Heath, Emma; (2003) Functional analysis of def-3, a novel dynamic nuclear RNA-binding protein. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Def-3 is a large hydrophilic nuclear RNA-binding protein, which contains a unique combination of functional domains conserved through evolution. These include a decamer repeat, two RNA-recognition motifs (RRMs), C4 and C2H2 type zinc-fingers and a G-patch domain. Biochemical analysis demonstrates that def-3 specifically binds to poly (G) RNA in vitro via the RRM/C4/RRM and G-patch domains. In addition, overexpression in Xenopus oocytes revealed that def-3 binds to the ribonucleoprotein (RNP) matrix of most transcription loops of the lampbrush chromosomes, suggesting that def-3 also interacts with RNA in vivo. Protein interaction studies reveal that the RRM/C4/RRM domain also facilitates interaction with the related RNA-binding protein luca-15 and the transcription factor Gfi-1, while the N-terminal domain is responsible for def-3 self-association. Together the results presented suggest def-3 is a component of one or more protein-complexes involved in the regulation of transcription and/or RNA processing. In mammalian cells endogenous def-3 protein is found diffuse in the nucleoplasm and localised to the splicing factor speckles, whereas exogenous def-3 protein is targeted to nuclear foci (def-3 bodies) which are always associated with the splicing factor speckles. Upon treatment with transcriptional inhibitors, a distinct sub-population of both, endogenous and exogenous def-3 protein relocalises to the nucleolar periphery and co-localises with paraspeckle protein 1. Fluorescence-Loss-In-Photobleaching analysis demonstrated a continuous exchange of def-3 between the nucleoli and nucleoplasmic compartments in transcriptionally active cells. Time-lapse microscopy confirmed the dynamic nature of the def-3 protein and suggests that def-3 movement is directional. Expression analysis showed that def-3 is differentially expressed in a spatially and temporally regulated manner during mouse embryogenesis. Early in development, def-3 expression was widespread, but became increasingly restricted as development progressed, consistent with the notion that def-3 function may be required in proliferating cells and is down regulated upon cellular differentiation.

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