المؤلفون: Genuth, Naomi R, Shi, Zhen, Kunimoto, Koshi, Hung, Victoria, Xu, Adele F, Kerr, Craig H, Tiu, Gerald C, Oses-Prieto, Juan A, Salomon-Shulman, Rachel EA, Axelrod, Jeffrey D, Burlingame, Alma L, Loh, Kyle M, Barna, Maria

المصدر: Nature Communications. 13(1)

مصطلحات موضوعية: Biochemistry and Cell Biology, Biological Sciences, Genetics, Stem Cell Research, Stem Cell Research - Nonembryonic - Non-Human, Underpinning research, 1.1 Normal biological development and functioning, Generic health relevance, Animals, Cell Differentiation, Humans, Mesoderm, Mice, Ribosomal Proteins, Ribosomes, Stem Cells, Wnt Signaling Pathway

الوصف: Recent findings suggest that the ribosome itself modulates gene expression. However, whether ribosomes change composition across cell types or control cell fate remains unknown. Here, employing quantitative mass spectrometry during human embryonic stem cell differentiation, we identify dozens of ribosome composition changes underlying cell fate specification. We observe upregulation of RPL10A/uL1-containing ribosomes in the primitive streak followed by progressive decreases during mesoderm differentiation. An Rpl10a loss-of-function allele in mice causes striking early mesodermal phenotypes, including posterior trunk truncations, and inhibits paraxial mesoderm production in culture. Ribosome profiling in Rpl10a loss-of-function mice reveals decreased translation of mesoderm regulators, including Wnt pathway mRNAs, which are also enriched on RPL10A/uL1-containing ribosomes. We further show that RPL10A/uL1 regulates canonical and non-canonical Wnt signaling during stem cell differentiation and in the developing embryo. These findings reveal unexpected ribosome composition modularity that controls differentiation and development through the specialized translation of key signaling networks.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/2fc9h5rxTest

المؤلفون: Tiu, Gerald C, Kerr, Craig H, Forester, Craig M, Krishnarao, Pallavi S, Rosenblatt, Hannah D, Raj, Nitin, Lantz, Travis C, Zhulyn, Olena, Bowen, Margot E, Shokat, Leila, Attardi, Laura D, Ruggero, Davide, Barna, Maria

المصدر: Developmental Cell. 56(14)

مصطلحات موضوعية: Biochemistry and Cell Biology, Biological Sciences, Biotechnology, Stem Cell Research - Nonembryonic - Non-Human, Genetics, Stem Cell Research, Rare Diseases, Generic health relevance, Adaptor Proteins, Signal Transducing, Animals, Body Patterning, Cell Cycle Proteins, Extremities, Gene Expression Regulation, Developmental, Haploinsufficiency, Mice, Mice, Knockout, Phenotype, Protein Biosynthesis, Protein Processing, Post-Translational, Ribosomal Proteins, Ribosomes, Tumor Suppressor Protein p53, 4E-BP1, limb development, nucleolar stress, p53, ribosomal protein haploinsufficiency, ribosome profiling, ribosomopathy, translational control, Medical and Health Sciences, Developmental Biology, Biochemistry and cell biology

الوصف: In ribosomopathies, perturbed expression of ribosome components leads to tissue-specific phenotypes. What accounts for such tissue-selective manifestations as a result of mutations in the ribosome, a ubiquitous cellular machine, has remained a mystery. Combining mouse genetics and in vivo ribosome profiling, we observe limb-patterning phenotypes in ribosomal protein (RP) haploinsufficient embryos, and we uncover selective translational changes of transcripts that controlling limb development. Surprisingly, both loss of p53, which is activated by RP haploinsufficiency, and augmented protein synthesis rescue these phenotypes. These findings are explained by the finding that p53 functions as a master regulator of protein synthesis, at least in part, through transcriptional activation of 4E-BP1. 4E-BP1, a key translational regulator, in turn, facilitates selective changes in the translatome downstream of p53, and this thereby explains how RP haploinsufficiency may elicit specificity to gene expression. These results provide an integrative model to help understand how in vivo tissue-specific phenotypes emerge in ribosomopathies.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/8gp8v1qxTest

المؤلفون: Nayak, Arabinda, Kim, Dong Young, Trnka, Michael J, Kerr, Craig H, Lidsky, Peter V, Stanley, David J, Rivera, Brianna Monique, Li, Kathy H, Burlingame, Alma L, Jan, Eric, Frydman, Judith, Gross, John D, Andino, Raul

المصدر: Cell Host & Microbe. 24(4)

مصطلحات موضوعية: Emerging Infectious Diseases, Genetics, Infectious Diseases, Biotechnology, Aetiology, 2.2 Factors relating to the physical environment, Infection, Animals, Argonaute Proteins, Cell Line, Dicistroviridae, Drosophila Proteins, Drosophila melanogaster, Humans, Mutation, Protein Binding, Protein Conformation, Protein Interaction Maps, RNA Interference, Ubiquitin-Protein Ligases, Viral Proteins, Virus Replication, Ago-2, Ago-2 degradation, CrPV, E3 ligase, RNAi, RNAi suppressor, antiviral immunity, insects, Microbiology, Medical Microbiology, Immunology

الوصف: The dicistrovirus, Cricket paralysis virus (CrPV) encodes an RNA interference (RNAi) suppressor, 1A, which modulates viral virulence. Using the Drosophila model, we combined structural, biochemical, and virological approaches to elucidate the strategies by which CrPV-1A restricts RNAi immunity. The atomic resolution structure of CrPV-1A uncovered a flexible loop that interacts with Argonaute 2 (Ago-2), thereby inhibiting Ago-2 endonuclease-dependent immunity. Mutations disrupting Ago-2 binding attenuates viral pathogenesis in wild-type but not Ago-2-deficient flies. CrPV-1A also contains a BC-box motif that enables the virus to hijack a host Cul2-Rbx1-EloBC ubiquitin ligase complex, which promotes Ago-2 degradation and virus replication. Our study uncovers a viral-based dual regulatory program that restricts antiviral immunity by direct interaction with and modulation of host proteins. While the direct inhibition of Ago-2 activity provides an efficient mechanism to establish infection, the recruitment of a ubiquitin ligase complex enables CrPV-1A to amplify Ago-2 inactivation to restrict further antiviral RNAi immunity.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/2368j8jgTest

المؤلفون: Leppek, Kathrin, Byeon, Gun Woo, Kladwang, Wipapat, Wayment-Steele, Hannah K., Kerr, Craig H., Xu, Adele F., Kim, Do Soon, Topkar, Ved V., Choe, Christian, Rothschild, Daphna, Tiu, Gerald C., Wellington-Oguri, Roger, Fujii, Kotaro, Sharma, Eesha, Watkins, Andrew M., Nicol, John J., Romano, Jonathan, Tunguz, Bojan, Diaz, Fernando, Cai, Hui, Guo, Pengbo, Wu, Jiewei, Meng, Fanyu, Shi, Shuai, Participants, Eterna, Dormitzer, Philip R., Solórzano, Alicia, Barna, Maria, Das, Rhiju

المصدر: Nature Communications ; volume 13, issue 1 ; ISSN 2041-1723

مصطلحات موضوعية: General Physics and Astronomy, General Biochemistry, Genetics and Molecular Biology, General Chemistry, Multidisciplinary

الوصف: Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop an RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that highly structured “superfolder” mRNAs can be designed to improve both stability and expression with further enhancement through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines.

الإتاحة: https://doi.org/10.1038/s41467-022-28776-wTest
https://www.nature.com/articles/s41467-022-28776-w.pdfTest
https://www.nature.com/articles/s41467-022-28776-wTest

المؤلفون: Raj, Nitin, Wang, Mengxiong, Seoane, Jose A., Zhao, Richard L., Kaiser, Alyssa M., Moonie, Nancie A., Demeter, Janos, Boutelle, Anthony M., Kerr, Craig H., Mulligan, Abigail S., Moffatt, Clare, Zeng, Shelya X., Lu, Hua, Barna, Maria, Curtis, Christina, Chang, Howard Y., Jackson, Peter K., Attardi, Laura D.

المصدر: Molecular Cell ; volume 82, issue 13, page 2370-2384.e10 ; ISSN 1097-2765

الإتاحة: https://doi.org/10.1016/j.molcel.2022.04.010Test
https://api.elsevier.com/content/article/PII:S1097276522003215?httpAccept=text/xmlTest
https://api.elsevier.com/content/article/PII:S1097276522003215?httpAccept=text/plainTest

المؤلفون: Skinnider, Michael A., Scott, Nichollas E., Prudova, Anna, Kerr, Craig H., Stoynov, Nikolay, Stacey, R. Greg, Chan, Queenie W.T., Rattray, David, Gsponer, Jörg, Foster, Leonard J.

المصدر: Cell ; volume 184, issue 15, page 4073-4089.e17 ; ISSN 0092-8674

مصطلحات موضوعية: General Biochemistry, Genetics and Molecular Biology

الإتاحة: https://doi.org/10.1016/j.cell.2021.06.003Test
https://api.elsevier.com/content/article/PII:S0092867421007042?httpAccept=text/xmlTest
https://api.elsevier.com/content/article/PII:S0092867421007042?httpAccept=text/plainTest

المؤلفون: Fujii, Kotaro, Zhulyn, Olena, Byeon, Gun Woo, Genuth, Naomi R., Kerr, Craig H., Walsh, Erin M., Barna, Maria

المساهمون: New York Stem Cell Foundation, Human Frontier Science Program, National Institutes of Health, Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology, Alfred P. Sloan Foundation

المصدر: Developmental Cell ; volume 56, issue 21, page 2928-2937.e9 ; ISSN 1534-5807

الإتاحة: https://doi.org/10.1016/j.devcel.2021.10.009Test
https://api.elsevier.com/content/article/PII:S1534580721008108?httpAccept=text/xmlTest
https://api.elsevier.com/content/article/PII:S1534580721008108?httpAccept=text/plainTest

المؤلفون: Kerr, Craig H., Skinnider, Michael A., Andrews, Daniel D. T., Madero, Angel M., Chan, Queenie W. T., Stacey, R. Greg, Stoynov, Nikolay, Jan, Eric, Foster, Leonard J.

المساهمون: Genome Canada, CIHR

المصدر: Genome Biology ; volume 21, issue 1 ; ISSN 1474-760X

الوصف: Background The type I interferon (IFN) response is an ancient pathway that protects cells against viral pathogens by inducing the transcription of hundreds of IFN-stimulated genes. Comprehensive catalogs of IFN-stimulated genes have been established across species and cell types by transcriptomic and biochemical approaches, but their antiviral mechanisms remain incompletely characterized. Here, we apply a combination of quantitative proteomic approaches to describe the effects of IFN signaling on the human proteome, and apply protein correlation profiling to map IFN-induced rearrangements in the human protein-protein interaction network. Results We identify > 26,000 protein interactions in IFN-stimulated and unstimulated cells, many of which involve proteins associated with human disease and are observed exclusively within the IFN-stimulated network. Differential network analysis reveals interaction rewiring across a surprisingly broad spectrum of cellular pathways in the antiviral response. We identify IFN-dependent protein-protein interactions mediating novel regulatory mechanisms at the transcriptional and translational levels, with one such interaction modulating the transcriptional activity of STAT1. Moreover, we reveal IFN-dependent changes in ribosomal composition that act to buffer IFN-stimulated gene protein synthesis. Conclusions Our map of the IFN interactome provides a global view of the complex cellular networks activated during the antiviral response, placing IFN-stimulated genes in a functional context, and serves as a framework to understand how these networks are dysregulated in autoimmune or inflammatory disease.

الإتاحة: https://doi.org/10.1186/s13059-020-02050-yTest

المؤلفون: Carlson, Michael Luke, Stacey, R Greg, Young, John William, Wason, Irvinder Singh, Zhao, Zhiyu, Rattray, David G, Scott, Nichollas, Kerr, Craig H, Babu, Mohan, Foster, Leonard J, Duong Van Hoa, Franck

المساهمون: Canadian Institutes of Health Research

المصدر: eLife ; volume 8 ; ISSN 2050-084X

الوصف: Protein-correlation-profiling (PCP), in combination with quantitative proteomics, has emerged as a high-throughput method for the rapid identification of dynamic protein complexes in native conditions. While PCP has been successfully applied to soluble proteomes, characterization of the membrane interactome has lagged, partly due to the necessary use of detergents to maintain protein solubility. Here, we apply the peptidisc, a ‘one-size fits all’ membrane mimetic, for the capture of the Escherichia coli cell envelope proteome and its high-resolution fractionation in the absence of detergent. Analysis of the SILAC-labeled peptidisc library via PCP allows generation of over 4900 possible binary interactions out of >700,000 random associations. Using well-characterized membrane protein systems such as the SecY translocon, the Bam complex and the MetNI transporter, we demonstrate that our dataset is a useful resource for identifying transient and surprisingly novel protein interactions. For example, we discover a trans-periplasmic supercomplex comprising subunits of the Bam and Sec machineries, including membrane-bound chaperones YfgM and PpiD. We identify RcsF and OmpA as bone fide interactors of BamA, and we show that MetQ association with the ABC transporter MetNI depends on its N-terminal lipid anchor. We also discover NlpA as a novel interactor of MetNI complex. Most of these interactions are largely undetected by standard detergent-based purification. Together, the peptidisc workflow applied to the proteomic field is emerging as a promising novel approach to characterize membrane protein interactions under native expression conditions and without genetic manipulation.

الإتاحة: https://doi.org/10.7554/elife.46615Test
https://cdn.elifesciences.org/articles/46615/elife-46615-v2.pdfTest
https://cdn.elifesciences.org/articles/46615/elife-46615-v2.xmlTest
https://elifesciences.org/articles/46615Test

المؤلفون: Romantsov, Tatyana, Gonzalez, Karen, Sahtout, Naheda, Culham, Doreen E., Coumoundouros, Chelsea, Garner, Jennifer, Kerr, Craig H., Chang, Limei, Turner, Raymond J., Wood, Janet M.

المساهمون: Natural Sciences and Engineering Research Council of Canada, Canadian Institutes of Health Research

المصدر: Molecular Microbiology ; volume 107, issue 5, page 623-638 ; ISSN 0950-382X 1365-2958

الوصف: Summary Osmosensing by transporter ProP is modulated by its cardiolipin (CL)‐dependent concentration at the poles of Escherichia coli cells. Other contributors to this phenomenon were sought with the BACterial Two‐Hybrid System (BACTH). The BACTH‐tagged variants T18‐ProP and T25‐ProP retained ProP function and localization. Their interaction confirmed the ProP homo‐dimerization previously established by protein crosslinking. YdhP, YjbJ and ClsA were prominent among the putative ProP interactors identified by the BACTH system. The functions of YdhP and YjbJ are unknown, although YjbJ is an abundant, osmotically induced, soluble protein. ClsA (CL Synthase A) had been shown to determine ProP localization by mediating CL synthesis. Unlike a deletion of clsA , deletion of ydhP or yjbJ had no effect on ProP localization or function. All three proteins were concentrated at the cell poles, but only ClsA localization was CL‐dependent. ClsA was shown to be N‐terminally processed and membrane‐anchored, with dual, cytoplasmic, catalytic domains. Active site amino acid replacements (H224A plus H404A) inactivated ClsA and compromised ProP localization. YdhP and YjbJ may be ClsA effectors, and interactions of YdhP, YjbJ and ClsA with ProP may reflect their colocalization at the cell poles. Targeted CL synthesis may contribute to the polar localization of CL, ClsA and ProP.

الإتاحة: https://doi.org/10.1111/mmi.13904Test