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Tuesday, August 14, 2007

MicroRNA 'sponges' could aid cancer studies

MIT researchers have developed a new way to study the function of microRNA, tiny strands of genetic material that help regulate at least 25 percent of a cell's genes.

The new technique could shed light on microRNA's hypothesized role in tumor development. Malfunctions in microRNA have been linked with cancer, but very few direct relationships have been established between specific microRNAs and the genes they regulate.

That could change, however, now that MIT Institute Professor Phillip Sharp and his colleagues have found a way to inhibit the activity of microRNA by genetically altering cells.

The technique, described in the August 12 online issue of Nature Methods, could "provide a tool to identify specific genes that are being regulated by microRNAs," said Sharp.

MicroRNA consists of short strings of about 22 nucleotides, the building blocks that make up RNA and DNA. MicroRNA binds to messenger RNA (mRNA), preventing it from delivering protein assembly instructions, thereby inhibiting gene expression.

Sharp, who is affiliated with MIT's Biology Department and Center for Cancer Research, said microRNA exists in every cell and controls a wide range of cell regulatory activities.

The MIT team has found a way to block microRNA activity by tricking cells into producing a microRNA "sponge," which soaks up microRNA and renders it ineffective. By de-activating microRNA, researchers can observe the resulting effects and determine which genes the microRNA is targeting.

The new technique could shed more light on microRNA's role in tumor development: Earlier studies have shown that a type of microRNA known as let-7 inhibits a cancer-inducing gene called RAS. Abnormally low levels of let-7 have been found in some types of tumor, said Sharp.

Sharp and MIT biology graduate student Margaret Ebert, lead author of the paper, decided to block microRNA activity by creating a gene that produces microRNA sponges and inserting it into their target cells. Each sponge can bind up to six microRNA molecules, but they could be engineered to bind more.

The sponge gene also includes a "reporter" gene that causes the cell to become fluorescent if it has taken up the gene, so the researchers can know for sure whether the microRNA sponge is being produced in a particular cell.

Ebert said the new sponge technique is an improvement over an older method that involves blocking microRNA activity with artificially synthesized strands of RNA, known as oligos. One advantage is the inclusion of the reporter gene; another is that the sponge genes can be expressed continuously, while oligos do not remain in the cell forever.

More importantly, the sponge technique could be used to create transgenic animals that express the sponge in all of their cells, allowing researchers to study microRNA function at the organismal level. With such animals, sponge genes could be designed so that the researchers can control when and where they are expressed.

Joel Neilson, a postdoctoral associate in the Center for Cancer Research, is also an author on the paper. The research was funded by the National Cancer Institute, the National Institutes of Health, a Howard Hughes Medical Institute Predoctoral Fellowship, a Paul and Cleo Schimmel Scholarship, and the Cancer Research Institute.

The MIT Center for Cancer Research was founded in 1974, and is one of eight National Cancer Institute-designated basic research centers. Its mission is to apply the tools of basic science and technology to determine how cancer is caused, progresses and responds to treatment.

About Rasearcher

Phillip A. Sharp

The Nobel Prize in Physiology or Medicine 1993

A sense of place was and remains an important part of my life. I was born in a rural community in the northern hill country of Kentucky. My earliest memories are those of a child playing around the house on our family farm, located in a bend of the Licking River near McKinneysburg. My mother, Kathrin Colvin Sharp, had grown up in that same house and her family had been part of this community for many generations. My father, Joseph Walter Sharp, grew up nearby within walking distance of the nearest town and county-seat, Falmouth. Both parents came from large families and I was surrounded by grandparents, aunts, uncles, siblings and cousins.

My formal education was entirely in the public schools of Pendleton County: McKinneysburg Elementary, Butler Elementary and High School and Pendleton County High School. Even though my studies never interfered with sports or fun, I managed to gain an appreciation of math and science.

All through my childhood, my parents strongly encouraged me to attend college. With that in mind, they taught me to save my money for college tuition, and, even more important, they allowed me to earn it by raising cattle for the market and growing tobacco. The rural background of my childhood made me feel more comfortable attending a small institution in a familiar environment. Therefore, I entered a small liberal arts school, Union College, in the foothills of eastern Kentucky. Union is in Barbourville, the county-seat of Knox County, and in those days it was one of the gateways for the youth from the mountains in the eastern part of the state to emerge into a larger world. While at Union, I majored in chemistry and mathematics and decided that I wanted to continue to study and learn about science, particularly chemistry. I also met and married a lovely girl from New Jersey, Ann Holcombe (Sharp).

A young professor at Union, Dr. Dan Foote, became a good friend and encouraged me to apply to the Department of Chemistry at the University of Illinois. This old and distinguished department must have recognized some hidden promise as I was offered a fellowship and soon began graduate studies under Victor Bloomfield in physical chemistry. Victor was an excellent mentor as he encouraged both my scientific as well as cultural growth. He provided funds for my participation in national scientific meetings and broadened my perspective on society and culture by being a long-haired liberal, well-read and artistic friend. Fortunately, I was deferred from the Vietnam draft for a number of years and was able to finish graduate school. My thesis dealt primarily with the description of DNA as a polymer using statistical and physical theories. My attempts at experimental science at this stage were juvenile. In spite of my youth on the farm, I was never very skilled in manual tasks; in fact, I soon lost interest in any complex "hands on" manipulations.

The 1966 volume of the Cold Spring Harbor Symposium on The Genetic Code stimulated my interest in molecular biology and genetics. A subsequent letter to Norman Davidson at the California Institute of Technology resulted in an offer of a postdoctoral position in 1969 and my immersion into a vibrant research program in molecular biology. Ron Davis, a graduate student in Norman's lab at the time, had previously developed the heteroduplex method for visualizing deletions in phage genomes with the electron microscope. Jerome Vinograd in an adjacent laboratory had discovered the superhelical structure of animal virus genomes. In this environment, I began the transition to experimental molecular biology by using the heteroduplex method and electron microscopy to study the structure of plasmids of the sex factors and drug resistant factors of bacteria. I was particularly interested in how sex factor plasmids acquired genomic sequences from the bacterial chromosome. We found that both sex and drug resistance factors contained transposable elements. This experience taught me many things, including the power of novel methodology and how a simple experiment can transform the understanding of an important problem.

At the end of my stay at Caltech, I opted to extend my postdoctoral period and begin to study the structure and pathway of expression of genes in human cells. The expression of genes of animal viruses with DNA genomes was the only experimentally approachable system at that time, and this led me to a further postdoctoral year at Cold Spring Harbor Laboratory under the mentorship of Jim Watson. Here, I entered a close-knit scientific commune of extremely talented people who lived and worked together in an isolated environment for nine months, and then were immersed in a continuous scientific meeting for the remaining three summer months.

At the Lab Joe Sambrook, a staff member, I, and others used hybridization techniques to map sequences in the simian virus 40 genome that were expressed as stable RNAs in both infected cells and oncogenic cells transformed by this virus. These were important results for understanding the biology of this papovavirus and helped move the laboratory into a very rapidly advancing field of research - the molecular and cell biology of tumor viruses. Luckily, or perhaps by design at a higher level, Ulf Pettersson, an expert in the growth of human adenovirus who had done graduate studies with Lennart Philipson in Uppsala, Sweden, was a fellow postdoctoral associate and my office mate at Cold Spring Harbor. Adenoviruses are common causes of respiratory and other types of infections in man; however, when infected into newborn rodents, they can cause tumors. The high levels of both replication and viral protein expression made the growth cycle of this virus ideal for the study of gene structure and regulation. Furthermore, the then recent discovery of restriction endonucleases offered the prospect of fragmenting the viral genome of 35,000 base pairs into tractable units. Ulf, I, and others generated the first restriction endonuclease maps of this virus, and Dr. S. Jane Flint and I began to analyze the regions of the genome expressed as mRNAs in both productively infected cells and in adenovirus transformed cells. This was an exciting period in the molecular biology of adenovirus with the discoveries (a) that only one specific fragment of the genome, the E1 region, was responsible for oncogenic transformation; (b) that restriction endonuclease length polymorphism could be utilized to generate genetic maps; (c) the mapping of specific genes on the viral genome; and (d) generation of a viral map of sequences expressed as stable RNAs.

Salvador Luria, the Director of the then recently established Center for Cancer Research at the Massachusetts Institute of Technology, called in 1974 to inquire if I would be interested in a position at the Center. After a brief visit to MIT I accepted. Fortunately, I was assigned laboratory space on the 5th floor, which was shared with David Baltimore, Nancy Hopkins, Robert Weinberg and David Housman. Salva was a visionary who protected his young faculty from unnecessary interruptions, thus allowing their research programs to flourish in an ideal scientific environment. He was also a role model for how a scientist could shape and lead a community. That summer, Jane Flint moved with me to MIT and we continued our analysis of adenovirus transcription, focusing on quantitating the levels of RNA from all parts of the genome in the nuclear and cytoplasmic compartments of the cell. We found that the nuclei of cells productively infected by adenovirus contained abundant sets of viral RNAs which were not transported to the cytoplasm. We speculated that these long nuclear RNAs were processed to generate the cytoplasmic mRNAs. This stimulated our interest in comparing the relative structures of nuclear precursor RNA and cytoplasmic mRNA from the adenovirus genome. We were joined in the latter part of these studies by a postdoctoral fellow, Dr. Susan Berget. For Sue, these studies were the beginning of a much more interesting series of experiments which form the first part of the lecture.

As mentioned above, Ann and I were married in 1964 while still undergraduates at Union College. Our three daughters arrived on a schedule which approximated a seven year itch: Christine Alynn was born in 1968, while I was still in graduate school, Sarah Katherin was born in 1974, just before we moved to New England, and Helena Holcombe was born in 1981. Ann teaches a preschool class in Newton, Massachusetts, the town we have made our home since moving from Cold Spring Harbor. My family and are deeply enamored with New England. We enjoy its rural towns, coastal beauty, and the changes of seasons.

Through the years at MIT my environment has remained relatively constant, though changes have occurred. David Baltimore and Robert Weinberg left the Center in 1983 to found the Whitehead Institute, which is associated with MIT. Salva retired from MIT in 1985 and I assumed his position as Director of the Center for Cancer Research. In 1991, I relinquished the Directorship to Richard Hynes and became the Head of the Department of Biology. The development of biotechnology has both enriched and complicated my work. Walter Gilbert of Harvard and I, along with a number of European colleagues, founded the biotechnology company Biogen in 1978 in Geneva, Switzerland. This organization and the friends who have worked for it have remained an important part of my life since.

My collaborators over the years have been: (listed in alphabetical order) A. S. Baldwin, S. M. Berget, A. J. Berk, K. Berkner, B. Blencowe, M. A. Brown, S. Buratowski, C. Carr, R. W. Carthew, C. Cepko, D. Chang, D. Chasman, L. A. Chodosh, G. Chu, R. G. Clerc, J. D. Crispino, D. J. Donoghue, A. Z. Fire, D. E. Fisher, S. J. Flint, M. Garcia-Blanco, A. Gil, S. Gilbert, P. J. Grabowski, H. Handa, U. Hansen, S. Hardy, S. Harper, T. Harrison, M. Horowitz, P. S. Jat, R. Kaufman, J. Kim, R. Kingston, J. Kjems, T. Kobayashi, M. M. Konarska, T. Kristie, A. I. Lamond, F. Laski, J. LeBowitz, K. LeClair, F. Lee, I. Lemischka, A. M. MacMillan, R. Marciniak, P. McCaw, R. Meyers, C. Moore, M. Moore, M. Morton, M. Murata, R. Padgett, J. Parvin, J. L. Pomerantz, C. Query, M. E. Samuels, J. Sedivy, S. Seiler, B. Shykind, H. Singh, H. Skolnik-David, M. Timmers, A. Virtanen, J. Weinberger, and Q. Zhou.

In 1980, Dr. Sharp received both the Eli Lilly Award in molecular biology and the U.S. Steel Award from the National Academy of Sciences. His awards are too numerous to list but some include MIT's James R. Killian, Jr., Faculty Achievement Award (1993), the John D. MacArthur Professorship (1987-1992), the first Salvador E. Luria Professorship (1992-), the New York Academy of Sciences Award in Biological and Medical Sciences, the General Motors Research Foundation Alfred P. Sloan, Jr., Prize for Cancer Research, the 1988 Louisa Gross Horwitz Prize, the 1988 Albert Lasker Basic Medical Research Award, the 1986 Gairdner Foundation International Award, Canada, and the 1980 Dickson Prize from the University of Pittsburgh. In 1985, he was the Harvey Society Lecturer and on May 4, 1991, he received the honorary degree of Doctor of Humane Letters from Union College, his Alma Mater. He is a member of the National Academy of Sciences, the American Academy of Arts and Sciences, associate member of the European Molecular Biology Organization and Fellow of the American Academy for the Advancement of Science, the American Philosophical Society, the Institute of Medicine of the National Academy of Sciences, and a member of the editorial board of the journal Cell. He is co-founder and Chairman of the Scientific Board of Biogen, Inc., and member of its Board of Directors.

Dr. Sharp has a distinguished record of public service, which partially includes having served as a member of the President's Advisory Council on Science and Technology, as co-chairman of the Director of NIH's Strategic Plan, as a member of the Committee on Science, Engineering, and Public Policy (COSEPUP), as a member of the Search Committee of Director, National Center for Human Genome Research, and more recently, as a member of the Search Committee for the Director, Office of AIDS Research, NIH. His career publications in peer reviewed and other journals are over 255.

From Les Prix Nobel. The Nobel Prizes 1993, Editor Tore Frängsmyr, [Nobel Foundation], Stockholm, 1994

This autobiography/biography was written at the time of the award and later published in the book series Les Prix Nobel/Nobel Lectures. The information is sometimes updated with an addendum submitted by the Laureate. To cite this document, always state the source as shown above.


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