Abstract
U-insertion/deletion RNA editing is a post-transcriptional mitochondrial RNA modification phenomenon required for viability of trypanosomatid parasites. Small guide RNAs encoded mainly by the thousands of catenated minicircles contain the information for this editing. We analyzed by NGS technology the mitochondrial genomes and transcriptomes of two strains, the old lab UC strain and the recently isolated LEM125 strain. PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions. Minicircles were identified by a characteristic size, the presence of three short conserved sequences, a region of inherently bent DNA and the presence of single gRNA genes at a fairly defined location. The LEM125 strain contained over 114 minicircles encoding different gRNAs and the UC strain only ~24 minicircles. Some LEM125 minicircles contained no identifiable gRNAs. Approximate copy numbers of the different minicircle classes in the network were determined by the number of PacBio CCS reads that assembled to each class. Mitochondrial RNA libraries from both strains were mapped against the minicircle and maxicircle sequences. Small RNA reads mapped to the putative gRNA genes but also to multiple regions outside the genes on both strands and large RNA reads mapped in many cases over almost the entire minicircle on both strands. These data suggest that minicircle transcription is complete and bidirectional, with 3’ processing yielding the mature gRNAs. Steady state RNAs in varying abundances are derived from all maxicircle genes, including portions of the repetitive divergent region. The relative extents of editing in both strains correlated with the presence of a cascade of cognate gRNAs. These data should provide the foundation for a deeper understanding of this dynamic genetic system as well as the evolutionary variation of editing in different strains.
Highlights
Kinetoplastid protists have an unusual mitochondrial genetic architecture: the mitochondrial genes + cryptogenes encoded in the 20–40 catenated maxicircles, and multiple guide RNAs encoded mainly in the ~10,000 catenated minicircles [1]
U-insertion/deletion RNA editing is a unique post-transcriptional mRNA modification process that occurs in trypanosomatid parasites and is required for viability
The participation of guide RNAs which are transcribed from the thousands of catenated minicircles in determining the precise sites and number of U’s inserted and deleted to create translatable mRNAs is novel and significant in terms of the recently realized importance of small RNAs in biology
Summary
Kinetoplastid protists have an unusual mitochondrial genetic architecture: the mitochondrial genes + cryptogenes encoded in the 20–40 catenated maxicircles, and multiple guide RNAs (gRNAs) encoded mainly in the ~10,000 catenated minicircles [1]. The gRNAs contain information for correction of DNA-encoded mRNA frameshifts at the RNA level [2] This unusual genetic organization reflects the evolution in these early protists of a post-transcriptional RNA modification phenomenon known as U-insertion/deletion RNA editing. The mechanism of this type of RNA editing involves hybridization of the 5’ end of the gRNA to the “pre-edited” mRNA sequence just downstream of the first editing site, endonuclease cleavage at the first upstream mismatch, followed either by the 3’ addition of U residues to the 5’ mRNA fragment which base pair with A or G “guiding nucleotides” in the gRNA and ligation of the two RNA fragments, or removal of unpaired 3’ U residues before RNA ligation [2]. Medium resolution cryoEM structures of the RECC particles from Leishmania tarentolae and Trypanosoma brucei have been published [17, 18] and several crystallographic structures of editing proteins are known [19,20,21,22,23,24,25,26]
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