Impacts of uORF codon identity and position on translation regulation

Abstract Translation regulation plays an important role in eukaryotic gene expression. Upstream open reading frames (uORFs) are potent regulatory elements located in 5′ mRNA transcript leaders. Translation of uORFs usually inhibit the translation of downstream main open reading frames, but some enhance expression. While a minority of uORFs encode conserved functional peptides, the coding regions of most uORFs are not conserved. Thus, the importance of uORF coding sequences on their regulatory functions remains largely unknown. We investigated the impact of an uORF coding region on gene regulation by assaying the functions of thousands of variants in the yeast YAP1 uORF. Varying uORF codons resulted in a wide range of functions, including repressing and enhancing expression of the downstream ORF. The presence of rare codons resulted in the most inhibitory YAP1 uORF variants. Inhibitory functions of such uORFs were abrogated by overexpression of complementary tRNA. Finally, regression analysis of our results indicated that both codon identity and position impact uORF function. Our results support a model in which a uORF coding sequence impacts its regulatory functions by altering the speed of uORF translation.

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Determinants of genome-wide distribution and evolution of uORFs in eukaryotes

Nature Communications ◽

10.1038/s41467-021-21394-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Hong Zhang ◽

Yirong Wang ◽

Xinkai Wu ◽

Xiaolu Tang ◽

Changcheng Wu ◽

...

Keyword(s):

Driving Forces ◽

Mrna Translation ◽

Purifying Selection ◽

Wide Distribution ◽

Open Reading Frames ◽

Translation Regulation ◽

Effective Population ◽

Upstream Open Reading Frames ◽

Functional Peptides ◽

Regulatory Functions

AbstractUpstream open reading frames (uORFs) play widespread regulatory functions in modulating mRNA translation in eukaryotes, but the principles underlying the genomic distribution and evolution of uORFs remain poorly understood. Here, we analyze ~17 million putative canonical uORFs in 478 eukaryotic species that span most of the extant taxa of eukaryotes. We demonstrate how positive and purifying selection, coupled with differences in effective population size (Ne), has shaped the contents of uORFs in eukaryotes. Besides, gene expression level is important in influencing uORF occurrences across genes in a species. Our analyses suggest that most uORFs might play regulatory roles rather than encode functional peptides. We also show that the Kozak sequence context of uORFs has evolved across eukaryotic clades, and that noncanonical uORFs tend to have weaker suppressive effects than canonical uORFs in translation regulation. This study provides insights into the driving forces underlying uORF evolution in eukaryotes.

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Exhaustive identification of conserved upstream open reading frames with potential translational regulatory functions from animal genomes

Scientific Reports ◽

10.1038/s41598-020-73307-6 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Hiro Takahashi ◽

Shido Miyaki ◽

Hitoshi Onouchi ◽

Taichiro Motomura ◽

Nobuo Idesako ◽

...

Keyword(s):

Regulatory Peptides ◽

Open Reading Frames ◽

Dependent Manner ◽

Wide Range ◽

Eukaryotic Mrnas ◽

Taxonomic Range ◽

Upstream Open Reading Frames ◽

Functional Peptides ◽

Reading Frames ◽

Animal Genomes

Abstract Upstream open reading frames (uORFs) are present in the 5′-untranslated regions of many eukaryotic mRNAs, and some peptides encoded by these regions play important regulatory roles in controlling main ORF (mORF) translation. We previously developed a novel pipeline, ESUCA, to comprehensively identify plant uORFs encoding functional peptides, based on genome-wide identification of uORFs with conserved peptide sequences (CPuORFs). Here, we applied ESUCA to diverse animal genomes, because animal CPuORFs have been identified only by comparing uORF sequences between a limited number of species, and how many previously identified CPuORFs encode regulatory peptides is unclear. By using ESUCA, 1517 (1373 novel and 144 known) CPuORFs were extracted from four evolutionarily divergent animal genomes. We examined the effects of 17 human CPuORFs on mORF translation using transient expression assays. Through these analyses, we identified seven novel regulatory CPuORFs that repressed mORF translation in a sequence-dependent manner, including one conserved only among Eutheria. We discovered a much higher number of animal CPuORFs than previously identified. Since most human CPuORFs identified in this study are conserved across a wide range of Eutheria or a wider taxonomic range, many CPuORFs encoding regulatory peptides are expected to be found in the identified CPuORFs.

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Exhaustive identification of conserved upstream open reading frames with potential translational regulatory functions from animal genomes

10.1101/672840 ◽

2019 ◽

Author(s):

Hiro Takahashi ◽

Shido Miyaki ◽

Hitoshi Onouchi ◽

Taichiro Motomura ◽

Nobuo Idesako ◽

...

Keyword(s):

Regulatory Peptides ◽

Open Reading Frames ◽

Dependent Manner ◽

Wide Range ◽

Eukaryotic Mrnas ◽

Taxonomic Range ◽

Upstream Open Reading Frames ◽

Functional Peptides ◽

Reading Frames ◽

Animal Genomes

AbstractUpstream open reading frames (uORFs) are present in the 5’-untranslated regions of many eukaryotic mRNAs, and some peptides encoded by these regions play important regulatory roles in controlling main ORF (mORF) translation. We previously developed a novel pipeline, ESUCA, to comprehensively identify plant uORFs encoding functional peptides, based on genome-wide identification of uORFs with conserved peptide sequences (CPuORFs). Here, we applied ESUCA to diverse animal genomes, because animal CPuORFs have been identified only by comparing uORF sequences between a limited number of species, and how many previously identified CPuORFs encode regulatory peptides is unclear. By using ESUCA, 1,517 (1,373 novel and 144 known) CPuORFs were extracted from four evolutionarily divergent animal genomes. We examined the effects of 17 human CPuORFs on mORF translation using transient expression assays. Through these analyses, we identified seven novel regulatory CPuORFs that repressed mORF translation in a sequence-dependent manner, including one conserved only among Eutheria. We discovered a much higher number of animal CPuORFs than previously identified. Since most human CPuORFs identified in this study are conserved across a wide range of Eutheria or a wider taxonomic range, many CPuORFs encoding regulatory peptides are expected to be found in the identified CPuORFs.

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Expanding the Diversity of the IS630-Tc1-mariner Superfamily: Discovery of a Unique DD37E Transposon and Reclassification of the DD37D and DD39D Transposons

Genetics ◽

10.1093/genetics/159.3.1103 ◽

2001 ◽

Vol 159 (3) ◽

pp. 1103-1115 ◽

Cited By ~ 1

Author(s):

Hongguang Shao ◽

Zhijian Tu

Keyword(s):

Phylogenetic Analyses ◽

Mosquito Species ◽

Open Reading Frames ◽

Inverted Repeats ◽

Sequence Comparisons ◽

Wide Range ◽

Multiple Copies ◽

Catalytic Motif ◽

Open The Doors ◽

Reading Frames

Abstract A novel transposon named ITmD37E was discovered in a wide range of mosquito species. Sequence analysis of multiple copies in three Aedes species showed similar terminal inverted repeats and common putative TA target site duplications. The ITmD37E transposases contain a conserved DD37E catalytic motif, which is unique among reported transposons of the IS630-Tc1-mariner superfamily. Sequence comparisons and phylogenetic analyses suggest that ITmD37E forms a novel family distinct from the widely distributed Tc1 (DD34E), mariner (DD34D), and pogo (DDxD) families in the IS630-Tc1-mariner superfamily. The inclusion in the phylogenetic analysis of recently reported transposons and transposons uncovered in our database survey provided revisions to previous classifications and identified two additional families, ITmD37D and ITmD39D, which contain DD37D and DD39D motifs, respectively. The above expansion and reorganization may open the doors to the discovery of related transposons in a broad range of organisms and help illustrate the evolution and structure-function relationships among these distinct transposases in the IS630-Tc1-mariner superfamily. The presence of intact open reading frames and highly similar copies in some of the newly characterized transposons suggests recent transposition. Studies of these novel families may add to the limited repertoire of transgenesis and mutagenesis tools for a wide range of organisms, including the medically important mosquitoes.

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Woot, an Active Gypsy-Class Retrotransposon in the Flour Beetle, Tribolium castaneum, is Associated With a Recent Mutation

Genetics ◽

10.1093/genetics/143.1.417 ◽

1996 ◽

Vol 143 (1) ◽

pp. 417-426

Author(s):

Richard W Beeman ◽

M Scott Thomson ◽

John M Clark ◽

Marco A DeCamillis ◽

Susan J Brown ◽

...

Keyword(s):

Sequence Analysis ◽

Tribolium Castaneum ◽

Frameshift Mutation ◽

Open Reading Frames ◽

Red Flour Beetle ◽

Intron Sequence ◽

Coding Region ◽

Long Terminal Repeats ◽

Flour Beetle ◽

Reading Frames

Abstract A recently isolated, lethal mutation of the homeotic Abdominal gene of the red flour beetle Tribolium castaneum is associated with an insertion of a novel retrotransposon into an intron. Sequence analysis indicates that this retrotransposon, named Woot, is a member of the gypsy family of mobile elements. Most strains of T. castaneum appear to harbor ~25-35 copies of Woot per genome. Woot is composed of long terminal repeats of unprecedented length (3.6 kb each), flanking an internal coding region 5.0 kb in length. For most copies of Woot, the internal region includes two open reading frames (ORFs) that correspond to the gag and pol genes of previously described retrotransposons and retroviruses. The copy of Woot inserted into Abdominal bears an apparent single frameshift mutation that separates the normal second ORF into two. Woot does not appear to generate infectious virions by the criterion that no envelop gene is discernible. The association of Woot with a recent mutation suggests that this retroelement is currently transpositionally active in at least some strains.

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Translation of ABCE1 Is Tightly Regulated by Upstream Open Reading Frames in Human Colorectal Cells

Biomedicines ◽

10.3390/biomedicines9080911 ◽

2021 ◽

Vol 9 (8) ◽

pp. 911

Author(s):

Joana Silva ◽

Pedro Nina ◽

Luísa Romão

Keyword(s):

Translational Regulation ◽

Regulatory Elements ◽

Ribosome Profiling ◽

Leader Sequence ◽

Open Reading Frames ◽

Regulatory Function ◽

Coding Sequence ◽

Abc Protein ◽

Upstream Open Reading Frames ◽

Reading Frames

ATP-binding cassette subfamily E member 1 (ABCE1) belongs to the ABC protein family of transporters; however, it does not behave as a drug transporter. Instead, ABCE1 actively participates in different stages of translation and is also associated with oncogenic functions. Ribosome profiling analysis in colorectal cancer cells has revealed a high ribosome occupancy in the human ABCE1 mRNA 5′-leader sequence, indicating the presence of translatable upstream open reading frames (uORFs). These cis-acting translational regulatory elements usually act as repressors of translation of the main coding sequence. In the present study, we dissect the regulatory function of the five AUG and five non-AUG uORFs identified in the human ABCE1 mRNA 5′-leader sequence. We show that the expression of the main coding sequence is tightly regulated by the ABCE1 AUG uORFs in colorectal cells. Our results are consistent with a model wherein uORF1 is efficiently translated, behaving as a barrier to downstream uORF translation. The few ribosomes that can bypass uORF1 (and/or uORF2) must probably initiate at the inhibitory uORF3 or uORF5 that efficiently repress translation of the main ORF. This inhibitory property is slightly overcome in conditions of endoplasmic reticulum stress. In addition, we observed that these potent translation-inhibitory AUG uORFs function equally in cancer and in non-tumorigenic colorectal cells, which is consistent with a lack of oncogenic function. In conclusion, we establish human ABCE1 as an additional example of uORF-mediated translational regulation and that this tight regulation contributes to control ABCE1 protein levels in different cell environments.

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The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences

Molecular and Cellular Biology ◽

10.1128/mcb.8.9.3827-3836.1988 ◽

1988 ◽

Vol 8 (9) ◽

pp. 3827-3836

Author(s):

N P Williams ◽

P P Mueller ◽

A G Hinnebusch

Keyword(s):

Translational Control ◽

Normal Growth ◽

Regulatory Elements ◽

Growth Conditions ◽

Open Reading Frames ◽

Regulatory Function ◽

Yeast Saccharomyces Cerevisiae ◽

Reading Frame ◽

Yeast Genes ◽

Reading Frames

Translational control of GCN4 expression in the yeast Saccharomyces cerevisiae is mediated by multiple AUG codons present in the leader of GCN4 mRNA, each of which initiates a short open reading frame of only two or three codons. Upstream AUG codons 3 and 4 are required to repress GCN4 expression in normal growth conditions; AUG codons 1 and 2 are needed to overcome this repression in amino acid starvation conditions. We show that the regulatory function of AUG codons 1 and 2 can be qualitatively mimicked by the AUG codons of two heterologous upstream open reading frames (URFs) containing the initiation regions of the yeast genes PGK and TRP1. These AUG codons inhibit GCN4 expression when present singly in the mRNA leader; however, they stimulate GCN4 expression in derepressing conditions when inserted upstream from AUG codons 3 and 4. This finding supports the idea that AUG codons 1 and 2 function in the control mechanism as translation initiation sites and further suggests that suppression of the inhibitory effects of AUG codons 3 and 4 is a general consequence of the translation of URF 1 and 2 sequences upstream. Several observations suggest that AUG codons 3 and 4 are efficient initiation sites; however, these sequences do not act as positive regulatory elements when placed upstream from URF 1. This result suggests that efficient translation is only one of the important properties of the 5' proximal URFs in GCN4 mRNA. We propose that a second property is the ability to permit reinitiation following termination of translation and that URF 1 is optimized for this regulatory function.

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Sequence and Organization of pXO1, the Large Bacillus anthracis Plasmid Harboring the Anthrax Toxin Genes

Journal of Bacteriology ◽

10.1128/jb.181.20.6509-6515.1999 ◽

1999 ◽

Vol 181 (20) ◽

pp. 6509-6515 ◽

Cited By ~ 250

Author(s):

R. T. Okinaka ◽

K. Cloud ◽

O. Hampton ◽

A. R. Hoffmaster ◽

K. K. Hill ◽

...

Keyword(s):

Bacillus Anthracis ◽

Regulatory Elements ◽

Open Reading Frames ◽

Significant Similarity ◽

Toxin Genes ◽

Theta Replication ◽

Large Plasmids ◽

High Degree ◽

Degree Of Similarity ◽

Reading Frames

ABSTRACT The Bacillus anthracis Sterne plasmid pXO1 was sequenced by random, “shotgun” cloning. A circular sequence of 181,654 bp was generated. One hundred forty-three open reading frames (ORFs) were predicted using GeneMark and GeneMark.hmm, comprising only 61% (110,817 bp) of the pXO1 DNA sequence. The overall guanine-plus-cytosine content of the plasmid is 32.5%. The most recognizable feature of the plasmid is a “pathogenicity island,” defined by a 44.8-kb region that is bordered by inverted IS1627 elements at each end. This region contains the three toxin genes (cya, lef, and pagA), regulatory elements controlling the toxin genes, three germination response genes, and 19 additional ORFs. Nearly 70% of the ORFs on pXO1 do not have significant similarity to sequences available in open databases. Absent from the pXO1 sequence are homologs to genes that are typically required to drive theta replication and to maintain stability of large plasmids in Bacillus spp. Among the ORFs with a high degree of similarity to known sequences are a collection of putative transposases, resolvases, and integrases, suggesting an evolution involving lateral movement of DNA among species. Among the remaining ORFs, there are three sequences that may encode enzymes responsible for the synthesis of a polysaccharide capsule usually associated with serotype-specific virulent streptococci.

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Depletion of the Trypanosome Pumilio Domain Protein PUF2 or of Some Other Essential Proteins Causes Transcriptome Changes Related to Coding Region Length

Eukaryotic Cell ◽

10.1128/ec.00018-14 ◽

2014 ◽

Vol 13 (5) ◽

pp. 664-674 ◽

Cited By ~ 19

Author(s):

Bhaskar Anand Jha ◽

Abeer Fadda ◽

Clementine Merce ◽

Elisha Mugo ◽

Dorothea Droll ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Mrna Translation ◽

Open Reading Frames ◽

Mrna Levels ◽

Coding Region ◽

Content Type ◽

Puf Proteins ◽

Reading Frames

ABSTRACT Pumilio domain RNA-binding proteins are known mainly as posttranscriptional repressors of gene expression that reduce mRNA translation and stability. Trypanosoma brucei has 11 PUF proteins. We show here that PUF2 is in the cytosol, with roughly the same number of molecules per cell as there are mRNAs. Although PUF2 exhibits a low level of in vivo RNA binding, it is not associated with polysomes. PUF2 also decreased reporter mRNA levels in a tethering assay, consistent with a repressive role. Depletion of PUF2 inhibited growth of bloodstream-form trypanosomes, causing selective loss of mRNAs with long open reading frames and increases in mRNAs with shorter open reading frames. Reexamination of published RNASeq data revealed the same trend in cells depleted of some other proteins. We speculate that these length effects could be caused by inhibition of the elongation phase of transcription or by an influence of translation status or polysomal conformation on mRNA decay.

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