Analysis of Biological Development (K. Kalthoff)

Updates to Topic 17: RNA Processing

Answers to Questions in Text

Splicing of doublesex pre-mRNA (pp. 439-441)

  1. Figure 17.12 shows the splicing patterns for doublesex pre-mRNA in wild-type and mutant XX individuals as predicted on the basis of the activation model versus the blockage model. What are the corresponding splicing patterns predicted for XY individuals? Answer: Mutant XY individuals should use the same splicing pattern as wild-type males.
  2. The splicing of doublesex pre-mRNA can be studied in cultured Drosophila cells transfected with plasmids containing an abbreviated doublesex+ gene and cDNAs encoding transformer-2 mRNA and the female-specific transformer mRNA (Hoshijima et al., 1991, Science 252: 833-836). In this experimental system, each of the three transgenes can be modified and the effects of the modifications studied with relative ease. Based on the analysis of Burtis and Baker reviewed on p. 439/440, what splicing pattern would you expect if the short pyrimidine tract in the splice acceptor site of exon 4 were extended to 13 or more pyrimidines? Answer: Female-specific splicing, irrespective of the presence of transformer and transformer-2 proteins.
  3. In contrast to “leaky” splicing of transformer pre-mRNA, where a lot of non-sex specific mRNA is produced along with female-specific mRNA (see Fig. 17.11), the alternative splicing of doublesex pre-mRNA is very precise. There is little if any female-specifc doublesex mRNA in males or vice versa. Why would you expect a positive selection for precision here? Answer: Leakyness would produce intersexes, which do not contribute to the reproductive success of a population.


Clarifications and Corrections

New Review Articles

Faustino N.A. and Cooper T.A. (2003) Pre-mRNA splicing and human disease. Genes & Devel. 17: 419-437

Hirose Y. and Manley J.L. (2000) RNA polymerase II and the integration of nuclear events. Genes & Devel. 14: 1415-1429

Maquat L.E. and Carmichael G.G. (2001) Quality control of mRNA function. Cell 104: 173-176

New Research Articles

Gallouzi I.E. and Steitz J.A. 2001) Delineation of mRNA export pathways by the use of cell-permeable peptides. Science 294: 1895-1901.
The transport of mRNAs from the nucleus to the cytoplasm involves adapter proteins that bind the mRNA as well as receptor proteins that interact with the nuclear pore complex. The investigators use inhibitory peptides to show that a given mRNA may use overlapping (partially redundant) pathways for leaving the nucleus. For example, HuR protein serves as an adapter for c-fos mRNA export through two pathways. One involves the HuR shuttling domain, HNS, which exhibits a heat shock-sensitive interaction with transportin 2 (Trn2); the other involves two protein ligands of HuR - pp32 and APRIL - which contain leucine-rich nuclear export signals (NES) recognized by the export receptor CRM1. Heterokaryon and in situ hybridization experiments reveal that the inhibitory peptides selectively block the nucleocytoplasmic shuttling of their respective adapter proteins without perturbing the overall cellular distribution of polyadenylated mRNAs.

Horabin J.I. and Schedl P. (1996) Splicing of the drosophila Sex-lethal early transcripts involves exon skipping that is independent of Sex-lethal protein. RNA 2: 1-10.

Nagengast A.A., Stitzinger S.M., Tseng C.H., Mount S.M. and Salz H.K. (2003) Sex-lethal splicing autoregulation in vitro: interactions between SEX-LETHAL, the U1 snRNP and UsAF underlie male exon skipping. Development 130: 463-471
The investigators provide genetic and biochemical evidence that the Sexlethal protein causes the skipping of exon 3 in Sxl pre-mRNA by binding to both the acceptor and the donor site of this exon.

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