It's in our DNA
At the Center for Nucleic Acids Science and Technology, interdisciplinary scientists develop tools to explore, monitor and control gene expression.
Since Watson and Crick unveiled the structure of DNA more than 50 years ago, the double helix has been in the limelight. But DNA is now playing costar while another molecule stands center stage - RNA. This multi-talented molecule plays a role in nearly every aspect of gene expression, from regulating the activity of genes to finetuning the production of proteins.
It is here - in this new RNA world - that the intricacies of many human diseases are being uncovered. Diseases like retinitis pigmentosa, myotonic dystrophy and cancer all have defects in how one form of RNA is processed to direct the construction of proteins. Severe anemias and certain cancers result from errors in the assembly of ribosomes, complexes of another form of RNA and protein that function as the cell's protein-producing machinery.
"The rapid influx of data from the human genome project has allowed scientists to identify more and more genes linked to disease and to explore in greater detail what happens at a molecular level. There is great optimism that genetic diseases will one day be treatable at the DNA or RNA level rather than at the traditional protein level," said Bruce Armitage, professor of chemistry and co-director of Carnegie Mellon's Center for Nucleic Acids Science and Technology (CNAST).
Treatments at this level should be far more precise and effective. Making advances in this area of RNA research requires two key things, according to Armitage - a deep understanding of how RNA regulates gene expression and the know-how to create new compounds that can report on RNA's activities and ultimately help scientists regulate biological processes involving RNA.
Genetic Cut and Paste
Although the genes encoded in DNA provide the instructions for making proteins, a gene does not actually build the protein. The genetic information from DNA is copied, or transcribed, into one form of RNA called messenger RNA (mRNA), which carries the genetic information to ribosomes, the cell's protein-building machinery. But mRNA is not a simple copy of DNA. The original mRNA transcript, called pre-mRNA, is copied letter for letter from DNA, but it is then cut and pasted back together to form a mature mRNA. Depending on which RNA segments are removed and which are kept - a process called alternative splicing - the transcripts from one gene can produce many varieties of mRNA and hence yield potentially thousands of different proteins. Javier Lopez, associate professor of biological sciences and a member of CNAST, has spent more than a decade investigating alternative splicing to understand the powerful and versatile ways it regulates gene expression. Alternative splicing contributes to major developmental decisions (like wing formation in fruit flies) and fine-tunes gene function.
Lopez carries out many studies on the fruit fly Drosophila melanogaster, an ideal model system for studying alternative pre-mRNA splicing. Many of the molecular mechanisms at play are the same in fruit flies and humans. Plus, certain instances of alternative splicing are stunningly obvious in fruit flies - if a specific gene is spliced one way, a male fly develops; if the gene is spliced another way, a female fly is born. Most alternative splicing events are not so easy to see in the lab, however, but their effects can be debilitating. Take, for example, a current project underway in the Lopez lab. They have uncovered genetic differences in an alternative splicing event that may influence risk for a human disease - schizophrenia. Lopez's collaborators at the University of Pittsburgh identified DNA sequence variations associated with schizophrenia in patients, and Lopez's preliminary evidence suggests that these variations alter the pre-mRNA splicing pattern of a specific gene. Lopez's first clue came when his lab found a previously undiscovered exon (a genetic region that is retained in mRNA during splicing) hidden inside an intron (a region that is removed from mRNA). Whether this exon is removed or kept during splicing is linked to the risk of having schizophrenia, Lopez discovered. When the gene sequence is the low-risk variant, the exon is removed from most pre-mRNA transcripts along with the intron. When the gene sequence is the high-risk variant, the exon is retained more frequently, resulting in disrupted protein production that could possibly explain the symptoms of schizophrenia.
"Our hypothesis right now is that some people are at higher risk for schizophrenia because their gene sequence creates stronger splicing signals, allowing the cell's splicing machinery to recognize the exon more efficiently," explained Lopez.
Lopez and his colleagues are working to understand exactly how splicing of this troublesome exon is regulated. Using a new method under development in collaboration with Armitage and Associate Professor of Chemistry Linda Peteanu, Lopez will soon be able to detect in real time within a cell when an individual molecule of RNA has been spliced and when it has not, something that hasn't been possible before now. To witness this difference in splicing, Lopez will employ peptide nucleic acids (PNAs) designed by Armitage and Peteanu. These synthetic molecules possess DNA-like properties, which allow the molecule to bind to a specific DNA or RNA sequence, and protein-like properties that make PNAs long-lived within cells. Armitage's PNAs can be tagged with a variety of fluorescent molecules whose light can be detected with a microscope. A PNA that binds to a pre-mRNA that's being spliced one way (exon is cut out) will be labeled with one color while another, differently colored fluorescent PNA will bind to a pre-mRNA that's being spliced the alternative way (exon is not cut out). If the exon is removed, red light is detected. If the exon is retained, orange light is detected. By analyzing the ratio of the colors in PNA-treated cells, Lopez will be able to use this new method to determine which cells have mRNAs that retained the miscreant exon.
Being able to determine the relative amounts of two alternatively spliced transcripts in a cell is critical to understanding the regulation and function of alternative splicing in specific genes, explains Lopez. Knowing how a gene's pre-mRNA transcript is spliced in healthy individuals is key to understanding how incorrect splicing could lead to diseases like schizophrenia.
Where Biologists and Chemists Meet
Lopez and other biologists working with CNAST are in a unique position. Their CNAST chemistry colleagues are experts in PNAs, versatile molecules that can bind to specific DNA and RNA targets inside cells, such as an RNA molecule that is undergoing splicing or a ribosomal RNA molecule producing a protein. With the largest and most diverse group of PNA researchers in the world, CNAST is poised to take the lead in developing new tools and technologies that can be applied not just to the study of RNA biology but also to potential therapeutics.
"What makes PNA unique is its synthetic flexibility," said Danith Ly, associate professor of chemistry and a member of CNAST. "We can design these molecules to have specific properties to serve a variety of needs."
First reported in 1991, PNA is like DNA and RNA but it contains a protein-like backbone, instead of a sugar backbone (see image this page). PNA contains the nucleobases found in DNA and RNA, enabling PNA to bind to DNA and RNA in a complementary, highly specific manner. Additionally, PNAs have neutral, pseudopeptide backbones - unlike DNA and RNA's negatively charged sugar-phosphate backbones - so they avoid detection and degradation within living cells.
Because PNAs have a high affinity for complementary DNA and RNA, and because they are very stable, long-lived molecules, scientists have high hopes for using PNA to study and manipulate cellular processes like gene expression. CNAST scientists have overcome a huge obstacle - the inability of PNAs to enter cells - thereby paving the way for applications of PNAs in the diagnosis and treatment of disease. With a little molecular plastic surgery, Ly developed PNAs that can be taken up by living cells.
Ly was inspired by an HIV protein called Tat, which is readily taken up by mammalian cells. He modified the PNA backbone so that it looked like the Tat protein, resulting in significantly improved delivery into human cells. Not only were the modified PNAs taken up by cells, but they also localized predominantly in the cell nucleus, the specialized compartment in the cell where mRNAs are made. Being able to localize to the nucleus is critical for using PNA to investigate, modify or block gene expression at the transcriptional or splicing stages, according to Ly.
Ly and his colleagues have already demonstrated that their modified PNAs can inhibit translation, the step in gene expression when the mature mRNA is used to build a protein. Ly treated human lung cancer cells with a PNA designed to bind to an mRNA that codes for a protein found on the cell surface. The protein, E-cadherin, usually holds cells together. In some types of cancer cells, defects in E-cadherin cause cells to detach from each other and spread, leading to metastatic cancer. In Ly's experiment, E-cadherin is detected on the cells not treated with PNA (see image this page). But in the PNA-treated cells, very little E-cadherin is detected, indicating that the PNA successfully stopped the mRNA from being translated into the protein.
Ly has also created PNAs with other exceptional properties, including the ability to bind to DNA and RNA much more strongly. According to Ly, this capability should make PNA an excellent tool for blocking transcription, processing or translation of RNA. The result could be a much more effective drug.
Added Armitage, "The molecular surgery Danith is doing on the structure of PNA, taking it far beyond what it was originally capable of doing, is making us excited about its biological potential."
While PNAs show extraordinary promise for medicine, Ly and Armitage also are using them in studies with biologists to explore fundamental scientific questions, such as determining the molecular steps involved in assembling a ribosome. Ribosomes are the cellular machines that take mRNAs and translate them into all the proteins in cells. Itself a complex of 79 different proteins and four different RNA molecules, ribosomes are critical to gene expression. Understanding how these complexes assemble to perform their protein-building duties is essential to appreciating their function, says John Woolford, professor, acting department head of biological sciences and co-director of CNAST. Woolford is working with Ly and Armitage to design PNAs that will bind to specific sites on ribosomal RNAs, tagging them with fluorescent molecules. Early stage experiments are revealing the spatial layout of RNA and protein components that comprise a ribosome. According to Woolford, these experiments will enable them to understand how functional ribosomes are formed in healthy individuals and how ribosome assembly goes awry in disease.
"RNA participates or is synthesized in different aspects of biology, and our research is represented in many of these areas. Because of the strong foundation we have here in RNA biology, we can be really intelligent in how we design our PNAs," said Woolford. "With these tools, there are fundamental questions in biology that can be answered and there can be practical applications in treating genetic and infectious diseases - those are boundless."