Trypanosomes can initiate nuclear export co-transcriptionally

Abstract

The nuclear envelope serves as important messenger RNA (mRNA) surveillance system. In yeast and human, several control systems act in parallel to prevent nuclear export of unprocessed mRNAs. Trypanosomes lack homologues to most of the involved proteins and their nuclear mRNA metabolism is non-conventional exemplified by polycistronic transcription and mRNA processing by trans-splicing. We here visualized nuclear export in trypanosomes by intra- and intermolecular multi-colour single molecule FISH. We found that, in striking contrast to other eukaryotes, the initiation of nuclear export requires neither the completion of transcription nor splicing. Nevertheless, we show that unspliced mRNAs are mostly prevented from reaching the nucleus-distant cytoplasm and instead accumulate at the nuclear periphery in cytoplasmic nuclear periphery granules (NPGs). Further characterization of NPGs by electron microscopy and proteomics revealed that the granules are located at the cytoplasmic site of the nuclear pores and contain most cytoplasmic RNA-binding proteins but none of the major translation initiation factors, consistent with a function in preventing faulty mRNAs from reaching translation. Our data indicate that trypanosomes regulate the completion of nuclear export, rather than the initiation. Nuclear export control remains poorly understood, in any organism, and the described way of control may not be restricted to trypanosomes.

Parallel monitoring of mRNA abundance, localisation and compactness with correlative single molecule FISH on LR White embedded samples

“…While the method appears to work relatively well in large, cultured mammalian cells, its limits become in particular apparent when working with smaller cells such as yeast and many protozoans, that have a cell wall or dense cytoskeleton underneath the plasma membrane. In trypanosomes for example, there is a huge variance in fluorescence intensities between mRNA molecules within the same cell 2,3 and the same is observed on recent smFISH images from Plasmodium 4 . Almost no yeast publications exist that use branched DNA technology.…”

“…High pressure freezing and LR White embedding of trypanosome cell pellets was done as previously described 3,21 . 100 nm sections of either PCF or BSF trypanosomes were cut with an 'ultra Jumbo Diamond Knife' (Diatome AG) and placed on a poly-lysine slide in the order of cutting, to allow subsequent 3D reconstruction (if required).…”

Unexpected diversity in eukaryotic transcription revealed by the retrotransposon hotspot family of Trypanosoma brucei

“…In yeast and humans, transcription and mRNA export are connected via the TREX complex (34), but, in trypanosomatids, no components of this complex have been identified apart from TbSub2 (33,35). It was recently shown, however, that mRNA export occurs cotranscriptionally (36). Another peculiarity of trypanosomatid Pol II is that the major subunit, RPB1, lacks the characteristic heptapeptide repeats found in the C-terminal domain (CTD) in other eukaryotes (37).…”

Characterising ISWI chromatin remodeler in Trypanosoma cruzi

“…Both proteins have been involved in processes associated with mRNA metabolism in T. brucei and T. cruzi (31,42) and are present in NPGs (nuclear periphery granules) in T. brucei. (33,43) These granules function as an mRNA nuclear export control system, avoiding unprocessed mRNAs to reach translation (33) . Strikingly, TcISWI orthologue in yeast (ISW1) was de-scribed as an mRNP nuclear export surveillance factor that retains export-incompetent transcripts near their transcription site.…”

“…(31) DHH1 is an RNA helicase that in eukaryotes, including trypanosomes, has been involved in multiple RNA metabolism-related processes and accumulates in stress granules, processing bodies and nuclear periphery granules (NPGs). (32,33) The two remaining TcISWI partners are proteins containing a domain characteristic of SMC proteins. Members of this protein family are implicated in several activities that modulate chromosome structure and participate in the segregation and condensation of these structures from bacteria to humans (review in (34) ).…”