PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “5' Untranslated Regions”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed2025

Machine learning-based analysis of the impact of 5' untranslated region on protein expression.

The 5' untranslated region (5'UTR) plays a crucial regulatory role in messenger RNA (mRNA), with modified 5'UTRs extensively utilized in vaccine production, gene therapy, etc. Nevertheless, manually optimizing 5'UTRs may encounter difficulties in balancing the effects of various cis-elements. Consequently, multiple 5'UTR libraries have been created, and machine learning models have been employed to analyze and predict translation efficiency (TE) and protein expression, providing insights into critical regulatory features. On the one hand, these screening libraries, based on TE and mean ribosome load, struggle to accurately quantify protein expression; on the other hand, a precise method for quantifying 5'UTRs necessitates a significantly costlier library. To resolve this dilemma, we constructed a library utilizing firefly luciferase as the reporter to measure accurate protein expression. In addition, we optimized the library construction method by clustering mRNA sequences to reduce redundant data and minimize the size of the dataset. This dual strategy by increasing accuracy and reducing dataset size was found to be effective in predicting the 5'UTRs from the PC3 cell line.

5' Untranslated Regions↗
PubMed2026

Functional analysis of stem-loop structures within the SARS-CoV-2 5' untranslated region using a plasmid-based reporter system.

The 5' untranslated region (5'UTR) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains highly conserved stem-loop structures that regulate viral gene expression. This study investigated the functional contributions of selected 5'UTR stem-loop elements to reporter gene expression using a plasmid-based mammalian expression system. Five constructs were tested using a non-integrating plasmid: the wild-type (WT) 5'UTR fused to GFP under the CMV promoter, and four deletion variants (&#x394;B, &#x394;C, &#x394;D, and &#x394;E) corresponding to deletions of stem-loop 4 (SL4), SL4.5, SL5, and SL5a, respectively. Following transfection into HEK293 cells, GFP fluorescence was quantified using a fluorescence microplate reader, and relative GFP transcript abundance was assessed by RT-qPCR. Deletion of SL4 (&#x394;B) resulted in marked reduction in both fluorescence and relative transcript abundance compared to WT construct, indicating substantially reduced reporter gene expression. In contrast, deletion of SL4.5, SL5, or SL5a did not produce the pronounced reduction observed for &#x394;B, although descriptive RT-qPCR analysis indicated differences in relative transcript abundance among these variants. Statistical analysis of fluorescence data demonstrated significant differences among constructs (one-way ANOVA, p&#x2009;<&#x2009;0.05). Because the reporter assay was based on plasmid expression, the observed differences likely reflect combined contributions from transcription, transcript abundance, RNA stability, and translation rather than translation alone. These findings demonstrate that the SL4 region contributes substantially to reporter gene expression in this experimental system, whereas the remaining stem-loop regions examined exert comparatively modest effects. This study provides additional insight into the functional organization of the SARS-CoV-2 5'UTR and establishes a framework for future investigations aimed at distinguished the transcriptional, post-transcriptional, and translational contributions of individual RNA structural elements.

5' Untranslated Regions↗
PubMed1977

The nucleotide sequences of the untranslated 5' regions of human alpha- and beta-globin mRNAs.

The complete sequences of the untranslated 5' regions of human alpha- and beta-globin mRNAs were determined by sequence analysis of full-length cDNAs. The single-stranded cDNAs were digested with the restriction endonuclease Hae III, and the two 3'-terminal fragments of 75 and 132 nucleotides, complementary to the 5' termini of the alpha- and beta-globin mRNAs, respectively, were isolated and sequenced. Including the initiation codon AUG, the untranslated 5' regions of human alpha- and beta-globin mRNAs contain 41 and 54 nucleotides, respectively, and exhibit striking homologies with the corresponding sequences in the rabbit. Human alpha- and beta-globin mRNAs have five bases in the region of the initiation codon that may form base pairs with the 3' terminus of 18S rRNA. Stable secondary structures with hairpin loops can be constructed in the untranslated 5' regions.

Base Sequence↗
PubMed1978

The nucleotide sequence of the 5' untranslated region of human gamma-globin mRNA.

The nucleotide sequence of the entire 5' untranslated region of human gamma-globin mRNA has been determined. This was accomplished by analyzing complementary DNA (cRNA) synthesized from the mRNA with reverse transcriptase. The CDNA was labeled at its 3' end with 32"p using terminal deoxynucleotidyl transferase, digested with the restriction endonuclease Hae III and the end-labeled fragment isolated ans sequenced by the method of Maxam and Gilbert. Including the initiation codon AUG, the 5' untranslated region of human gamma-globin mRNA contains 57 nucleotides, compared to 41 in alpha- and 54 in beta-globin mRNA. There is very little homology between alpha and gamma sequences in the 5' region. There is considerable homology between beta- and gamma-globin mRNAs in the regions proximal and distal to the initiation codon, but the entire sequence shows less homology than the human and rabbit beta-globin mRNAs. The hexanucleotide sequence CUUCUG is found near the 5' ends of all three human globin mRNAs, suggesting a possible role of this sequence or ribosomal binding. Both guanosine and cytidine were found at the 19th nucleotide position from the 5' end of the gamma mRNA. We believe this heterogeneity arises from the difference in nucleotide sequence between the A gamma and G gamma loci.

Base Sequence↗
PubMed2002

Pestivirus internal ribosome entry site (IRES) structure and function: elements in the 5' untranslated region important for IRES function.

The importance of certain structural features of the 5' untranslated region of classical swine fever virus (CSFV) RNA for the function of the internal ribosome entry site (IRES) was investigated by mutagenesis followed by in vitro transcription and translation. Deletions made from the 5' end of the CSFV genome sequence showed that the IRES boundary was close to nucleotide 65: thus, the IRES includes the whole of domain II but no sequences upstream of this domain. Deletions which invaded domain II even to a small extent reduced activity to about 20% that of the full-length structure, and this 20% residual activity persisted with more extensive deletions until the whole of domain II had been removed and the deletions invaded the pseudoknot, whereupon IRES activity fell to zero. The importance of both stems of the pseudoknot was verified by making mutations in both sides of each stem; this severely reduced IRES activity, but the compensating mutations which restored base pairing caused almost full IRES function to be regained. The importance of the length of the loop linking the two stems of the pseudoknot was demonstrated by the finding that a reduction in length from the wild-type AUAAAAUU to AUU almost completely abrogated IRES activity. Random A-->U substitutions in the wild-type sequence showed that IRES activity was fairly proportional to the number of A residues retained in this pseudoknot loop, with a preference for clustered neighboring A residues rather than dispersed As. Finally, it was found that the sequence of the highly conserved domain IIIa loop is, rather surprisingly, not important for the maintenance of full IRES activity, although amputation of the entire domain IIIa stem and loop was highly debilitating. These results are interpreted in the light of recent models, derived from cryo-electron microscopy, of the interaction of the closely related hepatitis C virus IRES with 40S ribosomal subunits.

5' Untranslated Regions↗
PubMed1979

Nucleotide sequence of the Hind-C fragment of simian virus 40 DNA. Comparison of the 5'-untranslated region of wild-type virus and of some deletion Mutants.

We report here the nucleotide sequence of the wild-type simian virus 40 (strain 776) restriction fragment Hind-C-P1 DNA and of the homologous region of various mutant DNAs which lack part of this fragment. During this work, we detected between EcoRII fragments N and G an additional, 17-base-pair EcoRII fragment, fragment P, which had previously been overlooked. Also, an additional dTpdG dinucleotide at residues L 339--340 was observed by sequence analysis of the DNA minus (E) strand; the presence of this dinucleotide was masked on sequencing patterns of the plus strand due to the persistence (during gel electrophoresis) of some secondary structures in the strand's 5'-terminal region. These nucleotide additions raise the total length of SV40 DNA to 5243 base pairs. The longest tandemly repeated segment in SV40 DNA now extends over 72 base pairs. SV40 deletion mutants dl 893 and dl 894 and SV40 strains Rh 911 and 1801 all lack an identical 72-base-pair-long DNA segment in the Hind-C region. This deletion corresponds precisely to one of the two aforementioned large tandemly repeated sequences. Mutant dl 895 lacks 66 base pairs, 63 of which are part of the former repetition. All these mutants, except dl 895, very probably were generated by an intramolecular, homologous recombination event. The 40-base-pair deletion in mutant dl 1811 includes the major capping site of SV40 late RNA. dl 1812 lacks only three base pairs, which are part of the overlapping HhaI and HpaII restriction sites at position 0.725--0.726.

Base Sequence↗
PubMed2026

Genome-wide detection of human 5' UTR variants that impact protein translation.

The 5' untranslated region (5' UTR) of messenger RNAs (mRNAs) plays a central role in regulating protein synthesis initiation, particularly through the Kozak sequence and upstream open reading frames (uORFs). Genetic variants within these regulatory elements could affect translation, altering gene expression and contributing to clinical phenotypes in humans. We developed a computational method called 5ULTRA (5' Untranslated Region Annotation) for analysis of whole-exome sequencing and whole-genome sequencing data to detect, annotate, and prioritize 5' UTR variants with potential translation impact. 5ULTRA identifies single-nucleotide variants, indels, and splicing variants that affect uORFs by creating or disrupting start/stop codons and that alter Kozak sequence strength of either the uORFs or the main coding sequence. 5ULTRA incorporates recent uORF databases and provides comprehensive annotations. 5ULTRA implements a machine-learning score to prioritize candidate variants with predicted effects on translation and also provides specific mechanistic predictions. The score correlates strongly with experimentally measured protein-level effects of 5' UTR variants. We applied 5ULTRA to multiple genetics datasets across diverse disease contexts, identifying candidate variants including potential cancer-driving somatic mutations predicted to decrease ABI1 level or increase NRAS abundance; common variants associated with traits such as multiple sclerosis, lung function, and cardiovascular function, by altering protein levels of TAGAP, VRTN, and SPAAR, respectively; and rare germline variants in our cohort, including a splicing variant of RPSA leading to 5' UTR sequence alteration that causes congenital asplenia and a variant of TNF that could predispose to tuberculosis.

Humans↗
PubMed2026

Rop-Mediated Suppression of RpoS Production Increases Resistance to Nitric Oxide.

We identified the RNA-binding protein Rop, encoded on the pOSAK1 plasmid of enterohaemorrhagic Escherichia coli (EHEC), as a novel factor that enhances nitric oxide (NO) resistance, although it has previously been reported to regulate plasmid copy number. The Rop-induced increase in NO resistance was significantly reduced in several small noncoding RNA (sRNA) gene-deficient EHEC mutants. Among these sRNAs, DsrA, ArcZ, and RprA were directly involved in the translational regulation of rpoS expression, suggesting that Rop modulates rpoS expression through sRNAs. To examine this mechanism, we generated sRNA gene-deficient mutants with an additional deletion of the 5' untranslated region (5' UTR) of rpoS, which is required for translational regulation. The increase in NO resistance by Rop was restored in the double mutant, suggesting that this phenotype is mediated by Rop-dependent interactions between sRNAs and the 5' UTR of rpoS mRNA. Furthermore, Rop promoted rpoS mRNA degradation, an effect that likely suppresses RpoS production and may thereby enhance NO resistance. Finally, an hfq-deficient EHEC mutant exhibited no increase in NO resistance in the presence of Rop, indicating that Hfq is essential for Rop-mediated NO resistance.

Nitric Oxide↗
PubMed2025

Post-transcriptional control of KRAS: functional roles of 5'UTR RNA G-quadruplexes, long noncoding RNA, and hnRNPA1.

Previous studies have shown that human KRAS expression is regulated at the transcriptional level by G-quadruplex DNA structures within its promoter. Here we show an additional level of regulation involving a post-transcriptional mechanism centred on the 5'-untranslated region (5'UTR) of the messenger RNA (mRNA) characterized by G4 structures (rG4s). Long noncoding RNAs (lncRNAs) and the protein hnRNPA1 are also involved in this mechanism. RIP-seq confirmed the presence of rG4s in the 5'UTR. Deletion of the rG4 region using CRISPR/Cas9 resulted in a significant increase in KRAS mRNA levels, indicating the role of the 5'UTR in controlling mRNA levels. RIP shows that hnRNPA1 is recruited to the 5'UTR, where it unfolds the rG4 structures and potentially affects mRNA stability. In addition, lncRNAs transcribed from the LINC01750 locus can hybridize to the rG4 region of 5'UTR and form RNA duplexes leading to RNase III-assisted degradation of the targeted mRNA. Activation of the LINC01750 locus with dCas9-VP64 resulted in downregulation of KRAS mRNA, whereas its suppression with dCas9-KRAB led to upregulation of both KRAS mRNA and protein. Since lncRNA-mediated regulation of mRNA appears to be a crucial aspect of cellular homeostasis and its disruption contributes to various diseases, understanding these mechanisms may&#xa0;reveal promising new therapeutic targets.

Humans↗
PubMed2026

Translational control by RPL22L1-specific ribosomes enhances DNA repair and chemoresistance.

Ribosome heterogeneity has emerged as a regulatory layer in gene expression, yet its biological roles in cancers remain poorly characterized. Here, we identify RPL22L1, a paralog of the ribosomal protein RPL22, as a key modulator of DNA damage response (DDR) in colorectal cancer cells. DNA damage induces RPL22L1 upregulation and ribosomal incorporation, forming RPL22L1-specific ribosomes. Ribosome profiling reveals that RPL22L1-containing ribosomes preferentially translate mRNAs with highly structured 5' untranslated region (5'UTR). In particular, RPL22L1 enhances the translation of ATRX through a cap-independent mechanism. ATRX subsequently recruits DNA-PKcs to DNA damage sites, thereby enhancing the DNA repair capacity. RPL22L1 loss creates exploitable DDR vulnerabilities, sensitizing cancer cells to cisplatin and PARP inhibitors in vitro and in vivo. Collectively, these findings uncover a specialized ribosome-mediated translational program in DDR and highlight RPL22L1 as a potential therapeutic target in DDR-based cancer therapy.

DNA Repair↗
PubMed2026

A-to-I RNA editing remodels 5'-UTR initiation codons to tune translational output.

A-to-I RNA editing is a prevalent post-transcriptional modification in higher eukaryotes that converts adenosine to inosine within RNA molecules. Because inosine is interpreted as guanosine during translation, editing can alter codon identity and potentially influence translation initiation signals. Here, we examined whether A-to-I editing within the 5' untranslated region (5'-UTR) can remodel upstream initiation codons and thereby tune downstream translation. Using luciferase-based reporter systems, we show that AUA-to-AUI editing generates an initiation-competent inosine-containing codon, whereas AUG-to-IUG editing markedly attenuates initiation and can relieve uORF-mediated repression. Quantitative in vitro and cellular assays establish the initiation hierarchy AUA&#x2009;<&#x2009;AUI&#x2009;<&#x2009;AUG, with IUG exhibiting strongly reduced initiation efficiency. Importantly, AUI-mediated upstream initiation did not behave like a canonical AUG-initiated uORF in the tested contexts; its effect on downstream ORF translation was modest and context-dependent. Transcriptome-wide bioinformatic analysis identified endogenous human transcripts whose 5'-UTRs harbor editing sites compatible with initiation-codon gain or attenuation. Reporter validation using native 5'-UTR sequences supports the possibility that editing-dependent initiation-codon remodeling can tune translational output in living cells, particularly through AUG-to-IUG-mediated derepression. Together, these findings establish a reporter-based framework in which A-to-I editing can remodel 5'-UTR initiation codons, while highlighting the need for endogenous protein-level and native-locus validation to determine physiological relevance.

RNA Editing↗
PubMed2026

Development of a rapid antiviral screening assay based on GFP reporter virus of bovine enterovirus.

In recent years, bovine enterovirus (BEV) has been increasingly associated with diarrhea in cattle in China, posing new challenges for disease control in the cattle industry. However, the mechanisms underlying BEV pathogenesis and virulence remain poorly understood. Infectious cDNA clones provide a powerful tool for dissecting viral replication and pathogenic mechanisms. In this study, we generated a full-length infectious cDNA clone of the BEV-F isolate HB19-1. Three overlapping fragments spanning the complete viral genome were amplified by RT-PCR and assembled downstream of a cytomegalovirus (CMV) promoter placed immediately upstream of the 5' untranslated region (5'UTR). To establish a reporter virus system, the green fluorescent protein (GFP) gene was inserted between the 5'UTR and the N terminus of VP4, followed by a 2A cleavage sequence (IKTAG) at the C terminus of GFP. The recombinant rHB19-GFP virus was successfully rescued. Growth curve analysis demonstrated that rHB19-GFP exhibited slower replication kinetics at early time points relative to the parental HB19-1 virus, with no significant difference in their peak viral titers. This GFP-expressing reporter virus enables convenient monitoring of BEV replication and provides a useful platform for antiviral screening. Using this system, we found that 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) inhibited BEV replication, suggesting its potential as an antiviral candidate. Overall, the rHB19-GFP infectious clone developed here offers a practical tool for studying BEV biology and for identifying antiviral compounds against BEV.

Animals↗
PubMed2025

Hexokinase 2 is an RNA-binding protein that regulates mRNA translation independently of glycolysis and induces melanoma cell proliferation.

Although metabolic benefits of glycolysis have been extensively described in tumor cells, the extra-metabolic functions linked to this energetic pathway in tumor growth and cell proliferation have not been clearly established yet. Recently, some key glycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase 2, were reported to regulate mRNA translation. Translational control of gene expression is considered as a critical effector in cancer biology, representing a highly promising area of research. Here, we report that Hexokinase 2 (HK2), a glucose kinase that catalyzes the first step of glycolysis at the outer mitochondrial membrane (OMM), is an RNA-binding protein (RBP) that regulates mRNA translation in melanoma cell lines. Polysome profiling experiments followed by RNA sequencing indicate that the translational regulation exerted by HK2 is partly independent of the metabolic status or the glycolytic pathway. We found that HK2 specifically regulates translation of the mRNA encoding SOX10, a transcription factor implicated in the regulation of tumor initiation, maintenance, and progression in melanoma. RNA-protein interaction assays, including CrossLinking ImmunoPrecipitation (CLIP), indicate that HK2 is an RBP whose interaction with RNA is independent of its enzymatic activity, its ability to bind glucose or its association with the OMM. HK2 directly interacts with the 5' untranslated region (5'UTR) of the SOX10 mRNA through a stem-loop RNA secondary structure. Using RNA-protein proximity ligation assays and a fluorescence-based ribosome-bound mRNA mapping method, we found that high glucose conditions, which promote the release of HK2 from the OMM, induce an increase in HK2-SOX10 mRNA interaction and SOX10 mRNA translation in the cytoplasm. We further showed that HK2-dependent SOX10 mRNA translation is involved in melanoma cell proliferation and colony formation. Collectively, our data highlight a nonmetabolic function of HK2 acting as an RBP and translation regulator.

Hexokinase↗
PubMed2026

A trainable language model with potential to modulate translation rates in non-model organisms by generating upstream untranslated region sequence libraries.

Tuning protein expression in non-model organisms is often constrained by the lack of validated genetic parts and predictive design tools. Translational tuning through the modulation of upstream untranslated regions (5'-UTRs) offers a potentially organism-agnostic route, but existing methods typically rely on mechanistic assumptions, prior knowledge that may not be available in non-model contexts, or the screening of sequence libraries. Here, we present a simple generative approach for creating synthetic 5'-UTR libraries based solely on the genomic sequence statistics of any desired organism. The method uses a sliding-window n-gram language model applied to native 5'-UTR sequences to produce novel sequences that preserve organism-specific base distributions and motifs without hard-coding specific motifs or mechanistic rules into inflexible statistical templates. We have applied this approach to the model bacterium Escherichia coli and the non-model probiotic Limosilactobacillus reuteri. Libraries of approximately 1,000 sequences were generated for each organism, from which about 100 unique sequences were experimentally tested for translation of a fluorescent reporter protein. In both organisms, the synthetic libraries yielded a broad range of translation levels from this relatively small number of tested variants. Sequences derived from an organism's own genomic statistics provided a more uniformly distributed range of translation rates in that organism than sequences derived from the other species. Correlations of individual sequence performance across the two species were weak, and thermodynamic predictions of ribosome binding strength showed very little predictive power, especially in the non-model L. reuteri. The results demonstrate that simple statistical language model approaches applied to genomic data can generate functional translational regulatory sequence libraries without detailed mechanistic knowledge or explicit reference to consensus motifs. The approach requires minimal computational resources, avoids reproducing native sequences, and can be readily applied to any organism with a sequenced genome. This strategy may lower technical barriers to expression tuning in non-model organisms.

5' Untranslated Regions↗
PubMed1978

Sequences of mouse immunoglobulin light chain genes before and after somatic changes.

We have determined the nucleotide sequences of the germ line gene as well as a corresponding somatically mutated and rearranged gene coding for a mouse immunoglobulin lambdaI type light chain. These sequencing studies were carried out on three Eco RI-DNA fragments which had been cloned from BALB/c mouse embryos or a lambdaI chainsecreting myeloma, H2020. The embryonic DNA clone Ig 99lambda contains two protein-encoding segments, one for the majority of the hydrophobic leader (L) and the other for the rest of the leader and the variable (V) region of the lambda0 chain (Cohn et al., 1974); these segments are separated by a 93 base pair (bp) intervening sequence (I-small). The coding of the V region ends with His at residue 97. The second embryonic DNA clone Ig 25lambda includes a 39 bp DNA segment (J) coding for the rest of the conventionally defined V region (that is, up to residue 110), and also contains the sequence coding for the constant (C) region approximately 1250 untranslated bp (I-large) away from the J sequence. The J sequence is directly linked with the V-coding sequence in the myeloma DNA clone, Ig 303lambda, which has the various DNA segments arranged in the following order: 5' untranslated region, L, l-small, V linked with J, l-large, C, 3' untranslated sequence. The lg 303lambda V DNA sequence codes for the V region synthesized by the H2020 myeloma and is different from the lg 99lambda V DNA sequence by only two bases. No silent base change was observed between the two DNA clones for the entire sequence spanning the 5' untranslated regions and the V-coding segments. These results confirm the previously drawn conclusion that an active complete lambdaI gene arises by somatic recombination that takes place at the ends of the V-coding DNA segment and the J sequence. No sequence homology was observed at or near the sites of the recombination.

Animals↗