scholarly journals Quality control of 40S ribosome head assembly ensures scanning competence

2020 ◽  
Vol 219 (11) ◽  
Author(s):  
Haina Huang ◽  
Homa Ghalei ◽  
Katrin Karbstein
Keyword(s):  
Quality Control ◽  
Conformational Changes ◽  
Translation Initiation ◽  
Site Selection ◽  
Start Codon ◽  
Start Site ◽  
Genetic Analyses ◽  
Head Assembly ◽  
Control Step ◽  
Assembly Intermediate

During translation initiation, 40S ribosomes scan the mRNA until they encounter the start codon, where conformational changes produce a translation-competent 80S complex. Destabilizing the scanning complex results in misinitiation at non-AUG codons, demonstrating its importance for fidelity. Here, we use a combination of biochemical and genetic analyses to demonstrate that the ability of the nascent subunit to adopt the scanning complex is tested during assembly via structural mimicry. Specifically, formation of the 80S-like assembly intermediate, which structurally resembles scanning complexes, requires the correct folding of two rRNA elements in the subunit head and the proper positioning of the universally conserved head proteins Rps3, Rps15, Rps20, and Rps29. rRNA misfolding impairs the formation of 80S-like ribosomes, and bypass of individual checkpoints using cancer-associated mutations produces ribosomes defective in accurate start-site selection. Thus, the formation of 80S-like assembly intermediates is a quality control step that ensures scanning competence of the nascent subunit.

scholarly journals Alternative translation initiation generates a functionally distinct isoform of the stress-activated kinase MK2

2018 ◽  
Author(s):  
Philipp Trulley ◽  
Goda Snieckute ◽  
Dorte Bekker-Jensen ◽  
Manoj B. Menon ◽  
Robert Freund ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Translation Initiation ◽  
Specific Binding ◽  
Protein Isoforms ◽  
Early Gene ◽  
Start Codon ◽  
Start Site ◽  
Alternative Translation ◽  
Transcriptional Gene Regulation ◽  
Translation Initiation Start

AbstractShaping of the proteome by alternative translation is an important mechanism of post-transcriptional gene regulation. It can lead to the expression of multiple protein isoforms originating from the same mRNA. Here we show that a novel, abundant and long isoform of the stress/p38MAPK-activated kinase MK2, a key regulator of transcription, migration, death signaling and post-transcriptional gene regulation, is constitutively translated from an alternative CUG translation initiation start site located in the 5′UTR of its mRNA. GC-rich sequences and putative G-quadruplex structures influence the usage of that codon as a translation initiation start site and the RNA helicase eIF4A1 is needed to ensure alternative isoform translation. We recapitulated the usage of the alternative start codon and determined the molecular properties of the short and a long MK2 isoforms. Phenotypically, only the short isoform phosphorylated Hsp27, supported migration and stress-induced immediate early gene (IEG) expression. Interaction profiling by quantitative mass-spectrometry revealed short isoform-specific binding partners that were associated with migration. In contrast, the long isoform contains additional putative phosphorylation sites in its unique N-terminus. In sum, our data reveal a longer and previously non-described isoform of MK2 with distinct physiological properties originating from alternative translation.

scholarly journals iRQC, a surveillance pathway for 40S ribosomal quality control during mRNA translation initiation

2021 ◽  
Author(s):  
Danielle Marie Garshott ◽  
Heeseon An ◽  
Elayanambi Sundaramoorthy ◽  
Marilyn Leonard ◽  
Alison Vicary ◽  
...  
Keyword(s):  
Quality Control ◽  
Stress Response ◽  
Ribosomal Protein ◽  
Protein Degradation ◽  
Ubiquitin Ligase ◽  
Translation Initiation ◽  
Mrna Translation ◽  
Start Codon ◽  
Independent Manner ◽  
Translation Elongation

Since multiple ribosomes can engage a single mRNA, nonuniform ribosome progression can result in collisions. Ribosome collisions during translation elongation elicit a multifaceted ribosome-associated quality control (RQC) response. Despite advanced mechanistic understanding of translation initiation, a parallel RQC pathway that acts on collided preinitiation complexes has not been described. Here, we show that blocking progression of scanning or elongating ribosomes past the start codon triggers uS3 and uS5 ribosomal ubiquitylation. We demonstrate that conditions that activate the integrated stress response can also induce preinitiation complex collisions. The ubiquitin ligase, RNF10, and the deubiquitylating enzyme, USP10, are the key regulators of uS3 and uS5 ubiquitylation. Prolonged uS3 and uS5 ubiquitylation results in 40S, but not 60S, ribosomal protein degradation in an autophagy-independent manner. This study identifies a distinct arm in the RQC pathway, initiation RQC (iRQC), that acts on pervasive ribosome collisions during translation initiation to modulate translation activity and capacity.

scholarly journals Borf: Improved ORF prediction in de-novo assembled transcriptome annotation

2021 ◽  
Author(s):  
Beth Signal ◽  
Tim Kahlke
Keyword(s):  
Site Selection ◽  
False Positive ◽  
De Novo ◽  
Start Codon ◽  
Antisense Strand ◽  
Upstream Sequence ◽  
Start Site ◽  
Rna Seq ◽  
Sense Strand ◽  
The Impact

ABSTRACTORF prediction in de-novo assembled transcriptomes is a critical step for RNA-Seq analysis and transcriptome annotation. However, current approaches do not appropriately account for factors such as strand-specificity and incompletely assembled transcripts. Strand-specific RNA-Seq libraries should produce assembled transcripts in the correct orientation, and therefore ORFs should only be annotated on the sense strand. Additionally, start site selection is more complex than appreciated as sequences upstream of the first start codon need to be correctly annotated as 5’ UTR in completely assembled transcripts, or part of the main ORF in incomplete transcripts. Both of these factors influence the accurate annotation of ORFs and therefore the transcriptome as a whole. We generated four de-novo transcriptome assemblies of well annotated species as a gold-standard dataset to test the impact strand specificity and start site selection have on ORF prediction in real data. Our results show that prediction of ORFs on the antisense strand in data from stranded RNA libraries results in false-positive ORFs with no or very low similarity to known proteins. In addition, we found that up to 23% of assembled transcripts had no stop codon upstream and in-frame of the first start codon, instead comprising a sequence of upstream codons. We found the optimal length cutoff of these upstream sequences to accurately classify these transcripts as either complete (upstream sequence is 5’ UTR) or 5’ incomplete (transcript is incompletely assembled and upstream sequence is part of the ORF). Here, we present Borf, the better ORF finder, specifically designed to minimise false-positive ORF prediction in stranded RNA-Seq data and improve annotation of ORF start-site prediction accuracy. Borf is written in Python3 and freely available at https://github.com/betsig/borf.

scholarly journals Evidence for involvement of trans-acting factors in selection of the AUG start codon during eukaryotic translational initiation.

1996 ◽  
Vol 16 (7) ◽  
pp. 3523-3534 ◽  
Author(s):  
S McBratney ◽  
P Sarnow
Keyword(s):  
Site Selection ◽  
Uv Light ◽  
Consensus Sequence ◽  
Structural Features ◽  
Start Codon ◽  
Translation System ◽  
Start Site ◽  
Translational Initiation ◽  
Rna Molecules ◽  
Reporter Mrna

The molecular mechanism with which an appropriate AUG codon is selected as the start site for translational initiation by eukaryotic ribosomes is not known. By using a cell-free translation system, small RNA molecules containing single AUG codons, surrounded by various nucleotide sequences, were tested for their abilities to interfere with the translation of a reporter mRNA. RNAs containing the AUG in an ACCAUGG context (Kozak consensus sequence) were able to inhibit translation of the reporter mRNA. In contrast, RNAs containing the AUG in a less favorable context for start site selection (for example, CAGAUGG) had no effect on the translation of the reporter mRNA. The effect mediated by the ACCAUGC-containing RNAs was not due to sequestration of ribosomal subunits or to particular structural features in these RNAs. To identify potential trans-acting factors that might be preferentially bound by ACCAUGG-containing RNAs, ACCAUGG- and CAGAUGC-containing RNAs with a single 4-thiouridine residue at the AUG were incubated with partially fractionated extracts, and AUG-binding proteins were identified after irradiation of the complexes with UV light and subsequent analysis by gel electrophoresis. The analysis (of such complexes in competition experiments revealed that proteins, approximately 50 and 100 kDa in size, were found to bind directly at the AUG codon embedded in the ACCAUGG motif. One of these proteins has been identified as the La autoantigen. These findings indicate that trans-acting factors may play a role in AUG start site selection during translational initiation.

Genetic selection for mutations that reduce or abolish ribosomal recognition of the HIS4 translational initiator region

1988 ◽  
Vol 8 (7) ◽  
pp. 2955-2963 ◽  
Author(s):  
T F Donahue ◽  
A M Cigan
Keyword(s):  
Translation Initiation ◽  
Genetic Selection ◽  
Start Codon ◽  
Direct Selection ◽  
Start Site ◽  
Functional Nature ◽  
Selection For ◽  
Sequence Requirements ◽  
Insight Into

A unique genetic selection was devised at the HIS4 locus to address the mechanism of translation initiation in Saccharomyces cerevisiae and to probe sequence requirements at the normal translational initiator region that might participate in ribosomal recognition of the AUG start codon. The first AUG codon at the 5' end of the HIS4 message serves as the start site for translation, and the -3 and +4 nucleotide positions flanking this AUG (AXXAUGG) correspond to a eucaryotic consensus start region. Despite this similarity, direct selection for mutations that reduce or abolish ribosomal recognition of this region does not provide any insight into the functional nature of flanking nucleotides. The only mutations identified that affected recognition of this region were alterations in the AUG start codon. Among 150 spontaneous isolates, 26 were shown to contain mutations in the AUG start codon, including all +1 changes (CUG, GUG, and UUG), all +3 changes (AUA, AUC, and AUU), and one +2 change (ACG). These seven mutations of the AUG start codon, as well as AAG and AGG constructed in vitro, were assayed for their ability to support HIS4 expression. No codon other than AUG is physiologically relevant to translation initiation at HIS4 as determined by growth tests and quantitated in his4-lacZ fusion strains. These data and analysis of other his4 alleles are consistent with a mechanism of initiation at HIS4 as proposed in the scanning model whereby the first AUG codon nearest the 5' end of the message serves as the start site for translation and points to the AUG codon in S. cerevisiae as an important component for ribosomal recognition of the initiator region.

scholarly journals Genetic selection for mutations that reduce or abolish ribosomal recognition of the HIS4 translational initiator region.

1988 ◽  
Vol 8 (7) ◽  
pp. 2955-2963 ◽  
Author(s):  
T F Donahue ◽  
A M Cigan
Keyword(s):  
Translation Initiation ◽  
Genetic Selection ◽  
Start Codon ◽  
Direct Selection ◽  
Start Site ◽  
Functional Nature ◽  
Selection For ◽  
Sequence Requirements ◽  
Insight Into

A unique genetic selection was devised at the HIS4 locus to address the mechanism of translation initiation in Saccharomyces cerevisiae and to probe sequence requirements at the normal translational initiator region that might participate in ribosomal recognition of the AUG start codon. The first AUG codon at the 5' end of the HIS4 message serves as the start site for translation, and the -3 and +4 nucleotide positions flanking this AUG (AXXAUGG) correspond to a eucaryotic consensus start region. Despite this similarity, direct selection for mutations that reduce or abolish ribosomal recognition of this region does not provide any insight into the functional nature of flanking nucleotides. The only mutations identified that affected recognition of this region were alterations in the AUG start codon. Among 150 spontaneous isolates, 26 were shown to contain mutations in the AUG start codon, including all +1 changes (CUG, GUG, and UUG), all +3 changes (AUA, AUC, and AUU), and one +2 change (ACG). These seven mutations of the AUG start codon, as well as AAG and AGG constructed in vitro, were assayed for their ability to support HIS4 expression. No codon other than AUG is physiologically relevant to translation initiation at HIS4 as determined by growth tests and quantitated in his4-lacZ fusion strains. These data and analysis of other his4 alleles are consistent with a mechanism of initiation at HIS4 as proposed in the scanning model whereby the first AUG codon nearest the 5' end of the message serves as the start site for translation and points to the AUG codon in S. cerevisiae as an important component for ribosomal recognition of the initiator region.

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