nMAT4, a maturase factor required for nad1 pre‐mRNA processing and maturation, is essential for holocomplex I biogenesis in Arabidopsis mitochondria

Abstract

SUMMARYGroup II introns are large catalytic RNAs that are found in bacteria and organellar genomes of lower eukaryotes, but are particularly prevalent within mitochondria in plants, where they are present in many critical genes. The excision of plant mitochondrial introns is essential for respiratory functions, and is facilitated in vivo by various protein cofactors. Typical group II introns are classified as mobile genetic elements, consisting of the self-splicing ribozyme and its own intron-encoded maturase protein. A hallmark of maturases is that they are intron-specific, acting as cofactors that bind their intron-containing pre-RNAs to facilitate splicing. However, the degeneracy of the mitochondrial introns in plants and the absence of cognate intronencoded maturase open reading frames suggest that their splicing in vivo is assisted by 'trans'-acting protein factors. Interestingly, angiosperms harbor several nuclear-encoded maturase-related (nMat) genes that contain N-terminal mitochondrial localization signals. Recently, we established the roles of two of these paralogs in Arabidopsis, nMAT1 and nMAT2, in the splicing of mitochondrial introns. Here we show that nMAT4 (At1g74350) is required for RNA processing and maturation of nad1 introns 1, 3 and 4 in Arabidopsis mitochondria. Seed germination, seedling establishment and development are strongly affected in homozygous nmat4 mutants, which also show modified respiration phenotypes that are tightly associated with complex I defects.

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

“…This phenomenon has been reported for other mutants, such as otp43 (Falcon de Longevialle et al, 2007), bir6 (Koprivova et al, 2010), mtsf1 (Haïli et al, 2013), nMat1 (Keren et al, 2012), nMat2 (Keren et al, 2009), nMat4 (Cohen et al, 2014), mcsf1 (Zmudjak et al, 2013), and indh (Wydro et al, 2013), as well as the Nicotiana sylvestris mutant cms2 (Gutierres et al, 1997) and the maize (Zea mays) nonchromosomal stripe1 (Karpova and Newton, 1999). The molecular defect in tang2 is much stronger than that of otp439, and this is reflected by the quantity of the assembled complex I in the mutants, which is very likely the reason behind the discrepancy between their phenotypes.…”

“…and M. polymorpha also contain several group I introns, the vast majority of the introns in flowering plants (with the exception of the cox1 intron in some species) belong to group II, with 23 in Arabidopsis and rice (Oryza sativa; Bonen, 2008). These introns seem to have lost their self-splicing ability, thus necessitating the help of maturases such as the nuclearly encoded nMAT1, nMAT2, nMAT3, and nMAT4 and possibly the mitochondrial MatR, whose function is still elusive (Keren et al, 2009(Keren et al, , 2012Brown et al, 2014;Cohen et al, 2014). Plant mitochondrial DNA is renowned for its high recombination rates and its ability to integrate foreign DNA sequences, explaining the difficulty of retracing the evolutionary story of group II introns (Bonen, 2008).…”

“…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.…”

Photoperiod Affects the Phenotype of Mitochondrial Complex I Mutants

“…Seedlings of Arabidopsis nMat1/4 (Keren et al, 2012;Cohen et al, 2014) and ndufv1 (Kühn et al, 2015) mutants barely survive unless grown in vitro with Suc supplementation. Differential impact of the mutations also was observed at the physiological and metabolic levels.…”

“…Mutants devoid of NAD subunits were characterized in maize (Zea mays; ncs2, deleted for NAD4; Marienfeld and Newton, 1994) and in Nicotiana sylvestris (CMSI and CMSII, deleted for NAD7; Pla et al, 1995). Mutations in genes controlling NAD cis/trans-processing have been described in N. sylvestris, maize, and Arabidopsis (Arabidopsis thaliana; for review, see Colas des Francs-Small and Small, 2014), and insertion mutants lacking nucleus-encoded subunits have been characterized in Arabidopsis (Meyer et al, 2009;Kühn et al, 2015;Soto et al, 2015). Impaired holocomplex assembly/activity are common features of all plant mutants characterized so far, but putative intermediate assembly forms have been observed in several cases (Gutierres et al, 1997;Karpova and Newton, 1999;Perales et al, 2005;Pineau et al, 2008;Meyer et al, 2009Meyer et al, , 2011Kühn et al, 2015).…”