scholarly journals Improving translation initiation site and stop codon recognition by using more than two classes

2014 ◽  
Vol 30 (19) ◽  
pp. 2702-2708
Author(s):  
J. Perez-Rodriguez ◽  
A. G. Arroyo-Pena ◽  
N. Garcia-Pedrajas
Keyword(s):  
Translation Initiation ◽  
Stop Codon ◽  
Initiation Site ◽  
Translation Initiation Site ◽  
Codon Recognition ◽  
Stop Codon Recognition

91 DISRUPTION OF TET1 DURING PORCINE EMBRYOGENESIS USING CRISPR/Cas9 SYSTEM

2017 ◽  
Vol 29 (1) ◽  
pp. 153
Author(s):  
K. Uh ◽  
J. Ryu ◽  
C. Ray ◽  
K. Lee
Keyword(s):  
Embryo Development ◽  
Translation Initiation ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Initiation Site ◽  
Translation Initiation Site ◽  
Epigenetic Marks ◽  
Hydroxymethyl Cytosine ◽  
Biallelic Mutations

Ten-eleven translocation (TET) enzymes catalyse oxidation of 5-methylcytosine to 5-hydroxymethyl cytosine. This TET-mediated conversion of 5-methylcytosine to 5-hydroxymethyl cytosine is implicated in initiating the DNA demethylation process, observed post-fertilization. Three members (TET1–3) of the TET family are differentially expressed during embryo development and appear to have different roles. Previous studies in mice suggest that TET1 is a key regulator in maintaining pluripotency in embryonic stem cells by managing epigenetic marks such as DNA methylation. This would imply that TET1 should be a regulator of epigenetic marks during embryo development, although this has not been demonstrated. Previously, we have cloned porcine TET1 from blastocysts (GenBank accession number KC137683) and demonstrated that the level of TET1 (mRNA and protein) was high in blastocysts. The protein level was greater in the inner cell mass compared with the trophectoderm. In this study, we generated TET1 knockout porcine embryos using CRISPR/Cas9 system to study the role of TET1 in controlling epigenetic marks during porcine embryo development. First, 2 sgRNA, immediately downstream of the presumable translation initiation site, were designed and synthesised; location of the sgRNA were nucleotide position at 2 to 21 bp and 23 to 42 bp, respectively (KC137683). Then, sgRNA (10 ng μL−1 each) and Cas9 mRNA (20 ng μL−1) were injected into the cytoplasm of IVF zygotes, and Day 7 blastocysts were genotyped. All embryos carried mutations on both alleles of TET1 (10/10), one homozygous and 9 biallelic mutations. However, immunocytochemistry analysis of other CRISPR/Cas9 injected embryos revealed that TET1 was not removed (10/10), indicating that the sgRNA may have not introduced a premature stop codon 3′ to the presumable translation initiation site. Therefore, 2 new sgRNA were designed to generate a premature stop codon at the 5′ side of a key functional domain, the 2-oxoglutarate-Fe(II)-dependent oxygenase domain (4690 to 5160 bp); the locations of the 2 sgRNA were 4450 to 4469 bp and 4501 to 4520 bp, respectively. Similarly, all of the embryos carried mutations in TET1 (7/7), 2 homozygous and 5 biallelic mutations. In addition, TET1 proteins were not detected in 11 of 16 blastocysts, confirmed by immunocytochemistry. In this study, we successfully generated embryos lacking TET1 by introducing designed CRISPR/Cas9 system during embryogenesis. Presence of TET1 from the first injection experiment suggests that the presumable translation initiation site is not accurate. Discrepancy between genotyping and immunocytochemistry results from the second injection experiment indicates that embryos possessing TET1 protein probably have mutations in triplets, thus no premature stop codon was synthesised. Further studies will focus on identifying the role of TET1 in maintaining pluripotency and epigenetic modification during pre-implantation stage using these embryos.

scholarly journals Deconstructing the individual steps of vertebrate translation initiation

2019 ◽  
Author(s):  
Adam Giess ◽  
Yamila N. Torres Cleuren ◽  
Håkon Tjeldnes ◽  
Maximilian Krause ◽  
Teshome Tilahun Bizuayehu ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Stop Codon ◽  
Small Subunit ◽  
Codon Recognition ◽  
Rate Determining Step ◽  
Expression Control ◽  
Gene Expression Control ◽  
Stop Codon Recognition ◽  
Specific Regulation ◽  
The Individual

AbstractTranslation initiation is often attributed as the rate determining step of eukaryotic protein synthesis and key to gene expression control 1. Despite this centrality the series of steps involved in this process are poorly understood 2,3. Here we capture the transcriptome-wide occupancy of ribosomes across all stages of translation initiation, enabling us to characterize the transcriptome-wide dynamics of ribosome recruitment to mRNAs, scanning across 5’ UTRs and stop codon recognition, in a higher eukaryote. We provide mechanistic evidence for ribosomes attaching to the mRNA by threading the mRNA through the small subunit. Moreover, we identify features regulating the recruitment and processivity of scanning ribosomes, redefine optimal initiation contexts and demonstrate endoplasmic reticulum specific regulation of initiation. Our approach enables deconvoluting translation initiation into separate stages and identifying the regulators at each step.

scholarly journals Molecular and Functional Differences between Heart mKv1.7 Channel Isoforms

2006 ◽  
Vol 128 (1) ◽  
pp. 133-145 ◽  
Author(s):  
Rocio K. Finol-Urdaneta ◽  
Nina Strüver ◽  
Heinrich Terlau
Keyword(s):  
Translation Initiation ◽  
Cell Function ◽  
Functional Divergence ◽  
Initiation Site ◽  
Translation Initiation Site ◽  
Inactivation Kinetics ◽  
Mouse Heart ◽  
Redox Stress ◽  
Redox Modulation ◽  
Stress States

Ion channels are membrane-spanning proteins that allow ions to permeate at high rates. The kinetic characteristics of the channels present in a cell determine the cell signaling profile and therefore cell function in many different physiological processes. We found that Kv1.7 channels from mouse heart muscle have two putative translation initiation start sites that generate two channel isoforms with different functional characteristics, mKv1.7L (489 aa) and a shorter mKv1.7S (457 aa). The electrophysiological analysis of mKv1.7L and mKv1.7S channels revealed that the two channel isoforms have different inactivation kinetics. The channel resulting from the longer protein (L) inactivates faster than the shorter channels (S). Our data supports the hypothesis that mKv1.7L channels inactivate predominantly due to an N-type related mechanism, which is impaired in the mKv1.7S form. Furthermore, only the longer version mKv1.7L is regulated by the cell redox state, whereas the shorter form mKv1.7S is not. Thus, expression starting at each translation initiation site results in significant functional divergence. Our data suggest that the redox modulation of mKv1.7L may occur through a site in the cytoplasmic N-terminal domain that seems to encompass a metal coordination motif resembling those found in many redox-sensitive proteins. The mRNA expression profile and redox modulation of mKv1.7 kinetics identify these channels as molecular entities of potential importance in cellular redox-stress states such as hypoxia.

scholarly journals Eukaryotic Release Factor 1 Phosphorylation by CK2 Protein Kinase Is Dynamic but Has Little Effect on the Efficiency of Translation Termination in Saccharomyces cerevisiae

2006 ◽  
Vol 5 (8) ◽  
pp. 1378-1387 ◽  
Author(s):  
Adam K. Kallmeyer ◽  
Kim M. Keeling ◽  
David M. Bedwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Synthesis ◽  
Protein Kinase ◽  
Stop Codon ◽  
Translation Termination ◽  
Release Factor ◽  
Codon Recognition ◽  
Number Of Factors ◽  
Stop Codon Recognition

ABSTRACT Protein synthesis requires a large commitment of cellular resources and is highly regulated. Previous studies have shown that a number of factors that mediate the initiation and elongation steps of translation are regulated by phosphorylation. In this report, we show that a factor involved in the termination step of protein synthesis is also subject to phosphorylation. Our results indicate that eukaryotic release factor 1 (eRF1) is phosphorylated in vivo at serine 421 and serine 432 by the CK2 protein kinase (previously casein kinase II) in the budding yeast Saccharomyces cerevisiae. Phosphorylation of eRF1 has little effect on the efficiency of stop codon recognition or nonsense-mediated mRNA decay. Also, phosphorylation is not required for eRF1 binding to the other translation termination factor, eRF3. In addition, we provide evidence that the putative phosphatase Sal6p does not dephosphorylate eRF1 and that the state of eRF1 phosphorylation does not influence the allosuppressor phenotype associated with a sal6Δ mutation. Finally, we show that phosphorylation of eRF1 is a dynamic process that is dependent upon carbon source availability. Since many other proteins involved in protein synthesis have a CK2 protein kinase motif near their extreme C termini, we propose that this represents a common regulatory mechanism that is shared by factors involved in all three stages of protein synthesis.

scholarly journals Link Between Short tandem Repeats and Translation Initiation Site Selection

2018 ◽  
Author(s):  
M Arabfard ◽  
K Kavousi ◽  
A Delbari ◽  
M Ohadi
Keyword(s):  
Amino Acids ◽  
Translation Initiation ◽  
Short Tandem Repeats ◽  
Tandem Repeats ◽  
Initiation Site ◽  
Translation Initiation Site ◽  
Vertebrate Species ◽  
Protein Coding ◽  
Human Specific ◽  
Short Tandem

AbstractRecent work in yeast and humans suggest that evolutionary divergence in cis-regulatory sequences impact translation initiation sites (TISs). Cis-elements can also affect the efficacy and amount of protein synthesis. Despite their vast biological implication, the landscape and relevance of short tandem repeats (STRs)/microsatellites to the human protein-coding gene TISs remain largely unknown. Here we characterized the STR distribution at the 120 bp cDNA sequence upstream of all annotated human protein-coding gene TISs based on the Ensembl database. Furthermore, we performed a comparative genomics study of all annotated orthologous TIS-flanking sequences across 47 vertebrate species (755,956 transcripts), aimed at identifying human-specific STRs in this interval. We also hypothesized that STRs may be used as genetic codes for the initiation of translation. The initial five amino acid sequences (excluding the initial methionine) that were flanked by STRs in human were BLASTed against the initial orthologous five amino acids in other vertebrate species (2,025,817 pair-wise TIS comparisons) in order to compare the number of events in which human-specific and non-specific STRs occurred with homologous and non-homologous TISs (i.e. ≥50% and <50% similarity of the five amino acids). We characterized human-specific STRs and a bias of this compartment in comparison to the overall (human-specific and non-specific) distribution of STRs (Mann Whitney p=1.4 × 10−11). We also found significant enrichment of non-homologous TISs flanked by human-specific STRs (p<0.00001). In conclusion, our data indicate a link between STRs and TIS selection, which is supported by differential evolution of the human-specific STRs in the TIS upstream flanking sequence.AbbreviationscDNAComplementary DNACDSCoding DNA sequenceSTRShort Tandem RepeatTISTranslation Initiation SiteTSSTranscription Start Site

scholarly journals utR.annotation: a tool for annotating genomic variants that could influence post-transcriptional regulation

2021 ◽  
Author(s):  
Yating Liu ◽  
Joseph Dougherty
Keyword(s):  
Stop Codon ◽  
R Package ◽  
Initiation Site ◽  
Open Reading Frames ◽  
Start Codon ◽  
Translation Initiation Site ◽  
Mouse Species ◽  
Translational Regulators ◽  
Post Transcriptional Regulation ◽  
Annotated Translation

Whole genome sequencing of patient populations is identifying thousands of new variants in UnTranslated Regions(UTRs). While the consequences of UTR mutations are not as easily predicted from primary sequence as coding mutations are, there are some known features of UTRs modulate their function. utR.annotation is an R package that can be used to annotate potential deleterious variants in the UTR regions for both human and mouse species. Given a CSV or VCF format variant file, utR.annotation provides information of each variant on whether and how it alters known translational regulators including:upstream Open Reading Frames (uORFs), upstream Kozak sequences, polyA signals, the Kozak sequence at the annotated translation initiation site, start codon, and stop codon, conservation scores in the variant position, and whether and how it changes ribosome loading based on a model from empirical data.

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