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- Materials and Methods
- Supporting Information
Mitochondrial genomes (mtDNAs) in angiosperms consist largely of noncoding sequences, within which c. 60 known functional genes are embedded (Unseld et al., 1997; Kubo et al., 2000; Adams et al., 2002; Notsu et al., 2002; Handa, 2003; Clifton et al., 2004; Ogihara et al., 2005; Sugiyama et al., 2005). These encode tRNAs, rRNAs, ribosomal proteins, subunits of the respiratory machinery (NADH:ubiquinone oxidoreductase (complex I), cytochrome bc1 complex (complex III), cytochrome c oxidase (complex IV), and ATP-synthase (complex V)), a twin-arginine translocation pathway component (TatC) and enzymes involved in cytochrome c biogenesis (Knoop, 2012). Yet the vast majority of mitochondrial proteins are encoded by nuclear genes, translated on cytosolic ribosomes, imported into the organelle and, in the case of the ribosomes and respiratory chain complexes, subsequently assembled together with organelle-encoded subunits. These processes require complex mechanisms to coordinate the expression and accumulation of proteins that are derived from two physically separate genetic systems (Barkan, 2011). Much of this control is thought to be at the post-transcriptional level, via nucleus-encoded RNA-binding cofactors, which are targeted to the mitochondria and function in the processing of the organellar transcripts.
The expression of mitochondrial genes in angiosperms is catalyzed by single subunit phage-type RNA polymerases, possibly in conjunction with accessory factors that aid promoter recognition (Kühn et al., 2009). The primary transcripts undergo extensive RNA processing steps, which include the splicing of numerous group II introns found within many essential genes (Unseld et al., 1997; Gagliardi & Binder, 2007; Bonen, 2008). The removal of these introns from the coding sequences they interrupt is essential for organellar function and is mediated by various protein cofactors.
Group II introns are large catalytic RNAs found in bacteria and in the organelles of fungi and certain protists, but they are particularly numerous within plant mitochondria (Gagliardi & Binder, 2007; Bonen, 2008). The organellar introns in plants are degenerate, lacking many intronic regions that were once considered to be essential for the splicing activity (Bonen, 2008). Several of these introns in angiosperms became fragmented, such that they are transcribed in pieces and are then spliced in ‘trans’ (Bonen, 2008). Given their degeneracy and the fact that none of the introns in plant mitochondria have been shown to self-splice in vitro, it is anticipated that they require the participation of protein cofactors for their efficient splicing in vivo. However, only a small number of such factors have been identified in plants, and even less is known about their specific roles in splicing. Proteins that function in the splicing of mitochondrial group II introns in plants include maturase-related proteins (Keren et al., 2009, 2012), pentatricopeptide repeat (PPR) proteins (Falcon de Longevialle et al., 2007; Koprivova et al., 2010; Liu et al., 2010), a homolog of ‘regulator of chromosome condensation’ (RUG3; Kühn et al., 2011), a ‘PORR-domain’ family member (WTF9; Colas des Francs-Small et al., 2011) and a DEAD-box RNA-helicase protein (PMH2) which influences the processing, or the stability, of many mitochondrial transcripts in Arabidopsis (Köhler et al., 2010). These proteins are diverse in origin and most probably also in their mechanism of action.
CRS1-YhbY domain (CRM; Pfam-PF01985) is a recently recognized RNA-binding motif of bacterial origin (Barkan et al., 2007; Keren et al., 2008) that is present in several group II intron splicing factors in plant chloroplasts: CRS2-associated factor 1 (CAF1), CAF2, CRM Family Member 2 (CFM2), CFM3 and CRS1 (Jenkins et al., 1997; Till et al., 2001; Ostersetzer et al., 2005; Asakura & Barkan, 2007; Barkan et al., 2007; Asakura et al., 2008). Interestingly, CAF1 and CAF2 are closely related paralogs, each containing two CRM domains, which form functional splicing complexes with a peptidyl-tRNA hydrolase homolog (CRS2) to promote the splicing of many group II introns in the chloroplasts (Jenkins & Barkan, 2001; Ostheimer et al., 2003; Asakura & Barkan, 2006; Asakura et al., 2008).
In angiosperms, the CRM family is represented by 14 orthologous gene groups, containing between one and four repeats of the conserved domain (Barkan et al., 2007), but these are largely uncharacterized. Two of these hypothetical proteins, annotated here as mitochondria CAF-like splicing factor 1 (mCSF1; At4 g31010) and mCSF2 (At5 g54890), are closely related to CAF factors and are predicted to be targeted to mitochondria (Ostheimer et al., 2006). These are therefore excellent candidates for mitochondrial group II intron splicing factors in plants.
In this study, we show the mitochondrial localization of mCSF1 and establish its roles in the splicing of group II introns in plants. The effects of lowering the expression of this novel mitochondria-localized CRM-associated factor on the phenotype and physiology of ‘knockdown’ mutants in Arabidopsis thaliana are discussed.