The dynamics of snRNP maturation and nuclear architecture in mammalian cells.
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Accurate mRNA splicing is vital for the correct expression of mammalian genes. snRNPs (small nuclear ribonucleoproteins) are essential splicing factors comprising a highly modified snRNA component and a number of additional proteins. Within the nucleus, splicing snRNPs show a complex localisation pattern, accumulating in Cajal bodies, nucleoli, speckles and a diffuse nucleoplasmic component. The initial stages of snRNP maturation, including the addition of the core snRNP, 'Sm', proteins, occur in the cytoplasm of the cell and require the protein 'survival of motor neurons' (SMN). Mutations in the human gene encoding SMN are responsible for the inherited muscular degenerative disease spinal muscular atrophy (SMA), although the mechanisms by which defects in snRNP assembly may lead to the disease are not understood. The localisation of snRNPs is dynamic, and certain to reflect molecular events underlying the control of the metabolic state of cells.

We are currently using stable cell lines expressing a variety of nuclear proteins, including SMN and Sm proteins, tagged with fluorescent proteins to examine the dynamics of a range of different factors. The behaviour of newly imported proteins entering the nucleus can be studied using a heterokaryon assay, in which cells from stable cell lines are fused to cells not expressing a fusion protein, or expressing a different 'colour' of fusion protein. Three-dimensional time-lapse imaging and photo-bleaching techniques such as FRAP (Fluorescence Recovery After Photo-bleaching) and FLIP (Fluorescence Loss In Photo-bleaching) are used to analyse the steady-state dynamics of nuclear proteins and the characteristics of their exchange between compartments.