The RCC1 family protein RUG3 is required for splicing of nad2 and complex I biogenesis in mitochondria of Arabidopsis thaliana

Abstract

SUMMARYWe have identified a mitochondrial protein (RUG3) that is required for accumulation of mitochondrial respiratory chain complex I. RUG3 is related to human REGULATOR OF CHROMOSOME CONDENSATION 1 (RCC1) and Arabidopsis UV-B RESISTANCE 8 (UVR8). Although the family of RCC1-like proteins in Arabidopsis has over 20 members, UVR8 is the sole plant representative of this family to have been functionally characterized. Mitochondria from Arabidopsis plants lacking a functional RUG3 gene showed greatly reduced complex I abundance and activity. In contrast, accumulation of complexes III, IV and V of the oxidative phosphorylation system and the capacity for succinate-dependent respiration were unaffected. A comprehensive study of processes contributing to complex I biogenesis in rug3 mutants revealed that RUG3 is required for efficient splicing of the nad2 mRNA, which encodes a complex I subunit. A comparison of the formation of complex I assembly intermediates between rug3 and wild type mitochondria indicated that NAD2 enters the assembly pathway at an early stage. Remarkably, rug3 mutants displayed increased capacities for import of nucleus-encoded mitochondrial proteins into the organelle and showed moderately increased mitochondrial transcript levels. This observation is consistent with global transcript changes indicating enhanced mitochondrial biogenesis in the rug3 mutant in response to the complex I defect.

Dual Location of the Mitochondrial Preprotein Transporters B14.7 and Tim23-2 in Complex I and the TIM17:23 Complex in Arabidopsis Links Mitochondrial Activity and Biogenesis

“…In the case of a variety of other respiratory chain components, the most notable effect observed was a >80% reduction in Ndufs4 protein abundance in the tim23-2 OE, rug, and rpotmp lines ( Figure 7C), supporting the previous report of this for mitochondria from rug lines (Kühn et al, 2011). Ndufs4 is a complex I subunit that is proposed to be present in the peripheral portion of complex I .…”

“…For example, while Tim23 and Tim17 from yeast and plants do not display high levels of sequence identity, the plant genes for these components can still complement yeast deletion strains and restore viability , and B14.7 does have a consensus PRAT domain, whereas Tim23-2 does not (Rassow et al, 1999). Finally even low levels of B14.7 are sufficient to maintain viability, as observed in the rug and rpotmp KO lines, where only low levels of B14.7 are observed ( Figure 7A) (Kühn et al, , 2011. The amount of the TIM23 complex is quite low compared with respiratory chain complexes, even in yeast, where genetic approaches had to be used to characterize TIM translocases, compared with the more abundant TOM components, which could be characterized by biochemical approaches .…”

The Pentatricopeptide Repeat Proteins TANG2 and ORGANELLE TRANSCRIPT PROCESSING439 Are Involved in the Splicing of the Multipartite nad5 Transcript Encoding a Subunit of Mitochondrial Complex I

“…Six-week-old tang2 plants displayed dark curled foliage (Fig. 1D), reminiscent of other complex I mutants such as otp43, NADH dehydrogenase (ubiquinone) fragment S subunit4, and rpoTmp (Falcon de Longevialle et al, 2007;Meyer et al, 2009;Kühn et al, 2011).…”

“…Expression of these organelle genes requires the participation of numerous nuclearly encoded factors that perform the posttranscriptional steps (editing, intron splicing, protection of the 39 and 59 ends, and stability) necessary for the production of translatable transcripts. Several families of such factors have been described, containing various RNA-binding domains such as CHLOROPLAST RNA SPLICING AND RIBOSOME MATURATION (CRM; Asakura and Barkan, 2006;Zmudjak et al, 2013), PLANT ORGANELLAR RNA RECOGNITION (Kroeger et al, 2009;Colas des Francs-Small et al, 2012), REG-ULATOR OF CHROMOSOME CONDENSATION (RCC; Kühn et al, 2011), MULTIPLE ORGANELLAR RNA EDITING FACTOR , and RNA EDITING-INTERACTING PROTEIN (Bentolila et al, 2012), but the pentatricopeptide repeat (PPR) protein family is by far the biggest, with around 450 genes found in Arabidopsis (Arabidopsis thaliana), where it was first described (Barkan and Small, 2014). These proteins are widely distributed among eukaryotes but particularly abundant in land plants.…”

“…The expression of the nad genes involves a combination of cis-and trans-splicing events that remove group II introns from the transcripts (Bonen, 2008). Several RNA-binding proteins are known to be involved in mitochondrial splicing events in nad transcripts: ORGANELLE TRANSCRIPT PROCESS-ING43 (OTP43; trans-splicing of nad1 intron 1; Falcon de Longevialle et al, 2007), ABSCISIC ACID OVERLY SENSITIVE5 (cis-splicing of nad2 intron 3; Liu et al, 2010), RCC1/UV-B RESISTANCE8/GUANINE NUCLEOTIDE EXCHANGE FACTOR3 (RUG3; (trans-splicing of nad2 intron 2 and cis-splicing of intron 3; Kühn et al, 2011), NUCLEAR MALE STERILE1 (cis-splicing of nad4 intron 1; Brangeon et al, 2000), BUTHIONINE SULFOMIXINE-INSENSITIVE ROOTS6 (BIR6; cis-splicing of nad7 intron 1; Koprivova et al, 2010), and other less-specific factors such as PUTATIVE MITOCHONDRIAL RNA HELI-CASE2 (PMH2; Köhler et al, 2010), maturases (nMAT1, nMAT2, and nMAT4; Keren et al, 2009Keren et al, , 2012Brown et al, 2014;Cohen et al, 2014), and the MITOCHONDRIAL CHLOROPLAST RNA SPLICING ASSOCIATED FACTORlike SPLICING FACTOR1 (mCSF1; Zmudjak et al, 2013) affecting numerous introns.…”