Analyze The Data

A specific protein kinase (PK) gene is speculated to be differentially spliced in muscle tissue. This gene comprises three exons and two intron sequences and in fibroblast cells encodes a 38.5-kD protein. Investigators have trans-fected various portions of a genomic or cDNA copy of the PK gene into both muscle and fibroblast cells (see the constructs in part (a) of the figure). The expression system utilizes a promoter active in both cell types and a C-terminal fusion to a small epitope tag called V5, which contributes ~5.5 kD to the fusion protein. Part (b) of the figure shows the results of an immunoprecipitation experiment designed to analyze the expression products of the transfected cells. A negative control (Neg) of untransfected muscle cells is included. Molecular weight markers in kD are indicated on the left. Immunoprecipitated proteins as shown in part (b) were then placed in protein kinase assays with two different substrates, A and B, to discern activity of the expressed proteins.

mRNA-exporter 514 mRNA surveillance 523 nuclear pore complex

(NPC) 509 poly(A) tail 496 pre-mRNA 494 pre-rRNA 525 Ran protein 510 ribozyme 527 RNA editing 508 RNA recognition motif (RRM) 495 RNA splicing 497 RNA-induced silencing complex (RISC) 518 small nuclear

RNAs (snRNAs) 499 spliceosome 499 SR proteins 501

Dreyfuss, G., V. N. Kim, and N. Kataoka. 2002. Messenger-RNA-binding proteins and the messages they carry. Nature Rev. Mol. Cell Biol. 3:195-205.

Moore, M. J. 2002. Nuclear RNA turnover. Cell 108:431-434.

Shatkin, A. J., and J. L. Manley. 2000. The ends of the affair: capping and polyadenylation. Nature Struct. Biol. 7:838-842.

Villa, T., J. A. Pleiss, and C. Guthrie. 2002. Spliceosomal snRNAs: Mg(2+)-dependent chemistry at the catalytic core? Cell 109:149-152.

Regulation of Pre-mRNA Processing

Black, D. L. 2003. Mechanisms of alternative pre-mRNA splicing. Ann. Rev. Biochem. 72:291-336.

Blanc, V., and N. O. Davidson. 2003. C-to-U RNA editing: mechanisms leading to genetic diversity. J. Biol. Chem. 278:1395-1398.

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

Maas, S., A. Rich, and K. Nishikura. 2003. A-to-I RNA editing: recent news and residual mysteries. J. Biol. Chem. 278:1391-1394.

Maniatis, T., and B. Tasic. 2002. Alternative pre-mRNA splicing and proteome expansion in metazoans. Nature 418: 236-243.

Macromolecular Transport Across the Nuclear Envelope

Chook, Y. M., and G. Blobel. 2001. Karyopherins and nuclear import. Curr. Opin. Struct. Biol. 11:703-715.

Conti, E., and E. Izaurralde. 2001. Nucleocytoplasmic transport enters the atomic age. Curr. Opin. Cell Biol. 13:310-319.

Johnson, A. W., E. Lund, and J. Dahlberg. 2002. Nuclear export of ribosomal subunits. Trends Biochem Sci. 27:580-585.

Reed, R., and E. Hurt. 2002. A conserved mRNA export machinery coupled to pre-mRNA splicing. Cell 108:523-531.

Ribbeck, K., and D. Gorlich. 2001. Kinetic analysis of translocation through nuclear pore complexes. Embo. J. 20:1320-1330.

Rout, M. P., and J. D. Aitchison. 2001. The nuclear pore complex as a transport machine. J. Biol. Chem. 276:16593-16596.

a. What can be concluded about differential splicing of the PK gene in fibroblast versus muscle cells? Are there other experiments that could confirm these results?

b. What sequence or sequences contribute to regulation of alternative splicing?

c. How does the presence or absence of exon 3 alter the catalytic activity of the encoded PK protein? Which data support your conclusions?

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