Mitotic kinetochore forms at centromere for proper chromosome segregation. Deposition of the centromere-specific histone H3 variant, spCENP-A/Cnp1, is vital for the formation of centromere-specific chromatin and the Mis17-Mis6 complex of the fission yeast Schizosaccharomyces pombe is required for the recruitment. Here we identified extragenic suppressors for a Mis17-Mis6 complex temperature sensitive (ts) mutant, mis17-S353P, using whole-genome sequencing. The large and small daughter nuclei phenotype observed in mis17-S353P was greatly rescued by these suppressors. Suppressor mutations in two ribonuclease genes involved in mRNA decay pathway, exo2 and pan2, may affect Mis17 protein level as mis17 mutant protein level was recovered in mis17 exo2 double mutant cells. Suppressor mutations in EKC/KEOPS complex genes might not regulate Mis17 protein level, but restored spCENP-A/Cnp1, Mis6 and Mis15’s centromeric localization in mis17-S353P. Therefore, EKC/KEOPS complex may prevent Cnp1/CENP-A recruitment by inhibiting Mis17-Mis6 complex formation or centromeric localization. Mutational analysis in protein structure indicated that suppressor mutations in EKC/KEOPS complex may interfere its kinase activity or complex formation. Our results suggest that mRNA decay pathway and EKC/KEOPS complex negatively regulate Mis17-Mis6 complex-mediated centromere formation by distinct and unexpected mechanisms.