Nonsense-mediated mRNA decay in collagen X

CHAPTER 5 ABSTRACT────────────────────────────────────────── 85 Genetic disorders are a major cause of human disability and disease. It has been estimated that 5-10% of the population is affected by an inherited disorder during their lifetime, and a startling 70% of pediatric admissions involve a genetic component. Considering this level of influence on human disability and the multifactorial genesis of most diseases, studies on the mechanisms of genetic disease, especially those relating to its fundamental mechanisms will have increasing relevance to almost every known disease. The mutations that can lead to genetic disorders are various, but one recurrent type is called a nonsense mutation. Nonsense mutations are known to be responsible for one third of all inherited diseases as well as many oncologic diseases. A nonsense mutation, caused by either a deletion, insertion or a point mutation, results in the generation of a stop codon in the mRNA before the genuine stop codon that signals the termination of translation of the mRNA into the appropriate full length protein. The theoretical consequence would be the synthesis of a shorter protein which could exert a dominant negative effect and have serious consequences for cell function. To avoid these possibly deleterious effects of nonsense mutations, the human body has developed an elegant quality control surveillance mechanism called nonsense mediated mRNA decay (NMD). This pathway detects the premature termination codons (PTC) inside the mRNA and renders the mutation-containing mRNA to NMD. Thus, the cells can be protected from the effect of a PTC. For the NMD machinery to be efficient, a signal is required that allows the machinery to distinguish between the normal and the premature stop codon. In previous studies on the NMD pathway, basic principles of NMD were developed. The signal to differentiate a PTC from the normal stop was discovered to be the connection point between exons where introns had been removed, the so called exon-exon junctions (EJC). They need to be found downstream of a PTC for the determination of prematurity. Since the last exon is not followed by an EJC any more, PTC‟s in the last exon of a gene cannot be detected by the NMD machinery and will therefore result in a truncated protein. However, when the genetic background of the cartilage disease Schmid Metaphyseal Chondrodysplasia (SMCD) was analyzed, it was discovered that the mutations in the last exon of collagen X that are causative for SMCD did indeed undergo NMD – impossible according to the known NMD rules. Furthermore, when the mutations were analyzed more closely it was found that the mutations lost this ability to NONSENSE-MEDIATED MRNA DECAY IN COLLAGEN X────────────FRIEDERIKE KREMER 86 trigger NMD in the last exon when they were located at a greater distance from the normal stop codon. These findings exposed a new complexity of the NMD principles. Obviously, lastexon NMD required a completely different mechanism to detect PTC‟s than did mutations in the other exons of genes. To identify this mechanism, the close by region of the 3‟untranslated region (3‟UTR) was analyzed. It was found that, upon deleting a small region of the 3‟UTR, NMD in the last exon of collagen X could no longer be triggered. This brought up an all new theory of 3‟UTR mediated NMD, that might still involve known NMD factors such as eIF4e or Stau1, but could also include yet unknown proteins. Further investigation into the 3‟UTR mediated NMD will offer insights into a broader spectrum of the principles of NMD. An approach to the new questions arising from this research would be to test the possibility of inhibition or enhancement of the 3‟UTR mediated NMD pathway in a mouse system and the resulting effects on the phenotype. Furthermore, investigation on those mutations in the last exon of collagen X that do not elicit NMD will shed light on the resulting phenotype of these mutations. Possibly, they result in a different disease that has yet to be connected with collagen X. The knowledge on the genetic background of this disease will greatly improve the scientific possibilities of research in this field. In the future, this research might allow us to intervene therapeutically in the genetic components of SMCD and other diseases caused by some form of NMD in humans and ameliorate or even prevent the detrimental effects arising from these genetic diseases.