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In this study pentatricopeptide repeat (PPR) proteins in filamentous ascomycetes are identified and functionally characterized. PPR proteins, which have in common a degenerated 35 amino acid motif often arranged in multiple tandems, are known to be implicated in various steps of RNA metabolism in mitochondria and chloroplasts. In filamentous ascomycetes we identified a common set of nine PPR proteins. For seven of these proteins, which were not yet characterized, knockout mutants of Neurospora crassa were analyzed. The knockout of three genes appeared to be lethal while four mutants showed different degrees of alterations in respiratory chain complexes. Two mutants are specifically affected in the assembly of a functional complex I while the other enzymes of the respiratory chain are present. Both mutants demonstrate the presence of a peripheral arm and the absence of a detectable membrane arm. Analysis of the mitochondrial RNA revealed distinct alterations of the transcript patterns for certain complex I subunits. Synthesis and/or stability of the transcript for ND2-ND3 is grossly impaired in one mutant while in the other mutant splicing of the transcript for ND1-ND4 is hampered. Our analysis provides the basis for a comprehensive characterization of PPR proteins in filamentous ascomycetes. [ABSTRACT FROM AUTHOR]