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Translatome and transcriptome analysis of TMA20 (MCT-1) and TMA64 (eIF2D) knockout yeast strains

ABSTRACT: We report ribosome profiling and RNA-Seq data of S.cerevisiae strains with TMA20, TMA64 single- and TMA20/TMA64 double-gene knockouts, grown in the rich YPD or minimal SD medium.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE122039 | GEO | 2018/11/01

REPOSITORIES: GEO

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Translatome and transcriptome analysis of TMA20 (MCT-1) and TMA64 (eIF2D) knockout yeast strains

Project description:Translatome and transcriptome analysis of TMA20 (MCT-1) and TMA64 (eIF2D) knockout yeast strains

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Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR) recycle post-termination 40S subunits in vivo

Project description:The recycling of ribosomal subunits after translation termination is critical for efficient gene expression. Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR) function as 40S recycling factors in vitro, but it is unknown whether they perform this function in vivo or serve as alternative initiation factors. Ribosome profiling of strains missing these factors revealed 80S ribosomes queued behind the stop codon, consistent with a block in 40S recycling. We found that unrecycled ribosomes could reinitiate translation at AUG codons in the 3’UTR, as evidenced by peaks in the footprint data and 3’UTR reporter analysis. In vitro translation experiments using reporter mRNAs containing upstream ORFs (uORFs) further established that reinitiation increased in the absence of these proteins. In some cases, 40S ribosomes appeared to rejoin with 60S subunits and undergo an alternative 80S reinitiation process in 3’UTRs. These results support a crucial role for Tma64, Tma20, and Tma22 in the recycling of 40S ribosomal subunits at stop codons and translation reinitiation.

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40S ribosome profiling reveals distinct roles for Tma20/Tma22 (MCT-1/DENR) and Tma64 (eIF2D) in 40S subunit recycling

Project description:The recycling of ribosomes at stop codons for use in further rounds of translation is critical for efficient protein synthesis. Removal of the 60S subunit is catalyzed by the ATPase Rli1 (ABCE1) while removal of the 40S is thought to require Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR). However, it remains unclear how these Tma proteins cause 40S removal and control reinitiation of downstream translation. Here we developed an enhanced 40S ribosome footprinting strategy to directly observe intermediate steps of ribosome recycling in cells. Deletion of the genes encoding these Tma proteins resulted in broad accumulation of unrecycled 40S subunits at stop codons, directly establishing their role in 40S recycling. Furthermore, the Tma20/Tma22 heterodimer was responsible for a majority of 40S recycling events while Tma64 played a minor role. Introduction of an autism-associated mutation into TMA22 resulted in a loss of 40S recycling activity, linking ribosome recycling and neurological disease.

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Translatome and transcriptome analysis of TMA20 (MCT-1) and TMA64 (eIF2D) knockout yeast strains.

Project description:TMA20 (MCT-1), TMA22 (DENR) and TMA64 (eIF2D) are eukaryotic translation factors involved in ribosome recycling and re-initiation. They operate with P-site bound tRNA in post-termination or (re-)initiation translation complexes, thus participating in the removal of 40S ribosomal subunit from mRNA stop codons after termination and controlling translation re-initiation on mRNAs with upstream open reading frames (uORFs), as well as de novo initiation on some specific mRNAs. Here we report ribosomal profiling data of S.cerevisiae strains with individual deletions of TMA20, TMA64 or both TMA20 and TMA64 genes. We provide RNA-Seq and Ribo-Seq data from yeast strains grown in the rich YPD or minimal SD medium. We illustrate our data by plotting differential distribution of ribosomal-bound mRNA fragments throughout uORFs in 5'-untranslated region (5' UTR) of GCN4 mRNA and on mRNA transcripts encoded in MAT locus in the mutant and wild-type strains, thus providing a basis for investigation of the role of these factors in the stress response, mating and sporulation. We also document a shift of transcription start site of the APC4 gene which occurs when the neighboring TMA64 gene is replaced by the standard G418-resistance cassette used for the creation of the Yeast Deletion Library. This shift results in dramatic deregulation of the APC4 gene expression, as revealed by our Ribo-Seq data, which can be probably used to explain strong genetic interactions of TMA64 with genes involved in the cell cycle and mitotic checkpoints. Raw RNA-Seq and Ribo-Seq data as well as all gene counts are available in NCBI Gene Expression Omnibus (GEO) repository under GEO accession GSE122039 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE122039).

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40S ribosome profiling reveals distinct roles for Tma20/Tma22 (MCT-1/DENR) and Tma64 (eIF2D) in 40S subunit recycling

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