Transcription and transport of a messenger RNP particle : Novel regulatory mechanisms

Sammanfattning: In all living cells gene expression is a central process that involves several steps, including transcription, processing, transport and translation. A fine-tuning of these events is a pre-requisite for proper cellular function. Many proteins are involved in this regulation and they are added to the nascent transcript concomitant with transcription, although they may exert their function much later. The messenger pre-mRNP particle formed needs accurate processing before exiting the cell nucleus through the nuclear pore complexes. The midge Chironomus tentans provides a unique experimental system for the structural analysis of the mRNP assembly and transport, due to the abundance and size of the RNP particles derived from the giant Balbiani ring (BR) genes. These granules can readily be identified in the electron microscope from early transcription in the nucleus to polysome formation in the cytoplasm. In this thesis, several different aspects of the BR RNP biogenesis have been investigated. We revealed that the RNA-binding protein hrp65-2 interacts directly with nuclear actin, and is present all along the active gene associated with nascent pre-mRNP. Disruption of this interaction leads to transcriptional inhibition. We propose that actin is a part of a chromatin remodeling complex, coupled to pre-mRNP via hrp65-2. We have identified and characterized a transcript-specific hnRNP protein, hrp130, present along the Balbiani ring 3 gene. The domain structure of hrp130 is similar to the human protein CA150 that works as a repressor of transcription elongation. We propose that hrp130 enables proper splicing of the intron-rich BR3 gene transcripts by regulating the elongation rate. We discovered a link between transcription and export of the BR particle, by inhibiting transcription and looking for transport defects over the nuclear envelope. Upon transcriptional inhibition, the binding of BR particles to the nuclear pore complexes was drastically reduced and the export was almost abolished. The passage of mRNPs and ribosomal particles through the nuclear pore complex was inhibited by the nucleoporin-binding agents wheat germ agglutinin and the monoclonal antibody mAb 414. There was an accumulation of BR and ribosomal particles in the nucleoplasm and remarkably also a zone free from rRNPs beneath the nuclear envelope. We suggest that normally the basket fibrils move freely and repel molecules not binding to nucleoporins. In summary, we have identified novel mechanisms for the regulation of BR RNP particle transcription and transport. We found an actin-hrp65-2 interaction essential for the transcription elongation process and a transcription elongation repressor, hrp130, enabling cotranscriptional splicing of an intron-rich gene. A close coupling between ongoing transcription and RNA export was detected, possibly taking place via regulation of the transport of the mRNPs to the nuclear pore complex. Finally, we identified an exclusion zone free from rRNPs beneath the nuclear envelope, the clearing probably accomplished by constantly moving basket fibrils.