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Conventional kinesin is a microtubule-based molecular motor that transports membranous and non-membranous cargoes towards the cellular periphery. The kinesin holoenzyme exists as a heterotetramer, consisting of two heavy chain and two light chain subunits and it is thought that one function of the light chains is to interact with the cargo. Kinesin was considered an ideal target to investigate the transport and assembly of keratin intermediate filament precursors following reports of a kinesin-vimentin intermediate filament interaction, and a series of GFP-tagged kinesin light chain mutants were generated to study this potential interaction. These mutants were shown not to act as dominant negatives in HaCaT, BHK-21 or PtK2 cell lines, and no mislocalisation of intermediate filaments or mitochondria was observed. However, mitochondrial transport was shown to be microtubule dependent and able to be disrupted in the cell lines studied. Detergent-based approaches established that only a small amount of mutant light chains were entering kinesin heterotetramers, thus confirming that the approach was not able to adequately address the question of a keratin-kinesin interaction. Alternative splicing of kinesin light chain pre-mRNA has been observed in lower organisms although evidence for alternative splicing of the human gene has not previously been reported. In this study, bioinformatic and molecular biological approaches identified 20 variants of the human KNS2 gene that are generated by alternative splicing of downstream exons but calculations suggest that KNS2 has the potential to produce over 285 000 spliceforms. The observation of multiple light chain isoforms is consistent with their proposed role in specific cargo attachment. Some of these variants are widely expressed whereas others show a more restricted pattern of expression. The alternative exons are all located 3' of exon 12 and the novel spliceforms produce both alternative carboxy termini and alternative 3' untranslated regions (3' UTRs). The alternative 3' UTRs were shown to vary in sequence and length, and a number of putative regulatory elements have been identified, including; ARE stability modulating elements, C/U rich elements and the LOX-DICE translation efficiency modulating element. The role of these elements in transcript stability was examined using a reporter-based assay following transcription inhibition and the half-lives of the tested KLC 3' UTRs demonstrated variable stability, mediated by specific sequence elements. Furthermore, the translational efficiency of these variants was also shown to be modulated by a number of cis-elements, using flow cytometry. This study further establishes the complexity of kinesin biology, particularly with respect to the diversity and highly regulated nature of the light chains.
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