Abstract
Transcription of the human mitochondrial genome and correct processing of the two long polycistronic transcripts are crucial for oxidative phosphorylation. According to the tRNA punctuation model, nucleolytic processing of these large precursor transcripts occurs mainly through the excision of the tRNAs that flank most rRNAs and mRNAs. However, some mRNAs are not punctuated by tRNAs, and it remains largely unknown how these non-canonical junctions are resolved. The FASTK family proteins are emerging as key players in non-canonical RNA processing. Here, we have generated human cell lines carrying single or combined knockouts of several FASTK family members to investigate their roles in non-canonical RNA processing. The most striking phenotypes were obtained with loss of FASTKD4 and FASTKD5 and with their combined double knockout. Comprehensive mitochondrial transcriptome analyses of these cell lines revealed a defect in processing at several canonical and non-canonical RNA junctions, accompanied by an increase in specific antisense transcripts. Loss of FASTKD5 led to the most severe phenotype with marked defects in mitochondrial translation of key components of the electron transport chain complexes and in oxidative phosphorylation. We reveal that the FASTK protein family members are crucial regulators of non-canonical junction and non-coding mitochondrial RNA processing.
Highlights
The circular 16.5 kbp human mitochondrial DNA encodes 37 genes including two rRNAs, 22 tRNAs, and 13 ORFs, all of which are essential for oxidative phosphorylation (OXPHOS) [1]
Mitochondrial transcription initiates at two distinct divergent promoters, one on each strand, leading to two almost genome-length polycistronic transcripts: the primary transcript generated from the heavy strand promoter (HSP) includes 8 monocistronic mRNAs, two bicistronic mRNAs (ND4L/4 and ATP8/6), 14 tRNAs, and two rRNAs, while the complementary primary transcript derived from the light strand promoter (LSP) encodes only one mRNA (ND6), 8 tRNAs, and long stretches of non-coding sequences [2,3]
The Fas-activated serine/threonine kinase (FASTK) family proteins regulate the levels of sense and antisense transcripts and non-canonical mitochondrial RNA (mtRNA) processing
Summary
The circular 16.5 kbp human mitochondrial DNA (mtDNA) encodes 37 genes including two rRNAs, 22 tRNAs, and 13 ORFs, all of which are essential for oxidative phosphorylation (OXPHOS) [1]. Most mRNAs and rRNAs within the polycistronic transcripts are flanked by tRNAs, and cleavage at the tRNA junctions by RNase P (MRPP1-3) and RNase Z (ELAC2) allows excision of the individual transcripts [7,8,9,10,11,12,13]. This is referred to as the ‘tRNA punctuation model’ [14,15], and the flanking tRNAs are named canonical processing junctions. Recent work has identified several candidates involved in the process, including the Fas-activated serine/threonine kinase (FASTK) family proteins [11,16,17,18,19]
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