Question 14.37

Alternative splicing takes place in more than 90% of the human genes that encode proteins. Researchers have found that how a pre-mRNA is spliced is affected by the pre-mRNA’s promoter sequence (D. Auboeuf et al. 2002. Science 298:416–419). In addition, factors that affect the rate of elongation of the RNA polymerase during transcription affect the type of splicing that takes place. These findings suggest that the process of transcription affects splicing. Propose one or more mechanisms that would explain how transcription might affect alternative splicing.

Question 14.38

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disease caused by mutations in the gene that encodes dystrophin, a large protein that plays an important role in the development of normal muscle fibers. The dystrophin gene is immense, spanning 2.5 million base pairs, and includes 79 exons and 78 introns. Many of the mutations that cause DMD produce premature stop codons, which bring protein synthesis to a halt, resulting in a greatly shortened and nonfunctional form of dystrophin. Some geneticists have proposed treating DMD patients by introducing small RNA molecules that cause the spliceosome to skip the exon containing the stop codon. The introduction of the small RNAs will produce a protein that is somewhat shortened (because an exon is skipped and some amino acids are missing) but may still result in a protein that has some function (A. Goyenvalle et al. 2004. Science 306:1796–1799). The small RNAs used for exon skipping are complementary to bases in the pre-mRNA. If you were designing small RNAs to bring about exon skipping for the treatment of DMD, what sequences should the small RNAs contain?

410

Question 14.39

In eukaryotic cells, a poly(A) tail is normally added to pre-mRNA molecules but not to rRNA or tRNA. With the use of recombinant DNA techniques, a protein-encoding gene (which is normally transcribed by RNA polymerase II) can be connected to a promoter for RNA polymerase I. This hybrid gene is subsequently transcribed by RNA polymerase I and the appropriate pre-mRNA is produced, but this pre-mRNA is not cleaved at the 3′ end and a poly(A) tail is not added.

Propose a mechanism to explain how the type of promoter found at the 5′ end of a gene can affect whether a poly(A) tail is added to the 3′ end.

Question 14.40

SR proteins are essential to proper spliceosome assembly and are known to take part in the regulation of alternative splicing. Surprisingly, the role of SR proteins in splice-site selection and alternative splicing is affected by the promoter used for the transcription of the pre-mRNA. For example, through genetic engineering, RNA polymerase II promoters that have somewhat different sequences can be created. When pre-mRNAs with exactly the same sequences are transcribed from two different RNA polymerase II promoters that differ slightly in sequence, which promoter is used can affect how the pre-mRNA is spliced.

Propose a mechanism for how the DNA sequence of an RNA polymerase II promoter could affect alternative splicing of the pre-mRNA.