FASTKD1 and FASTKD4 have opposite effects on expression of specific mitochondrial RNAs, depending upon their endonuclease-like RAP domain.
FASTK family proteins have been identified as regulators of mitochondrial RNA homeostasis linked to mitochondrial diseases, but much remains unknown about these proteins. We show that CRISPR-mediated disruption of FASTKD1 increases ND3 mRNA level, while disruption of FASTKD4 reduces the level of ND3 and of other mature mRNAs including ND5 and CYB, and causes accumulation of ND5-CYB precursor RNA. Disrupting both FASTKD1 and FASTKD4 in the same cell results in decreased ND3 mRNA similar to the effect of depleting FASTKD4 alone, indicating that FASTKD4 loss is epistatic. Interestingly, very low levels of FASTKD4 are sufficient to prevent ND3 loss and ND5-CYB precursor accumulation, suggesting that FASTKD4 may act catalytically. Furthermore, structural modeling predicts that each RAP domain of FASTK proteins contains a nuclease fold with a conserved aspartate residue at the putative active site. Accordingly, mutation of this residue in FASTKD4 abolishes its function. Experiments with FASTK chimeras indicate that the RAP domain is essential for the function of the FASTK proteins, while the region upstream determines RNA targeting and protein localization. In conclusion, this paper identifies new aspects of FASTK protein biology and suggests that the RAP domain function depends on an intrinsic nucleolytic activity.
Cell Biology Department, University of Geneva, 1211 Geneva 4, Switzerland.
Erik
E
Boehm
Cell Biology Department, University of Geneva, 1211 Geneva 4, Switzerland.
Sofia
S
Zaganelli
Cell Biology Department, University of Geneva, 1211 Geneva 4, Switzerland.
Kinsey
K
Maundrell
Cell Biology Department, University of Geneva, 1211 Geneva 4, Switzerland.
Alexis A
AA
Jourdain
INSERM U-1212, CNRS UMR 5320, Université de Bordeaux, ARNA Laboratory, Bordeaux 33000, France.
Stéphane
S
Thore
Cell Biology Department, University of Geneva, 1211 Geneva 4, Switzerland.
Jean-Claude
JC
Martinou
D000595:Amino Acid Sequence; D064113:CRISPR-Cas Systems; D045303:Cytochromes b / Q000235:genetics*; D042967:Electron Transport Complex I / Q000235:genetics*; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D008958:Models, Molecular; D011487:Protein Conformation; D000072417:Protein Domains; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D000077278:RNA, Mitochondrial; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D016415:Sequence Alignment; D017385:Sequence Homology; D014158:Transcription, Genetic
C000622952:FASTKD1 protein, human; D024101:Mitochondrial Proteins; D012333:RNA, Messenger; D000077278:RNA, Mitochondrial; D016601:RNA-Binding Proteins; C000622953:TBRG4 protein, human; D012313:RNA; D045303:Cytochromes b; C481838:MT-ND5 protein, human; D042967:Electron Transport Complex I; C114961:MT-ND3 protein, human