Identification of a Pentatricopeptide Repeat Protein Implicated in Splicing of Intron 1 of Mitochondrial nad7 Transcripts

Abstract

Splicing of plant organellar transcripts is facilitated by members of a large protein family, the pentatricopeptide repeat proteins. We have identified a pentatricopeptide repeat protein in a genetic screen for mutants resistant to inhibition of root growth by buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis and consequently named BIR6 (BSO-insensitive roots 6). BIR6 is involved in splicing of intron 1 of the mitochondrial nad7 transcript. Loss-of-function mutations in BIR6 result in a strongly reduced accumulation of fully processed nad7 transcript. This affects assembly of Complex I and results in moderate growth retardation. In agreement with disruption of Complex I function, the genes encoding alternative NADH oxidizing enzymes are induced in the mutant, and the mutant plants are less sensitive to mannitol and salt stress. Mutation in the BIR6 gene allowed normal root growth in presence of BSO and strongly attenuated depletion of glutathione content at these conditions. The same phenotype was observed with other mutants affected in function of Complex I, thus reinforcing the importance of Complex I function for cellular redox homeostasis.

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

“…Both mutants exhibited defects in the splicing of nad5 introns (introns 1 and 2-3), but the splicing efficiency of intron 2-3 was about 8-fold lower for tang2 than for otp439. Both also appeared affected in the splicing of nad2 intron 1, but the latter could be a pleiotropic effect, as it has been reported previously for mutants impaired in complex I (Falcon de Longevialle et al, 2007;Koprivova et al, 2010). The splicing efficiency of other transcripts was not significantly altered in either mutant (Fig.…”

“…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.…”

“…For this test, two sets of primers are used, one set specifically amplifying spliced RNA and the other set specifically amplifying unspliced RNA. Splicing efficiency is calculated as a ratio of spliced to unspliced forms of each transcript in the mutant normalized to the same ratio in the complemented plants (Falcon de Longevialle et al, 2007;Koprivova et al, 2010;Colas des Francs-Small et al, 2012). Due to its small size, nad5 exon 3 was not amplified individually; for this initial screen, the primers amplifying the spliced transcript were placed on exons 2 and 4, and thus the splicing efficiencies of introns 2 and 3 are presented together in the histogram as intron 2-3 (Fig.…”

“…The primers used for the splicing test quantitative RT-PCR assays were described previously (Koprivova et al, 2010;Haïli et al, 2013). Quantitative RT-PCR was performed using LightCycler 480 SYBR Green I Master Mix (Roche Diagnostics) and a Roche LightCycler 480 real-time PCR system with the following thermal cycling program: 95°C for 10 min, followed by 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 10 s. The data were analyzed using the LightCycler 480 software version 1.5 (Roche Diagnostics).…”

“…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.…”