nMAT1, a nuclear-encoded maturase involved in the trans-splicing of nad1 intron 1, is essential for mitochondrial complex I assembly and function

Mitochondrial genomes (mtDNAs) in angiosperms contain numerous group II-type introns that reside mainly within protein-coding genes that are required for organellar genome expression and respiration. While splicing of group II introns in non-plant systems is facilitated by proteins encoded within the introns themselves (maturases), the mitochondrial introns in plants have diverged and have lost the vast majority of their intron-encoded ORFs. Only a single maturase gene (matR) is retained in plant mtDNAs, but its role(s) in the splicing of mitochondrial introns is currently unknown. In addition to matR, plants also harbor four nuclear maturase genes (nMat 1 to 4) encoding mitochondrial proteins that are expected to act in the splicing of group II introns. Recently, we established the role of one of these proteins, nMAT2, in the splicing of several mitochondrial introns in Arabidopsis. Here, we show that nMAT1 is required for trans-splicing of nad1 intron 1 and also functions in cis-splicing of nad2 intron 1 and nad4 intron 2. Homozygous nMat1 plants show retarded growth and developmental phenotypes, modified respiration activities and altered stress responses that are tightly correlated with mitochondrial complex I defects.     

AtnMat2, a nuclear-encoded maturase required for splicing of group-II introns in Arabidopsis mitochondria

Mitochondria (mt) in plants house about 20 group-II introns, which lie within protein-coding genes required in both organellar genome expression and respiration activities. While in nonplant systems the splicing of group-II introns is mediated by proteins encoded within the introns themselves (known as “maturases”), only a single maturase ORF (matR) has retained in the mitochondrial genomes in plants; however, its putative role(s) in the splicing of organellar introns is yet to be established. Clues to other proteins are scarce, but these are likely encoded within the nucleus as there are no obvious candidates among the remaining ORFs within the mtDNA. Intriguingly, higher plants genomes contain four maturase-related genes, which exist in the nucleus as self-standing ORFs, out of the context of their evolutionary-related group-II introns “hosts.” These are all predicted to reside within mitochondria and may therefore act “in-trans” in the splicing of organellar-encoded introns. Here, we analyzed the intracellular locations of the four nuclear-encoded maturases in Arabidopsis and established the roles of one of these genes, At5g46920 (AtnMat2), in the splicing of several mitochondrial introns, including the single intron within cox2, nad1 intron2, and nad7 intron2.     

The DEAD-box RNA helicase ZmRH48 is required for the splicing of multiple mitochondrial introns,mitochondrial complex biosynthesis,and seed development in maize

RNA helicases participate in nearly all aspects of RNA metabolism by rearranging RNAs or RNA–protein complexes in an adenosine triphosphate-dependent manner. Due to the large RNA helicase families in plants, the precise roles of many RNA helicases in plant physiology and development remain to be clarified. Here, we show that mutations in maize (Zea mays) DEAD-box RNA helicase 48 (ZmRH48) impair the splicing of mitochondrial introns, mitochondrial complex biosynthesis, and seed development. Loss of ZmRH48 function severely arrested embryogenesis and endosperm development, leading to defective kernel formation. ZmRH48 is targeted to mitochondria, where its deficiency dramatically reduced the splicing efficiency of five cis-introns (nad5 intron 1; nad7 introns 1, 2, and 3; and ccmFc intron 1) and one trans-intron (nad2 intron 2), leading to lower levels of mitochondrial complexes I and III. ZmRH48 interacts with two unique pentatricopeptide repeat (PPR) proteins, PPR-SMR1 and SPR2, which are required for the splicing of over half of all mitochondrial introns. PPR-SMR1 interacts with SPR2, and both proteins interact with P-type PPR proteins and Zm-mCSF1 to facilitate intron splicing. These results suggest that ZmRH48 is likely a component of a splicing complex and is critical for mitochondrial complex biosynthesis and seed development.     

A PORR domain protein required for rpl2 and ccmF(C) intron splicing and for the biogenesis of c-type cytochromes in Arabidopsis mitochondria

Angiosperm mitochondria encode approximately 20 group II introns, which interrupt genes involved in the biogenesis and function of the respiratory chain. Nucleus‐encoded splicing factors have been identified for approximately half of these introns. The splicing factors derive from several protein families defined by atypical RNA binding domains that function primarily in organelles. We show here that the Arabidopsis protein WTF9 is essential for the splicing of group II introns in two mitochondrial genes for which splicing factors had not previously been identified: rpl2 and ccmF_C. WTF9 harbors a recently recognized RNA binding domain, the PORR domain, which was originally characterized in the chloroplast splicing factor WTF1. These findings show that the PORR domain family also functions in plant mitochondria, and highlight the parallels between the machineries for group II intron splicing in plant mitochondria and chloroplasts. In addition, we used the splicing defects in wtf9 mutants as a means to functionally characterize the mitochondrial rpl2 and ccmF_C genes. Loss of ccmF_C expression correlates with the loss of cytochromes c and c₁, confirming a role for ccmF_C in cytochrome biogenesis. By contrast, our results strongly suggest that splicing is not essential for the function of the mitochondrial rpl2 gene, and imply that the Rpl2 fragment encoded by rpl2 exon 1 functions in concert with a nuclear gene product that provides the remainder of this essential ribosomal protein in trans.     

The yeast mitochondrial leucyl-tRNA synthetase is a splicing factor for the excision of several group I introns

Summary The Saccharomyces cerevisiae nuclear gene NAM2 codes for mitochondrial leucyl-tRNA synthetase (mLRS). Herbert et al. (1988, EMBO J 7:473–483) proposed that this protein is involved in mitochondrial RNA splicing. Here we present the construction and analyses of nine mutations obtained by creating two-codon insertions within the NAM2 gene. Three of these prevent respiration while maintaining the mitochondrial genome. These three mutants: (1) display in vitro a mLRS activity ranging from 0%–50% that of the wild type: (2) allow in vivo the synthesis of several mitochondrially encoded proteins; (3) prevent the synthesis of the COXII protein but not of its mRNA; (4) abolish the splicing of the group I introns bI4 and aI4; and (5) affect significantly the excision of the group I introns bI2, bI3 and aI3. Importation of the bI4 maturase from the cytoplasm into mitochondria in a nam2 ^– mutant strain does not restore the excision of the introns bI4 and aI4 implying that the splicing deficiency does not result from the absence of the bI4 maturase. We conclude that the mLRS is a splicing factor essential for the excision of the group I introns bI4 and aI4 and probably important for the excision of other group I introns.     

Pentamidine inhibits mitochondrial intron splicing and translation in Saccharomyces cerevisiae

Pentamidine inhibits in vitro splicing of nuclear group I introns from rRNA genes of some pathogenic fungi and is known to inhibit mitochondrial function in yeast. Here we report that pentamidine inhibits the self-splicing of three group I and two group II introns of yeast mitochondria. Comparison of yeast strains with different configurations of mitochondrial introns (12, 5, 4, or 0 introns) revealed that strains with the most introns were the most sensitive to growth inhibition by pentamidine on glycerol medium. Analysis of blots of RNA from yeast strains grown in raffinose medium in the presence or absence of pentamidine revealed that the splicing of seven group I and two group II introns that have intron reading frames was inhibited by the drug to varying extents. Three introns without reading frames were unaffected by the drug in vivo, and two of these were inhibited in vitro, implying that the drug affects splicing by acting directly on RNA in vitro, but on another target in vivo. Because the most sensitive introns in vivo are the ones whose splicing depends on a maturase encoded by the intron reading frames, we tested pentamidine for effects on mitochondrial translation. We found that the drug inhibits mitochondrial but not cytoplasmic translation in cells at concentrations that inhibit mitochondrial intron splicing. Therefore, pentamidine is a potent and specific inhibitor of mitochondrial translation, and this effect explains most or all of its effects on respiratory growth and on in vivo splicing of mitochondrial introns.     

A maturase-encoding group IIA intron of yeast mitochondria self-splices in vitro.

Intron 1 of the coxI gene of yeast mitochondrial DNA (aI1) is a group IIA intron that encodes a maturase function required for its splicing in vivo. It is shown here to self-splice in vitro under some reaction conditions reported earlier to yield efficient self-splicing of group IIB introns of yeast mtDNA that do not encode maturase functions. Unlike the group IIB introns, aI1 is inactive in 10 mM Mg2+ (including spermidine) and requires much higher levels of Mg2+ and added salts (1M NH4Cl or KCl or 2M (NH4)2SO4) for ready detection of splicing activity. In KCl-stimulated reactions, splicing occurs with little normal branch formation; a post-splicing reaction of linear excised intron RNA that forms shorter lariat RNAs with branches at cryptic sites was evident in those samples. At low levels of added NH4Cl or KCl, the precursor RNA carries out the first reaction step but appears blocked in the splicing step. AI1 RNA is most reactive at 37-42 degrees C, as compared with 45 degrees C for the group IIB introns; and it lacks the KCl- or NH4Cl-dependent spliced-exon reopening reaction that is evident for the self-splicing group IIB introns of yeast mitochondria. Like the group IIB intron aI5 gamma, the domain 4 of aI1 can be largely deleted in cis, without blocking splicing; also, trans-splicing of half molecules interrupted in domain 4 occurs. This is the first report of a maturase-encoding intron of either group I or group II that self-splices in vitro.     

Leucyl-tRNA Synthetase-dependent and -independent Activation of a Group I Intron

Leucyl-tRNA synthetase (LeuRS) is an essential RNA splicing factor for yeast mitochondrial introns. Intracellular experiments have suggested that it works in collaboration with a maturase that is encoded within the bI4 intron. RNA deletion mutants of the large bI4 intron were constructed to identify a competently folded intron for biochemical analysis. The minimized bI4 intron was active in RNA splicing and contrasts with previous proposals that the canonical core of the bI4 intron is deficient for catalysis. The activity of the minimized bI4 intron was enhanced in vitro by the presence of the bI4 maturase or LeuRS.Although the aminoacyl-tRNA synthetases (aaRSs)⁶ are best known for their role in protein synthesis, many have functionally expanded and are essential to a wide range of other cellular activities that are unrelated to tRNA aminoacylation (1). The class I aaRSs, leucyl- (LeuRS or NAM2) and tyrosyl-tRNA synthetase (TyrRS or CYT-18) are required for RNA splicing of cognate group I introns in the mitochondria of certain lower eukaryotes (2). In yeast, processing of two related group I introns called bI4 and aI4α (Fig. 1) from the cob and cox1α genes, respectively, require yeast mitochondrial LeuRS (3, 4). Likewise, expression of Neurospora crassa mitochondrial genes, such as those for the large ribosomal RNA, is dependent on TyrRS for excising group I introns (5).Open in a separate windowFIGURE 1.Predicted secondary structures of the bI4 and aI4α group I introns. The secondary structure of the canonical core was based on previous predictions (19). Solid bold lines indicate linear connectivities of the nucleic acid strand with arrowheads oriented in the 5′ to 3′ direction. The dashed lines represent putative tertiary interactions. Dotted lines with numbers identify insertions where secondary structures were ambiguous. Arrows in the P1 and P9 domain show splice sites, whereas boxed nucleotides are paired regions.LeuRS facilitates RNA splicing in concert with a bI4 maturase that is encoded within the bI4 intron. Genetic investigations showed that an inactivated bI4 maturase resulting in deficient splicing activity of the bI4 and aI4α group I introns can be rescued by a suppressor mutation of LeuRS to restore mitochondrial respiration (4, 6). In addition, the splicing defect can be compensated by a mutant aI4α DNA endonuclease that is closely related to the bI4 maturase (7, 8).Previously, we used intracellular three-hybrid assays to demonstrate that LeuRS and bI4 maturase can independently bind to the bI4 intron and stimulate RNA splicing activity in the non-physiological yeast nucleus compartment (9). RNA-dependent two-hybrid assays also supported that the bI4 intron could simultaneously bind both the bI4 maturase and LeuRS. In this case, the RNA was co-expressed with LeuRS and bI4 maturase that was fused to either LexA or B42 to generate a two-hybrid response. This suggested that the bI4 intron was bridging these two protein splicing factors. In either the RNA-dependent two-hybrid or three-hybrid assays, bI4 intron splicing occurred only in the presence of LeuRS or bI4 maturase or both.We hypothesized that the bI4 maturase and LeuRS bind to distinct sites of the bI4 intron to form a ternary complex and promote efficient splicing activity. However, the functional basis of the collaboration between these two splicing cofactors or how either of them promotes RNA splicing remains unclear.We sought to characterize the respective splicing roles of the bI4 maturase and LeuRS via biochemical investigations. Previous attempts to develop an in vitro splicing assay for the bI4 intron or its closely related aI4α intron have failed (10, 11). It was hypothesized that the long length of the bI4 intron (∼1600 nucleotides) and its highly A:U-rich content (∼80%) hindered RNA folding in vitro as well as stabilization of its competent structure.Efforts to produce an active form of the bI4 intron have relied on building chimeric group I introns by interchanging RNA domains with the more stable Tetrahymena thermophila group I intron (11). Based on these results, it was proposed that the catalytic core of the bI4 group I intron was inherently defective (11). In this case, the group I intron would be expected to be completely dependent on its protein splicing factors similar to the bI3 intron that relies on the bI3 maturase and Mrs1 for activity (12). Thus, it was hypothesized that the bI4 maturase and/or LeuRS splicing factors aided the bI4 group I intron by targeting its core region to compensate for these deficiencies.We focused our efforts on re-designing the bI4 intron to develop a minimized molecule that might be competent for splicing. Because both the bI4 and aI4α group I introns rely on the bI4 maturase and LeuRS for their splicing activity, we compared their secondary structures to identify and eliminate peripheral regions outside of their catalytic cores. A small active derivative of the bI4 intron, comprised of just 380 nucleotides primarily from the canonical core, was generated. Thus, we show that, in and of itself, the canonical core of this group I intron is competent for splicing. Both the bI4 maturase and LeuRS enhance the splicing activity of the minimized bI4 intron. However, it is possible that protein-dependent splicing of the bI4 intron represents an intermediate evolutionary step in which the RNA activity is becoming increasingly dependent on its protein splicing factors.