Trans-splicing and RNA editing of LSU rRNA in Diplonema mitochondria.

Mitochondrial ribosomal RNAs (rRNAs) often display reduced size and deviant secondary structure, and sometimes are fragmented, as are their corresponding genes. Here we report a mitochondrial large subunit rRNA (mt-LSU rRNA) with unprecedented features. In the protist Diplonema, the rnl gene is split into two pieces (modules 1 and 2, 534- and 352-nt long) that are encoded by distinct mitochondrial chromosomes, yet the rRNA is continuous. To reconstruct the post-transcriptional maturation pathway of this rRNA, we have catalogued transcript intermediates by deep RNA sequencing and RT-PCR. Gene modules are transcribed separately. Subsequently, transcripts are end-processed, the module-1 transcript is polyuridylated and the module-2 transcript is polyadenylated. The two modules are joined via trans-splicing that retains at the junction ∼ 26 uridines, resulting in an extent of insertion RNA editing not observed before in any system. The A-tail of trans-spliced molecules is shorter than that of mono-module 2, and completely absent from mitoribosome-associated mt-LSU rRNA. We also characterize putative antisense transcripts. Antisense-mono-modules corroborate bi-directional transcription of chromosomes. Antisense-mt-LSU rRNA, if functional, has the potential of guiding concomitantly trans-splicing and editing of this rRNA. Together, these findings open a window on the investigation of complex regulatory networks that orchestrate multiple and biochemically diverse post-transcriptional events.

RNA-level unscrambling of fragmented genes in Diplonema mitochondria.

We previously reported a unique genome with systematically fragmented genes and gene pieces dispersed across numerous circular chromosomes, occurring in mitochondria of diplonemids. Genes are split into up to 12 short fragments (modules), which are separately transcribed and joined in a way that differs from known trans-splicing. Further, cox1 mRNA includes six non-encoded uridines indicating RNA editing. In the absence of recognizable cis-elements, we postulated that trans-splicing and RNA editing are directed by trans-acting molecules. Here, we provide insight into the post-transcriptional processes by investigating transcription, RNA processing, trans-splicing and RNA editing in cox1 and at a newly discovered site in cob. We show that module precursor transcripts are up to several thousand nt long and processed accurately at their 5' and 3' termini to yield the short coding-only regions. Processing at 5' and 3' ends occurs independently, and a processed terminus engages in trans-splicing even if the module's other terminus is yet unprocessed. Moreover, only cognate module transcripts join, though without directionality. In contrast, module transcripts requiring RNA editing only trans-splice when editing is completed. Finally, experimental and computational analyses suggest the existence of RNA trans-factors with the potential for guiding both trans-splicing and RNA editing.

Evolutionarily conserved cox1 trans-splicing without cis-motifs.

In the protist Diplonema papillatum (Diplonemea, Euglenozoa), mitochondrial genes are systematically fragmented with each nonoverlapping piece (module) encoded individually on a distinct circular chromosome. Gene modules are transcribed separately, and precursor transcripts are assembled to mature mRNA by a trans-splicing process of yet unknown mechanism. Expression of the cox1 gene that consists of nine modules, also involves RNA editing by which six uridines are added between Modules 4 and 5. Here, we investigate whether the unusual features of cox1 are shared by all Diplonemea and what the mechanism of trans-splicing might be. We examine three additional species representing both Diplonemea genera, namely D. papillatum described before, and D. ambulator, Diplonema sp.2, and Rhynchopus euleeides and discover that in all Diplonemea, the cox1 gene is discontinuous and split up into nine modules that each reside on a distinct chromosome. Positions of gene breakpoints vary by up to two nucleotides. Further, all taxa have six nonencoded uridines inserted in cox1 mRNA at exactly the same position as D. papillatum. In silico searches do not detect signatures of introns known to engage in trans-splicing, in particular Group I, Group II, spliceosomal, and transfer RNA introns. Nor did we find statistically significant reverse-complementary motifs between adjacent modules and their flanking regions, or residues conserved within or across species. This provides compelling evidence that trans-splicing in Diplonemea mitochondria does not rely on sequence elements in cis but rather proceeds by a mechanism employing matchmaking trans factors, such as RNAs or proteins.