RNA Polymerases and the Transcription Event

Transcription Products

Introns, Exons, and Splicing hn RNA

Alternate Splicing of Exons

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Alternate Splicing of Exons

A classic example of alternate splicing is the rat muscle protein, troponin T. The gene consists of five exons, each representing a domain of a final protein. These exons are each separated by an intron. The five exons are W, X, Alpha, Beta, and Z. Two types of protein are found. The alpha form consists of exons W, X, alpha and Z. The beta form consists of the W, X, Beta and Z exons. The two different types of the protein are produced by alternative splicing of the same gene. The two different gene products are produced by selective splicing such that introns three and four and the fourth exon are spliced as one unit. In some manner the 5' GT sequence of intron 3 and the 3' AG sequence of the fourth intron are used during the splcing event.

Alternate Splicing of a Plant mRNA [Werneke et al., The Plant Cell 1:815 (1989)]

The plant example of alternate is for the gene encoding the enzyme RUBISCO activate. RUBISCO activase is a soluble, nuclear-encoded chloroplast enzyme. This abundant protein, is about 2% of the soluble leaf protein in spinach. Two molecular forms of the protein are recognized. In spinach, the proteins are 41 kd and 45 kd polypedtides, and the proteins are 44 kd and 47 kd polypeptides in Arabidopsis. The monocot corn is different and has only one protein. This enzyme is required to activate the enzymatic activity of RUBISCO, the first step in the conversion of the electromagnetic energy collected during the light reaction of photosynthesis in chemical energy in the forms of sugars.

The RUBISCO activase gene has been cloned. cDNA clones had been isolated previously and revealed two nucleotide binding sites. When the cDNA was expressed in E. coli only one polypeptide was detected. Antibodies from each of the 41 and 45 kd strongly crossed reacted with each other suggesting a high degree of homology between the proteins. Furthermore genomic southerns with the cDNAs showed that both spinach and Arabidopis had a single gene. How can this be explained ?

Additional cDNA clones were isolated and sequenced for both spinach and Arabadopsis. The following table describes the differences between the two types of cDNAs.

Species 3' Sequence Comparison Internal Sequences
Spinach Identical 22-bp insertion; introduced a new stop codon
Arabadopsis Identical 11-bp insertion at same location; introduced a new stop

Genomic clones were then isolated for each of the two species and sequenced. The sequence data revealed that each of the genes contained seven exons and six introns. Comparison of the sequences of the genomic and cDNA clones of spinach revealed the following manner in which the two different types of cDNAs could be generated. The full length mRNA that gave rise to the larger protein was generated by splicing at the terminal-most 5' and 3' splicing sites. 22-bp into intron 6 is another 5' splice junction sequence. Alternate splicing occurs at this site to generate the mRNAs that are translated into the shorter proteins. This 22-bp sequence can be considered an extension of exon 6 and is called an auxon for alternative exon. Alternative splicing events can form different mRNAs from a single primary transcript. Thus an exon can actually be represented as an intron in another transcript.

Copyright © 1998. Phillip McClean