Genome-wide RNAi screen identifies novel players in human 60S subunit biogenesis including key enzymes of polyamine metabolism.

Ribosome assembly is an essential process that is linked to human congenital diseases and tumorigenesis. While great progress has been made in deciphering mechanisms governing ribosome biogenesis in eukaryotes, an inventory of factors that support ribosome synthesis in human cells is still missing, in particular regarding the maturation of the large 60S subunit. Here, we performed a genome-wide RNAi screen using an imaging-based, single cell assay to unravel the cellular machinery promoting 60S subunit assembly in human cells. Our screen identified a group of 310 high confidence factors. These highlight the conservation of the process across eukaryotes and reveal the intricate connectivity of 60S subunit maturation with other key cellular processes, including splicing, translation, protein degradation, chromatin organization and transcription. Intriguingly, we also identified a cluster of hits comprising metabolic enzymes of the polyamine synthesis pathway. We demonstrate that polyamines, which have long been used as buffer additives to support ribosome assembly in vitro, are required for 60S maturation in living cells. Perturbation of polyamine metabolism results in early defects in 60S but not 40S subunit maturation. Collectively, our data reveal a novel function for polyamines in living cells and provide a rich source for future studies on ribosome synthesis.

Air2p is critical for the assembly and RNA-binding of the TRAMP complex and the KOW domain of Mtr4p is crucial for exosome activation.

Trf4/5p-Air1/2p-Mtr4p polyadenylation complex (TRAMP) is an essential component of nuclear RNA surveillance in yeast. It recognizes a variety of nuclear transcripts produced by all three RNA polymerases, adds short poly(A) tails to aberrant or unstable RNAs and activates the exosome for their degradation. Despite the advances in understanding the structural features of the isolated complex subunits or their fragments, the details of complex assembly, RNA recognition and exosome activation remain poorly understood. Here we provide the first understanding of the RNA binding mode of the complex. We show that Air2p is an RNA-binding subunit of TRAMP. We identify the zinc knuckles (ZnK) 2, 3 and 4 as the RNA-binding domains, and reveal the essentiality of ZnK4 for TRAMP4 polyadenylation activity. Furthermore, we identify Air2p as the key component of TRAMP4 assembly providing bridging between Mtr4p and Trf4p. The former is bound via the N-terminus of Air2p, while the latter is bound via ZnK5, the linker between ZnK4 and 5 and the C-terminus of the protein. Finally, we uncover the RNA binding part of the Mtr4p arch, the KOW domain, as the essential component for TRAMP-mediated exosome activation.

Advanced Cell Classifier: User-Friendly Machine-Learning-Based Software for Discovering Phenotypes in High-Content Imaging Data.

High-content, imaging-based screens now routinely generate data on a scale that precludes manual verification and interrogation. Software applying machine learning has become an essential tool to automate analysis, but these methods require annotated examples to learn from. Efficiently exploring large datasets to find relevant examples remains a challenging bottleneck. Here, we present Advanced Cell Classifier (ACC), a graphical software package for phenotypic analysis that addresses these difficulties. ACC applies machine-learning and image-analysis methods to high-content data generated by large-scale, cell-based experiments. It features methods to mine microscopic image data, discover new phenotypes, and improve recognition performance. We demonstrate that these features substantially expedite the training process, successfully uncover rare phenotypes, and improve the accuracy of the analysis. ACC is extensively documented, designed to be user-friendly for researchers without machine-learning expertise, and distributed as a free open-source tool at www.cellclassifier.org.

Genome-wide RNAi Screening Identifies Protein Modules Required for 40S Subunit Synthesis in Human Cells.

Ribosome biogenesis is a highly complex process requiring many assisting factors. Studies in yeast have yielded comprehensive knowledge of the cellular machinery involved in this process. However, many aspects of ribosome synthesis are different in higher eukaryotes, and the global set of mammalian ribosome biogenesis factors remains unexplored. We used an imaging-based, genome-wide RNAi screen to find human proteins involved in 40S ribosomal subunit biogenesis. Our analysis identified ∼ 300 factors, many part of essential protein modules such as the small subunit (SSU) processome, the eIF3 and chaperonin complexes, and the ubiquitin-proteasome system. We demonstrate a role for the vertebrate-specific factor RBIS in ribosome synthesis, uncover a requirement for the CRL4 E3 ubiquitin ligase in nucleolar ribosome biogenesis, and reveal that intracellular glutamine synthesis supports 40S subunit production.