Identification and characterization of hippuristanol-resistant mutants reveals eIF4A1 dependencies within mRNA 5′ leader regions

Abstract Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targeting eukaryotic initiation factor (eIF) 4A, a DEAD-box RNA helicase required for ribosome recruitment to mRNA templates. Hipp binds to the carboxyl-terminal domain of eIF4A, locks it in a closed conformation, and inhibits its RNA binding. The dependencies of mRNAs for eIF4A during initiation is contingent on the degree of secondary structure within their 5′ leader region. Interest in targeting eIF4A therapeutically in cancer and viral-infected settings stems from the dependencies that certain cellular (e.g. pro-oncogenic, pro-survival) and viral mRNAs show towards eIF4A. Using a CRISPR/Cas9-based variomics screen, we identify functional EIF4A1 Hipp-resistant alleles, which in turn allowed us to link the translation-inhibitory and cytotoxic properties of Hipp to eIF4A1 target engagement. Genome-wide translational profiling in the absence or presence of Hipp were undertaken and our validation studies provided insight into the structure-activity relationships of eIF4A-dependent mRNAs. We find that mRNA 5′ leader length, overall secondary structure and cytosine content are defining features of Hipp-dependent mRNAs.

[1]  Mohamed A. M. Ali.  The DEAD-box protein family of RNA helicases: sentinels for a myriad of cellular functions with emerging roles in tumorigenesis , 2021, International Journal of Clinical Oncology.

[2]  Patrick B. F. O'Connor,et al.  Rocaglates Induce Gain-of-Function Alterations to eIF4A and eIF4F , 2020, Cell reports.

[3]  J. Pelletier,et al.  Modulation of RNA Condensation by the DEAD-Box Protein eIF4A , 2020, Cell.

[4]  P. Bevilacqua,et al.  mRNA structural elements immediately upstream of the start codon dictate dependence upon eIF4A helicase activity , 2019, Genome Biology.

[5]  S. Vajda,et al.  Amidino-Rocaglates: A Potent Class of eIF4A Inhibitors. , 2019, Cell chemical biology.

[6]  N. Sonenberg,et al.  The Organizing Principles of Eukaryotic Ribosome Recruitment. , 2019, Annual review of biochemistry.

[7]  Jan Kolomazník,et al.  G4Hunter web application: a web server for G-quadruplex prediction , 2019, Bioinform..

[8]  Mark Gerstein,et al.  GENCODE reference annotation for the human and mouse genomes , 2018, Nucleic Acids Res..

[9]  G. Portella,et al.  RNA G-quadruplexes at upstream open reading frames cause DHX36- and DHX9-dependent translation of human mRNAs , 2018, Genome Biology.

[10]  Leah L. Zagore,et al.  The helicase Ded1p controls use of near-cognate translation initiation codons in 5′UTRs , 2018, Nature.

[11]  E. Baloglu,et al.  Target identification of small molecules using large-scale CRISPR-Cas mutagenesis scanning of essential genes , 2018, Nature Communications.

[12]  Colin Echeverría Aitken,et al.  Yeast eIF4A enhances recruitment of mRNAs regardless of their structural complexity , 2017, eLife.

[13]  Nicholas T Ingolia,et al.  Transcriptome-wide measurement of translation by ribosome profiling. , 2017, Methods.

[14]  Antoine de Weck,et al.  Project DRIVE: A Compendium of Cancer Dependencies and Synthetic Lethal Relationships Uncovered by Large-Scale, Deep RNAi Screening , 2017, Cell.

[15]  C. Fraser,et al.  A helicase-independent activity of eIF4A in promoting mRNA recruitment to the human ribosome , 2017, Proceedings of the National Academy of Sciences.

[16]  Masahiro Ito,et al.  Discovery and Characterization of a Eukaryotic Initiation Factor 4A-3-Selective Inhibitor That Suppresses Nonsense-Mediated mRNA Decay. , 2017, ACS chemical biology.

[17]  Nicholas T. Ingolia,et al.  Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor , 2016, Nature.

[18]  R. Cencic,et al.  CRISPR-Mediated Drug-Target Validation Reveals Selective Pharmacological Inhibition of the RNA Helicase, eIF4A , 2016, Cell reports.

[19]  A. Heger,et al.  UMI-tools: modeling sequencing errors in Unique Molecular Identifiers to improve quantification accuracy , 2016, bioRxiv.

[20]  J. Pelletier,et al.  nanoCAGE reveals 5′ UTR features that define specific modes of translation of functionally related MTOR-sensitive mRNAs , 2016, Genome research.

[21]  J. Pelletier,et al.  eIF4AII is dispensable for miRNA-mediated gene silencing , 2015, RNA.

[22]  Nicholas T. Ingolia,et al.  Genome-wide analysis of translational efficiency reveals distinct but overlapping functions of yeast DEAD-box RNA helicases Ded1 and eIF4A , 2015, Genome research.

[23]  T. Tokuyasu,et al.  Differential Requirements for eIF4E Dose in Normal Development and Cancer , 2015, Cell.

[24]  Paul J. Groot-Kormelink,et al.  Variomics Screen Identifies the Re-entrant Loop of the Calcium-activated Chloride Channel ANO1 That Facilitates Channel Activation* , 2014, The Journal of Biological Chemistry.

[25]  E. Trotta,et al.  On the Normalization of the Minimum Free Energy of RNAs by Sequence Length , 2014, PloS one.

[26]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[27]  Konstantinos J. Mavrakis,et al.  RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer , 2014, Nature.

[28]  N. Sonenberg,et al.  Single-molecule kinetics of the eukaryotic initiation factor 4AI upon RNA unwinding. , 2014, Structure.

[29]  A. Kinghorn,et al.  Rocaglamide, silvestrol and structurally related bioactive compounds from Aglaia species. , 2014, Natural product reports.

[30]  Josée Dostie,et al.  Repurposing CRISPR/Cas9 for in situ functional assays , 2013, Genes & development.

[31]  P. Nordlund,et al.  Monitoring Drug Target Engagement in Cells and Tissues Using the Cellular Thermal Shift Assay , 2013, Science.

[32]  Wei Li,et al.  A functional variomics tool for discovering drug-resistance genes and drug targets. , 2013, Cell reports.

[33]  Alfonso Valencia,et al.  APPRIS: annotation of principal and alternative splice isoforms , 2012, Nucleic Acids Res..

[34]  R. Cencic,et al.  A cellular response linking eIF4AI activity to eIF4AII transcription. , 2012, RNA.

[35]  Pedro L. Fernandes,et al.  Report of the EMBnet AGM 2011 Workshop , 2011 .

[36]  Peter F. Stadler,et al.  ViennaRNA Package 2.0 , 2011, Algorithms for Molecular Biology.

[37]  David J. Rawlings,et al.  Tracking genome engineering outcome at individual DNA breakpoints , 2011, Nature Methods.

[38]  Marcel Martin.  Cutadapt removes adapter sequences from high-throughput sequencing reads , 2011 .

[39]  Christos G. Gkogkas,et al.  mRNA helicases: the tacticians of translational control , 2011, Nature Reviews Molecular Cell Biology.

[40]  S. Vajda,et al.  Reversing chemoresistance by small molecule inhibition of the translation initiation complex eIF4F , 2010, Proceedings of the National Academy of Sciences.

[41]  J. Pelletier,et al.  Efficient synthetic approach to potent antiproliferative agent hippuristanol via Hg(II)-catalyzed spiroketalization. , 2010, Organic letters.

[42]  Feng Ding,et al.  On the significance of an RNA tertiary structure prediction. , 2010, RNA.

[43]  Cole Trapnell,et al.  Ultrafast and memory-efficient alignment of short DNA sequences to the human genome , 2009, Genome Biology.

[44]  S. Lowe,et al.  Therapeutic suppression of translation initiation modulates chemosensitivity in a mouse lymphoma model. , 2008, The Journal of clinical investigation.

[45]  Ronny Lorenz,et al.  The Vienna RNA Websuite , 2008, Nucleic Acids Res..

[46]  R. Cencic,et al.  Selective Pharmacological Targeting of a DEAD Box RNA Helicase , 2008, PloS one.

[47]  Tao Wang,et al.  Therapeutic suppression of translation initiation factor eIF4E expression reduces tumor growth without toxicity. , 2007, The Journal of clinical investigation.

[48]  F. Niesen,et al.  The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability , 2007, Nature Protocols.

[49]  A. Degterev,et al.  Small-Molecule Inhibition of the Interaction between the Translation Initiation Factors eIF4E and eIF4G , 2007, Cell.

[50]  R. Cencic,et al.  RNA-mediated sequestration of the RNA helicase eIF4A by Pateamine A inhibits translation initiation. , 2006, Chemistry & biology.

[51]  J. Pelletier,et al.  Functional characterization of IRESes by an inhibitor of the RNA helicase eIF4A , 2006, Nature chemical biology.

[52]  N. Tanner,et al.  The DEAD-box protein family of RNA helicases. , 2006, Gene.

[53]  Jun O. Liu,et al.  Inhibition of eukaryotic translation initiation by the marine natural product pateamine A. , 2005, Molecular cell.

[54]  N. Sonenberg,et al.  Stimulation of mammalian translation initiation factor eIF4A activity by a small molecule inhibitor of eukaryotic translation. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[55]  C. Wagner,et al.  Synthesis and evaluation of potential inhibitors of eIF4E cap binding to 7-methyl GTP. , 2005, Bioorganic & medicinal chemistry letters.

[56]  N. Sonenberg,et al.  The requirement for eukaryotic initiation factor 4A (elF4A) in translation is in direct proportion to the degree of mRNA 5' secondary structure. , 2001, RNA.

[57]  R. Rodriguez,et al.  A noncommercial dual luciferase enzyme assay system for reporter gene analysis. , 2000, Analytical biochemistry.

[58]  N. Sonenberg,et al.  The HRIGRXXR region of the DEAD box RNA helicase eukaryotic translation initiation factor 4A is required for RNA binding and ATP hydrolysis , 1993, Molecular and cellular biology.

[59]  J. Tanaka,et al.  HIPPURISTANOLS, CYTOTOXIC POLYOXYGENATED STEROIDS FROM THE GORGONIAN ISIS HIPPURIS , 1981 .