SUMMARY Ribosome biogenesis is a highly regulated, essential cellular process. Although studies in yeast have established some of the biological principles of ribosome biogenesis, many of the intricacies of its regulation in higher eukaryotes remain unknown. To understand how ribosome biogenesis is globally integrated in human cells, we conducted a genome-wide siRNA screen for regulators of nucleolar number. We found 139 proteins whose depletion changed the number of nucleoli per nucleus from 2–3 to only 1 in human MCF10A cells. Follow-up analyses on 20 hits found many (90%) to be essential for the nucleolar functions of rDNA transcription (7), pre-ribosomal RNA (pre-rRNA) processing (16), and/or global protein synthesis (14). This genome-wide analysis exploits the relationship between nucleolar number and function to discover diverse cellular pathways that regulate the making of ribosomes and paves the way for further exploration of the links between ribosome biogenesis and human disease.

No abstract

Nucleoli are dynamic nuclear condensates in eukaryotic cells that originate through ribosome biogenesis at loci that harbor the ribosomal DNA. These loci are known as nucleolar organizer regions (NORs) and there are 10 in a human diploid genome. While there are 10 NORs, however, the number of nucleoli observed in cells is variable. Furthermore, changes in number are associated with disease, with increased numbers and size common in aggressive cancers. In the near-diploid human breast epithelial cell line, MCF10A, the most frequently observed number of nucleoli is 2-3 per cell. Here, to identify novel regulators of ribosome biogenesis we used high-throughput quantitative imaging of MCF10A cells to identify proteins that, when depleted, increase the percentage of nuclei with ≥5 nucleoli. Unexpectedly, this unique screening approach led to identification of proteins associated with the cell cycle. Functional analysis on a subset of hits further revealed not only proteins required for progression through S and G2/M phase, but also proteins required explicitly for the regulation of RNA polymerase I transcription and protein synthesis. Thus, results from this screen for increased nucleolar number highlight the significance of the nucleolus in human cell cycle regulation, linking RNA polymerase I transcription to cell cycle progression.

Purpose To produce a physician and scientific workforce capable of delivering high quality, culturally competent health care and research, academic medical centers must assess their capacity for diversity and inclusion and respond to identified opportunities. Thus, the Diversity Engagement Survey (DES) is presented as a diagnostic and benchmarking tool. Method The 22-item DES connects workforce engagement theory with inclusion and diversity constructs. Face and content validity were established based on decades of previous work to promote institutional diversity. The survey was pilot tested at a single academic medical center and subsequently administered at 13 additional academic medical centers. Cronbach alphas assessed internal consistency and Confirmatory Factor Analysis (CFA) established construct validity. Criterion validity was assessed by observed separation in scores for groups traditionally recognized to have less workforce engagement. Results The sample consisted of 13,694 individuals at 14 medical schools from across the U.S. who responded to the survey administered between 2011– 2012. The Cronbach alphas for inclusion and engagement factors (range: 0.68 to 0.85), CFA fit indices, and item correlations with latent constructs, indicated an acceptable model fit and that questions measured the intended concepts. DES scores clearly distinguished higher and lower performing institutions. The DES detected important disparities for black, women, and those who did not have heterosexual orientation. Conclusions This study demonstrated that the DES is a reliable and valid instrument for internal assessment and evaluation or external benchmarking of institutional progress in building inclusion and engagement.