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

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Regulation of Pre-mRNA Processing

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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.

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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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