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Next-generation libraries for robust RNA interference-based genome-wide screens (2015)

Kampmann, Martin ; Horlbeck, Max A. ; Chen, Yuwen ; [et al.]

National Academy of Sciences

In: PNAS - Proceedings of the National Academy of Sciences of the United States of America. 2015; 112(26): E3384-E3391. Published 2015 Jun 15. doi: 10.1073/pnas.1508821112.

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Publication Date: 2015-06-15

Description: Genetic screening based on loss-of-function phenotypes is a powerful discovery tool in biology. Although the recent development of clustered regularly interspaced short palindromic repeats (CRISPR)-based screening approaches in mammalian cell culture has enormous potential, RNA interference (RNAi)-based screening remains the method of choice in several biological contexts. We previously demonstrated that ultracomplex pooled short-hairpin RNA (shRNA) libraries can largely overcome the problem of RNAi off-target effects in genome-wide screens. Here, we systematically optimize several aspects of our shRNA library, including the promoter and microRNA context for shRNA expression, selection of guide strands, and features relevant for postscreen sample preparation for deep sequencing. We present next-generation high-complexity libraries targeting human and mouse protein-coding genes, which we grouped into 12 sublibraries based on biological function. A pilot screen suggests that our next-generation RNAi library performs comparably to current CRISPR interference (CRISPRi)-based approaches and can yield complementary results with high sensitivity and high specificity.

Print ISSN: 0027-8424

Electronic ISSN: 1091-6490

Topics: Biology , Medicine , Natural Sciences in General

Published by National Academy of Sciences

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Chaperone-mediated reflux of secretory proteins to the cytosol during endoplasmic reticulum stress (2019)

Igbaria, Aeid ; Merksamer, Philip I. ; Trusina, Ala ; [et al.]

National Academy of Sciences

In: PNAS - Proceedings of the National Academy of Sciences of the United States of America. 2019; 116(23): 11291-11298. Published 2019 May 17. doi: 10.1073/pnas.1904516116.

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Publication Date: 2019-05-17

Description: Diverse perturbations to endoplasmic reticulum (ER) functions compromise the proper folding and structural maturation of secretory proteins. To study secretory pathway physiology during such “ER stress,” we employed an ER-targeted, redox-responsive, green fluorescent protein—eroGFP—that reports on ambient changes in oxidizing potential. Here we find that diverse ER stress regimes cause properly folded, ER-resident eroGFP (and other ER luminal proteins) to “reflux” back to the reducing environment of the cytosol as intact, folded proteins. By utilizing eroGFP in a comprehensive genetic screen in Saccharomyces cerevisiae, we show that ER protein reflux during ER stress requires specific chaperones and cochaperones residing in both the ER and the cytosol. Chaperone-mediated ER protein reflux does not require E3 ligase activity, and proceeds even more vigorously when these ER-associated degradation (ERAD) factors are crippled, suggesting that reflux may work in parallel with ERAD. In summary, chaperone-mediated ER protein reflux may be a conserved protein quality control process that evolved to maintain secretory pathway homeostasis during ER protein-folding stress.

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Electronic ISSN: 1091-6490

Topics: Biology , Medicine , Natural Sciences in General

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Htm1p–Pdi1p is a folding-sensitive mannosidase that marks N-glycoproteins for ER-associated protein degradation (2016)

Liu, Yi-Chang ; Fujimori, Danica Galonić ; Weissman, Jonathan S.

National Academy of Sciences

In: PNAS - Proceedings of the National Academy of Sciences of the United States of America. 2016; 113(28): E4015-E4024. Published 2016 Jun 28. doi: 10.1073/pnas.1608795113.

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Publication Date: 2016-06-28

Description: Our understanding of how the endoplasmic reticulum (ER)-associated protein degradation (ERAD) machinery efficiently targets terminally misfolded proteins while avoiding the misidentification of nascent polypeptides and correctly folded proteins is limited. For luminal N-glycoproteins, demannosylation of their N-glycan to expose a terminal α1,6-linked mannose is necessary for their degradation via ERAD, but whether this modification is specific to misfolded proteins is unknown. Here we report that the complex of the mannosidase Htm1p and the protein disulfide isomerase Pdi1p (Htm1p–Pdi1p) acts as a folding-sensitive mannosidase for catalyzing this first committed step in Saccharomyces cerevisiae. We reconstitute this step in vitro with Htm1p–Pdi1p and model glycoprotein substrates whose structural states we can manipulate. We find that Htm1p–Pdi1p is a glycoprotein-specific mannosidase that preferentially targets nonnative glycoproteins trapped in partially structured states. As such, Htm1p–Pdi1p is suited to act as a licensing factor that monitors folding in the ER lumen and preferentially commits glycoproteins trapped in partially structured states for degradation.

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Electronic ISSN: 1091-6490

Topics: Biology , Medicine , Natural Sciences in General

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Combined chemical–genetic approach identifies cytosolic HSP70 dependence in rhabdomyosarcoma (2016)

Sabnis, Amit J. ; Guerriero, Christopher J. ; Olivas, Victor ; [et al.]

National Academy of Sciences

In: PNAS - Proceedings of the National Academy of Sciences of the United States of America. 2016; 113(32): 9015-9020. Published 2016 Jul 22. doi: 10.1073/pnas.1603883113.

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Publication Date: 2016-07-22

Description: Cytosolic and organelle-based heat-shock protein (HSP) chaperones ensure proper folding and function of nascent and injured polypeptides to support cell growth. Under conditions of cellular stress, including oncogenic transformation, proteostasis components maintain homeostasis and prevent apoptosis. Although this cancer-relevant function has provided a rationale for therapeutically targeting proteostasis regulators (e.g., HSP90), cancer-subtype dependencies upon particular proteostasis components are relatively undefined. Here, we show that human rhabdomyosarcoma (RMS) cells, but not several other cancer cell types, depend upon heat-shock protein 70 kDA (HSP70) for survival. HSP70-targeted therapy (but not chemotherapeutic agents) promoted apoptosis in RMS cells by triggering an unfolded protein response (UPR) that induced PRKR-like endoplasmic reticulum kinase (PERK)–eukaryotic translation initiation factor α (eIF2α)–CEBP homologous protein (CHOP) signaling and CHOP-mediated cell death. Intriguingly, inhibition of only cytosolic HSP70 induced the UPR, suggesting that the essential activity of HSP70 in RMS cells lies at the endoplasmic reticulum–cytosol interface. We also found that increased CHOP mRNA in clinical specimens was a biomarker for poor outcomes in chemotherapy-treated RMS patients. The data suggest that, like human epidermal growth factor receptor 2 (HER2) amplification in breast cancer, increased CHOP in RMS is a biomarker of decreased response to chemotherapy but enhanced response to targeted therapy. Our findings identify the cytosolic HSP70–UPR axis as an unexpected regulator of RMS pathogenesis, revealing HSP70-targeted therapy as a promising strategy to engage CHOP-mediated apoptosis and improve RMS treatment. Our study highlights the utility of dissecting cancer subtype-specific dependencies on proteostasis networks to uncover unanticipated cancer vulnerabilities.

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Electronic ISSN: 1091-6490

Topics: Biology , Medicine , Natural Sciences in General

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A scalable strategy for high-throughput GFP tagging of endogenous human proteins (2016)

Leonetti, Manuel D. ; Sekine, Sayaka ; Kamiyama, Daichi ; [et al.]

National Academy of Sciences

In: PNAS - Proceedings of the National Academy of Sciences of the United States of America. 2016; 113(25): E3501-E3508. Published 2016 Jun 06. doi: 10.1073/pnas.1606731113.

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Publication Date: 2016-06-06

Description: 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.

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Electronic ISSN: 1091-6490

Topics: Biology , Medicine , Natural Sciences in General

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Versatile in vivo regulation of tumor phenotypes by dCas9-mediated transcriptional perturbation (2016)

Braun, Christian J. ; Bruno, Peter M. ; Horlbeck, Max A. ; [et al.]

National Academy of Sciences

In: PNAS - Proceedings of the National Academy of Sciences of the United States of America. 2016; 113(27): E3892-E3900. Published 2016 Jun 20. doi: 10.1073/pnas.1600582113.

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Publication Date: 2016-06-20

Description: Targeted transcriptional regulation is a powerful tool to study genetic mediators of cellular behavior. Here, we show that catalytically dead Cas9 (dCas9) targeted to genomic regions upstream or downstream of the transcription start site allows for specific and sustainable gene-expression level alterations in tumor cells in vitro and in syngeneic immune-competent mouse models. We used this approach for a high-coverage pooled gene-activation screen in vivo and discovered previously unidentified modulators of tumor growth and therapeutic response. Moreover, by using dCas9 linked to an activation domain, we can either enhance or suppress target gene expression simply by changing the genetic location of dCas9 binding relative to the transcription start site. We demonstrate that these directed changes in gene-transcription levels occur with minimal off-target effects. Our findings highlight the use of dCas9-mediated transcriptional regulation as a versatile tool to reproducibly interrogate tumor phenotypes in vivo.

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Electronic ISSN: 1091-6490

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The general mode of translation inhibition by macrolide antibiotics (2014)

Kannan, Krishna ; Kanabar, Pinal ; Schryer, David ; [et al.]

National Academy of Sciences

In: PNAS - Proceedings of the National Academy of Sciences of the United States of America. 2014; 111(45): 15958-15963. Published 2014 Oct 27. doi: 10.1073/pnas.1417334111.

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Publication Date: 2014-10-27

Print ISSN: 0027-8424

Electronic ISSN: 1091-6490

Topics: Biology , Medicine , Natural Sciences in General

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Dynamic translation regulation inCaulobactercell cycle control (2016)

Schrader, Jared M. ; Li, Gene-Wei ; Childers, W. Seth ; [et al.]

National Academy of Sciences

In: PNAS - Proceedings of the National Academy of Sciences of the United States of America. 2016; 113(44): E6859-E6867. Published 2016 Oct 17. doi: 10.1073/pnas.1614795113.

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Publication Date: 2016-10-17

Description: Progression of theCaulobactercell cycle requires temporal and spatial control of gene expression, culminating in an asymmetric cell division yielding distinct daughter cells. To explore the contribution of translational control, RNA-seq and ribosome profiling were used to assay global transcription and translation levels of individual genes at six times over the cell cycle. Translational efficiency (TE) was used as a metric for the relative rate of protein production from each mRNA. TE profiles with similar cell cycle patterns were found across multiple clusters of genes, including those in operons or in subsets of operons. Collections of genes associated with central cell cycle functional modules (e.g., biosynthesis of stalk, flagellum, or chemotaxis machinery) have consistent but different TE temporal patterns, independent of their operon organization. Differential translation of operon-encoded genes facilitates precise cell cycle-timing for the dynamic assembly of multiprotein complexes, such as the flagellum and the stalk and the correct positioning of regulatory proteins to specific cell poles. The cell cycle-regulatory pathways that produce specific temporal TE patterns are separate from—but highly coordinated with—the transcriptional cell cycle circuitry, suggesting that the scheduling of translational regulation is organized by the same cyclical regulatory circuit that directs the transcriptional control of theCaulobactercell cycle.

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Topics: Biology , Medicine , Natural Sciences in General

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