Signals in many biological processes can be amplified by recruiting multiple copies of regulatory proteins to a site of action. Harnessing this principle, we have developed a protein scaffold, a ...repeating peptide array termed SunTag, which can recruit multiple copies of an antibody-fusion protein. We show that the SunTag can recruit up to 24 copies of GFP, thereby enabling long-term imaging of single protein molecules in living cells. We also use the SunTag to create a potent synthetic transcription factor by recruiting multiple copies of a transcriptional activation domain to a nuclease-deficient CRISPR/Cas9 protein and demonstrate strong activation of endogenous gene expression and re-engineered cell behavior with this system. Thus, the SunTag provides a versatile platform for multimerizing proteins on a target protein scaffold and is likely to have many applications in imaging and controlling biological outputs.
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•SunTag allows controlled protein multimerization on a protein scaffold•SunTag enables long-term single-molecule imaging in living cells•SunTag greatly improves CRISPR-based activation of gene expression
A protein tagging system, SunTag, is developed to enable long-term single-molecule imaging and to enhance CRISPR-based transcriptional activation in living cells.
Detailed phylogenies of tumor populations can recount the history and chronology of critical events during cancer progression, such as metastatic dissemination. We applied a Cas9-based, single-cell ...lineage tracer to study the rates, routes, and drivers of metastasis in a lung cancer xenograft mouse model. We report deeply resolved phylogenies for tens of thousands of cancer cells traced over months of growth and dissemination. This revealed stark heterogeneity in metastatic capacity, arising from preexisting and heritable differences in gene expression. We demonstrate that these identified genes can drive invasiveness and uncovered an unanticipated suppressive role for
We also show that metastases disseminated via multidirectional tissue routes and complex seeding topologies. Overall, we demonstrate the power of tracing cancer progression at subclonal resolution and vast scale.
p97 is a AAA-ATPase with multiple cellular functions, one of which is critical regulation of protein homeostasis pathways. We describe the characterization of CB-5083, a potent, selective, and orally ...bioavailable inhibitor of p97. Treatment of tumor cells with CB-5083 leads to accumulation of poly-ubiquitinated proteins, retention of endoplasmic reticulum-associated degradation (ERAD) substrates, and generation of irresolvable proteotoxic stress, leading to activation of the apoptotic arm of the unfolded protein response. In xenograft models, CB-5083 causes modulation of key p97-related pathways, induces apoptosis, and has antitumor activity in a broad range of both hematological and solid tumor models. Molecular determinants of CB-5083 activity include expression of genes in the ERAD pathway, providing a potential strategy for patient selection.
•CB-5083 is a potent and selective small-molecule inhibitor of the cancer target p97•CB-5083 induced a strong unfolded protein response leading to cancer cell death•CB-5083 has antitumor effects in vivo in multiple myeloma and solid tumor models
Anderson et al. characterize CB-5083 as a potent, selective, and orally bioavailable inhibitor of p97, a AAA-ATPase critical for regulating protein homeostasis pathways. CB-5083 activates the apoptotic arm of the unfolded protein response and has antitumor activity in several hematological and solid tumor models.
Ribosome profiling suggests that ribosomes occupy many regions of the transcriptome thought to be noncoding, including 5′ UTRs and long noncoding RNAs (lncRNAs). Apparent ribosome footprints outside ...of protein-coding regions raise the possibility of artifacts unrelated to translation, particularly when they occupy multiple, overlapping open reading frames (ORFs). Here, we show hallmarks of translation in these footprints: copurification with the large ribosomal subunit, response to drugs targeting elongation, trinucleotide periodicity, and initiation at early AUGs. We develop a metric for distinguishing between 80S footprints and nonribosomal sources using footprint size distributions, which validates the vast majority of footprints outside of coding regions. We present evidence for polypeptide production beyond annotated genes, including the induction of immune responses following human cytomegalovirus (HCMV) infection. Translation is pervasive on cytosolic transcripts outside of conserved reading frames, and direct detection of this expanded universe of translated products enables efforts at understanding how cells manage and exploit its consequences.
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•Assembled 80S ribosomes occupy many 5′ UTRs and some lncRNAs•Fragment length metric (FLOSS) identifies true ribosome footprints bioinformatically•Large ribosome subunit pull-down confirms ribosome footprints experimentally•Noncanonical translation produces peptides, including novel antigens
Ribosome profiling, an experimental approach for detecting and quantifying translation, detected apparent ribosome footprints outside of annotated protein-coding genes, including many 5′ UTRs and some lncRNAs. Here, Ingolia et al. verify the presence of assembled 80S ribosomes on these RNAs through computational and experimental approaches. Furthermore, they show that peptides are translated from these regions in human cytomegalovirus (HCMV) and serve as HCMV-specific antigens, suggesting that endogenous human 5′ UTRs and lncRNAs may encode self-antigens and cancer neoantigens as well.
Maintenance of endoplasmic reticulum (ER) function is achieved in part through Ire1 (inositol-requiring enzyme 1), a transmembrane protein activated by protein misfolding in the ER. The cytoplasmic ...nuclease domain of Ire1 cleaves the messenger RNA (mRNA) encoding XBP-1 (X-box-binding protein 1), enabling splicing and production of this active transcription factor. We recently showed that Ire1 activation independently induces the rapid turnover of mRNAs encoding membrane and secreted proteins in Drosophila melanogaster cells through a pathway we call regulated Ire1-dependent decay (RIDD). In this study, we show that mouse fibroblasts expressing wild-type Ire1 but not an Ire1 variant lacking nuclease activity also degrade mRNAs in response to ER stress. Using a second variant of Ire1 that is activated by a small adenosine triphosphate analogue, we show that although XBP-1 splicing can be artificially induced in the absence of ER stress, RIDD appears to require both Ire1 activity and ER stress. Our data suggest that cells use a multitiered mechanism by which different conditions in the ER lead to distinct outputs from Ire1.
Protein synthesis by ribosomes takes place on a linear substrate but at non-uniform speeds. Transient pausing of ribosomes can affect a variety of co-translational processes, including protein ...targeting and folding. These pauses are influenced by the sequence of the messenger RNA. Thus, redundancy in the genetic code allows the same protein to be translated at different rates. However, our knowledge of both the position and the mechanism of translational pausing in vivo is highly limited. Here we present a genome-wide analysis of translational pausing in bacteria by ribosome profiling--deep sequencing of ribosome-protected mRNA fragments. This approach enables the high-resolution measurement of ribosome density profiles along most transcripts at unperturbed, endogenous expression levels. Unexpectedly, we found that codons decoded by rare transfer RNAs do not lead to slow translation under nutrient-rich conditions. Instead, Shine-Dalgarno-(SD)-like features within coding sequences cause pervasive translational pausing. Using an orthogonal ribosome possessing an altered anti-SD sequence, we show that pausing is due to hybridization between the mRNA and 16S ribosomal RNA of the translating ribosome. In protein-coding sequences, internal SD sequences are disfavoured, which leads to biased usage, avoiding codons and codon pairs that resemble canonical SD sites. Our results indicate that internal SD-like sequences are a major determinant of translation rates and a global driving force for the coding of bacterial genomes.
Ribosome profiling has revealed pervasive but largely uncharacterized translation outside of canonical coding sequences (CDSs). In this work, we exploit a systematic CRISPR-based screening strategy ...to identify hundreds of noncanonical CDSs that are essential for cellular growth and whose disruption elicits specific, robust transcriptomic and phenotypic changes in human cells. Functional characterization of the encoded microproteins reveals distinct cellular localizations, specific protein binding partners, and hundreds of microproteins that are presented by the human leukocyte antigen system. We find multiple microproteins encoded in upstream open reading frames, which form stable complexes with the main, canonical protein encoded on the same messenger RNA, thereby revealing the use of functional bicistronic operons in mammals. Together, our results point to a family of functional human microproteins that play critical and diverse cellular roles.
Many, perhaps most, proteins, are capable of forming self-propagating, β-sheet (amyloid) aggregates. Amyloid-like aggregates are found in a wide range of diseases and underlie prion-based ...inheritance. Despite intense interest in amyloids, structural details have only recently begun to be revealed as advances in biophysical approaches, such as hydrogen-deuterium exchange, X-ray crystallography, solid-state nuclear magnetic resonance (SSNMR), and cryoelectron microscopy (cryoEM), have enabled high-resolution insights into their molecular organization. Initial studies found that despite the highly divergent primary structure of different amyloid-forming proteins, amyloids from different sources share many structural similarities. With higher-resolution information, however, it has become clear that, on the molecular level, amyloids comprise a wide diversity of structures. Particularly surprising has been the finding that identical polypeptides can fold into multiple, distinct amyloid conformations and that this structural diversity can lead to distinct heritable prion states or strains.
A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a ...native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context.
Ribosome Profiling: Global Views of Translation Ingolia, Nicholas T; Hussmann, Jeffrey A; Weissman, Jonathan S
Cold Spring Harbor perspectives in biology,
05/2019, Letnik:
11, Številka:
5
Journal Article
Recenzirano
Odprti dostop
The translation of messenger RNA (mRNA) into protein and the folding of the resulting protein into an active form are prerequisites for virtually every cellular process and represent the single ...largest investment of energy by cells. Ribosome profiling-based approaches have revolutionized our ability to monitor every step of protein synthesis in vivo, allowing one to measure the rate of protein synthesis across the proteome, annotate the protein coding capacity of genomes, monitor localized protein synthesis, and explore cotranslational folding and targeting. The rich and quantitative nature of ribosome profiling data provides an unprecedented opportunity to explore and model complex cellular processes. New analytical techniques and improved experimental protocols will provide a deeper understanding of the factors controlling translation speed and its impact on protein function and cell physiology as well as the role of ribosomal RNA and mRNA modifications in regulating translation.
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