RNA Processing and Gene Therapy
Every year, new discoveries are made about ribonucleic acid, or RNA. In fact, RNA is such a fascinating and important molecule that many scientists believe that life on Earth started out in an "RNA World". How can we understand how RNA works? Is it possible to use RNA therapeutically? Our group studies the basic molecular biology of RNA. In addition, we have devised novel RNA strategies for gene therapy, in order to alleviate diseases such as beta-thalassemia, muscular dystrophy and AIDS.
RNA is the crucial molecule that allows different genes (composed of DNA) to be made into proteins that carry out diverse functions in animal, plant or bacterial cells. DNA is first copied, or transcribed into RNA, which then acts as a template for protein production. RNA is not always immediately functional after it has been transcribed. Often, it must first be processed (see sidebar) and only then can it be translated into the corresponding protein. The regulation of RNA processing is a fundamental mechanism by which cells ensure that the correct proteins are made at the right time, in the right place.
Our lab studies the processing of a special group of RNAs, i.e. those that encode histone proteins. Histones are used to package the DNA into chromosomes. Histone RNAs must be cleaved, or processed, at the downstream end in order for the RNA to become competent for translation into protein. A long-standing mystery has been the identity of the proteins involved in histone RNA processing. In recent years we have discovered several proteins that are specifically involved in this process. One of these binds to an RNA hairpin structure near the cleavage site of the histone RNA and hence was called hairpin binding protein. Two other proteins (called Lsm10 and Lsm 1) were found to be components of the U7 snRNP, a complex that is itself required for histone RNA processing, and which contains other proteins and the short U7 RNA. Interestingly, Lsm10 and Lsm11 are both new member of the "Sm and Sm-like" protein family. Members of this family have been found in all three major domains of life (including all types of bacteria, fungi, plants and animals). A common feature of Sm and Sm-like proteins is their ability to form RNA-protein complexes and to control various aspects of RNA metabolism.
For gene therapy, our lab has developed a novel antisense RNA strategy, to target diseases such as beta-thalassemia. Beta-thalassemia is characterized by anemia, a lack of red blood cells, due to insufficient production of the blood protein beta-globin. In several types of beta-thalassemia, mutations in the beta-globin RNA cause abnormal RNA splicing (see sidebar). This results in a deficiency in normal beta-globin protein, and thus in potentially fatal anemia. The idea behind "our kind" of RNA gene therapy is that carefully designed antisense, or complementary RNA sequences can physically block abnormal splice sites, and therefore force the splicing machinery to use the normal splice sites instead. Our lab has tested this strategy using a "modified U7" antisense RNA to correct abnormal beta-globin splicing in cultured cell lines, and results so far indicate that correct splicing can be restored. The current challenge is to express the therapeutic U7 RNA gene in red blood cell precursors which are found in the bone marrow. For this purpose gene therapy vectors derived from the HIV virus are being used. We are also studying how interfering with the splicing of specific RNAs can be used to treat other diseases such as muscular dystrophy and AIDS.
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RNA is first transcribed from DNA as a long precursor, or pre-mRNA, which must be further modified, or processed, in order for translation into protein to occur. A common modification of pre-mRNAs is splicing. Splicing means that parts of the RNA not directly relevant for making protein (introns), are removed. The remaining parts (exons) are linked together to make a mature mRNA. The control of splicing depends on signals within the RNA sequence itself (splice sites). Histone pre-mRNAs are unusual in that they are not spliced. Instead, histone mRNA is matured by a specific cleaving off of the unnecessary downstream part of the pre-mRNA. The regulation of this cleavage is complex and depends on many different proteins, some of which are still unknown.
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What is gene therapy? This term refers to strategies designed to cure or ameliorate disease by the replacement of a disease gene with a new functional gene, by the additional expression of a functional gene, or of a gene that repairs the disease gene or its faulty products.
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