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^ Differential splicing ^

E1

E2

E1

Translation

Figure 4.3 Differential splicing of mRNA can yield different polypeptide products. Transcription of a gene sequence yields a 'primary transcript' RNA. This contains coding regions (exons) and non-coding regions (introns). A major feature of the subsequent processing of the primary transcript is 'splicing', the process by which introns are removed, leaving the exons in a contiguous sequence. Although most eukaryotic primary transcripts produce only one mature mRNA (and hence code for a single polypeptide), some can be differentially spliced, yielding two or more mature mRNAs. The latter can, therefore, code for two or more polypeptides. E: exon; I: intron

Additionally, the cellular location at which the resultant polypeptide will function often cannot be predicted from RNA delection/sequences nor can detailed information regarding how the polypeptide product's functional activity will be regulated (e.g. via post-translational mechanisms such as phosphorylation, partial proteolysis, etc.). Therefore, protein-based drug leads/targets are often more successfully identified by direct examination of the expressed protein complement of the cell, i.e. its proteome. Like the transcriptome (total cellular RNA content), and in contrast to the genome, the proteome is not static, with changes in cellular conditions triggering changes in cellular protein profiles/concentrations. This field of study is termed proteomics.

Proteomics, therefore, is closely aligned to functional genomics and entails the systematic and comprehensive analysis of the proteins expressed in the cell and their function. Classical proteomic studies generally entailed initial extraction of the total protein content from the target cell/tissue, followed by separation of the proteins therein using two-dimensional electrophoresis (Chapter 7). Isolated protein 'spots' could then be eluted from the electrophoretic gel and subjected to further analysis; mainly to Edman degradation, in order to generate partial amino acid sequence data. The sequence data could then be used to interrogate protein sequence databanks in order to, for example, assign putative function by sequence homology searches (Figure 4.4). Two-dimensional electrophoresis, however, is generally capable of resolving no more than 2000 different proteins, and proteins expressed at low levels may not be detected at all if their gel concentration is below

Figure 4.4 The proteomics approach. Refer to text for details

the (protein) staining threshold. The latter point can be particularly significant in the context of drug/target identification, as most such targets are likely to be kinases and other regulatory proteins that are generally expressed within cells at very low levels.

More recently, high-resolution chromatographic techniques (particularly reverse-phase and ion exchanged-based high-performance liquid chromatography (HPLC)) have been applied in the separation of proteome proteins and high-resolution mass spectrometry is being employed to aid high-throughput sequence determination.

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