scholarly journals Tunable protein synthesis by transcript isoforms in human cells

2015 ◽  
Author(s):  
Stephen N Floor ◽  
Jennifer A Doudna
Keyword(s):  
Translational Control ◽  
Protein Production ◽  
Dynamic Range ◽  
Human Cells ◽  
Untranslated Regions ◽  
Specific Expression ◽  
Transcript Isoforms ◽  
Protein Levels ◽  
Eukaryotic Genes ◽  
Control Protein

Eukaryotic genes generate multiple mRNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5′ and 3′ untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5′ untranslated regions exert robust translational control between cell lines, while 3′ untranslated regions can confer cell-type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels.

scholarly journals Tunable protein synthesis by transcript isoforms in human cells

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Stephen N Floor ◽  
Jennifer A Doudna
Keyword(s):  
Translational Control ◽  
Protein Production ◽  
Dynamic Range ◽  
Human Cells ◽  
Untranslated Regions ◽  
Specific Expression ◽  
Transcript Isoforms ◽  
Protein Levels ◽  
Eukaryotic Genes ◽  
Control Protein

Eukaryotic genes generate multiple RNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5′ and 3′ untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5′ untranslated regions exert robust translational control between cell lines, while 3′ untranslated regions can confer cell type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels.

scholarly journals Widespread Effects of Chemokine 3′ Untranslated Regions on mRNA Degradation and Protein Production in Human Cells

2018 ◽  
Vol 201 (3) ◽  
pp. 1053-1061 ◽  
Author(s):  
Wenxue Zhao ◽  
David J. Erle
Keyword(s):  
Protein Production ◽  
Mrna Degradation ◽  
Human Cells ◽  
Untranslated Regions

Forebrain and midbrain development requires epiblast-restrictedOtx2translational control mediated by its 3′ UTR

Development ◽  
2001 ◽  
Vol 128 (15) ◽  
pp. 2989-3000 ◽  
Author(s):  
Pietro Pilo Boyl ◽  
Massimo Signore ◽  
Dario Acampora ◽  
Juan Pedro Martinez-Barbera ◽  
Cristina Ilengo ◽  
...  
Keyword(s):  
Brain Development ◽  
Translational Control ◽  
Transcriptional Control ◽  
Molecular Level ◽  
Regulatory Element ◽  
Untranslated Regions ◽  
Visceral Endoderm ◽  
Protein Levels ◽  
Neuronal Progenitor ◽  
Anterior Neural Plate

Otx genes play an important role in brain development. Previous mouse models suggested that the untranslated regions (UTRs) of Otx2 mRNA may contain regulatory element(s) required for its post-transcriptional control in epiblast and neuroectoderm. In order to study this, we have perturbed the 3′ UTR of Otx2 by inserting a small fragment of DNA from the λ phage. Otx2λ mutants exhibited proper gastrulation and normal patterning of the early anterior neural plate, but from 8.5 days post coitum they developed severe forebrain and midbrain abnormalities. OTX2 protein levels in Otx2λ mutants were heavily reduced in the epiblast, axial mesendoderm and anterior neuroectoderm but not in the visceral endoderm. At the molecular level, we found out that the ability of the Otx2λ mRNA to form efficient polyribosome complexes was impaired. Sequence analysis of the Otx2-3′ UTR revealed a 140 bp long element that is present only in vertebrate Otx2 genes and conserved in identity by over 80%. Our data provide experimental evidence that murine brain development requires accurate translational control of Otx2 mRNA in epiblast and neuronal progenitor cells. This leads us to hypothesise that this control might have important evolutionary implications.

scholarly journals Six mouse alpha-tubulin mRNAs encode five distinct isotypes: testis-specific expression of two sister genes.

1986 ◽  
Vol 6 (7) ◽  
pp. 2409-2419 ◽  
Author(s):  
A Villasante ◽  
D Wang ◽  
P Dobner ◽  
P Dolph ◽  
S A Lewis ◽  
...  
Keyword(s):  
Duplication Event ◽  
Developmental Expression ◽  
Untranslated Regions ◽  
Coding Region ◽  
Specific Expression ◽  
Translational Initiation ◽  
Alpha Tubulin ◽  
Tubulin Isotypes ◽  
Tubulin Isotype ◽  
Genes Encoding

Five mouse alpha-tubulin isotypes are described, each distinguished by the presence of unique amino acid substitutions within the coding region. Most, though not all of these isotype-specific amino acids, are clustered at the carboxy terminus. One of the alpha-tubulin isotypes described is expressed exclusively in testis and is encoded by two closely related genes (M alpha 3 and M alpha 7) which have homologous 3' untranslated regions but which differ at multiple third codon positions and in their 5' untranslated regions. We show that a subfamily of alpha-tubulin genes encoding the same testis-specific isotype also exists in humans. Thus, we conclude that the duplication event leading to a pair of genes encoding a testis-specific alpha-tubulin isotype predated the mammalian radiation, and both members of the duplicated sequence have been maintained since species divergence. A second alpha-tubulin gene, M alpha 6, is expressed ubiquitously at a low level, whereas a third gene, M alpha 4, is unique in that it does not encode a carboxy-terminal tyrosine residue. This gene yields two transcripts: a 1.8-kilobase (kb) mRNA that is abundant in muscle and a 2.4-kb mRNA that is abundant in testis. Whereas the 1.8-kb mRNA encodes a distinct alpha-tubulin isotype, the 2.4-kb mRNA is defective in that the methionine residue required for translational initiation is missing. Patterns of developmental expression of the various alpha-tubulin isotypes are presented. Our data support the view that individual tubulin isotypes are capable of conferring functional specificity on different kinds of microtubules.

scholarly journals Improved ribosome-footprint and mRNA measurements provide insights into dynamics and regulation of yeast translation

2015 ◽  
Author(s):  
David E Weinberg ◽  
Premal Shah ◽  
Stephen W Eichhorn ◽  
Jeffrey A Hussmann ◽  
Joshua B Plotkin ◽  
...  
Keyword(s):  
Translational Control ◽  
Nucleotide Composition ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Untranslated Regions ◽  
Coding Regions ◽  
Genome Wide ◽  
Upstream Open Reading Frames ◽  
Improved Methods ◽  
Reading Frames

Ribosome-footprint profiling provides genome-wide snapshots of translation, but technical challenges can confound its analysis. Here, we use improved methods to obtain ribosome-footprint profiles and mRNA abundances that more faithfully reflect gene expression in Saccharomyces cerevisiae. Our results support proposals that both the beginning of coding regions and codons matching rare tRNAs are more slowly translated. They also indicate that emergent polypeptides with as few as three basic residues within a 10-residue window tend to slow translation. With the improved mRNA measurements, the variation attributable to translational control in exponentially growing yeast was less than previously reported, and most of this variation could be predicted with a simple model that considered mRNA abundance, upstream open reading frames, cap-proximal structure and nucleotide composition, and lengths of the coding and 5′- untranslated regions. Collectively, our results reveal key features of translational control in yeast and provide a framework for executing and interpreting ribosome- profiling studies.

scholarly journals Actively translated uORFs reduce translation and mRNA stability independent of NMD in Arabidopsis

2021 ◽  
Author(s):  
Hsin-Yen Larry Wu ◽  
Polly Yingshan Hsu
Keyword(s):  
Gene Expression ◽  
Mrna Stability ◽  
Gene Expression Regulation ◽  
Regulatory Elements ◽  
Expression Regulation ◽  
Ribosome Profiling ◽  
Translation Efficiency ◽  
Nonsense Mediated Decay ◽  
Protein Levels ◽  
Eukaryotic Genes

ABSTRACTUpstream ORFs (uORFs) are widespread cis-regulatory elements in the 5’ untranslated regions of eukaryotic genes. Translation of uORFs could negatively regulate protein synthesis by repressing main ORF (mORF) translation and by reducing mRNA stability presumably through nonsense-mediated decay (NMD). While the above expectations were supported in animals, they have not been extensively tested in plants. Using ribosome profiling, we systematically identified 2093 Actively Translated uORFs (ATuORFs) in Arabidopsis seedlings and examined their roles in gene expression regulation by integrating multiple genome-wide datasets. Compared with genes without uORFs, we found ATuORFs result in 38%, 14%, and 43% reductions in translation efficiency, mRNA stability, and protein levels, respectively. The effects of predicted but not actively translated uORFs are much weaker than those of ATuORFs. Interestingly, ATuORF-containing genes are also expressed at higher levels and encode longer proteins with conserved domains, features that are common in evolutionarily older genes. Moreover, we provide evidence that uORF translation in plants, unlike in vertebrates, generally does not trigger NMD. We found ATuORF-containing transcripts are degraded through 5’ to 3’ decay, while NMD targets are degraded through both 5’ to 3’ and 3’ to 5’ decay, suggesting uORF-associated mRNA decay and NMD have distinct genetic requirements. Furthermore, we showed ATuORFs and NMD repress translation through separate mechanisms. Our results reveal that the potent inhibition of uORFs on mORF translation and mRNA stability in plants are independent of NMD, highlighting a fundamental difference in gene expression regulation by uORFs in the plant and animal kingdoms.

scholarly journals Elevated cJUN expression and an ATF/CRE site within the ATF3 promoter contribute to activation of ATF3 transcription by the amino acid response

2013 ◽  
Vol 45 (4) ◽  
pp. 127-137 ◽  
Author(s):  
Lingchen Fu ◽  
Michael S. Kilberg
Keyword(s):  
Amino Acid ◽  
Transcriptional Activation ◽  
Translational Control ◽  
Mammalian Cells ◽  
Transcriptional Control ◽  
Present Report ◽  
Binding Activity ◽  
Maximal Response ◽  
Protein Levels ◽  
Amino Acid Response

Mammalian cells respond to amino acid deprivation through multiple signaling pathways referred to as the amino acid response (AAR). Transcription factors mediate the AAR after their activation by several mechanisms; examples include translational control (activating transcription factor 4, ATF4), phosphorylation (p-cJUN), and transcriptional control (ATF3). ATF4 induces ATF3 transcription through a promoter-localized C/EBP-ATF response element (CARE). The present report characterizes an ATF/CRE site upstream of the CARE that also contributes to AAR-induced ATF3 transcription. ATF4 binds to the ATF/CRE and CARE sequences and both are required for a maximal response to ATF4 induction. ATF3, which antagonizes ATF4 and represses its own gene, also exhibited binding activity to the ATF/CRE and CARE sequences. The AAR resulted in elevated total cJUN and p-cJUN protein levels and both forms exhibited binding activity to the ATF/CRE and CARE ATF3 sequences. Knockdown of AAR-enhanced cJUN expression blocked induction of the ATF3 gene and mutation of either the ATF/CRE or the CARE site prevented the cJUN-dependent increase in ATF3-driven luciferase activity. The results indicate that both increased cJUN and the cis-acting ATF/CRE sequence within the ATF3 promoter contribute to the transcriptional activation of the gene during the AAR.

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