mCSF1, a nucleus‐encoded CRM protein required for the processing of many mitochondrial introns, is involved in the biogenesis of respiratory complexes I and IV in Arabidopsis

Abstract

SummaryThe coding regions of many mitochondrial genes in plants are interrupted by intervening sequences that are classified as group II introns. Their splicing is essential for the expression of the genes they interrupt and hence for respiratory function, and is facilitated by various protein cofactors. Despite the importance of these cofactors, only a few of them have been characterized.CRS1-YhbY domain (CRM) is a recently recognized RNA-binding domain that is present in several characterized splicing factors in plant chloroplasts. The Arabidopsis genome encodes 16 CRM proteins, but these are largely uncharacterized.Here, we analyzed the intracellular location of one of these hypothetical proteins in Arabidopsis, mitochondrial CAF-like splicing factor 1 (mCSF1; At4 g31010), and analyzed the growth phenotypes and organellar activities associated with mcsf1 mutants in plants.Our data indicated that mCSF1 resides within mitochondria and its functions are essential during embryogenesis. Mutant plants with reduced mCSF1 displayed inhibited germination and retarded growth phenotypes that were tightly associated with reduced complex I and IV activities. Analogously to the functions of plastid-localized CRM proteins, analysis of the RNA profiles in wildtype and mcsf1 plants showed that mCSF1 acts in the splicing of many of the group II intron RNAs in Arabidopsis mitochondria.

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.…”

“…As nad5 introns 2 and 3 are both spliced in trans and introns 1 and 4 are spliced in cis, we can expect to find at least six specific factors for nad5 alone, as well as maturases and more general splicing factors. mCSF1, a CRM protein, is involved in splicing introns 1, 2, 3, and possibly 4 (Zmudjak et al, 2013). PPR proteins, maturases, CRM proteins, as well as the PMH2 helicase, an RNA chaperone required for the formation or maintenance of complex RNA secondary structures of introns, are likely to be part of a heteromultimeric splicing complex, as suggested for mitochondrial (Köhler et al, 2010) and chloroplast group IIB (Asakura et al, 2008) introns.…”

“…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.…”

The MTL1 Pentatricopeptide Repeat Protein Is Required for Both Translation and Splicing of the Mitochondrial NADH DEHYDROGENASE SUBUNIT7 mRNA in Arabidopsis

“…A comparison of published Arabidopsis mitochondrial splicing mutants indicates that splicing reductions of at least 5003 compared with the wild type seems necessary to result in a nearly complete loss of complex I on Blue Native gels (Koprivova et al, 2010;Kühn et al, 2011;Cohen et al, 2014;Colas des Francs-Small et al, 2014;Hsieh et al, 2015). The decrease in splicing efficiency can be much milder, though, when several complex I introns are concomitantly affected in a same mutant (Keren et al, 2012;Zmudjak et al, 2013). The manner by which PPR proteins assist group II intron splicing is currently unknown at the molecular level.…”