The nucleolus is a dynamic structure that has roles in various processes, from ribosome biogenesis to regulation of the cell cycle and the cellular stress response. Such functions are frequently mediated by the sequestration or release of nucleolar proteins. Our understanding of protein targeting to the nucleolus is much less complete than our knowledge of membrane-spanning translocation systems-such as those involved in nuclear targetingand the experimental evidence reveals that few parallels exist with these better-characterized systems. Here, we discuss the current understanding of nucleolar targeting, explore the types of sequence that control the localization of a protein to the nucleolus, and speculate that certain subsets of nucleolar proteins might act as hub proteins that are able to bind to multiple protein targets. In parallel to other subnuclear structures, such as PML bodies, the proteins that are involved in the formation and maintenance of the nucleolus are inexorably linked to nucleolar trafficking. Keywords: hub protein; nucleolar localization; nucleolin; nucleolus; nucleophosmin EMBO reports (2009) 10, 231-238. doi:10.1038/embor.2009.14 See Glossary for abbreviations used in this article. IntroductionThe nucleus is a highly ordered structure that contains non-membranebound subcompartments-including PML bodies, splicing speckles, Cajal bodies and the nucleolus-that have specific functions. The nucleolus is the largest subnuclear structure (Fig 1) and is easily visible under the light microscope owing to its high refractive index. It is centred on rDNA repeats within the chromosomes and is traditionally assoc iated with ribosome biogenesis. In mammalian cells, the number and activity of nucleoli vary during the cell cycle according to differing metabolic conditions and cell types.The mammalian nucleolus can be morphologically divided into several discrete regions-the fibrillar centre (FC), the dense fibrillar centre (DFC) and the granular component (GC)-that have roles in the various steps of rRNA synthesis. The FC contains the trans cription factor UBF and is rich in RNA polymerase I. The DFCs are associated with, and surround, the FCs and contain fibrillarin, an RNA methyltransferase and nucleolin-a protein that has multiple roles in nucleolar and cellular biology . Surrounding both the FC and the DFC is the GC, which is the site of the partial maturation and assembly of pre-ribosomes, accumulates NPM, and is enriched with ribosomal proteins and assembly factors. The GC might also contain regions that comprise protein complexes that are devoid of RNA (Politz et al, 2005).Our understanding of nucleolar function changed markedly with the formulation of the plurifunctional nucleolus hypothesis (Pederson, 1998), which has now been underpinned by proteomic analysis (Boisvert et al, 2007), and proposes that the nucleolus has multiple functions in health and disease (Matthews & Olson, 2006;Stark & Taliansky, 2009). For example, there is extensive evidence that the nucleolus is involved in the respo...

Peer-reviewed journal publication is the main means for academic researchers in the life sciences to create a permanent public record of their work. These publications are also the de facto currency for career progress, with a strong link between journal brand recognition and perceived value. The current peer-review process can lead to long delays between submission and publication, with cycles of rejection, revision, and resubmission causing redundant peer review. This situation creates unique challenges for early career researchers (ECRs), who rely heavily on timely publication of their work to gain recognition for their efforts. Today, ECRs face a changing academic landscape, including the increased interdisciplinarity of life sciences research, expansion of the researcher population, and consequent shifts in employer and funding demands. The publication of preprints, publicly available scientific manuscripts posted on dedicated preprint servers prior to journal-managed peer review, can play a key role in addressing these ECR challenges. Preprinting benefits include rapid dissemination of academic work, open access, establishing priority or concurrence, receiving feedback, and facilitating collaborations. Although there is a growing appreciation for and adoption of preprints, a minority of all articles in life sciences and medicine are preprinted. The current low rate of preprint submissions in life sciences and ECR concerns regarding preprinting need to be addressed. We provide a perspective from an interdisciplinary group of ECRs on the value of preprints and advocate their wide adoption to advance knowledge and facilitate career development.

Human respiratory syncytial virus (HRSV) is a major cause of pediatric lower respiratory tract disease to which there is no vaccine or efficacious chemotherapeutic strategy. Although RNA synthesis and virus assembly occur in the cytoplasm, HRSV is known to induce nuclear responses in the host cell as replication alters global gene expression. Quantitative proteomics was used to take an unbiased overview of the protein changes in transformed human alveolar basal epithelial cells infected with HRSV. Underpinning this was the use of stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS, which allowed the direct and simultaneous identification and quantification of both cellular and viral proteins. To reduce sample complexity and increase data return on potential protein localization, cells were fractionated into nuclear and cytoplasmic extracts. This resulted in the identification of 1,140 cellular proteins and six viral proteins. The proteomics data were analyzed using Ingenuity Pathways Analysis to identify defined canonical pathways and functional groupings. Selected data were validated using Western blot, direct and indirect immunofluorescence confocal microscopy, and functional assays. The study served to validate and expand upon known HRSV-host cell interactions, including those associated with the antiviral response and alterations in subnuclear structures such as the nucleolus and ND10 (promyelocytic leukemia bodies). In addition, novel changes were observed in mitochondrial proteins and functions, cell cycle regulatory molecules, nuclear pore complex proteins and nucleocytoplasmic trafficking proteins. These data shed light into how the cell is potentially altered to create conditions more favorable for infection. Additionally, the study highlights the application and advantage of stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS for the analysis of virus-host interactions.

A major limitation to applying quantitative LC-MS/MS proteomics to small samples, such as single cells, are the losses incured during sample cleanup. To relieve this limitation, we developed a Minimal ProteOmic sample Preparation (mPOP) method for culture-grown mammalian cells. mPOP obviates cleanup and thus eliminates cleanup-related losses while expediting sample preparation and simplifying its automation. Bulk SILAC samples processed by mPOP or by conventional urea-based methods indicated that mPOP results in complete cell lysis and accurate relative quantification. We integrated mPOP lysis with the Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS) sample preparation, and benchmarked the quantification of such samples on a Q-exactive instrument. The results demonstrate low noise and high technical reproducibility. Then, we FACS sorted single U-937, HEK-293, and mouse ES cells into 96-well plates and analyzed them by automated mPOP and SCoPE-MS. The quantified proteins enabled separating the single cells by cell-type and cell-division-cycle phase.discussions and constructive comments.

SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigens S and N: the main targets for vaccine and antibody testing efforts. We discover significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases. We show that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, show a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19.

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Virus-host interactions involve complex interplay between

Background: Noroviruses use a virus-encoded protein, VPg, covalently linked to the viral RNA for translation.Results: The direct interaction of VPg with the central domain of eIF4G is required for norovirus translation.Conclusion: eIF4G plays a central role in norovirus VPg-dependent translation initiation.Significance: The VPg-eIF4G interaction may provide a suitable target for the specific inhibition of norovirus translation.