Discussion

In trypanosomes, mRNAs are transcribed as long polycistrons and processed by trans-splicing. Inhibition of trans-splicing causes the formation of a novel type of RNP granule at the cytoplasmic site of the nucleus, nuclear periphery granules (NPGs). Here, we have determined the granule�s proteome by co-purifying the granules with trypanosome nuclei followed by validation via eYFP tagging. We have identified 45 novel NPG granule proteins and a further 58 NPG candidate proteins. NPGs contain almost exclusively proteins with a known connection to mRNA metabolism. The absence of translation factors, together with the granule�s perinuclear localisation, dependency on active transcription and insensitivity to cycloheximide indicates that NPGs are cytoplasmic RNP complexes prior to entry into translation: the presence or absence of a certain protein in the NPG proteome can therefore serve as a prediction tool for its entry-time into mRNA metabolism. Single mRNA FISH showed no enrichment of unspliced mRNAs at the nuclear periphery, but a large fraction of unspliced mRNA in the cytoplasm. This argues against a function of NPGs in mRNA quality control and we discuss alternative reasons for NPG formation. While the exact function and nature of NPGs still remains unknown, their formation must result from an interruption of the mRNA nuclear export pathway. The further analysis of such a unique �frozen state� can contribute to a better understanding of nuclear export mechanisms.