Determinants of Translation Efficiency of Specific mRNAs in Mammalian Cells

1990 ◽  
pp. 99-113
Author(s):  
David S. Peabody
Keyword(s):  
Mammalian Cells ◽  
Translation Efficiency ◽  
Specific Mrnas

scholarly journals m5U54 tRNA Hypomodification by Lack of TRMT2A Drives the Generation of tRNA-Derived Small RNAs

2021 ◽  
Vol 22 (6) ◽  
pp. 2941
Author(s):  
Marisa Pereira ◽  
Diana R. Ribeiro ◽  
Miguel M. Pinheiro ◽  
Margarida Ferreira ◽  
Stefanie Kellner ◽  
...  
Keyword(s):  
Stress Response ◽  
Small Rnas ◽  
Mammalian Cells ◽  
Cellular Stress ◽  
Cellular Stress Response ◽  
Translation Regulation ◽  
Translation Efficiency ◽  
Down Regulation ◽  
Gene Expression Control ◽  
The Impact

Transfer RNA (tRNA) molecules contain various post-transcriptional modifications that are crucial for tRNA stability, translation efficiency, and fidelity. Besides their canonical roles in translation, tRNAs also originate tRNA-derived small RNAs (tsRNAs), a class of small non-coding RNAs with regulatory functions ranging from translation regulation to gene expression control and cellular stress response. Recent evidence indicates that tsRNAs are also modified, however, the impact of tRNA epitranscriptome deregulation on tsRNAs generation is only now beginning to be uncovered. The 5-methyluridine (m5U) modification at position 54 of cytosolic tRNAs is one of the most common and conserved tRNA modifications among species. The tRNA methyltransferase TRMT2A catalyzes this modification, but its biological role remains mostly unexplored. Here, we show that TRMT2A knockdown in human cells induces m5U54 tRNA hypomodification and tsRNA formation. More specifically, m5U54 hypomodification is followed by overexpression of the ribonuclease angiogenin (ANG) that cleaves tRNAs near the anticodon, resulting in accumulation of 5′tRNA-derived stress-induced RNAs (5′tiRNAs), namely 5′tiRNA-GlyGCC and 5′tiRNA-GluCTC, among others. Additionally, transcriptomic analysis confirms that down-regulation of TRMT2A and consequently m5U54 hypomodification impacts the cellular stress response and RNA stability, which is often correlated with tiRNA generation. Accordingly, exposure to oxidative stress conditions induces TRMT2A down-regulation and tiRNA formation in mammalian cells. These results establish a link between tRNA hypomethylation and ANG-dependent tsRNAs formation and unravel m5U54 as a tRNA cleavage protective mark.

Effect of upstream reading frames on translation efficiency in simian virus 40 recombinants

1986 ◽  
Vol 6 (7) ◽  
pp. 2704-2711 ◽  
Author(s):  
D S Peabody ◽  
S Subramani ◽  
P Berg
Keyword(s):  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Initiation Codon ◽  
Efficient Production ◽  
Translation Efficiency ◽  
Reading Frame ◽  
Recombinant Viruses ◽  
Internal Translation ◽  
Late Region

In a previous report (S. Subramani, R. Mulligan, and P. Berg, Mol. Cell. Biol. 1:854-864, 1981), it was shown that mouse dihydrofolate reductase (DHFR) could be efficiently expressed from simian virus 40 recombinant viruses containing the DHFR cDNA in different locations in the viral late region. This was true even in the case of the SVGT7dhfr26 recombinant, which had the DHFR coding sequence 700 to 800 nucleotides from the 5' end of the mRNA, where it was preceded by the VP2 and VP3 initiator AUGs and a number of other noninitiator AUGs. To investigate the process of internal translation initiation in mammalian cells, we constructed a series of SVGT7dhfr recombinants in which the upstream VP2 and VP3 reading frame was terminated in various positions relative to the DHFR initiation codon. The efficient production of DHFR in infected CV1 cells depended on having the terminators of the VP2-VP3 reading frame positioned upstream or nearby downstream from the DHFR initiation codon. These results reinforce the notion that mammalian ribosomes are capable of translational reinitiation.

scholarly journals Trf4 and Trf5 Proteins of Saccharomyces cerevisiae Exhibit Poly(A) RNA Polymerase Activity but No DNA Polymerase Activity

2005 ◽  
Vol 25 (22) ◽  
pp. 10183-10189 ◽  
Author(s):  
Lajos Haracska ◽  
Robert E. Johnson ◽  
Louise Prakash ◽  
Satya Prakash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Polymerase ◽  
Protein Complexes ◽  
Polymerase Activity ◽  
Yeast Cells ◽  
Translation Efficiency ◽  
Eukaryotic Dna ◽  
Specific Mrnas ◽  
Rna Polymerase Activity ◽  
The Stability

ABSTRACT The Saccharomyces cerevisiae Trf4 and Trf5 proteins are members of a distinct family of eukaryotic DNA polymerase β-like nucleotidyltransferases, and a template-dependent DNA polymerase activity has been reported for Trf4. To define the nucleotidyltransferase activities associated with Trf4 and Tr5, we purified these proteins from yeast cells and show that whereas both proteins exhibit a robust poly(A) polymerase activity, neither of them shows any evidence of a DNA polymerase activity. The poly(A) polymerase activity, as determined for Trf4, is strictly Mn2+ dependent and highly ATP specific, incorporating AMP onto the free 3′-hydroxyl end of an RNA primer. Unlike the related poly(A) polymerases from other eukaryotes, which are located in the cytoplasm and regulate the stability and translation efficiency of specific mRNAs, the Trf4 and Trf5 proteins are nuclear, and a multiprotein complex associated with Trf4 has been recently shown to polyadenylate a variety of misfolded or inappropriately expressed RNAs which activate their degradation by the exosome. To account for the effects of Trf4/Trf5 proteins on the various aspects of DNA metabolism, including chromosome condensation, DNA replication, and sister chromatid cohesion, we suggest an additional and essential role for the Trf4 and Trf5 protein complexes in generating functional mRNA poly(A) tails in the nucleus.

scholarly journals Human 4E-T represses translation of bound mRNAs and enhances microRNA-mediated silencing

2013 ◽  
Vol 42 (5) ◽  
pp. 3298-3313 ◽  
Author(s):  
Anastasiia Kamenska ◽  
Wei-Ting Lu ◽  
Dorota Kubacka ◽  
Helen Broomhead ◽  
Nicola Minshall ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Translational Regulation ◽  
Binding Protein ◽  
Quantitative Polymerase Chain Reaction ◽  
Mrna Translation ◽  
Developmental Model ◽  
Model Systems ◽  
P Bodies ◽  
Specific Mrnas

Abstract A key player in translation initiation is eIF4E, the mRNA 5′ cap-binding protein. 4E-Transporter (4E-T) is a recently characterized eIF4E-binding protein, which regulates specific mRNAs in several developmental model systems. Here, we first investigated the role of its enrichment in P-bodies and eIF4E-binding in translational regulation in mammalian cells. Identification of the conserved C-terminal sequences that target 4E-T to P-bodies was enabled by comparison of vertebrate proteins with homologues in Drosophila (Cup and CG32016) and Caenorhabditis elegans by sequence and cellular distribution. In tether function assays, 4E-T represses bound mRNA translation, in a manner independent of these localization sequences, or of endogenous P-bodies. Quantitative polymerase chain reaction and northern blot analysis verified that bound mRNA remained intact and polyadenylated. Ectopic 4E-T reduces translation globally in a manner dependent on eIF4E binding its consensus Y30X4Lϕ site. In contrast, tethered 4E-T continued to repress translation when eIF4E-binding was prevented by mutagenesis of YX4Lϕ, and modestly enhanced the decay of bound mRNA, compared with wild-type 4E-T, mediated by increased binding of CNOT1/7 deadenylase subunits. As depleting 4E-T from HeLa cells increased steady-state translation, in part due to relief of microRNA-mediated silencing, this work demonstrates the conserved yet unconventional mechanism of 4E-T silencing of particular subsets of mRNAs.

scholarly journals Na,K-ATPase β1-Subunit Increases the Translation Efficiency of the α1-Subunit in MSV-MDCK Cells

2004 ◽  
Vol 15 (7) ◽  
pp. 3224-3232 ◽  
Author(s):  
Sigrid A. Rajasekaran ◽  
Jegan Gopal ◽  
Dianna Willis ◽  
Cromwell Espineda ◽  
Jeffery L. Twiss ◽  
...  
Keyword(s):  
Half Life ◽  
Mammalian Cells ◽  
Mdck Cells ◽  
Short Pulse ◽  
Ectopic Expression ◽  
Translation Efficiency ◽  
Mrna Levels ◽  
Translation Rate ◽  
Subunit Protein ◽  
Subunit Mrna

The Na,K-ATPase consists of an α- and β-subunit. Moloney sarcoma virus-transformed MDCK cells (MSV-MDCK) express low levels of Na,K-ATPase β1-subunit. Ectopic expression of Na,K-ATPase β1-subunit in these cells increased the protein levels of the α1-subunit of Na,K-ATPase. This increase was not due to altered transcription of the α1-subunit gene or half-life of the α1-subunit protein because both α1-subunit mRNA levels and half-life of the α1-subunit protein were comparable in MSV-MDCK and β1-subunit expressing MSV-MDCK cells. However, short pulse labeling revealed that the initial translation rate of the α1-subunit in β1-subunit expressing MSV-MDCK cells was six- to sevenfold higher compared with MSV-MDCK cells. The increased translation was specific to α1-subunit because translation rates of occludin and β-catenin, membrane and cytosolic proteins, respectively, were not altered. In vitro cotranslation/translocation experiments using rabbit reticulocyte lysate and rough microsomes revealed that the α1-subunit mRNA is more efficiently translated in the presence of β1-subunit. Furthermore, sucrose density gradient analysis revealed significantly more α1-subunit transcript associated with the polysomal fraction in β1-subunit expressing MSV-MDCK cells compared with MSV-MDCK cells, indicating that in mammalian cells the Na,K-ATPase β1-subunit is involved in facilitating the translation of the α1-subunit mRNA in the endoplasmic reticulum.

scholarly journals ADAR1 A-to-I RNA editing alters codon usage

2018 ◽  
Author(s):  
Pavla Brachova ◽  
Nehemiah S. Alvarez ◽  
Xiaoman Hong ◽  
Kailey A. Vincent ◽  
Keith E. Latham ◽  
...  
Keyword(s):  
Codon Usage ◽  
Rna Editing ◽  
Mammalian Cells ◽  
Germinal Vesicle ◽  
Translation Efficiency ◽  
Nucleotide Substitutions ◽  
Cellular Processes ◽  
Wobble Position ◽  
A Genome ◽  
Transcriptional Gene Regulation

AbstractBackgroundFully grown mammalian oocytes and eggs are transcriptionally quiescent, and therefore have a unique RNA environment in which cellular processes depend on post-transcriptional regulation. RNA editing of adenosines into inosines (A-to-I) by adenosine deaminases acting on RNA (ADARs) is a common post-transcriptional gene regulatory mechanism, yet it has not been systematically studied in oocytes.ResultsA genome-wide RNA editing analysis of transcriptionally active growing oocytes from postnatal day 12 (PND12) mice, fully grown germinal vesicle (GV) oocytes, and transcriptionally quiescent metaphase II (MII) eggs indicates an abundant amount of A-to-I editing of mRNA transcripts. Editing of mRNA was greatest in GV oocyte and MII eggs compared to the PND12 immature oocytes, this was consistent with ADAR1 levels within these cells. Compared to somatic tissues, oocytes exhibited a different pattern of RNA editing, with a high proportion of RNA edits occurring in the coding regions. These edits resulted in nucleotide substitutions that were enriched at the third nucleotide of the codon (wobble position). Codon usage can affect mRNA stability and translation efficiency.ConclusionsRNA editing in mouse oocytes is distinct from RNA editing in somatic cells due to increased frequencies of coding sequence RNA edits. We provide evidence in support of a previously unreported phenomenon of selective ADAR1 editing of the codon wobble position. Editing of the wobble position has the potential to fine tune post-transcriptional gene regulation through altering codon usage. This important observation advances our current understanding of RNA editing in mammalian cells.

scholarly journals Systematic use of synthetic 5′-UTR RNA structures to tune protein translation improves yield and quality of complex proteins in mammalian cell factories

2020 ◽  
Vol 48 (20) ◽  
pp. e119-e119
Author(s):  
Peter Eisenhut ◽  
Aman Mebrahtu ◽  
Mona Moradi Barzadd ◽  
Niklas Thalén ◽  
Gerald Klanert ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Gc Content ◽  
Protein Translation ◽  
Translation Efficiency ◽  
Rna Structures ◽  
Expression Levels ◽  
Mammalian Cell Lines

Abstract Predictably regulating protein expression levels to improve recombinant protein production has become an important tool, but is still rarely applied to engineer mammalian cells. We therefore sought to set-up an easy-to-implement toolbox to facilitate fast and reliable regulation of protein expression in mammalian cells by introducing defined RNA hairpins, termed ‘regulation elements (RgE)’, in the 5′-untranslated region (UTR) to impact translation efficiency. RgEs varying in thermodynamic stability, GC-content and position were added to the 5′-UTR of a fluorescent reporter gene. Predictable translation dosage over two orders of magnitude in mammalian cell lines of hamster and human origin was confirmed by flow cytometry. Tuning heavy chain expression of an IgG with the RgEs to various levels eventually resulted in up to 3.5-fold increased titers and fewer IgG aggregates and fragments in CHO cells. Co-expression of a therapeutic Arylsulfatase-A with RgE-tuned levels of the required helper factor SUMF1 demonstrated that the maximum specific sulfatase activity was already attained at lower SUMF1 expression levels, while specific production rates steadily decreased with increasing helper expression. In summary, we show that defined 5′-UTR RNA-structures represent a valid tool to systematically tune protein expression levels in mammalian cells and eventually help to optimize recombinant protein expression.

Signalling to translation: how signal transduction pathways control the protein synthetic machinery

2007 ◽  
Vol 403 (2) ◽  
pp. 217-234 ◽  
Author(s):  
Christopher G. Proud
Keyword(s):  
Protein Synthesis ◽  
Protein Kinases ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mrna Translation ◽  
Signalling Pathways ◽  
Cancer Tissue ◽  
Energy Status ◽  
Specific Mrnas

Recent advances in our understanding of both the regulation of components of the translational machinery and the upstream signalling pathways that modulate them have provided important new insights into the mechanisms by which hormones, growth factors, nutrients and cellular energy status control protein synthesis in mammalian cells. The importance of proper control of mRNA translation is strikingly illustrated by the fact that defects in this process or its control are implicated in a number of disease states, such as cancer, tissue hypertrophy and neurodegeneration. Signalling pathways such as those involving mTOR (mammalian target of rapamycin) and mitogen-activated protein kinases modulate the phosphorylation of translation factors, the activities of the protein kinases that act upon them and the association of RNA-binding proteins with specific mRNAs. These effects contribute both to the overall control of protein synthesis (which is linked to cell growth) and to the modulation of the translation or stability of specific mRNAs. However, important questions remain about both the contributions of individual regulatory events to the control of general protein synthesis and the mechanisms by which the translation of specific mRNAs is controlled.

scholarly journals Dicer-dependent and -independent Argonaute2 Protein Interaction Networks in Mammalian Cells

2012 ◽  
Vol 11 (11) ◽  
pp. 1442-1456 ◽  
Author(s):  
Anne Frohn ◽  
H. Christian Eberl ◽  
Julia Stöhr ◽  
Elke Glasmacher ◽  
Sabine Rüdel ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Isotope Labeling ◽  
Current View ◽  
Knock Out ◽  
Functional Studies ◽  
Proteomics Approach ◽  
Small Regulatory Rnas ◽  
Specific Mrnas ◽  
Mrna Binding

Argonaute (Ago) proteins interact with small regulatory RNAs such as microRNAs (miRNAs) and facilitate gene-silencing processes. miRNAs guide Ago proteins to specific mRNAs leading to translational silencing or mRNA decay. In order to understand the mechanistic details of miRNA function, it is important to characterize Ago protein interactors. Although several proteomic studies have been performed, it is not clear how the Ago interactome changes on miRNA or mRNA binding. Here, we report the analysis of Ago protein interactions in miRNA-containing and miRNA-depleted cells. Using stable isotope labeling in cell culture in conjunction with Dicer knock out mouse embryonic fibroblasts, we identify proteins that interact with Ago2 in the presence or the absence of Dicer. In contrast to our current view, we find that Ago-mRNA interactions can also take place in the absence of miRNAs. Our proteomics approach provides a rich resource for further functional studies on the cellular roles of Ago proteins.

scholarly journals Effect of upstream reading frames on translation efficiency in simian virus 40 recombinants.

1986 ◽  
Vol 6 (7) ◽  
pp. 2704-2711 ◽  
Author(s):  
D S Peabody ◽  
S Subramani ◽  
P Berg
Keyword(s):  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Initiation Codon ◽  
Efficient Production ◽  
Translation Efficiency ◽  
Reading Frame ◽  
Recombinant Viruses ◽  
Internal Translation ◽  
Late Region

In a previous report (S. Subramani, R. Mulligan, and P. Berg, Mol. Cell. Biol. 1:854-864, 1981), it was shown that mouse dihydrofolate reductase (DHFR) could be efficiently expressed from simian virus 40 recombinant viruses containing the DHFR cDNA in different locations in the viral late region. This was true even in the case of the SVGT7dhfr26 recombinant, which had the DHFR coding sequence 700 to 800 nucleotides from the 5' end of the mRNA, where it was preceded by the VP2 and VP3 initiator AUGs and a number of other noninitiator AUGs. To investigate the process of internal translation initiation in mammalian cells, we constructed a series of SVGT7dhfr recombinants in which the upstream VP2 and VP3 reading frame was terminated in various positions relative to the DHFR initiation codon. The efficient production of DHFR in infected CV1 cells depended on having the terminators of the VP2-VP3 reading frame positioned upstream or nearby downstream from the DHFR initiation codon. These results reinforce the notion that mammalian ribosomes are capable of translational reinitiation.

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