This work focused on the regulation of Mitochondrial RNA by the FASTK proteins. CRISPR KOs were used to investigate the effects of the loss of FASTKD1, FASTKD3, and FASTKD4, while work with FASTK and FASTKD2 was done with siRNAs and MEF KOs. All FASTK proteins were found to affect mitochondrial RNA, but the specificity and effect on RNA was varied. In particular loss of FASTKD1 or FASTKD3 had opposite effects to FASTKD4 on the loss of the ND3 RNA. FASTKD4 loss showed a striking phenotype in which there was a loss of mature ND5 and CYB RNA and a large increase in the ND5-CYB precursor. Structural modeling and mutagenesis studies suggested a similarity of a domain of the FASTK proteins to an endonuclease. In contrast the N terminal region appears to resemble PPR proteins and determines if the protein is incorporated into mitochondrial RNA granules or not.