scholarly journals The energy landscape of −1 ribosomal frameshifting

2020 ◽  
Vol 6 (1) ◽  
pp. eaax6969 ◽  
Author(s):  
Junhong Choi ◽  
Sinéad O’Loughlin ◽  
John F. Atkins ◽  
Joseph D. Puglisi
Keyword(s):  
Single Molecule ◽  
Messenger Rna ◽  
Information Transfer ◽  
Mechanistic Model ◽  
High Rate ◽  
Coding Region ◽  
Ribosomal Frameshifting ◽  
Reading Frame

Maintenance of translational reading frame ensures the fidelity of information transfer during protein synthesis. Yet, programmed ribosomal frameshifting sequences within the coding region promote a high rate of reading frame change at predetermined sites thus enriching genomic information density. Frameshifting is typically stimulated by the presence of 3′ messenger RNA (mRNA) structures, but how these mRNA structures enhance −1 frameshifting remains debatable. Here, we apply single-molecule and ensemble approaches to formulate a mechanistic model of ribosomal −1 frameshifting. Our model suggests that the ribosome is intrinsically susceptible to frameshift before its translocation and this transient state is prolonged by the presence of a precisely positioned downstream mRNA structure. We challenged this model using temperature variation in vivo, which followed the prediction made based on in vitro results. Our results provide a quantitative framework for analyzing other frameshifting enhancers and a potential approach to control gene expression dynamically using programmed frameshifting.

scholarly journals Revealing the host antiviral protein ZAP-S as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting

2021 ◽  
Author(s):  
Matthias Michael Zimmer ◽  
Anuja Nitin Kibe ◽  
Ulfert Rand ◽  
Lukas Pekarek ◽  
Luca Cicin-Sain ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Specific Inhibitor ◽  
Antiviral Protein ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Rna Molecules ◽  
Cellular Factors ◽  
Infected Cells

Programmed ribosomal frameshifting (PRF) is a fundamental gene expression event in many viruses including SARS-CoV-2, which allows production of essential structural and replicative enzymes from an alternative reading frame. Despite the importance of PRF for the viral life cycle, it is still largely unknown how and to what extent cellular factors alter mechanical properties of frameshifting RNA molecules and thereby impact virulence. This prompted us to comprehensively dissect the interplay between the host proteome and the SARS-CoV-2 frameshift element. Here, we reveal that zinc-finger antiviral protein (ZAP-S) is a direct and specific regulator of PRF in SARS-CoV-2 infected cells. ZAP-S overexpression strongly impairs frameshifting and viral replication. Using in vitro ensemble and single-molecule techniques, we further demonstrate that ZAP-S directly interacts with the SARS-CoV-2 RNA and ribosomes and interferes with the folding of the frameshift RNA. Together these data illuminate ZAP-S as de novo host-encoded specific inhibitor of SARS-CoV-2 frameshifting and expand our understanding of RNA-based gene regulation.

Identification and characterization of CKLiK, a novel granulocyte Ca++/calmodulin-dependent kinase

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3215-3223 ◽  
Author(s):  
Sandra Verploegen ◽  
Jan-Willem J. Lammers ◽  
Leo Koenderman ◽  
Paul J. Coffer
Keyword(s):  
Messenger Rna ◽  
Kinase Activity ◽  
Myeloid Cell ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Reading Frame ◽  
Effector Functions ◽  
Calcium Calmodulin Dependent

Human granulocytes are characterized by a variety of specific effector functions involved in host defense. Several widely expressed protein kinases have been implicated in the regulation of these effector functions. A polymerase chain reaction–based strategy was used to identify novel granulocyte-specific kinases. A novel protein kinase complementary DNA with an open reading frame of 357 amino acids was identified with homology to calcium-calmodulin–dependent kinase I (CaMKI). This has been termed CaMKI-like kinase (CKLiK). Analysis of CKLiK messenger RNA (mRNA) expression in hematopoietic cells demonstrated an almost exclusive expression in human polymorphonuclear leukocytes (PMN). Up-regulation of CKLiK mRNA occurs during neutrophilic differentiation of CD34+ stem cells. CKLiK kinase activity was dependent on Ca++ and calmodulin as analyzed by in vitro phosphorylation of cyclic adenosine monophosphate responsive element modulator (CREM). Furthermore, CKLiK- transfected cells treated with ionomycin demonstrated an induction of CRE- binding protein (CREB) transcriptional activity compared to control cells. Additionally, CaMK-kinaseα enhanced CKLiK activity. In vivo activation of CKLiK was shown by addition of interleukin (IL)-8 to a myeloid cell line stably expressing CKLiK. Furthermore inducible activation of CKLiK was sufficient to induce extracellular signal-related kinase (ERK) mitogen-activated protein (MAP) kinase activity. These data identify a novel Ca++/calmodulin-dependent PMN- specific kinase that may play a role in Ca++-mediated regulation of human granulocyte functions.

scholarly journals Correction of Frameshift Mutations in the atpB Gene by Translational Recoding in Chloroplasts of Oenothera and Tobacco

2021 ◽  
Author(s):  
Irina Malinova ◽  
Arkadiusz Zupok ◽  
Amid Massouh ◽  
Mark Aurel Schöttler ◽  
Etienne H Meyer ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Coding Region ◽  
Functional Protein ◽  
Ribosomal Frameshifting ◽  
Photoautotrophic Growth ◽  
Reading Frame ◽  
Evening Primrose ◽  
Frameshift Mutations ◽  
Transplastomic Tobacco ◽  
Albino Phenotype

Abstract Translational recoding, also known as ribosomal frameshifting, is a process that causes ribosome slippage along the messenger RNA, thereby changing the amino acid sequence of the synthesized protein. Whether the chloroplast employs recoding is unknown. I-iota, a plastome mutant of Oenothera (evening primrose), carries a single adenine insertion in an oligoA stretch [11A] of the atpB coding region (encoding a β-subunit of the ATP synthase). The mutation is expected to cause synthesis of a truncated, non-functional protein. We report that a full-length AtpB protein is detectable in I-iota leaves, suggesting operation of a recoding mechanism. To characterize the phenomenon, we generated transplastomic tobacco lines in which the atpB reading frame was altered by insertions or deletions in the oligoA motif. We observed that insertion of two adenines was more efficiently corrected than insertion of a single adenine, or deletion of one or two adenines. We further show that homopolymeric composition of the oligoA stretch is essential for recoding, as an additional replacement of AAA lysine codon by AAG resulted in an albino phenotype. Our work provides evidence for the operation of translational recoding in chloroplasts. Recoding enables correction of frameshift mutations and can restore photoautotrophic growth in the presence of mutation that otherwise would be lethal.

scholarly journals The short isoform of the host antiviral protein ZAP acts as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Matthias M. Zimmer ◽  
Anuja Kibe ◽  
Ulfert Rand ◽  
Lukas Pekarek ◽  
Liqing Ye ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Molecule ◽  
Antiviral Protein ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Cellular Factors ◽  
Infected Cells ◽  
Direct Regulator ◽  
Alternative Reading

AbstractProgrammed ribosomal frameshifting (PRF) is a fundamental gene expression event in many viruses, including SARS-CoV-2. It allows production of essential viral, structural and replicative enzymes that are encoded in an alternative reading frame. Despite the importance of PRF for the viral life cycle, it is still largely unknown how and to what extent cellular factors alter mechanical properties of frameshift elements and thereby impact virulence. This prompted us to comprehensively dissect the interplay between the SARS-CoV-2 frameshift element and the host proteome. We reveal that the short isoform of the zinc-finger antiviral protein (ZAP-S) is a direct regulator of PRF in SARS-CoV-2 infected cells. ZAP-S overexpression strongly impairs frameshifting and inhibits viral replication. Using in vitro ensemble and single-molecule techniques, we further demonstrate that ZAP-S directly interacts with the SARS-CoV-2 RNA and interferes with the folding of the frameshift RNA element. Together, these data identify ZAP-S as a host-encoded inhibitor of SARS-CoV-2 frameshifting and expand our understanding of RNA-based gene regulation.

Identification and characterization of CKLiK, a novel granulocyte Ca++/calmodulin-dependent kinase

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3215-3223 ◽  
Author(s):  
Sandra Verploegen ◽  
Jan-Willem J. Lammers ◽  
Leo Koenderman ◽  
Paul J. Coffer
Keyword(s):  
Messenger Rna ◽  
Kinase Activity ◽  
Myeloid Cell ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Reading Frame ◽  
Effector Functions ◽  
Calcium Calmodulin Dependent

Abstract Human granulocytes are characterized by a variety of specific effector functions involved in host defense. Several widely expressed protein kinases have been implicated in the regulation of these effector functions. A polymerase chain reaction–based strategy was used to identify novel granulocyte-specific kinases. A novel protein kinase complementary DNA with an open reading frame of 357 amino acids was identified with homology to calcium-calmodulin–dependent kinase I (CaMKI). This has been termed CaMKI-like kinase (CKLiK). Analysis of CKLiK messenger RNA (mRNA) expression in hematopoietic cells demonstrated an almost exclusive expression in human polymorphonuclear leukocytes (PMN). Up-regulation of CKLiK mRNA occurs during neutrophilic differentiation of CD34+ stem cells. CKLiK kinase activity was dependent on Ca++ and calmodulin as analyzed by in vitro phosphorylation of cyclic adenosine monophosphate responsive element modulator (CREM). Furthermore, CKLiK- transfected cells treated with ionomycin demonstrated an induction of CRE- binding protein (CREB) transcriptional activity compared to control cells. Additionally, CaMK-kinaseα enhanced CKLiK activity. In vivo activation of CKLiK was shown by addition of interleukin (IL)-8 to a myeloid cell line stably expressing CKLiK. Furthermore inducible activation of CKLiK was sufficient to induce extracellular signal-related kinase (ERK) mitogen-activated protein (MAP) kinase activity. These data identify a novel Ca++/calmodulin-dependent PMN- specific kinase that may play a role in Ca++-mediated regulation of human granulocyte functions.

scholarly journals Thermodynamic control of −1 programmed ribosomal frameshifting

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Lars V. Bock ◽  
Neva Caliskan ◽  
Natalia Korniy ◽  
Frank Peske ◽  
Marina V. Rodnina ◽  
...  
Keyword(s):  
Energy Difference ◽  
Mrna Sequence ◽  
Thermodynamic Control ◽  
Base Pairs ◽  
Free Energy Difference ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Slippery Sequence

Abstract mRNA contexts containing a ‘slippery’ sequence and a downstream secondary structure element stall the progression of the ribosome along the mRNA and induce its movement into the −1 reading frame. In this study we build a thermodynamic model based on Bayesian statistics to explain how −1 programmed ribosome frameshifting can work. As training sets for the model, we measured frameshifting efficiencies on 64 dnaX mRNA sequence variants in vitro and also used 21 published in vivo efficiencies. With the obtained free-energy difference between mRNA-tRNA base pairs in the 0 and −1 frames, the frameshifting efficiency of a given sequence can be reproduced and predicted from the tRNA−mRNA base pairing in the two frames. Our results further explain how modifications in the tRNA anticodon modulate frameshifting and show how the ribosome tunes the strength of the base-pair interactions.

scholarly journals Revealing the host antiviral protein ZAP-S as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting

2021 ◽  
Author(s):  
Matthias Zimmer ◽  
Anuja Kibe ◽  
Ulfert Rand ◽  
Lukas Pekarek ◽  
Luka Cicin-Sain ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Specific Inhibitor ◽  
Antiviral Protein ◽  
Ribosomal Frameshifting ◽  
Reading Frame ◽  
Rna Molecules ◽  
Cellular Factors ◽  
Infected Cells

Abstract Programmed ribosomal frameshifting (PRF) is a fundamental gene expression event in many viruses including SARS-CoV-2, which allows production of essential structural and replicative enzymes from an alternative reading frame. Despite the importance of PRF for the viral life cycle, it is still largely unknown how and to what extent cellular factors alter mechanical properties of frameshifting RNA molecules and thereby impact virulence. This prompted us to comprehensively dissect the interplay between the host proteome and the SARS-CoV-2 frameshift element. Here, we reveal that zinc-finger antiviral protein (ZAP-S) is a direct and specific regulator of PRF in SARS-CoV-2 infected cells. ZAP-S overexpression strongly impairs frameshifting and viral replication. Using in vitro ensemble and single-molecule techniques, we further demonstrate that ZAP-S directly interacts with the SARS-CoV-2 RNA and ribosomes and interferes with the folding of the frameshift RNA. Together these data illuminate ZAP-S as de novo host-encoded specific inhibitor of SARS-CoV-2 frameshifting and expand our understanding of RNA-based gene regulation.

scholarly journals The structure of an alternate form of complement C3 that displays costimulatory growth factor activity for B lymphocytes.

1994 ◽  
Vol 180 (6) ◽  
pp. 2079-2088 ◽  
Author(s):  
Y Cahen-Kramer ◽  
I L Mårtensson ◽  
F Melchers
Keyword(s):  
B Cells ◽  
Interleukin 2 ◽  
Immunoglobulin M ◽  
Complement C3 ◽  
In Vitro Assays ◽  
Leader Peptide ◽  
Coding Region ◽  
Reading Frame

In this study, the structure of a novel 1.9-kb transcript coding for complement component 3 (C3) is described. This alternate C3 is identical to the 3' end of the C3 message beginning at position 3300 of the C3 cDNA. Its transcription appears to be driven by an alternate promoter located within intron 8 of the C3 gene. This alternate C3 message contains an open reading frame that may encode a 536-amino acid-long protein identical to the 3' part of the C3 alpha chain. The resulting protein contains the complement receptor CR2 binding site. The suggested 5' end of coding region of the alternate C3 includes information for a potential hydrophobic leader peptide that would allow secretion of the protein. In vitro assays with macrophage-depleted mouse splenic B cells indicate that an activity is secreted from cell lines transfected with the alternate C3 cDNA. Together with Sepharose-bound immunoglobulin M-specific monoclonal antibodies and interleukin 2, it costimulates the proliferation of B cells. Implications for possible in vivo functions are discussed.

scholarly journals STEM-26. SMALL MOLECULE DRUG CBL0137 INHIBITS THE FACT COMPLEX AND SENSITIZES GLIOBLASTOMA CANCER STEM CELLS TO RADIOTHERAPY

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii201-ii202
Author(s):  
Miranda Tallman ◽  
Abigail Zalenski ◽  
Amanda Deighen ◽  
Morgan Schrock ◽  
Sherry Mortach ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
High Rate ◽  
Orthotopic Model ◽  
In Vivo Models ◽  
Specific Cytotoxicity ◽  
Treatment Paradigm ◽  
Cancer Agent

Abstract Glioblastoma (GBM) is a malignant brain tumor with nearly universal recurrence. GBM cancer stem cells (CSCs), a subpopulation of radio- and chemo-resistant cancer cells capable of self-renewal, contribute to the high rate of recurrence. The anti-cancer agent, CBL0137, inhibits the FACT (facilitates chromatin transcription) complex leading to cancer cell specific cytotoxicity. Here, we show that CBL0137 sensitized GBM CSCs to radiotherapy using both in vitro and in vivo models. Treatment of CBL0137 combined with radiotherapy led to increased DNA damage in GBM patient specimens and failure to resolve the damage led to decreased cell viability. Using clonogenic assays, we confirmed that CBL0137 radiosensitized the CSCs. To validate that combination therapy impacted CSCs, we used an in vivo subcutaneous model and showed a decrease in the frequency of cancer stem cells present in tumors as well as decreased tumor volume. Using an orthotopic model of GBM, we confirmed that treatment with CBL0137 followed by radiotherapy led to significantly increased survival compared to either treatment alone. Radiotherapy remains a critical component of patient care for GBM, even though there exists a resistant subpopulation. Radio-sensitizing agents, including CBL0137, pose an exciting treatment paradigm to increase the efficacy of irradiation, especially by inclusively targeting CSCs.

scholarly journals The Design and Preclinical Evaluation of a Single-Label Bimodal Nanobody Tracer for Image-Guided Surgery

Biomolecules ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 360
Author(s):  
Pieterjan Debie ◽  
Noemi B. Declerck ◽  
Danny van Willigen ◽  
Celine M. Huygen ◽  
Bieke De Sloovere ◽  
...  
Keyword(s):  
Single Molecule ◽  
Gamma Ray ◽  
Nuclear Imaging ◽  
Primary Amines ◽  
Resection Margins ◽  
Fluorescent Light ◽  
Single Structure ◽  
Fluorescence Images

Intraoperative guidance using targeted fluorescent tracers can potentially provide surgeons with real-time feedback on the presence of tumor tissue in resection margins. To overcome the limited depth penetration of fluorescent light, combining fluorescence with SPECT/CT imaging and/or gamma-ray tracing has been proposed. Here, we describe the design and preclinical validation of a novel bimodal nanobody-tracer, labeled using a “multifunctional single attachment point” (MSAP) label, integrating a Cy5 fluorophore and a diethylenetriaminepentaacetic acid (DTPA) chelator into a single structure. After conjugation of the bimodal MSAP to primary amines of the anti-HER2 nanobody 2Rs15d and 111In-labeling of DTPA, the tracer’s characteristics were evaluated in vitro. Subsequently, its biodistribution and tumor targeting were assessed by SPECT/CT and fluorescence imaging over 24 h. Finally, the tracer’s ability to identify small, disseminated tumor lesions was investigated in mice bearing HER2-overexpressing SKOV3.IP1 peritoneal lesions. [111In]In-MSAP.2Rs15d retained its affinity following conjugation and remained stable for 24 h. In vivo SPECT/CT and fluorescence images showed specific uptake in HER2-overexpressing tumors with low background. High tumor-to-muscle ratios were obtained at 1h p.i. and remained 19-fold on SPECT/CT and 3-fold on fluorescence images over 24 h. In the intraperitoneally disseminated model, the tracer allowed detection of larger lesions via nuclear imaging, while fluorescence enabled accurate removal of submillimeter lesions. Bimodal nuclear/fluorescent nanobody-tracers can thus be conveniently designed by conjugation of a single-molecule MSAP-reagent carrying a fluorophore and chelator for radioactive labeling. Such tracers hold promise for clinical applications.

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