Transcriptome-wide analysis of the function of Ded1 in translation preinitiation complex assembly in a reconstituted in vitro system

We have developed a deep sequencing-based approach, Rec-Seq, that allows simultaneous monitoring of ribosomal 48S preinitiation complex (PIC) formation on every mRNA in the translatome in an in vitro reconstituted system. Rec-Seq isolates key early steps in translation initiation in the absence of a...
Ausführliche Beschreibung

Gespeichert in:

Autor*in:	Fujun Zhou [verfasserIn] Julie M Bocetti [verfasserIn] Meizhen Hou [verfasserIn] Daoming Qin [verfasserIn] Alan G Hinnebusch [verfasserIn] Jon R Lorsch [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2024

Schlagwörter:	translation protein synthesis initiation ribosome profiling yeast in vitro

Übergeordnetes Werk:	In: eLife - eLife Sciences Publications Ltd, 2013, 13(2024)
Übergeordnetes Werk:	volume:13 ; year:2024

Links:	Link aufrufen Link aufrufen Link aufrufen Journal toc

DOI / URN:	10.7554/eLife.93255

Katalog-ID:	DOAJ097361585

Internformat


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allfields	10.7554/eLife.93255 doi (DE-627)DOAJ097361585 (DE-599)DOAJ379dbd1d0a6640ed96c6e0b8d84b47d5 DE-627 ger DE-627 rakwb eng QH301-705.5 Fujun Zhou verfasserin aut Transcriptome-wide analysis of the function of Ded1 in translation preinitiation complex assembly in a reconstituted in vitro system 2024 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier We have developed a deep sequencing-based approach, Rec-Seq, that allows simultaneous monitoring of ribosomal 48S preinitiation complex (PIC) formation on every mRNA in the translatome in an in vitro reconstituted system. Rec-Seq isolates key early steps in translation initiation in the absence of all other cellular components and processes. Using this approach, we show that the DEAD-box ATPase Ded1 promotes 48S PIC formation on the start codons of >1000 native mRNAs, most of which have long, structured 5′-untranslated regions (5′UTRs). Remarkably, initiation measured in Rec-Seq was enhanced by Ded1 for most mRNAs previously shown to be highly Ded1-dependent by ribosome profiling of ded1 mutants in vivo, demonstrating that the core translation functions of the factor are recapitulated in the purified system. Our data do not support a model in which Ded1acts by reducing initiation at alternative start codons in 5′UTRs and instead indicate it functions by directly promoting mRNA recruitment to the 43S PIC and scanning to locate the main start codon. We also provide evidence that eIF4A, another essential DEAD-box initiation factor, is required for efficient PIC assembly on almost all mRNAs, regardless of their structural complexity, in contrast to the preferential stimulation by Ded1 of initiation on mRNAs with long, structured 5′UTRs. translation protein synthesis initiation ribosome profiling yeast in vitro Medicine R Science Q Biology (General) Julie M Bocetti verfasserin aut Meizhen Hou verfasserin aut Daoming Qin verfasserin aut Alan G Hinnebusch verfasserin aut Jon R Lorsch verfasserin aut In eLife eLife Sciences Publications Ltd, 2013 13(2024) (DE-627)728518384 (DE-600)2687154-3 2050084X nnns volume:13 year:2024 https://doi.org/10.7554/eLife.93255 kostenfrei https://doaj.org/article/379dbd1d0a6640ed96c6e0b8d84b47d5 kostenfrei https://elifesciences.org/articles/93255 kostenfrei https://doaj.org/toc/2050-084X Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_171 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4335 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 13 2024
spelling	10.7554/eLife.93255 doi (DE-627)DOAJ097361585 (DE-599)DOAJ379dbd1d0a6640ed96c6e0b8d84b47d5 DE-627 ger DE-627 rakwb eng QH301-705.5 Fujun Zhou verfasserin aut Transcriptome-wide analysis of the function of Ded1 in translation preinitiation complex assembly in a reconstituted in vitro system 2024 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier We have developed a deep sequencing-based approach, Rec-Seq, that allows simultaneous monitoring of ribosomal 48S preinitiation complex (PIC) formation on every mRNA in the translatome in an in vitro reconstituted system. Rec-Seq isolates key early steps in translation initiation in the absence of all other cellular components and processes. Using this approach, we show that the DEAD-box ATPase Ded1 promotes 48S PIC formation on the start codons of >1000 native mRNAs, most of which have long, structured 5′-untranslated regions (5′UTRs). Remarkably, initiation measured in Rec-Seq was enhanced by Ded1 for most mRNAs previously shown to be highly Ded1-dependent by ribosome profiling of ded1 mutants in vivo, demonstrating that the core translation functions of the factor are recapitulated in the purified system. Our data do not support a model in which Ded1acts by reducing initiation at alternative start codons in 5′UTRs and instead indicate it functions by directly promoting mRNA recruitment to the 43S PIC and scanning to locate the main start codon. We also provide evidence that eIF4A, another essential DEAD-box initiation factor, is required for efficient PIC assembly on almost all mRNAs, regardless of their structural complexity, in contrast to the preferential stimulation by Ded1 of initiation on mRNAs with long, structured 5′UTRs. translation protein synthesis initiation ribosome profiling yeast in vitro Medicine R Science Q Biology (General) Julie M Bocetti verfasserin aut Meizhen Hou verfasserin aut Daoming Qin verfasserin aut Alan G Hinnebusch verfasserin aut Jon R Lorsch verfasserin aut In eLife eLife Sciences Publications Ltd, 2013 13(2024) (DE-627)728518384 (DE-600)2687154-3 2050084X nnns volume:13 year:2024 https://doi.org/10.7554/eLife.93255 kostenfrei https://doaj.org/article/379dbd1d0a6640ed96c6e0b8d84b47d5 kostenfrei https://elifesciences.org/articles/93255 kostenfrei https://doaj.org/toc/2050-084X Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_171 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4335 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 13 2024
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allfieldsGer	10.7554/eLife.93255 doi (DE-627)DOAJ097361585 (DE-599)DOAJ379dbd1d0a6640ed96c6e0b8d84b47d5 DE-627 ger DE-627 rakwb eng QH301-705.5 Fujun Zhou verfasserin aut Transcriptome-wide analysis of the function of Ded1 in translation preinitiation complex assembly in a reconstituted in vitro system 2024 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier We have developed a deep sequencing-based approach, Rec-Seq, that allows simultaneous monitoring of ribosomal 48S preinitiation complex (PIC) formation on every mRNA in the translatome in an in vitro reconstituted system. Rec-Seq isolates key early steps in translation initiation in the absence of all other cellular components and processes. Using this approach, we show that the DEAD-box ATPase Ded1 promotes 48S PIC formation on the start codons of >1000 native mRNAs, most of which have long, structured 5′-untranslated regions (5′UTRs). Remarkably, initiation measured in Rec-Seq was enhanced by Ded1 for most mRNAs previously shown to be highly Ded1-dependent by ribosome profiling of ded1 mutants in vivo, demonstrating that the core translation functions of the factor are recapitulated in the purified system. Our data do not support a model in which Ded1acts by reducing initiation at alternative start codons in 5′UTRs and instead indicate it functions by directly promoting mRNA recruitment to the 43S PIC and scanning to locate the main start codon. We also provide evidence that eIF4A, another essential DEAD-box initiation factor, is required for efficient PIC assembly on almost all mRNAs, regardless of their structural complexity, in contrast to the preferential stimulation by Ded1 of initiation on mRNAs with long, structured 5′UTRs. translation protein synthesis initiation ribosome profiling yeast in vitro Medicine R Science Q Biology (General) Julie M Bocetti verfasserin aut Meizhen Hou verfasserin aut Daoming Qin verfasserin aut Alan G Hinnebusch verfasserin aut Jon R Lorsch verfasserin aut In eLife eLife Sciences Publications Ltd, 2013 13(2024) (DE-627)728518384 (DE-600)2687154-3 2050084X nnns volume:13 year:2024 https://doi.org/10.7554/eLife.93255 kostenfrei https://doaj.org/article/379dbd1d0a6640ed96c6e0b8d84b47d5 kostenfrei https://elifesciences.org/articles/93255 kostenfrei https://doaj.org/toc/2050-084X Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_171 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4335 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 13 2024
allfieldsSound	10.7554/eLife.93255 doi (DE-627)DOAJ097361585 (DE-599)DOAJ379dbd1d0a6640ed96c6e0b8d84b47d5 DE-627 ger DE-627 rakwb eng QH301-705.5 Fujun Zhou verfasserin aut Transcriptome-wide analysis of the function of Ded1 in translation preinitiation complex assembly in a reconstituted in vitro system 2024 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier We have developed a deep sequencing-based approach, Rec-Seq, that allows simultaneous monitoring of ribosomal 48S preinitiation complex (PIC) formation on every mRNA in the translatome in an in vitro reconstituted system. Rec-Seq isolates key early steps in translation initiation in the absence of all other cellular components and processes. Using this approach, we show that the DEAD-box ATPase Ded1 promotes 48S PIC formation on the start codons of >1000 native mRNAs, most of which have long, structured 5′-untranslated regions (5′UTRs). Remarkably, initiation measured in Rec-Seq was enhanced by Ded1 for most mRNAs previously shown to be highly Ded1-dependent by ribosome profiling of ded1 mutants in vivo, demonstrating that the core translation functions of the factor are recapitulated in the purified system. Our data do not support a model in which Ded1acts by reducing initiation at alternative start codons in 5′UTRs and instead indicate it functions by directly promoting mRNA recruitment to the 43S PIC and scanning to locate the main start codon. We also provide evidence that eIF4A, another essential DEAD-box initiation factor, is required for efficient PIC assembly on almost all mRNAs, regardless of their structural complexity, in contrast to the preferential stimulation by Ded1 of initiation on mRNAs with long, structured 5′UTRs. translation protein synthesis initiation ribosome profiling yeast in vitro Medicine R Science Q Biology (General) Julie M Bocetti verfasserin aut Meizhen Hou verfasserin aut Daoming Qin verfasserin aut Alan G Hinnebusch verfasserin aut Jon R Lorsch verfasserin aut In eLife eLife Sciences Publications Ltd, 2013 13(2024) (DE-627)728518384 (DE-600)2687154-3 2050084X nnns volume:13 year:2024 https://doi.org/10.7554/eLife.93255 kostenfrei https://doaj.org/article/379dbd1d0a6640ed96c6e0b8d84b47d5 kostenfrei https://elifesciences.org/articles/93255 kostenfrei https://doaj.org/toc/2050-084X Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_171 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4335 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 13 2024
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callnumber-first	Q - Science
author	Fujun Zhou
spellingShingle	Fujun Zhou misc QH301-705.5 misc translation misc protein synthesis misc initiation misc ribosome profiling misc yeast misc in vitro misc Medicine misc R misc Science misc Q misc Biology (General) Transcriptome-wide analysis of the function of Ded1 in translation preinitiation complex assembly in a reconstituted in vitro system
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topic_title	QH301-705.5 Transcriptome-wide analysis of the function of Ded1 in translation preinitiation complex assembly in a reconstituted in vitro system translation protein synthesis initiation ribosome profiling yeast in vitro
topic	misc QH301-705.5 misc translation misc protein synthesis misc initiation misc ribosome profiling misc yeast misc in vitro misc Medicine misc R misc Science misc Q misc Biology (General)
topic_unstemmed	misc QH301-705.5 misc translation misc protein synthesis misc initiation misc ribosome profiling misc yeast misc in vitro misc Medicine misc R misc Science misc Q misc Biology (General)
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title	Transcriptome-wide analysis of the function of Ded1 in translation preinitiation complex assembly in a reconstituted in vitro system
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title_full	Transcriptome-wide analysis of the function of Ded1 in translation preinitiation complex assembly in a reconstituted in vitro system
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title_sort	transcriptome-wide analysis of the function of ded1 in translation preinitiation complex assembly in a reconstituted in vitro system
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title_auth	Transcriptome-wide analysis of the function of Ded1 in translation preinitiation complex assembly in a reconstituted in vitro system
abstract	We have developed a deep sequencing-based approach, Rec-Seq, that allows simultaneous monitoring of ribosomal 48S preinitiation complex (PIC) formation on every mRNA in the translatome in an in vitro reconstituted system. Rec-Seq isolates key early steps in translation initiation in the absence of all other cellular components and processes. Using this approach, we show that the DEAD-box ATPase Ded1 promotes 48S PIC formation on the start codons of >1000 native mRNAs, most of which have long, structured 5′-untranslated regions (5′UTRs). Remarkably, initiation measured in Rec-Seq was enhanced by Ded1 for most mRNAs previously shown to be highly Ded1-dependent by ribosome profiling of ded1 mutants in vivo, demonstrating that the core translation functions of the factor are recapitulated in the purified system. Our data do not support a model in which Ded1acts by reducing initiation at alternative start codons in 5′UTRs and instead indicate it functions by directly promoting mRNA recruitment to the 43S PIC and scanning to locate the main start codon. We also provide evidence that eIF4A, another essential DEAD-box initiation factor, is required for efficient PIC assembly on almost all mRNAs, regardless of their structural complexity, in contrast to the preferential stimulation by Ded1 of initiation on mRNAs with long, structured 5′UTRs.
abstractGer	We have developed a deep sequencing-based approach, Rec-Seq, that allows simultaneous monitoring of ribosomal 48S preinitiation complex (PIC) formation on every mRNA in the translatome in an in vitro reconstituted system. Rec-Seq isolates key early steps in translation initiation in the absence of all other cellular components and processes. Using this approach, we show that the DEAD-box ATPase Ded1 promotes 48S PIC formation on the start codons of >1000 native mRNAs, most of which have long, structured 5′-untranslated regions (5′UTRs). Remarkably, initiation measured in Rec-Seq was enhanced by Ded1 for most mRNAs previously shown to be highly Ded1-dependent by ribosome profiling of ded1 mutants in vivo, demonstrating that the core translation functions of the factor are recapitulated in the purified system. Our data do not support a model in which Ded1acts by reducing initiation at alternative start codons in 5′UTRs and instead indicate it functions by directly promoting mRNA recruitment to the 43S PIC and scanning to locate the main start codon. We also provide evidence that eIF4A, another essential DEAD-box initiation factor, is required for efficient PIC assembly on almost all mRNAs, regardless of their structural complexity, in contrast to the preferential stimulation by Ded1 of initiation on mRNAs with long, structured 5′UTRs.
abstract_unstemmed	We have developed a deep sequencing-based approach, Rec-Seq, that allows simultaneous monitoring of ribosomal 48S preinitiation complex (PIC) formation on every mRNA in the translatome in an in vitro reconstituted system. Rec-Seq isolates key early steps in translation initiation in the absence of all other cellular components and processes. Using this approach, we show that the DEAD-box ATPase Ded1 promotes 48S PIC formation on the start codons of >1000 native mRNAs, most of which have long, structured 5′-untranslated regions (5′UTRs). Remarkably, initiation measured in Rec-Seq was enhanced by Ded1 for most mRNAs previously shown to be highly Ded1-dependent by ribosome profiling of ded1 mutants in vivo, demonstrating that the core translation functions of the factor are recapitulated in the purified system. Our data do not support a model in which Ded1acts by reducing initiation at alternative start codons in 5′UTRs and instead indicate it functions by directly promoting mRNA recruitment to the 43S PIC and scanning to locate the main start codon. We also provide evidence that eIF4A, another essential DEAD-box initiation factor, is required for efficient PIC assembly on almost all mRNAs, regardless of their structural complexity, in contrast to the preferential stimulation by Ded1 of initiation on mRNAs with long, structured 5′UTRs.
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title_short	Transcriptome-wide analysis of the function of Ded1 in translation preinitiation complex assembly in a reconstituted in vitro system
url	https://doi.org/10.7554/eLife.93255 https://doaj.org/article/379dbd1d0a6640ed96c6e0b8d84b47d5 https://elifesciences.org/articles/93255 https://doaj.org/toc/2050-084X
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Transcriptome-wide analysis of the function of Ded1 in translation preinitiation complex assembly in a reconstituted in vitro system

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