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Pol5 is required for recycling of small subunit biogenesis factors and for formation of the polypeptide exit tunnel of the large ribosomal subunit

ABSTRACT: Pol5 is required for recycling of small subunit biogenesis factors and for formation of the polypeptide exit tunnel of the large ribosomal subunit

PROVIDER: PRJNA549607 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR9326371.fastq.gz	Fastqsanger.gz
	SRR9326372.fastq.gz	Fastqsanger.gz

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Similar Datasets

Pol5 is required for recycling of small subunit biogenesis factors and for formation of the polypeptide exit tunnel of the large ribosomal subunit

Project description:More than 200 assembly factors (AFs) are required for the production of ribosomes in yeast. The stepwise association and dissociation of these AFs with the pre-ribosomal subunits occurs in a strictly hierarchical manner to ensure correct maturation of the pre-rRNAs and assembly of the ribosomal proteins. Although decades of research have provided a wealth of insights into the functions of many AFs, others remain poorly characterized. Pol5 was initially classified with B-type DNA polymerases, however, several lines of evidence indicate the involvement of this protein in ribosome assembly. Here, we show that depletion of Pol5 affects the processing of pre-rRNAs destined for the both the large and small subunits. Furthermore, we identify binding sites for Pol5 in the 5’ external transcribed spacer and within domain III of the 25S rRNA sequence. Consistent with this, we reveal that Pol5 is required for recruitment of ribosomal proteins that form the polypeptide exit tunnel in the LSU and that depletion of Pol5 impairs the release of 5’ ETS fragments from early pre-40S particles. The dual functions of Pol5 in 60S assembly and recycling of pre-40S AFs suggest that this factor could contribute to ensuring the stoichiometric production of ribosomal subunits.

2019-11-05 | GSE132973 | GEO

Nascent polypeptide within the exit tunnel stabilizes the ribosome to counteract risky translation

Project description:Continuous translation elongation, irrespective of amino acid sequences, is a prerequisite for living organisms to produce their proteomes. However, the risk of elongation abortion is concealed within nascent polypeptide products. For example, negatively charged sequences with occasional intermittent prolines, termed intrinsic ribosome destabilization (IRD) sequences, destabilizes the translating ribosomal complex. Thus, some nascent chain sequences lead to premature translation cessation. Here, we show that most potential IRD sequences in the middle of open reading frames remain cryptic by two mechanisms: the nascent polypeptide itself that spans the exit tunnel and its bulky amino acid residues that occupy the tunnel entrance region. Thus, nascent polypeptide products have a built-in ability to ensure elongation continuity by serving as a bridge and thus by protecting the large and small ribosomal subunits from dissociation.

2021-12-08 | GSE184331 | GEO

Nascent polypeptide within the exit tunnel stabilizes the ribosome to counteract risky translation

Project description:Nascent polypeptide within the exit tunnel stabilizes the ribosome to counteract risky translation

| PRJNA764085 | ENA

The Ribosome Assembly Factor Reh1 is Released from the Polypeptide Exit Tunnel in the Pioneer Round of Translation

Project description:The Ribosome Assembly Factor Reh1 is Released from the Polypeptide Exit Tunnel in the Pioneer Round of Translation

| PRJNA1031690 | ENA

Cysteine oxidation and disulfide formation in the ribosomal exit tunnel

Project description:Understanding the conformational sampling of translation-arrested ribosome nascent chain complexes is key to understand co-translational folding. Up to now, coupling of cysteine oxidation, disulfide bond formation and structure formation in nascent chains has remained elusive. Here, we investigate the eye-lens protein γB-crystallin in the ribosomal exit tunnel. Using mass spectrometry, theoretical simulations, dynamic nuclear polarization-enhanced solid-state nuclear magnetic resonance and cryo-electron microscopy, we show that thiol groups of cysteine residues undergo S-glutathionylation and S-nitrosylation and form non-native disulfide bonds. Thus, covalent modification chemistry occurs already prior to nascent chain release as the ribosome exit tunnel provides sufficient space even for disulfide bond formation which can guide protein folding.

2020-11-09 | PXD021574 | Pride

Hcr1/eIF3j is a 60S ribosomal subunit recycling accessory factor in vivo

Project description:Hcr1/eIF3j is a sub-stoichiometric subunit of eukaryotic initiation factor 3 (eIF3) that can dissociate the post-termination 40S ribosomal subunit from mRNA in vitro. We examined this ribosome recycling role in vivo by ribosome profiling and reporter assays and found that loss of Hcr1 led to reinitiation of translation in 3’UTRs, consistent with a defect in recycling. However, the defect appeared to be in recycling of the 60S subunit, rather than the 40S subunit, because reinitiation did not require an AUG codon and was suppressed by overexpression of the 60S dissociation factor Rli1/ABCE1. Consistent with a 60S recycling role, overexpression of Hcr1 could not compensate for loss of 40S recycling factors Tma64/eIF2D and Tma20/MCT-1. Intriguingly, loss of Hcr1 triggered higher expression of RLI1 via an apparent feedback loop. These findings suggest Hcr1/eIF3j is recruited to ribosomes at stop codons and may coordinate the transition to a new round of translation.

2019-05-23 | GSE124204 | GEO

Protein synthesis in the developing neocortex at near-atomic resolution reveals Ebp1-mediated neuronal proteostasis at the 60S tunnel exit

Project description:Proteostasis must be finely tuned in the nervous system, as it represents an essential feature of neurodevelopment, and dominant pathology in neurodegenerative disease. At the gateway of proteostasis is the ribosome; however, the architecture of ribosomal complexes in the developing mammalian brain has not been visualized. In this study, we analyze the molecular architecture of ex vivo embryonic and perinatal mouse neocortical ribosomes, revealing Ebp1 as a 60S peptide tunnel exit binding factor at near-atomic resolution by multiparticle cryo-electron microscopy. The impact of Ebp1 on the neuronal proteome was analyzed by pSILAC and BONCAT coupled mass spectrometry, implicating Ebp1 in neurite outgrowth proteostasis, with in vivo embryonic Ebp1 knockdown resulting in neurite outgrowth dysregulation. This study constitutes the first near-atomic resolution structure of actively translating ribosomes ex vivo from the nervous system, and visualizes Ebp1 as a chaperone of protein synthesis at the 60S peptide tunnel exit in neocortical neurogenesis.

2021-01-04 | PXD014841 | Pride

Protein synthesis in the developing neocortex at near-atomic resolution reveals Ebp1-mediated neuronal proteostasis at the 60S tunnel exit

2021-01-04 | PXD014740 | Pride

Ribosome profiling reveals sequence-independent post-initiation pausing as a signature of translation

Project description:The journey of a newly synthesized polypeptide starts in the peptidyltransferase center of the ribosome, from where it traverses the exit tunnel. The interior of the ribosome exit tunnel is neither straight nor smooth. How the ribosome dynamics in vivo is influenced by the exit tunnel is poorly understood. Genome-wide ribosome profiling in mammalian cells reveals elevated ribosome density at the start codon and surprisingly the downstream 5th codon position as well. We found that the highly focused ribosomal pausing shortly after initiation is attributed to the geometry of the exit tunnel, as deletion of the loop region from ribosome protein L4 diminishes translational pausing at the 5th codon position. Unexpectedly, the ribosome variant undergoes translational abandonment shortly after initiation, suggesting that there exists an obligatory step between initiation and elongation commitment. We propose that the post-initiation pausing of ribosomes represents an inherent signature of the translation machinery to ensure productive translation.

2021-02-17 | GSE166874 | GEO

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.

2021-03-01 | GSE145904 | GEO

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