A ribonuclease III domain protein functions in group II intron splicing in maize chloroplasts.
Chloroplast genomes in land plants harbor approximately 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.
Watkins
Kenneth P
KP
Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA.
Kroeger
Tiffany S
TS
Cooke
Amy M
AM
Williams-Carrier
Rosalind E
RE
Friso
Giulia
G
Belcher
Susan E
SE
van Wijk
Klaas J
KJ
Barkan
Alice
A
D000483:Alleles; D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000235:genetics* / Q000378:metabolism; D047468:Immunoprecipitation; D007438:Introns / Q000235:genetics*; D008024:Ligands; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics*; D018749:RNA, Plant / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D043244:Ribonuclease III / Q000737:chemistry*; D012270:Ribosomes / Q000378:metabolism; D003313:Zea mays / Q000235:genetics*
D008024:Ligands; D010940:Plant Proteins; D012322:RNA Precursors; D018749:RNA, Plant; D011994:Recombinant Proteins; D043244:Ribonuclease III
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