Novel modes of RNA editing in mitochondria

Abstract

Gene structure and expression in diplonemid mitochondria are unparalleled. Genes are fragmented in pieces (modules) that are separately transcribed, followed by the joining of module transcripts to contiguous RNAs. Some instances of unique uridine insertion RNA editing at module boundaries were noted, but the extent and potential occurrence of other editing types remained unknown. Comparative analysis of deep transcriptome and genome data from Diplonema papillatum mitochondria reveals ∼220 post-transcriptional insertions of uridines, but no insertions of other nucleotides nor deletions. In addition, we detect in total 114 substitutions of cytosine by uridine and adenosine by inosine, amassed into unusually compact clusters. Inosines in transcripts were confirmed experimentally. This is the first report of adenosine-to-inosine editing of mRNAs and ribosomal RNAs in mitochondria. In mRNAs, editing causes mostly amino-acid additions and non-synonymous substitutions; in ribosomal RNAs, it permits formation of canonical secondary structures. Two extensively edited transcripts were compared across four diplonemids. The pattern of uridine-insertion editing is strictly conserved, whereas substitution editing has diverged dramatically, but still rendering diplonemid proteins more similar to other eukaryotic orthologs. We posit that RNA editing not only compensates but also sustains, or even accelerates, ultra-rapid evolution of genome structure and sequence in diplonemid mitochondria.

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Euglena Transcript Processing

“…Following expression the module transcripts require processing through endonucleolytic cleavage, polyadenylation of the 3′ module, and trans-splicing in order to form mature full length transcripts (Kiethega et al 2013). The mechanism through which this transsplicing occurs is not yet understood, though it has been proposed that small guide RNAs may help in facilitating this process (Kiethega et al 2013;Moreira et al 2016). Additional editing of modules may also occur, including addition of short uridine stretches (1-3 nucleotides) to module ends, as well as both C-to-U and A-to-I editing (Moreira et al 2016).…”

“…The mechanism through which this transsplicing occurs is not yet understood, though it has been proposed that small guide RNAs may help in facilitating this process (Kiethega et al 2013;Moreira et al 2016). Additional editing of modules may also occur, including addition of short uridine stretches (1-3 nucleotides) to module ends, as well as both C-to-U and A-to-I editing (Moreira et al 2016). In the second major Euglenozoan group, the kinetoplastids, mitochondrial DNA (termed kinetoplast or kDNA) is also arranged into two classes of circular chromosomes.…”

“…In Diplonema papillatum, a 26 nucleotide poly-U stretch is found separating the 5′ and 3′ portions of the mature spliced LSU that is not encoded in the genes for either LSU fragment indicating that a process related to uridine insertion into mRNA modules can also occur to diplonemid rRNA. A short 366 nt RNA has been proposed as a potential mitochondrial SSU rRNA, but as of yet it is unclear if this represents the entire SSU rRNA or an individual fragment (Moreira et al 2016). Small rRNAs are not unheard of in Euglenozoans.…”

Unbiased Mitoproteome Analyses Confirm Non-canonical RNA, Expanded Codon Translations

“…Transcription is not always perfectly accurate, but in some cases, RNA–DNA differences (RDDs) are not random and are systematically detected at some specific positions, either in the form of nucleotide substitutions [24], also observed on mitochondrion-encoded RNAs [25], [26], [27]) or deletions [28]. These punctual differences between transcript and DNA occur shortly after transcripts exit the RNA polymerase, suggesting posttranscriptional RNA editing [29].…”