A Ribonuclease III Domain Protein Functions in Group II Intron Splicing in Maize Chloroplasts

Abstract

Chloroplast genomes in land plants harbor ;20 group II introns. Genetic approaches have identified proteins involved in the splicing of many of these introns, but the proteins identified to date cannot account for the large size of intron ribonucleoprotein complexes and are not sufficient to reconstitute splicing in vitro. Here, we describe an additional protein that promotes chloroplast group II intron splicing in vivo. This protein, RNC1, was identified by mass spectrometry analysis of maize (Zea mays) proteins that coimmunoprecipitate with two previously identified chloroplast splicing factors, CAF1 and CAF2. RNC1 is a plant-specific protein that contains two ribonuclease III (RNase III) domains, the domain that harbors the active site of RNase III and Dicer enzymes. However, several amino acids that are essential for catalysis by RNase III and Dicer are missing from the RNase III domains in RNC1. RNC1 is found in complexes with a subset of chloroplast group II introns that includes but is not limited to CAF1-and CAF2-dependent introns. The splicing of many of the introns with which it associates is disrupted in maize rnc1 insertion mutants, indicating that RNC1 facilitates splicing in vivo. Recombinant RNC1 binds both single-stranded and double-stranded RNA with no discernible sequence specificity and lacks endonuclease activity. These results suggest that RNC1 is recruited to specific introns via protein-protein interactions and that its role in splicing involves RNA binding but not RNA cleavage activity.

Mutations in SUPPRESSOR OF VARIEGATION1, a Factor Required for Normal Chloroplast Translation, Suppress var2-Mediated Leaf Variegation in Arabidopsis

“…Several of these genes have been cloned, including DCL and DAL, which code for novel chloroplast proteins (Bellaoui et al, 2003;Bisanz et al, 2003), and RNC1, which codes for a maize group II splicing factor that mediates RNA binding but does not have endonuclease activity (Watkins et al, 2007). Mutants deficient in RNC1 (rnc1) are illustrative of the principle that mutants with defects in chloroplast rRNA processing need not define genes whose products are directly involved in this process; rather, processing defects are frequently found in mutants with primary lesions in ribosome assembly and/or function (Keus et al, 1984;Barkan, 1993;Leal-Klevezas et al, 2000;Bellaoui et al, 2003;Williams and Barkan, 2003;Bollenbach et al, 2005;Schmitz-Linneweber et al, 2006).…”

“…Other factors that affect chloroplast rRNA maturation are defined by a handful of mutants that accumulate rRNA intermediates. These include the following: (1) maize high chlorophyll fluorescence7 (Barkan, 1993), maize rnc1 (Watkins et al, 2007), and Arabidopsis white cotyledon (Yamamoto et al, 2000), all of which accumulate primarily 16S rRNA precursors; (2) Chlamydomonas ac20, which is defective in 23S rRNA maturation (Holloway and Herrin, 1998); (3) Arabidopsis dal (for dag-like, from the differentiation and greening mutant of snapdragon [Antirrhinum majus]) (Chatterjee et al, 1996), which accumulates 16S and 23S precursor rRNAs (Babiychuk et al, 1997;Bisanz et al, 2003); and (4) tomato dcl (for defective chloroplasts and leaves), in which 4.5S rRNA processing is defective (Bellaoui et al, 2003); 4.5S rRNA processing is also impaired in Arabidopsis mutants with downregulated DCL gene expression (Bellaoui and Gruissem, 2004). (C) Determination of the T-DNA insertion site in ClpR1/SVR2.…”

APO1 Promotes the Splicing of Chloroplast Group II Introns and Harbors a Plant-Specific Zinc-Dependent RNA Binding Domain

“…The ndhA and ndhB introns are not designated as APO1 ligands because the splicing defects in apo1 mutants were mild. Results are summarized from this work and from previously published work (Jenkins et al, 1997;Vogel et al, 1999;Jenkins and Barkan, 2001;Till et al, 2001;Ostheimer et al, 2003;Schmitz-Linneweber et al, 2006;Asakura and Barkan, 2007;Watkins et al, 2007;Asakura et al, 2008;Beick et al, 2008;de Longevialle et al, 2008;Prikryl et al, 2008;Kroeger et al, 2009). …”

A pentatricopeptide repeat gene of rice is required for splicing of chloroplast transcripts and RNA editing of ndhA

“…CRS1 is required for the splicing of atpF (Till et al 2001), and rice CFM3 is required for the splicing of ndhB, petB, petD, rpl16, rps16, and trnG transcripts (Asakura et al 2008). The maize PORR/DUF860 protein WTF1 functions in concert with RNC1, a ribonuclease III domain protein, to promote the splicing of group II introns (Watkins et al 2007). On the contrary, little is known regarding the functions and mechanisms of action of PPR proteins in splicing.…”

“…Previously, some nucleus-encoded proteins, such as PPR proteins, CRM proteins, and domain of unknown function 860 (DUF860) proteins, have been identified as being required for splicing in most plastid introns in plants (Falcon de Longevialle et al 2010). The complexes CAF1/ CRS2 and CAF2/CRS2 are required for the splicing of overlapping subsets of nine plastid transcripts in maize (Ostheimer et al 2003), and maize RNC1 is required for the splicing of ten introns (Watkins et al 2007). Arabidopsis CFM2 with four CRM domains is required for the splicing of four introns (Asakura and Barkan 2007 …”