Search

The majority of metazoan genes encode pre-mRNAs that are subject to alternative splicing. For example, it has recently been estimated that as many as 74% of human genes encode alternatively spliced mRNAs (1). An alternatively spliced gene can generate anywhere from 2 different isoforms to as many as 38,016 isoforms in the case of the Drosophila Dscam gene (2). Thus, alternative splicing serves to greatly expand the diversity of the proteins encoded by a genome (3). [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) is a useful tool for degrading targeted messenger RNAs (mRNAs) and thus "knocking down" the abundance of the encoded protein. We have been using RNAi in cultured Drosophila cells to evaluate the effect of "knocking down" numerous mRNA processing factors on the alternative splicing of specific pre-mRNAs. This relatively simple technique has allowed us to identify a number of splicing factors that impact the alternative splicing of particular alternatively spliced exons. This approach can be extended to examine the splicing of nearly any gene. [ABSTRACT FROM AUTHOR]

Single-strand conformational polymorphism analysis has been used successfully to identify single nucleotide changes within sequences based on the fact that multidetection enhancement gels will separate molecules based on their conformation rather than their size. We have expanded the utility of this technique to analyze easily the alternative splicing of pre-mRNAs containing multiple mutually exclusive exons of the same size. We have used this technique to study the Caenorhabditis elegans let-2 gene containing two alternative exons and the Drosophilia melanogaster Dscam gene, which contains 12 mutually exclusive exons. The ease and the quantitative nature of this technique should be very useful. [ABSTRACT FROM AUTHOR]