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28. In addition to snRNAs, the spliceosome contains a number of proteins. Some of these proteins are associated with the snRNAs to form snRNPs. Other proteins are associated with the spliceosome but are not associated with any specific snRNA.

One group of spliceosomal proteins comprises the precursor RNA-processing (PRP) proteins. Three PRP proteins that directly take part in splicing are PRP2, PRP16, and PRP22. The results of studies have shown that PRP2 is required for the first step of the splicing reaction, PRP16 acts accumulate if the following components were omitted from the splicing system? Explain your reasoning.

25. The splicing system introduced in Problem 24 is used to splice an RNA molecule containing two exons and one intron. This time, however, the U2 snRNA used in the splicing reaction contains several mutations in the sequence that pairs with the U6 snRNA. What would be the effect of these mutations on the splicing process?

26. A geneticist isolates a gene that contains five exons. He then isolates the mature mRNA produced by this gene. After making the DNA single stranded, he mixes the single-stranded DNA and RNA. Some of the single-stranded DNA hybridizes (pairs) with the complementary mRNA. Draw a picture of what the DNA - RNA hybrids would look like under the electron microscope.

27. The chemical reagent psoralen can be used to elucidate nucleic acid structure. This chemical attaches itself to nucleic acids and, on exposure to UV light, forms covalent bonds between closely associated nucleotide sequences. Such cross-links provide information about the proximity of RNA molecules to one another in complex structures.

Psoralen cross-linking has been used to examine the structure of the spliceosome. In one study, the following cross-linked structures were obtained during splicing. U1, U2, U5, and U6 became cross-linked to pre-mRNA. U2 was cross-linked to U6 and to pre-mRNA. The U1, U5, and U6 cross-links with pre-mRNA were mapped to sequences near the 5' splice site, whereas the U2 snRNA cross-links with pre-mRNA were mapped to the branch site. After splicing, U2, U5, and U6 were cross-linked to the excised lariat.

Explain these results in regard to what is known about the structure of the spliceosome and how it functions in RNA splicing. (Based on D. A. Wassarman, and J. A. Steitz, 1992, Interactions of small nuclear RNAs with precursor messenger RNA during in vitro splicing, Science 257:1918-1925.)

at the second step, and PRP22 is required for the release of the mRNA from the spliceosome. Other studies have found that these PRP proteins have amino acid sequences similar to the sequences found in RNA helicase enzymes—enzymes that are capable of unwinding two paired RNA molecules. On the basis of this information, propose a functional role for PRP2, PRP16, and PRP22 in RNA splicing.

29. Propose a scenario by which spliceosomal-mediated splicing might have evolved from the splicing of group II introns.

SUGGESTED READINGS]_

Bjork, G. R., J. U. Erikson, C. E. D. Gustafsson, T. G. Hagervall, Y. H. Jonsson, and P. M. Wikstrom. 1987. Transfer RNA modification. Annual Review of Biochemistry 56:263 - 288. A review of how tRNA is processed. Broker, T. R., L. T. Chow, A. R. Dunn, R. E. Gelinas, J. A. Hassel, D. F. Klessig, J. B. Lewis, R. J. Roberts, and B. S. Zain. 1978. Adenovirus-2 messengers: an example of baroque molecular architecture. Cold Spring Harbor Symposium on Quantatative Biology 42:531 -534.

One of the first reports of introns in eukaryotic genes.

Gott, J. M., and R. B. Emerson. 2000. Functions and mechanisms of RNA editing. Annual Review of Genetics 34:499 - 531.

An extensive review of the different types of RNA editing and their mechanisms. Hurst, L. D. 1994. The uncertain origin of introns. Nature 371:381 - 382.

A discussion of some of the ideas about when and how introns first arose.

Keller, W. 1995. No end yet to messenger RNA 3' processing. Cell 81:829-832.

An excellent review of processing at the 3' end of eukaryotic pre-mRNA.

Lake, J. A. 1981. The ribosome. Scientific American 245(2):84 - 97.

A review of the structure of ribosomes. Landweber, L. F., P. J. Simon, and T. A. Wagner. 1998. Ribozyme engineering and early evolution. Bioscience 48:94-103. A nice review of the idea that early life may have consisted of an RNA world.

McKeown, M. 1992. Alternative mRNA splicing. Annual Review of Cell Biology 8:133 -155.

An extensive review that discusses the different types of alternative splicing with specific examples of each type.

Misteli, T., J. F. Caceres, and D. L. Spector. 1997. The dynamics of a pre-mRNA splicing factor in living cells. Nature 387:523 - 527.

Reports that pre-mRNA splicing and transcription take place at the same sites in the nucleus.

Nilsen, T. W. 1994. RNA - RNA interactions in the spliceosome: unraveling the ties that bind. Cell 78: 1 -4. An excellent summary of how RNA - RNA interactions play an important role in the splicing of nuclear pre-mRNAs.

Noller, H. F. 1984. Structure of ribosomal RNA. Annual Review of Biochemistry 53:119 -162. A review of rRNA.

Rich, A., and S. H. Kim. 1978. The three-dimensional structure of transfer RNA. Scientific American 238(1):52 -62. Discusses the structure of tRNA.

Scott, J. 1995. A place in the world for RNA editing. Cell 81:833-836.

A good, succinct review of RNA editing.

Smith, C. M., and J. A. Steitz. 1997. Sno storm in the nucleolus: new roles for myriad small RNPs. Cell 89:669 -672. A good review of the role of snoRNAs in rRNA processing.

Volkin, E., and L. Astrachan. 1956. Phosphorous incorporation in Escherichia coli ribonucleic acid after infections with bacteriophage T2. Virology 2:149-161.

Reports the discovery of short-lived RNA after phage infection.

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