move both into germline and muscle cells. In my opinion, one of the major weaknesses of this paper was its lack of detail for the techniques and results found. More could have been added to describe exactly what they did and more raw data could have backed up the conclusions. There is even one part where the authors refer to unpublished observations. However, this lack of detail could also be seen as a strength of the paper. The paper was very straightforward and uncomplicated. Because of the fact that Mello and Fire were presenting a new discovery, it was probably best to not speculate too much on

10 10 mechanisms and other information before performing further experiments. Another strength of the paper is that Mello and Fire covered every possible angle before assuming RNAi is an active biological process in the organism. Throughout the paper, its states possible processes that could have caused the given results, so a variety of experiments were performed to back their claims. Summary In my opinion, the primary paper was the main reason for Mello to receive the Nobel Prize. RNAi was a major discovery that changed the way we understand the flow of genetic information. However, the simplicity and usefulness of the technique used for many experiments in countless papers by Craig Mello himself and other researchers in the years after the primary paper was published (and still to this day) could have also contributed to Mello winning the Noble Prize. From an inquisitive child to Harvard to winning the Nobel Prize, Craig Mello has led a life of exploration inside and outside of his research. His discovery of the active biological process RNA interference found that double stranded RNA could specifically target a gene and turn off its expression. The discovery gave us a potent new technique for gene silencing and studying the function of genes. It also holds promise as a medical treatment for a variety of illnesses. To this day, Mello is still doing research on RNAi to further our understanding of it and the world around us.

11 11 References Bender, E. (2014). The Second Coming of RNAi. The Scientist. Craig C. Mello, (2007). Marquis Who's Who, Marquis Who's Who. Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., Mello, C.C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 19, Mello, C., Fire, A., (1995) DNA transformation. Methods Cell Biol. 48, Mello, C.C. (2007). Craig C. Mello-The Nobel Prize in Physiology or Medicine 2006: Autobiography. Nobelprize.org. Mello, C.C. (2014). Return to the RNAi world: rethinking gene expression and evolution. Cell Death Differ. 14(12): No author. (2009) RNA Interference and Development in C. elegans. Howard Hughes Medical Institute. No author. (2014). RXi Pharmaceuticals. Bloomberg BusinessWeek. No author. (2007). The Nobel Prize in Physiology or Medicine Nobelprize.org. Siomi, H., Siomi, M.C. (2009). On the Road to Reading the RNA-Interference Code. Nature. 457 (7228) (2888 words)