Mature ribosomal RNAs (rRNAs) are produced from polycistronic precursors following complex processing. Precursor (pre)-rRNA processing has been extensively characterized in yeast and was assumed to be conserved in humans. We functionally characterized 625 nucleolar proteins in HeLa cells and identified 286 required for processing, including 74 without a yeast homolog. For selected candidates, we demonstrated that pre-rRNA processing defects are conserved in different cell types (including primary cells), defects are not due to activation of a p53-dependent nucleolar tumor surveillance pathway, and they precede cell-cycle arrest and apoptosis. We also investigated the exosome's role in processing internal transcribed spacers (ITSs) and report that 3' end maturation of 18S rRNA involves EXOSC10/Rrp6, a yeast ITS2 processing factor. We conclude that human cells adopt unique strategies and recruit distinct trans-acting factors to carry out essential processing steps, posing fundamental implications for understanding ribosomopathies at the molecular level and developing effective therapeutic agents.

A proteome-wide mapping of interactions between hepatitis C virus (HCV) and human proteins was performed to provide a comprehensive view of the cellular infection. A total of 314 protein-protein interactions between HCV and human proteins was identified by yeast two-hybrid and 170 by literature mining. Integration of this data set into a reconstructed human interactome showed that cellular proteins interacting with HCV are enriched in highly central and interconnected proteins. A global analysis on the basis of functional annotation highlighted the enrichment of cellular pathways targeted by HCV. A network of proteins associated with frequent clinical disorders of chronically infected patients was constructed by connecting the insulin, Jak/STAT and TGFb pathways with cellular proteins targeted by HCV. CORE protein appeared as a major perturbator of this network. Focal adhesion was identified as a new function affected by HCV, mainly by NS3 and NS5A proteins.

The ease of construction of multiple mutant strains in Schizosaccharomyces pombe is limited by the number of available genetic markers. We describe here three new cassettes for PCR-mediated gene disruption that can be used in combination with commonly used fission yeast markers to make multiple gene deletions. The natMX6, hphMX6 and bleMX6 markers give rise to resistance towards the antibiotics nourseothricin (NAT), hygromycin B and phleomycin, respectively. The cassettes are composed of exogenous sequences to increase the frequency of integration at targeted loci, and have a structure similar to the commonly used pFA6a-kanMX6 modular plasmid system. This allows a simple exchange of the kanMX6 marker in existing strains with any of the three new cassettes. Alternatively, oligonucleotide primers designed for the modular kanMX6 cassettes can be used to make the transforming PCR fragments for gene disruption. We illustrate the construction of a mutant strain with six independent gene disruptions, using the novel antibiotic cassettes in combination with existing genetic markers.