Analyze the data 10-2: Alternative Splicing of a Protein Kinase A specific prote

Question

Analyze the data 10-2: Alternative Splicing of a Protein Kinase

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 transfected 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.

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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Explanation / Answer

a) Alternative splicing is a process that enable a mRNA to direct synthesis of different protein variants, they may have different cellular functions or properties. It occur by rearranging the pattern of intron and exon elements that are joined by splicing to alter the mRNA coding sequence. In fibroblast , differential splicing produce three different mRNA and different product. In immunoprecipitation data you can see that three protein of different molecular weight whereas in muscle cells, two different protein product form by differential splicing. In muscle cell, 1 and 3 mRNA product have same molecular weight and same function.

b) Cis acting elements in protein kinase pre mRNA lead to altered tissue specific choices that create alternate mature transcript. Exonic and intronic splicing enhancer site regulate alternative splicing. These are cis acting sequence bound to trans factor.

c) Presence of exon 3 , in muscles cell have affinity toward substrate B where is the absence result in no catalytic activity for both the substrate. In fibroblast, exon 3 presence result in different specificity toward substrate A and B. It indicate that for catalytic action of protein kinase , presence of exon 3 is necessary. Exon 3 form a catalytic domain of protein. Data C support this result.

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