Untranslated Region Length Research Articles

Characterization and evolution of 5′ and 3′ untranslated regions in eukaryotes

Untranslated regions (UTRs) in eukaryotes play a significant role in the regulation of translation and mRNA half-life, as well as interacting with specific RNA-binding proteins. However, UTRs receive less attention than more crucial elements such as genes, and the basic structural and evolutionary characteristics of UTRs of different species, and the relationship between these UTRs and the genome size and species gene number is not well understood. To address these questions, we performed a comparative analysis of 5′ and 3′ untranslated regions of different species by analyzing the basic characteristics of 244,976 UTRs from three eukaryote kingdoms (Plantae, Fungi, and Protista). The results showed that the UTR lengths and SSR frequencies in UTRs increased significantly with increasing species gene number while the length and G+C content in 5′ UTRs and different types of repetitive sequences in 3′ UTRs increased with the increase of genome size. We also found that the sequence length of 5′ UTRs was significantly positively correlated with the presence of transposons and SSRs while the sequence length of 3′ UTRs was significantly positively correlated with the presence of tandem repeat sequences. These results suggested that evolution of species complexity from lower organisms to higher organisms is accompanied by an increase in the regulatory complexity of UTRs, mediated by increasing UTR length, increasing G+C content of 5′ UTRs, and insertion and expansion of repetitive sequences.

Genome-wide Analysis of Pre-mRNA 3′ End Processing Reveals a Decisive Role of Human Cleavage Factor I in the Regulation of 3′ UTR Length

Through alternative polyadenylation, human mRNAs acquire longer or shorter 3' untranslated regions, the latter typically associated with higher transcript stability and increased protein production. To understand the dynamics of polyadenylation site usage, we performed transcriptome-wide mapping of both binding sites of 3' end processing factors CPSF-160, CPSF-100, CPSF-73, CPSF-30, Fip1, CstF-64, CstF-64τ, CF I(m)25, CF I(m)59, and CF I(m)68 and 3' end processing sites in HEK293 cells. We found that although binding sites of these factors generally cluster around the poly(A) sites most frequently used in cleavage, CstF-64/CstF-64τ and CFI(m) proteins have much higher positional specificity compared to CPSF components. Knockdown of CF I(m)68 induced a systematic use of proximal polyadenylation sites, indicating that changes in relative abundance of a single 3' end processing factor can modulate the length of 3' untranslated regions across the transcriptome and suggesting a mechanism behind the previously observed increase in tumor cell invasiveness upon CF I(m)68 knockdown.

A unifying model for mTORC1-mediated regulation of mRNA translation

The mTOR Complex 1 (mTORC1) kinase nucleates a pathway that promotes cell growth and proliferation and is the target of rapamycin, a drug with many clinical uses1. mTORC1 regulates mRNA translation, but the overall translational program is poorly defined and no unifying model exists to explain how mTORC1 differentially controls the translation of specific mRNAs. Here we use high-resolution transcriptome-scale ribosome profiling to monitor translation in cells acutely treated with the mTOR inhibitor Torin1, which, unlike rapamycin, fully inhibits mTORC12. These data reveal a surprisingly simple view of the mRNA features and mechanisms that confer mTORC1-dependent translation control. The subset of mRNAs that are specifically regulated by mTORC1 consists almost entirely of transcripts with established 5′ terminal oligopyrimidine (TOP) motifs, or, like Hsp90ab1 and Ybx1, with previously unrecognized TOP or related TOP-like motifs that we identified. We find no evidence to support proposals that mTORC1 preferentially regulates mRNAs with increased 5′ UTR length or complexity3. mTORC1 phosphorylates a myriad of translational regulators, but how it controls TOP mRNA translation is unknown4. Remarkably, loss of just the well-characterized mTORC1 substrates, the 4E-BP family of translational repressors, is sufficient to render TOP and TOP-like mRNA translation resistant to Torin1. The 4E-BPs inhibit translation initiation by interfering with the interaction between the cap-binding protein eIF4E and eIF4G1. Loss of this interaction diminishes the capacity of eIF4E to bind TOP and TOP-like mRNAs much more than other mRNAs, explaining why mTOR inhibition selectively suppresses their translation. Our results clarify the translational program controlled by mTORC1 and identify 4E-BPs and eIF4G1 as its master effectors.

Analysis of Nonsense‐Mediated mRNA Decay in Saccharomyces cerevisiae

Nonsense-mediated mRNA decay is a highly conserved pathway that degrades mRNAs with premature termination codons. These mRNAs include mRNAs transcribed from nonsense or frameshift alleles as well as wild-type mRNA with signals that direct ribosomes to terminate prematurely. This unit describes techniques to monitor steady-state mRNA levels, decay rates, and structural features of mRNAs targeted by this pathway, as well as in vivo analysis of nonsense suppression and allosuppression in the yeast Saccharomyces cerevisiae. Protocols for the structural features of mRNA include analysis of cap status, 5' and 3' untranslated region (UTR) lengths, and poly(A) tail length.

Global Patterns of Tissue-Specific Alternative Polyadenylation in Drosophila

We analyzed the usage and consequences of alternative cleavage and polyadenylation (APA) in Drosophila melanogaster by using >1 billion reads of stranded mRNA-seq across a variety of dissected tissues. Beyond demonstrating that a majority of fly transcripts are subject to APA, we observed broad trends for 3' untranslated region (UTR) shortening in the testis and lengthening in the central nervous system (CNS); the latter included hundreds of unannotated extensions ranging up to 18 kb. Extensive northern analyses validated the accumulation of full-length neural extended transcripts, and in situ hybridization indicated their spatial restriction to the CNS. Genes encoding RNA binding proteins (RBPs) and transcription factors were preferentially subject to 3' UTR extensions. Motif analysis indicated enrichment of miRNA and RBP sites in the neural extensions, and their termini were enriched in canonical cis elements that promote cleavage and polyadenylation. Altogether, we reveal broad tissue-specific patterns of APA in Drosophila and transcripts with unprecedented 3' UTR length in the nervous system.

Target gene expression levels and competition between transfected and endogenous microRNAs are strong confounding factors in microRNA high-throughput experiments

BackgroundMicroRNA (miRNA) target genes tend to have relatively long and conserved 3' untranslated regions (UTRs), but to what degree these characteristics contribute to miRNA targeting is poorly understood. Different high-throughput experiments have, for example, shown that miRNAs preferentially regulate genes with both short and long 3' UTRs and that target site conservation is both important and irrelevant for miRNA targeting.ResultsWe have analyzed several gene context-dependent features, including 3' UTR length, 3' UTR conservation, and messenger RNA (mRNA) expression levels, reported to have conflicting influence on miRNA regulation. By taking into account confounding factors such as technology-dependent experimental bias and competition between transfected and endogenous miRNAs, we show that two factors - target gene expression and competition - could explain most of the previously reported experimental differences. Moreover, we find that these and other target site-independent features explain about the same amount of variation in target gene expression as the target site-dependent features included in the TargetScan model.ConclusionsOur results show that it is important to consider confounding factors when interpreting miRNA high throughput experiments and urge special caution when using microarray data to compare average regulatory effects between groups of genes that have different average gene expression levels.

Evolution of 5' Untranslated Region Length and Gene Expression Reprogramming in Yeasts

The sequences of the untranslated regions (UTRs) of mRNAs play important roles in posttranscriptional regulation, but whether a change in UTR length can significantly affect the regulation of gene expression is not clear. In this study, we examined the connection between UTR length and Expression Correlation with cytosolic ribosomal proteins (CRP) genes (ECC), which measures the level of expression similarity of a group of genes with CRP genes under various growth conditions. We used data from the aerobic fermentation yeast Saccharomyces cerevisiae and the aerobic respiration yeast Candida albicans. To reduce statistical fluctuations, we computed the ECC for the genes in a Gene Ontology (GO) functional group. We found that in both species, ECC is strongly correlated with the 5' UTR length but not with the 3' UTR length and that the 5' UTR length is evolutionarily better conserved than the 3' UTR length. Interestingly, we found 11 GO groups that have had a substantial increase in 5' UTR length in the S. cerevisiae lineage and that the length increase was associated with a substantial decrease in ECC. Moreover, 9 of the 11 GO groups of genes are involved in mitochondrial respiration function, whose expression reprogramming has been shown to be a major factor for the evolution of aerobic fermentation. Finally, we found that an increase in 5' UTR length may decrease the +1 nucleosome occupancy. This study provides a new angle to understand the role of 5' UTR in gene expression regulation and evolution.

Is There a Classical Nonsense-Mediated Decay Pathway in Trypanosomes?

In many eukaryotes, messenger RNAs with premature termination codons are destroyed by a process called “nonsense-mediated decay”, which requires the RNA helicase Upf1 and also, usually, an interacting factor, Upf2. Recognition of premature termination codons may rely on their distance from either a splice site or the polyadenylation site, and long 3′-untranslated regions can trigger mRNA decay. The protist Trypanosoma brucei relies heavily on mRNA degradation to determine mRNA levels, and 3′-untranslated regions play a major role in control of mRNA decay. We show here that trypanosomes have a homologue of Upf1, TbUPF1, which interacts with TbUPF2 and (in an RNA-dependent fashion) with poly(A) binding protein 1, PABP1. Introduction of a premature termination codon in either an endogenous gene or a reporter gene decreased mRNA abundance, as expected for nonsense-mediated decay, but a dependence of this effect on TbUPF1 could not be demonstrated, and depletion of TbUPF1 by over 95% had no effect on parasite growth or the mRNA transcriptome. Further investigations of the reporter mRNA revealed that increases in open reading frame length tended to increase mRNA abundance. In contrast, inhibition of translation, either using 5′-secondary structures or by lengthening the 5′-untranslated region, usually decreased reporter mRNA abundance. Meanwhile, changing the length of the 3′-untranslated region had no consistent effect on mRNA abundance. We suggest that in trypanosomes, translation per se may inhibit mRNA decay, and interactions with multiple RNA-binding proteins preclude degradation based on 3′-untranslated region length alone.

Copper tolerance of Saccharomyces cerevisiae nonsense-mediated mRNA decay mutants

The eukaryotic nonsense-mediated mRNA (NMD) is a specialized pathway that leads to the recognition and rapid degradation of mRNAs with premature termination codons, and importantly some natural mRNAs as well. Natural mRNAs with atypically long 3'-untranslated regions (UTRs) are degraded by NMD in Saccharomyces cerevisiae. A number of S. cerevisiae mRNAs undergo alternative 3'-end processing producing mRNA isoforms that differ in their 3'-UTR lengths. Some of these alternatively 3'-end processed mRNA isoforms have atypically long 3'-UTRs and would be likely targets for NMD-mediated degradation. Here, we investigated the role NMD plays in the regulation of expression of CTR2, which encodes a vacuolar membrane copper transporter. CTR2 pre-mRNA undergoes alternative 3'-end processing to produce two mRNA isoforms with 300-nt and 2-kb 3'-UTRs. We show that both CTR2 mRNA isoforms are differentially regulated by NMD. The regulation of CTR2 mRNA by NMD has physiological consequences, since nmd mutants are more tolerant to toxic levels of copper relative to wild-type yeast cells and the copper tolerance of nmd mutants is dependent on the presence of CTR2.

RNA polymerase II kinetics inpolopolyadenylation signal selection

Regulated alternative polyadenylation is an important feature of gene expression, but how gene transcription rate affects this process remains to be investigated. polo is a cell-cycle gene that uses two poly(A) signals in the 3' untranslated region (UTR) to produce alternative messenger RNAs that differ in their 3'UTR length. Using a mutant Drosophila strain that has a lower transcriptional elongation rate, we show that transcription kinetics can determine alternative poly(A) site selection. The physiological consequences of incorrect polo poly(A) site choice are of vital importance; transgenic flies lacking the distal poly(A) signal cannot produce the longer transcript and die at the pupa stage due to a failure in the proliferation of the precursor cells of the abdomen, the histoblasts. This is due to the low translation efficiency of the shorter transcript produced by proximal poly(A) site usage. Our results show that correct polo poly(A) site selection functions to provide the correct levels of protein expression necessary for histoblast proliferation, and that the kinetics of RNA polymerase II have an important role in the mechanism of alternative polyadenylation.

The 3′-untranslated region length and AU-rich RNA location modulate RNA–protein interaction and translational control of β2-adrenergic receptor mRNA

Posttranscriptional controls play a major role in β(2)-adrenergic receptor (β(2)-AR) expression. We recently reported that β(2)-AR mRNA translation is suppressed by elements in its 3'-untranslated region (UTR). We also identified T-cell-restricted intracellular antigen-related protein (TIAR) and HuR as prominent AU-rich (ARE) RNA-binding proteins that associate with β(2)-AR mRNA 3'-UTR. In this study, we identified a poly(U) region at the distal end of the 3'-UTR as critical for TIAR binding to β(2)-AR mRNA and for translational suppression. Here, we also report that the locations of the poly(U) and ARE sequences within the 3'-UTR are important determinants that control the translation of β(2)-AR mRNA. Consistent with this finding, a 20-nucleotide ARE RNA from the proximal 3'-UTR that did not inhibit mRNA translation in its native position was able to suppress translation when re-located to the distal 3'-UTR of the receptor mRNA. Immunoprecipitation and polysome profile analysis demonstrated the importance of 3'-UTR length and the ARE RNA location within the 3'-UTR, as key determinants of RNA/protein interactions and translational control of β(2)-AR mRNA. Further, the importance of 3'-UTR length and ARE location in TIAR and HuR association with mRNA and translational suppression was demonstrated using a chimeric luciferase reporter gene.

Patterns of 5′ Untranslated Region Length Distribution in Encephalitozoon cuniculi: Implications for Gene Regulation and Potential Links Between Transcription and Splicing

Encephalitozoon cuniculi, a eukaryotic intracellular parasite belonging to the group Microsporidia, has a highly reduced and compacted genome. Its mRNA transcripts have been found to differ between the two life stages, the spore and meront, of the parasite. Spore transcripts generally have more transcription start sites, longer 5' untranslated regions (UTRs), and overlap more frequently with upstream genes than those of meronts. A previous analysis of 31 meront gene transcripts showed that most have short 5'UTRs, and intron-containing genes, mostly ribosomal protein genes, exclusively have very short 5'UTRs. Here we analyzed a larger set of transcripts from meronts, and we find a pattern of 5'UTR length distribution similar to other reduced genomes. There is an abundance of very short 5'UTRs that are <20 bp in length, and very few 5'UTRs that are much longer. We also find a relationship between gene categories and 5'UTR length: intron-containing genes and ribosomal protein genes have exclusively short 5'UTRs. We suggest that the abundance of short 5'UTRs may be related to a class of highly expressed genes that benefit the parasite's growth cycle. Also, the longer 5'UTRs may be playing a role in down-regulating expression of genes that require temporal or environment-induced expression.

Spatiotemporal Regulation of Multiple Overlapping Sense and Novel Natural Antisense Transcripts at the Nrgn and Camk2n1 Gene Loci during Mouse Cerebral Corticogenesis

Nrgn and Camk2n1 are highly expressed in the brain and play an important role in synaptic long-term potentiation via regulation of Ca(2+)/calmodulin-dependent protein kinase II. We have shown that the gene loci for these 2 proteins are actively transcribed in the adult cerebral cortex and feature multiple overlapping transcripts in both the sense and antisense orientations with alternative polyadenylation. These transcripts were upregulated in the adult compared with embryonic and P1.5 mouse cerebral cortices, and transcripts with different 3' untranslated region lengths showed differing expression profiles. In situ hybridization (ISH) analysis revealed spatiotemporal regulation of the Nrgn and Camk2n1 sense and natural antisense transcripts (NATs) throughout cerebral corticogenesis. In addition, we also demonstrated that the expression of these transcripts was organ-specific. Both Nrgn and Camk2n1 sense and NATs were also upregulated in differentiating P19 teratocarcinoma cells. RNA fluorescent ISH analysis confirmed the capability of these NATs to form double-stranded RNA aggregates with the sense transcripts in the cytoplasm of cells obtained from the brain. We propose that the differential regulation of multiple sense and novel overlapping NATs at the Nrgn and Camk2n1 loci will increase the diversity of posttranscriptional regulation, resulting in cell- and time-specific regulation of their gene products during cerebral corticogenesis and function.

Expression of oestrogen receptor β isoforms is regulated by transcriptional and post-transcriptional mechanisms

Although ERs (oestrogen receptors) mediate breast tumour behaviour, the precise role of ERbeta remains unclear. This is mainly because analyses have been complicated by the presence in breast tissue of three ERbeta protein variants (ERbeta1, ERbeta2 and ERbeta5) that derive from differential 3' splicing. We have recently identified the first known mechanisms responsible for the differential control of isoform expression, involving regulation of translation via 5'-UTRs (untranslated regions). In the present study, we have uncovered further complexity involving the influence of multiple promoters and cross-talk between 5'- and 3'-UTRs. We demonstrate that full-length ERbeta mRNAs are transcribed from three separate promoters; two promoters are well-established within the literature, whereas the third represents a novel finding. Each promoter produces transcripts with distinct 5'-UTRs. The differential 3' splicing that produces transcripts coding for the ERbeta isoforms also defines isoform-specific 3'-UTRs. We identified exact 3'-UTR sequences for each isoform, and have shown that alternative polyadenylation sites are used in a cell-type specific manner to produce transcripts with 3'-UTRs of different lengths. Critically, we show that 5'- and 3'-UTRs combine to specify the efficiencies with which individual transcripts are translated, with 3'-UTR length having a key influence. In addition, we demonstrate how 17beta-oestradiol, a key driver of breast cancer development, affects the regulation of ERbeta expression at both transcriptional and translational levels.

Genome-wide functional analysis of human 5' untranslated region introns

Genes with short 5'UTR introns have higher expression than genes with no or long 5'UTR introns. Complex evolutionary forces act on these introns.

An Abundance of Ubiquitously Expressed Genes Revealed by Tissue Transcriptome Sequence Data

The parts of the genome transcribed by a cell or tissue reflect the biological processes and functions it carries out. We characterized the features of mammalian tissue transcriptomes at the gene level through analysis of RNA deep sequencing (RNA-Seq) data across human and mouse tissues and cell lines. We observed that roughly 8,000 protein-coding genes were ubiquitously expressed, contributing to around 75% of all mRNAs by message copy number in most tissues. These mRNAs encoded proteins that were often intracellular, and tended to be involved in metabolism, transcription, RNA processing or translation. In contrast, genes for secreted or plasma membrane proteins were generally expressed in only a subset of tissues. The distribution of expression levels was broad but fairly continuous: no support was found for the concept of distinct expression classes of genes. Expression estimates that included reads mapping to coding exons only correlated better with qRT-PCR data than estimates which also included 3′ untranslated regions (UTRs). Muscle and liver had the least complex transcriptomes, in that they expressed predominantly ubiquitous genes and a large fraction of the transcripts came from a few highly expressed genes, whereas brain, kidney and testis expressed more complex transcriptomes with the vast majority of genes expressed and relatively small contributions from the most expressed genes. mRNAs expressed in brain had unusually long 3′UTRs, and mean 3′UTR length was higher for genes involved in development, morphogenesis and signal transduction, suggesting added complexity of UTR-based regulation for these genes. Our results support a model in which variable exterior components feed into a large, densely connected core composed of ubiquitously expressed intracellular proteins.

Control of Glutamate Receptor 2 (GluR2) Translational Initiation by Its Alternative 3′ Untranslated Regions

Four major glutamate receptor 2 (GluR2) transcripts differing in size (approximately 4 and approximately 6 kilobases) due to alternative 3' untranslated regions (UTRs), and also containing alternative 5'UTRs, exist in the brain. Both the long 5'UTR and long 3'UTR repress translation of GluR2 mRNA; repression by the 3'UTR is relieved after seizures. To understand the mechanism of translational repression, we used rabbit reticulocyte lysates as an in vitro translation system to examine the expression profiles of firefly reporter mRNAs bearing alternative combinations of GluR2 5'UTR and 3'UTR in the presence of inhibitors of either translational elongation or initiation. Translation of reporter mRNAs bearing the long GluR2 3'UTR was insensitive to low concentrations of the translation elongation inhibitors cycloheximide (0.7-70 nM) and anisomycin (7.5-750 nM), in contrast to a reporter bearing the short 3'UTR, which was inhibited. These data suggest that the rate-limiting step for translation of GluR2 mRNA bearing the long 3'UTR is not elongation. Regardless of the GluR2 UTR length, translation of all reporter mRNAs was equally sensitive to desmethyl-desamino-pateamine A (0.2-200 nM), an initiation inhibitor. Kasugamycin, which can facilitate recognition of certain mRNAs by ribosomes leading to alternative initiation, had no effect on translation of a capped reporter bearing both short 5'UTR and short 3'UTR, but increased the translation rate of reporters bearing either the long GluR2 5'UTR or long 3'UTR. Our findings suggest that both the long 5'UTR and long 3'UTR of GluR2 mRNA repress translation at the initiation step.

Genome-Wide Identification of Alternative Splice Forms Down-Regulated by Nonsense-Mediated mRNA Decay in Drosophila

Alternative mRNA splicing adds a layer of regulation to the expression of thousands of genes in Drosophila melanogaster. Not all alternative splicing results in functional protein; it can also yield mRNA isoforms with premature stop codons that are degraded by the nonsense-mediated mRNA decay (NMD) pathway. This coupling of alternative splicing and NMD provides a mechanism for gene regulation that is highly conserved in mammals. NMD is also active in Drosophila, but its effect on the repertoire of alternative splice forms has been unknown, as has the mechanism by which it recognizes targets. Here, we have employed a custom splicing-sensitive microarray to globally measure the effect of alternative mRNA processing and NMD on Drosophila gene expression. We have developed a new algorithm to infer the expression change of each mRNA isoform of a gene based on the microarray measurements. This method is of general utility for interpreting splicing-sensitive microarrays and high-throughput sequence data. Using this approach, we have identified a high-confidence set of 45 genes where NMD has a differential effect on distinct alternative isoforms, including numerous RNA–binding and ribosomal proteins. Coupled alternative splicing and NMD decrease expression of these genes, which may in turn have a downstream effect on expression of other genes. The NMD–affected genes are enriched for roles in translation and mitosis, perhaps underlying the previously observed role of NMD factors in cell cycle progression. Our results have general implications for understanding the NMD mechanism in fly. Most notably, we found that the NMD–target mRNAs had significantly longer 3′ untranslated regions (UTRs) than the nontarget isoforms of the same genes, supporting a role for 3′ UTR length in the recognition of NMD targets in fly.

Molecular characterization and expression profiles of cyclin A and cyclin B during ovarian development of the giant tiger shrimp Penaeus monodon

The meiotic maturation of oocytes is regulated by maturation promoting factor (MPF), a complex of cdc2 (Cdk1) and cyclin B and other Cdk/cyclin complexes. To better understand molecular aspects governing reproductive maturation of the giant tiger shrimp ( Penaeus monodon), the full length cDNAs and genomic organization of cyclins A and B ( PMCyA and PMCyB) were characterized. A single form of PMCyA contained an open reading frame (ORF) of 1326 bp corresponding to a deduced protein of 441 amino acids. Its genomic sequence contained 5 exons, 4 introns and untranslated regions (UTRs) spanning 2586 bp in length. In contrast, PMCyB possessed three isoforms with an identical ORF of 1206 bp (401 amino acids) but three different 3′ UTR lengths of 416, 543 and 1117 bp, respectively. Their respective genomic sequences were composed of 8 exons, 7 introns and UTRs covering 4181, 4307 and 4940 bp. Expression levels of both PMCyA and PMCyB in ovaries of broodstock were much greater than those of juveniles ( P < 0.05). During ovarian development and after spawning of normal shrimp broodstock, PMCyA was not differentially expressed ( P > 0.05) whereas the level of PMCyB in stage IV was greater than that of stage I ovaries ( P < 0.05). Unilateral eyestalk ablation, a technique commonly used to induce spawning in P. monodon female brooders, had no effects on transcription of PMCyB ( P > 0.05) but resulted in a lower expression of PMCyA at stage IV of ovarian development of this economically important species ( P < 0.05).

A Test of the Null Model for 5' UTR Evolution Based on GC Content

Eukaryotic mRNAs are headed by a stretch of noncoding sequence, the 5' untranslated region (UTR). It has been proposed that the length of 5' UTRs is selectively neutral and evolves under a process of stochastic destruction and recruitment of core promoter elements, combined with selection against the premature initiation of translation. We test this null model by investigating whether 5' UTR length varies with genomic GC content, an implicit prediction of the model. Using simulations, we show that the null model predicts a positive relationship between GC content and UTR length for genes regulated by a TATA box. Although this prediction is borne out qualitatively in genomic data from yeast, fruit flies, and humans, we find marked quantitative discrepancies. We conclude that UTR length may be shaped to some degree by the forces considered in the null model but that the model fails to provide a complete explanation for UTR length evolution.