Splicing sites

In addition to affecting the efficiency of promoter utilization, eukaryotic cells employ alternative RNA processing to control gene expression. This can result when alternative promoters, intron-exon splice sites, or polyadenylation sites are used. Occasionally, heterogeneity within a cell results, but more commonly the same primary transcript is processed differendy in different tissues. A few examples of each of these types of regulation are presented below. 

P-Thalassemla (MIM 141900) A very wide variety of mutations in the p-globin gene, including deletions, nonsense and frameshift mutations, and others affecting every aspect of its structure (eg, splice sites, promoter mutants)... 

Nielsen, D. A., Jenkins, G. L., Stefanisko, K. M., Jefferson, K. K. Goldman, D. (1997). Sequence, splice site and population frequency distribution analyses of the polymorphic human tryptophan hydroxylase intron 7. Brain Res. Mol. Brain Res., 45, 145-8. 

GSH-S deficiency is a more frequent cause of GSH deficiency (HI7), and more than 20 families with this enzyme deficiency have been reported since the first report by Oort et al. (05). There are two distinct types of GSH-S deficiency with different clinical pictures. In the red blood cell type, the enzyme defect is limited to red blood cells and the only clinical presentation is mild hemolysis. In the generalized type, the deficiency is also found in tissues other than red blood cells, and the patients show not only chronic hemolytic anemia but also metabolic acidosis with marked 5-oxoprolinuria and neurologic manifestations including mental retardation. The precise mechanism of these two different phenotypes remains to be elucidated, because the existence of tissue-specific isozymes is not clear. Seven mutations at the GSH-S locus on six alleles—four missense mutations, two deletions, and one splice site mutation—have been identified (S14). 

A4. Arredondo-Vega, F. X., Santisteban, I., Kelly, S., Schlossman, C., Umetsu, D and Hershfield, M. S., Correct splicing despite a G- A mutation at the invariant first nucleotide of a 5 splice site A possible basis for disparate clinical phenotypes in siblings with adenosine deaminase (ADA) deficiency. Am. J. Hum. Genet. 54,820-830 (1994). 

K9. Kanno, H., Wei, D. C. C., Miwa, S., Chan, L. C., and Fujii, H., Identification of a 5 -splice site mutation and a missense mutation in homozygous pyruvate kinase deficiency cases found in Hong Kong. Blood 82 (Suppl. 1), 97a (1993). 

N2. Nakajima, H., Kono, N Yamasaki, T Hotta, K., Kawachi, M Kuwajima, M., Noguchi, T., Tanaka, T., and Tarui, S Genetic defect in muscle phosphofructokinase deficiency Abnormal splicing of the muscle phosphofructokinase gene due to a point mutation at the 5 -splice site. J. Biol. Chem. 265, 9392-9395 (1990). 

Santisteban, I., Arredondo-Vega, F. X., Kelly, S., Debre, M., Fischer, A., Pdrignon, J. L., Hilman, B., Eldahr, J., Dreyfus, D. H., Howell, P. L., and Hershfield, M. S Four new adenosine deaminase mutations, altering a zinc-binding histidine, two conserved alanine, and a 5 splice site. Hum. Mutat. 5,243-250 (1995). 

To make mRNA, the primary transcript must be spliced to bring the protein-coding sequences (exons) together and to remove the intervening sequences (introns). The splice signals consist of a 5 and a 3 set of sequences that are always found at splice junctions. However, this is generally believed to provide too little information to recognize a splice site specifically and correctly. Some sequences in the intron are also important. 

Hutton, M., Lendon, C. L., Rizzu, P. et al. Association of missense and 5 -splice-site mutations in tau with the inherited dementia FTDP-17. Nature 393 702-705,1998. 

Frank deletions of the gene are not common. A frequent mutation among northern Europeans (=40%) is a G to A transition at the 5 donor splice site in intron 12, resulting in absence of the C terminus. Another relatively common (=20%) mutation in northern Europeans involves a C to T transition in exon 12, resulting in substitution of a tryptophan for an arginine residue [21]. Over 70 different mutations have been described to date in the American population [22]. 

It is noteworthy that there is another limiting factor in the choice of amino acid types at the junction sites which affect the enzymatic process of the intein. For example, in the case of SceVMA (also called PI-Seel) from the IMPACT system, proline, cysteine, asparagine, aspartic acid, and arginine cannot be at the C-terminus of the N-terminal target protein just before the intein sequence. The presence of these residues at this position would either slow down the N-S acyl shift dramatically or lead to immediate hydrolysis of the product from the N-S acyl shift [66]. The compatibility of amino acid types at the proximal sites depends on the specific inteins and needs to be carefully considered during the design of the required expression vectors. The specific amino acid requirements at a particular splicing site depends on the specific intein used and is thus a crucial point in this approach. 

Ishikawa F, Matunis MJ, Dreyfuss G, Cech TR (1993) Nuclear proteins diat bind die pre-mRNA 3 splice site sequence r(UUAG/G) and die human telomeric DNA sequence d(TTAGGG)n. Mol Cell... 

Figure 2. Genetic aberrations observed in HAT genes, (a) Schematic representation of a balanced chromosomal translocation. These translocations result in the formation two new fusion genes, which can give rise to one or two fusion proteins, (b) Examples of nonsense (RTS patient RT163.1), missense (RT209.1), deletion (followed by frame shift RT231.1) mutations, as well as sphee site acceptor (RT211.3) or splice site donor (RT39.1) mutations...

Shown are nonsense, missense, deletion/insertion and splice site mutations. The amino acid changes are predictions in most cases. 

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