Most factors known to function in mRNA turnover are not essential for cell viability. To identify essential factors,~4000 temperature-sensitive yeast strains were screened for an increase in the level of the unstable CYH2 pre-mRNA. At the non-permissive temperature, five mutants exhibited decreased decay rates of the CYH2 pre-mRNA and mRNA, and the STE2, URA5 and PAB1 mRNAs. Of these, the mutant ts1159 had the most extensive phenotype. Expression of the TIF51A gene (encoding eIF-5A) complemented the temperature-sensitive growth and mRNA decay phenotypes of ts1159. The tif51A allele was rescued from these cells and shown to encode a serine to proline change within a predicted α-helical segment of the protein. ts1159 also exhibited an~30% decrease in protein synthesis at the restrictive temperature. Measurement of amino acid incorporation in wild-type cells incubated with increasing amounts of cycloheximide demonstrated that a decrease in protein synthesis of this magnitude could not account for the full extent of the mRNA decay defects observed in ts1159. Interestingly, the ts1159 cells accumulated uncapped mRNAs at the non-permissive temperature. These results suggest that eIF-5A plays a role in mRNA turnover, perhaps acting downstream of decapping.
Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder resulting in progressive degeneration of the muscle. It affects about 1 in 3,500 male children. Becker's muscular dystrophy is a less severe disease allelic to DMD. Some 30% of DMD patients suffer from various degrees of mental retardation. The giant DMD gene spans about 2,000 kilobases and codes for a 14-kilobase messenger RNA and a protein of molecular weight 427,000. DMD mRNA is most abundant in skeletal and cardiac muscle and less so in smooth muscle. We reported that the expression of the gene is developmentally regulated during the differentiation of primary muscle cultures and in myogenic cell lines in a way similar to the expression of muscle-specific genes such as myosin light chain 2 and skeletal muscle actin. Similar results have been obtained with human primary myogenic cells. Significant levels of DMD mRNA are found in brain tissue. Here we show that the transcript of the DMD gene and the amino terminal of the encoded protein differ in brain and muscle. The 5' ends of these mRNA species are derived from different exons. The results suggest that the two mRNA types are transcribed from different promoters.
The regulation of genes for acetylcholine receptor (AChR), myogenic factors and other muscle‐specific proteins has been analyzed in experimental autoimmune myasthenia gravis (EAMG) and following denervation. The levels of the transcripts for the myogenic factors, MyoDl, myogenin and MRF4, were measured using Northern blot analysis. Myogenin and MRF4 transcript levels were observed to be 3.1 ‐ and 2.6‐fold higher in muscle of rats with EAMG than in controls, respectively. MyoDl levels, however, remained unchanged. The increases in AChR, myogenin and MRF4 mRNAs were one order of magnitude higher in 2‐week denervated muscle than in the myasthenic muscle. The levels of muscle creatine kinase (MCK), α‐actin and muscle dystrophin transcripts were also analyzed. Dystrophin levels were found to be 1.7‐ and 4.7‐fold higher in EAMG and denervated muscle, respectively, than in controls; in contrast, MCK and α‐actin levels remained unchanged.
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