Selective Translation Complex Profiling Reveals Staged Initiation and Co-translational Assembly of Initiation Factor Complexes

Overview

Journal Mol Cell

Publisher Cell Press

Specialty Cell Biology

Date 2020 Jun 27

PMID 32589964

Citations 65

Authors

Susan Wagner

Anna Herrmannova

Vladislava Hronova

Stanislava Gunisova

Neelam D Sen

Ross D Hannan

Alan G Hinnebusch

Nikolay E Shirokikh

Thomas Preiss

Leos Shivaya Valasek

Affiliations

Soon will be listed here.

Abstract

Translational control targeting the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast translation complex profile sequencing (TCP-seq), related to ribosome profiling, and adapted it for mammalian cells. Human TCP-seq, capable of capturing footprints of 40S subunits (40Ss) in addition to 80S ribosomes (80Ss), revealed that mammalian and yeast 40Ss distribute similarly across 5'TRs, indicating considerable evolutionary conservation. We further developed yeast and human selective TCP-seq (Sel-TCP-seq), enabling selection of 40Ss and 80Ss associated with immuno-targeted factors. Sel-TCP-seq demonstrated that eIF2 and eIF3 travel along 5' UTRs with scanning 40Ss to successively dissociate upon AUG recognition; notably, a proportion of eIF3 lingers on during the initial elongation cycles. Highlighting Sel-TCP-seq versatility, we also identified four initiating 48S conformational intermediates, provided novel insights into ATF4 and GCN4 mRNA translational control, and demonstrated co-translational assembly of initiation factor complexes.

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Structural basis for translational control by the human 48S initiation complex.

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Differential effects of 40S ribosome recycling factors on reinitiation at regulatory uORFs in GCN4 mRNA are not dictated by their roles in bulk 40S recycling.

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References

Nielsen K, Valasek L, Sykes C, Jivotovskaya A, Hinnebusch A . Interaction of the RNP1 motif in PRT1 with HCR1 promotes 40S binding of eukaryotic initiation factor 3 in yeast. Mol Cell Biol. 2006; 26(8):2984-98. PMC: 1446953. DOI: 10.1128/MCB.26.8.2984-2998.2006. View

Hellen C . Translation Termination and Ribosome Recycling in Eukaryotes. Cold Spring Harb Perspect Biol. 2018; 10(10). PMC: 6169810. DOI: 10.1101/cshperspect.a032656. View

Sokabe M, Fraser C, Hershey J . The human translation initiation multi-factor complex promotes methionyl-tRNAi binding to the 40S ribosomal subunit. Nucleic Acids Res. 2011; 40(2):905-13. PMC: 3258154. DOI: 10.1093/nar/gkr772. View

Lin Y, Li F, Huang L, Polte C, Duan H, Fang J . eIF3 Associates with 80S Ribosomes to Promote Translation Elongation, Mitochondrial Homeostasis, and Muscle Health. Mol Cell. 2020; 79(4):575-587.e7. DOI: 10.1016/j.molcel.2020.06.003. View

Shiber A, Doring K, Friedrich U, Klann K, Merker D, Zedan M . Cotranslational assembly of protein complexes in eukaryotes revealed by ribosome profiling. Nature. 2018; 561(7722):268-272. PMC: 6372068. DOI: 10.1038/s41586-018-0462-y. View

Hronova V, Mohammad M, Wagner S, Panek J, Gunisova S, Zeman J . Does eIF3 promote reinitiation after translation of short upstream ORFs also in mammalian cells?. RNA Biol. 2017; 14(12):1660-1667. PMC: 5731805. DOI: 10.1080/15476286.2017.1353863. View

Park H, Himmelbach A, Browning K, Hohn T, Ryabova L . A plant viral "reinitiation" factor interacts with the host translational machinery. Cell. 2001; 106(6):723-33. DOI: 10.1016/s0092-8674(01)00487-1. View

Valasek L, Nielsen K, Hinnebusch A . Direct eIF2-eIF3 contact in the multifactor complex is important for translation initiation in vivo. EMBO J. 2002; 21(21):5886-98. PMC: 131060. DOI: 10.1093/emboj/cdf563. View

Bohlen J, Fenzl K, Kramer G, Bukau B, Teleman A . Selective 40S Footprinting Reveals Cap-Tethered Ribosome Scanning in Human Cells. Mol Cell. 2020; 79(4):561-574.e5. DOI: 10.1016/j.molcel.2020.06.005. View

10.

Gunisova S, Valasek L . Fail-safe mechanism of GCN4 translational control--uORF2 promotes reinitiation by analogous mechanism to uORF1 and thus secures its key role in GCN4 expression. Nucleic Acids Res. 2014; 42(9):5880-93. PMC: 4027193. DOI: 10.1093/nar/gku204. View

11.

Anders S, Pyl P, Huber W . HTSeq--a Python framework to work with high-throughput sequencing data. Bioinformatics. 2014; 31(2):166-9. PMC: 4287950. DOI: 10.1093/bioinformatics/btu638. View

12.

Skabkin M, Skabkina O, Hellen C, Pestova T . Reinitiation and other unconventional posttermination events during eukaryotic translation. Mol Cell. 2013; 51(2):249-64. PMC: 4038429. DOI: 10.1016/j.molcel.2013.05.026. View

13.

Zeman J, Itoh Y, Kukacka Z, Rosulek M, Kavan D, Kouba T . Binding of eIF3 in complex with eIF5 and eIF1 to the 40S ribosomal subunit is accompanied by dramatic structural changes. Nucleic Acids Res. 2019; 47(15):8282-8300. PMC: 6735954. DOI: 10.1093/nar/gkz570. View

14.

Robichaud N, Sonenberg N . Translational control and the cancer cell response to stress. Curr Opin Cell Biol. 2017; 45:102-109. DOI: 10.1016/j.ceb.2017.05.007. View

15.

Dennis M, Person M, Browning K . Phosphorylation of plant translation initiation factors by CK2 enhances the in vitro interaction of multifactor complex components. J Biol Chem. 2009; 284(31):20615-28. PMC: 2742826. DOI: 10.1074/jbc.M109.007658. View

16.

Langmead B, Trapnell C, Pop M, Salzberg S . Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009; 10(3):R25. PMC: 2690996. DOI: 10.1186/gb-2009-10-3-r25. View

17.

Pisarev A, Hellen C, Pestova T . Recycling of eukaryotic posttermination ribosomal complexes. Cell. 2007; 131(2):286-99. PMC: 2651563. DOI: 10.1016/j.cell.2007.08.041. View

18.

Cigan A, Foiani M, Hannig E, Hinnebusch A . Complex formation by positive and negative translational regulators of GCN4. Mol Cell Biol. 1991; 11(6):3217-28. PMC: 360174. DOI: 10.1128/mcb.11.6.3217-3228.1991. View

19.

Shirokikh N, Archer S, Beilharz T, Powell D, Preiss T . Translation complex profile sequencing to study the in vivo dynamics of mRNA-ribosome interactions during translation initiation, elongation and termination. Nat Protoc. 2017; 12(4):697-731. DOI: 10.1038/nprot.2016.189. View

20.

Ingolia N, Brar G, Rouskin S, McGeachy A, Weissman J . The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat Protoc. 2012; 7(8):1534-50. PMC: 3535016. DOI: 10.1038/nprot.2012.086. View