Ribosome biogenesis (RB) is an intricate and evolutionarily conserved process that takes place mainly in the nucleolus and is required for eukaryotic cells to maintain homeostasis, grow in size, and divide. Our laboratory has identified the NUF2 protein, part of the mitotic kinetochore, in a genome-wide siRNA screen for proteins required for making ribosomes in MCF10A human breast epithelial cells (Farley-Barnes, 2018). After rigorous validation and using several biochemical and cell-based assays, we find a role for NUF2 in pre-rRNA transcription, the primary and rate-limiting step of RB. siRNA depletion of other components of the NUF2 kinetochore sub-complex, NDC80, SPC24, and SPC25, also reduce pre-rRNA transcription. Interestingly, essential protein components for pre-rRNA transcription, including the largest subunit of RNA polymerase I, POLR1A, are reduced upon siRNA depletion of NUF2 and its protein partners. Their reduced levels are a likely mechanism for the decrease in pre-rRNA transcription. siRNA depletion of NUF2 and NDC80 also cause increased TP53 and CDKN1A (p21) mRNA levels, which can be restored by co-depletion of RPL5, indicating activation of the nucleolar stress pathway. These results reveal a new connection between proteins with a known role in mitosis to the function of the nucleolus in RB during interphase.