Biology subjects

Lorsch, J. R.

Publications and source records attributed to Lorsch, J. R..

5 recordsLinked to original sources

bioRxiv2023

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 pre-initiation 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 (5UTRs). 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 5UTRs 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 5UTRs.

biochemistry↗

bioRxiv2019

Temperature-Dependent Regulation of Upstream Open Reading Frame Translation in S. Cerevisiae

BackgroundTranslation of an mRNA in eukaryotes starts at AUG in most cases. Near-cognate codons (NCCs) such as UUG, ACG and AUU are also used as start sites at low levels in S. cerevisiae. Initiation from NCCs or AUGs in the 5-untranslated regions (UTRs) of mRNAs can lead to translation of upstream open reading frames (uORFs) that might regulate expression of the main ORF (mORF). Although there is some circumstantial evidence that the translation of uORFs can be affected by environmental conditions, little is known about how it is affected by changes in growth temperature.\n\nResultsUsing reporter assays, we found that changes in growth temperature can affect translation from NCC start sites in yeast cells, suggesting the possibility that gene expression could be regulated by temperature by altering use of different uORF start codons. Using ribosome profiling, we provide evidence that growth temperature regulates the efficiency of translation of nearly 200 uORFs in S. cerevisiae. Of these uORFs, most that start with an AUG codon have increased translational efficiency at 37 {degrees}C relative to 30 {degrees}C and decreased efficiency at 20 {degrees}C. For translationally regulated uORFs starting with NCCs, we did not observe a general trend for the direction of regulation as a function of temperature, suggesting mRNA-specific features can determine the mode of temperature-dependent regulation. Consistent with this conclusion, the position of the uORFs in the 5-leader relative to the 5-cap and the start codon of the main ORF correlates with the direction of temperature-dependent regulation of uORF translation. We have identified several novel cases in which changes in uORF translation are inversely correlated with changes in the translational efficiency of the downstream main ORF. Our data suggest that translation of these mRNAs is subject to temperature-dependent, uORF-mediated regulation.\n\nConclusionsOverall, our data suggest that alterations in the translation of specific uORFs by temperature can regulate gene expression in S. cerevisiae.

genomics↗

bioRxiv2018

Translational initiation factor eIF5 replaces eIF1 on the 40S ribosomal subunit to promote start-codon recognition

In eukaryotic translation initiation AUG recognition of the mRNA requires accommodation of Met-tRNAi in a \"PIN\" state, which is antagonized by the factor eIF1. eIF5 is a GTPase activating protein (GAP) of eIF2 that additionally promotes stringent AUG selection, but the molecular basis of its dual function was unknown. We present a cryo-electron microscopy (cryo-EM) reconstruction of a 48S pre-initiation complex (PIC), at an overall resolution of 3.0 [A], featuring the N-terminal domain (NTD) of eIF5 bound to the 40S subunit at the location vacated by eIF1. eIF5 interacts with and allows a more accommodated orientation of Met-tRNAi. Substitutions of eIF5 residues involved in the eIF5-NTD/tRNAi interaction influenced initiation at near-cognate UUG codons in vivo, and the closed/open PIC conformation in vitro, consistent with direct stabilization of the codon:anticodon duplex by the wild-type eIF5-NTD. The present structure reveals the basis for a key role of eIF5 in start-codon selection.

biophysics↗

bioRxiv2018

Yeast Ded1 promotes 48S translation pre-initiation complex assembly in an mRNA-specific and eIF4F-dependent manner

DEAD-box RNA helicase Dedl is thought to resolve secondary structures in mRNA 5'-untranslated regions (5'-UTRs) that impede 48S preinitiation complex (PIC) formation at the initiation codon. We reconstituted Ded1 acceleration of 48S PIC assembly on native mRNAs in a pure system, and recapitulated increased Ded1-dependence of mRNAs that are Ded1-hyperdependent in vivo. Stem-loop (SL) structures in 5'-UTRs of native and synthetic mRNAs increased the Ded1 requirement to overcome their intrinsically low rates of 48S PIC recruitment. Ded1 acceleration of 48S assembly was greater in the presence of eIF4F, and domains mediating one or more Ded1 interactions with eIF4G or helicase eIF4A were required for efficient recruitment of all mRNAs; however, the relative importance of particular Ded1 and eIF4G domains were distinct for each mRNA. Our results account for the Ded1 hyper-dependence of mRNAs with structure-prone 5'-UTRs, and implicate an eIF4E{middle dot}eIF4G{middle dot}eIF4A{middle dot}Ded1 complex in accelerating 48S PIC assembly on native mRNAs.

biochemistry↗

bioRxiv2017

eIF4A is stimulated by the pre-initiation complex and enhances recruitment of mRNAs regardless of structural complexity

eIF4A is a DEAD-box RNA-dependent ATPase thought to unwind RNA secondary structure in the 5'-untranslated regions (UTRs) of mRNAs to promote their recruitment to the eukaryotic translation pre-initiation complex (PIC). We show that eIF4As ATPase activity is markedly stimulated in the presence of the PIC, independently of eIF4E*eIF4G, but dependent on subunits i and g of the heteromeric eIF3 complex. Surprisingly, eIF4A accelerated the rate of recruitment of all mRNAs tested, regardless of their degree of structural complexity. Structures in the 5'-UTR and 3' of the start codon synergistically inhibit mRNA recruitment in a manner relieved by eIF4A, indicating that the factor does not act solely to melt hairpins in 5'-UTRs. Our findings that eIF4A functionally interacts with the PIC and plays important roles beyond unwinding 5-UTR structure is consistent with a recent proposal that eIF4A modulates the conformation of the 40S ribosomal subunit to promote mRNA recruitment.

biophysics↗