UTR Structures Research Articles

5′UTR G-quadruplex structure enhances translation in size dependent manner

Translation initiation in bacteria is frequently regulated by various structures in the 5′ untranslated region (5′UTR). Previously, we demonstrated that G-quadruplex (G4) formation in non-template DNA enhances transcription. In this study, we aim to explore how G4 formation in mRNA (RG4) at 5′UTR impacts translation using a T7-based in vitro translation system and in E. coli. We show that RG4 strongly promotes translation efficiency in a size-dependent manner. Additionally, inserting a hairpin upstream of the RG4 further enhances translation efficiency, reaching up to a 12-fold increase. We find that the RG4-dependent effect is not due to increased ribosome affinity, ribosome binding site accessibility, or mRNA stability. We propose a physical barrier model in which bulky structures in 5′UTR biases ribosome movement toward the downstream start codon, thereby increasing the translation output. This study provides biophysical insights into the regulatory role of 5′UTR structures in in vitro and bacterial translation, highlighting their potential applications in tuning gene expression.

A novel splicing variant in MICAL-1 gene is associated with epilepsy

Germline MICAL1 defects have been rarely reported in patients with epilepsy and the genotype-phenotype association remains unclear. In this study, the patient was a 4.6 years old girl who presented with onset of recurrent focal seizures with onset at age 3.4 years. EEG showed abnormal δ-wave activity in the right central and middle temporal lobe. Trio WES showed a novel heterozygous variant c.-43-1G > A in the MICAL1 gene in the patient and her normal mother. Minigene verified two abnormal transcripts due to the mutation, which was predicted to interrupt 5′UTR structures of MICAL1. The patient was clinically diagnosed with benign childhood epilepsy with centrotemporal spike (BECTS). As far as we know, this is the first BECTS case with documented MICAL1 mutation. Novel MICAL1 variant c.-43-1G > A putatively interrupted MICAL1 translation by changing 5′UTR structures and, however, further functioning study is needed.

A novel mechanism of 5' UTR structure in promoting prokaryotic translational initiation.

Gene expression involves consecutive and delicate regulation of transcription and translation. One of the mechanisms that controls the initiation of translation is the structure of the 5’ untranslated region (5’ UTR) of mRNA. In prokaryotes, the initiation process depends on the ribosome binding site (RBS), which is a complementary sequence to the ribosomal RNA and provides an anchor of ribosome assembly. Current research has demonstrated that releasing the RBS from an aptamer or hairpin structure could resume translation. However, how structures upstream of the RBS 5’ UTR are involved in the process of translational control is poorly understood. Here we applied a cell-free in vitro translation system to investigate additional upstream 5’ UTR structures such as hairpins and RNA G-quadruplexes (rG4) located far from RBS. First, we discovered that rG4 strongly promotes translation efficiency compared to non-structural RNA, and the enhancement increased with addition of a hairpin structure. After systematically evaluating the rG4 structures, we found a strong correlation (r = 0.88) between translation efficiency and the bulkiness of rG4. We also elucidated that the increase in translation is unlikely to be caused by providing additional RBS or altering the accessibility of RBS. Finally, we demonstrated that the formation of structure is essential to enhance the translation. Consequently, our results support a novel mechanism where structures in the 5’ UTR can serve as a physical blockade to force ribosome to move forward instead of falling off from 5’ site of mRNA.

Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation

How the eukaryotic 43S preinitiation complex scans along the 5' untranslated region (5' UTR) of a capped mRNA to locate the correct start codon remains elusive. Here, we directly track yeast 43S-mRNA binding, scanning, and 60S subunit joining by real-time single-molecule fluorescence spectroscopy. 43S engagement with mRNA occurs through a slow, ATP-dependent process driven by multiple initiation factors including the helicase eIF4A. Once engaged, 43S scanning occurs rapidly and directionally at ∼100 nucleotides per second, independent of multiple cycles of ATP hydrolysis by RNA helicases post ribosomal loading. Scanning ribosomes can proceed through RNA secondary structures, but 5' UTR hairpin sequences near start codons drive scanning ribosomes at start codons backward in the 5' direction, requiring rescanning to arrive once more at a start codon. Direct observation of scanning ribosomes provides a mechanistic framework for translational regulation by 5' UTR structures and upstream near-cognate start codons.

Phase separation of Ddx3xb helicase regulates maternal-to-zygotic transition in zebrafish.

Vertebrate embryogenesis involves a conserved and fundamental process, called the maternal-to-zygotic transition (MZT), which marks the switch from a maternal factors-dominated state to a zygotic factors-driven state. Yet the precise mechanism underlying MZT remains largely unknown. Here we report that the RNA helicase Ddx3xb in zebrafish undergoes liquid-liquid phase separation (LLPS) via its N-terminal intrinsically disordered region (IDR), and an increase in ATP content promotes the condensation of Ddx3xb during MZT. Mutant form of Ddx3xb losing LLPS ability fails to rescue the developmental defect of Ddx3xb-deficient embryos. Interestingly, the IDR of either FUS or hnRNPA1 can functionally replace the N-terminal IDR in Ddx3xb. Phase separation of Ddx3xb facilitates the unwinding of 5' UTR structures of maternal mRNAs to enhance their translation. Our study reveals an unprecedent mechanism whereby the Ddx3xb phase separation regulates MZT by promoting maternal mRNA translation.

A database of flavivirus RNA structures with a search algorithm for pseudoknots and triple base interactions.

MotivationThe Flavivirus genus includes several important pathogens, such as Zika, dengue and yellow fever virus. Flavivirus RNA genomes contain a number of functionally important structures in their 3′ untranslated regions (3′UTRs). Due to the diversity of sequences and topologies of these structures, their identification is often difficult. In contrast, predictions of such structures are important for understanding of flavivirus replication cycles and development of antiviral strategies.ResultsWe have developed an algorithm for structured pattern search in RNA sequences, including secondary structures, pseudoknots and triple base interactions. Using the data on known conserved flavivirus 3′UTR structures, we constructed structural descriptors which covered the diversity of patterns in these motifs. The descriptors and the search algorithm were used for the construction of a database of flavivirus 3′UTR structures. Validating this approach, we identified a number of domains matching a general pattern of exoribonuclease Xrn1-resistant RNAs in the growing group of insect-specific flaviviruses.Availability and implementationThe Leiden Flavivirus RNA Structure Database is available at https://rna.liacs.nl. The search algorithm is available at https://github.com/LeidenRNA/SRHS.Supplementary information Supplementary data are available at Bioinformatics online.

The archaeal RNA chaperone TRAM0076 shapes the transcriptome and optimizes the growth of Methanococcus maripaludis.

TRAM is a conserved domain among RNA modification proteins that are widely distributed in various organisms. In Archaea, TRAM occurs frequently as a standalone protein with in vitro RNA chaperone activity; however, its biological significance and functional mechanism remain unknown. This work demonstrated that TRAM0076 is an abundant standalone TRAM protein in the genetically tractable methanoarcheaon Methanococcus maripaludis. Deletion of MMP0076, the gene encoding TRAM0076, markedly reduced the growth and altered transcription of 55% of the genome. Substitution mutations of Phe39, Phe42, Phe63, Phe65 and Arg35 in the recombinant TRAM0076 decreased the in vitro duplex RNA unfolding activity. These mutations also prevented complementation of the growth defect of the MMP0076 deletion mutant, indicating that the duplex RNA unfolding activity was essential for its physiological function. A genome-wide mapping of transcription start sites identified many 5′ untranslated regions (5′UTRs) of 20–60 nt which could be potential targets of a RNA chaperone. TRAM0076 unfolded three representative 5′UTR structures in vitro and facilitated the in vivo expression of a mCherry reporter system fused to the 5′UTRs, thus behaving like a transcription anti-terminator. Flag-tagged-TRAM0076 co-immunoprecipitated a large number of cellular RNAs, suggesting that TRAM0076 plays multiple roles in addition to unfolding incorrect RNA structures. This work demonstrates that the conserved archaeal RNA chaperone TRAM globally affects gene expression and may represent a transcriptional element in ancient life of the RNA world.

Functional interplay between DEAD-box RNA helicases Ded1 and Dbp1 in preinitiation complex attachment and scanning on structured mRNAs in vivo.

RNA structures that impede ribosome binding or subsequent scanning of the 5'-untranslated region (5'-UTR) for the AUG initiation codon reduce translation efficiency. Yeast DEAD-box RNA helicase Ded1 appears to promote translation by resolving 5'-UTR structures, but whether its paralog, Dbp1, performs similar functions is unknown. Furthermore, direct in vivo evidence was lacking that Ded1 or Dbp1 resolves 5'-UTR structures that impede attachment of the 43S preinitiation complex (PIC) or scanning. Here, profiling of translating 80S ribosomes reveals that the translational efficiencies of many more mRNAs are reduced in a ded1-ts dbp1Δ double mutant versus either single mutant, becoming highly dependent on Dbp1 or Ded1 only when the other helicase is impaired. Such 'conditionally hyperdependent' mRNAs contain unusually long 5'-UTRs with heightened propensity for secondary structure and longer transcript lengths. Consistently, overexpressing Dbp1 in ded1 cells improves the translation of many such Ded1-hyperdependent mRNAs. Importantly, Dbp1 mimics Ded1 in conferring greater acceleration of 48S PIC assembly in a purified system on mRNAs harboring structured 5'-UTRs. Profiling 40S initiation complexes in ded1 and dbp1 mutants provides direct evidence that Ded1 and Dbp1 cooperate to stimulate both PIC attachment and scanning on many Ded1/Dbp1-hyperdependent mRNAs in vivo.

Flaviviruses Produce a Subgenomic Flaviviral RNA That Enhances Mosquito Transmission.

Mosquito-borne flaviviruses (MBFVs) are a global public health burden. MBFVs have several unique 3'UTR structures that inhibit the host RNA decay machinery to produce subgenomic flaviviral RNAs (sfRNAs). Number of sfRNA species and their relative quantities are dependent on the 3'UTR tertiary structures and can vary between tissues. Two recent in vivo studies demonstrated that sfRNA enhances mosquito transmission, resulting in increased infection rate of saliva. Transmission efficiency is determined by the immune response. First evidence points to sfRNA interference with the Toll and RNAi immune pathways. However, a more complex picture that includes flexibility in sfRNA production and interaction with immune-related proteins remains to be explored.

DDX3 Modulates Neurite Development via Translationally Activating an RNA Regulon Involved in Rac1 Activation.

The RNA helicase DDX3 is a component of neuronal granules, and its gene mutations are linked to intellectual disability (ID). Here we demonstrate that DDX3 depletion in neurons impairs neurite development by downregulating Rac1 level and activation. Moreover, DDX3 activates the translation of functionally coherent mRNAs involved in Rac1 activation including Rac1 Among the DDX3 regulon, Prkaca encodes the catalytic subunit of PKA, a potential activator of Rac1 in neurons. DDX3-modulated PKAcα and Rac1 expression tunes the strength of PKA-Rac1 signaling and thereby contributes to neurite outgrowth and dendritic spine formation. Inhibition of DDX3 activity or expression in neonatal mice impaired dendritic outgrowth and spine formation of hippocampal neurons, echoing neuronal deficits underling DDX3 mutation-associated ID. Finally, we provide evidence that DDX3 activates local protein synthesis through a 5' UTR-dependent mechanism in neurons. The novel DDX3 regulon may conduct a spatial and temporal control of Rac1 signaling to regulate neurite development. DDX3X mutations are linked to intellectual disability (ID). We provide first evidence that DDX3 is required for neurite outgrowth and dendritic spine formation in vitro and in vivo We identified a DDX3 regulon constituting functionally cohesive mRNAs involved in Rac1 signaling, which contributes to DDX3-modulated neurite development. Inhibition or ablation of DDX3 in vivo shortened neurite lengths and impaired dendritic spine formation in hippocampal neurons, reflecting the prevalence of ID-associated DDX3X mutations in the helicase domain. Mechanistically, DDX3 activates local protein synthesis of mRNAs sharing similar 5' UTR structures and therefore controls Rac1 signaling strength in neurites.

EIF4B stimulates translation of long mRNAs with structured 5' UTRs and low closed-loop potential but weak dependence on eIF4G.

DEAD-box RNA helicases eukaryotic translation initiation factor 4A (eIF4A) and Ded1 promote translation by resolving mRNA secondary structures that impede preinitiation complex (PIC) attachment to mRNA or scanning. Eukaryotic translation initiation factor 4B (eIF4B) is a cofactor for eIF4A but also might function independently of eIF4A. Ribosome profiling of mutants lacking eIF4B or with impaired eIF4A or Ded1 activity revealed that eliminating eIF4B reduces the relative translational efficiencies of many more genes than does inactivation of eIF4A, despite comparable reductions in bulk translation, and few genes display unusually strong requirements for both factors. However, either eliminating eIF4B or inactivating eIF4A preferentially impacts mRNAs with longer, more structured 5' untranslated regions (UTRs). These findings reveal an eIF4A-independent role for eIF4B in addition to its function as eIF4A cofactor in promoting PIC attachment or scanning on structured mRNAs. eIF4B, eIF4A, and Ded1 mutations also preferentially impair translation of longer mRNAs in a fashion mitigated by the ability to form closed-loop messenger ribonucleoprotein particles (mRNPs) via eIF4F-poly(A)-binding protein 1 (Pab1) association, suggesting cooperation between closed-loop assembly and eIF4B/helicase functions. Remarkably, depleting eukaryotic translation initiation factor 4G (eIF4G), the scaffold subunit of eukaryotic translation initiation factor 4F (eIF4F), preferentially impacts short mRNAs with strong closed-loop potential and unstructured 5' UTRs, exactly the opposite features associated with hyperdependence on the eIF4B/helicases. We propose that short, highly efficient mRNAs preferentially depend on the stimulatory effects of eIF4G-dependent closed-loop assembly.

Bioactive Peptides in Cancer: Therapeutic Use and Delivery Strategies

Bioactive Peptides in Cancer: Therapeutic Use and Delivery Strategies

Role of HIV-1 RNA and protein determinants for the selective packaging of spliced and unspliced viral RNA and host U6 and 7SL RNA in virus particles.

HIV-1 particles contain RNA species other than the unspliced viral RNA genome. For instance, viral spliced RNAs and host 7SL and U6 RNAs are natural components that are non-randomly incorporated. To understand the mechanism of packaging selectivity, we analyzed the content of a large panel of HIV-1 variants mutated either in the 5′UTR structures of the viral RNA or in the Gag-nucleocapsid protein (GagNC). In parallel, we determined whether the selection of host 7SL and U6 RNAs is dependent or not on viral RNA and/or GagNC. Our results reveal that the polyA hairpin in the 5′UTR is a major packaging determinant for both spliced and unspliced viral RNAs. In contrast, 5′UTR RNA structures have little influence on the U6 and 7SL RNAs, indicating that packaging of these host RNAs is independent of viral RNA packaging. Experiments with GagNC mutants indicated that the two zinc-fingers and N-terminal basic residues restrict the incorporation of the spliced RNAs, while favoring unspliced RNA packaging. GagNC through the zinc-finger motifs also restricts the packaging of 7SL and U6 RNAs. Thus, GagNC is a major contributor to the packaging selectivity. Altogether our results provide new molecular insight on how HIV selects distinct RNA species for incorporation into particles.

A potential regulatory role for mRNA secondary structures within the prothrombin 3'UTR

The distal 3'UTR of prothrombin mRNA exhibits significant sequence heterogeneity reflecting an inexact 3′-cleavage/polyadenylation reaction. This same region encompasses a single-nucleotide polymorphism that enhances the normal post-transcriptional processing of nascent prothrombin transcripts. Both observations indicate the importance of 3'UTR structures to physiologically relevant properties of prothrombin mRNA. Using a HepG2-based model system, we mapped both the primary structures of reporter mRNAs containing the prothrombin 3'UTR, as well as the secondary structures of common, informative 3'UTR processing variants. A chromatographic method was subsequently employed to assess the effects of structural heterogeneities on the binding of candidate trans-acting regulatory factors. We observed that prothrombin 3'UTRs are constitutively polyadenylated at seven or more positions, and can fold into at least two distinct stem-loop conformations. These alternate structures expose/sequester a consensus binding site for hnRNP-I/PTB-1, a trans-acting factor with post-transcriptional regulatory properties. hnRNP-I/PTB-1 exhibits different affinities for the alternate 3'UTR secondary structures in vitro, predicting a corresponding regulatory role in vivo. These analyses demonstrate a critical link between the structure of the prothrombin 3'UTR and its normal function, providing a basis for further investigations into the molecular pathophysiology of naturally occurring polymorphisms within this region.

RNA secondary structure of the feline immunodeficiency virus 5′UTR and Gag coding region

The 5′ untranslated region (5′UTR) of lentiviral genomic RNA is highly structured, and is the site of multiple RNA–RNA and RNA–protein interactions throughout the viral life cycle. The 5′UTR plays a critical role during transcription, translational regulation, genome dimerization, reverse transcription priming and encapsidation. The 5′UTR structures of human lentiviruses have been extensively studied, yet the respective role and conformation of each domain is still controversial. To gain insight into the structure-function relationship of lentiviral 5′UTRs, we modelled the RNA structure of the feline immunodeficiency virus (FIV), a virus that is evolutionarily distant from the primate viruses. Through combined chemical and enzymatic structure probing and a thorough phylogenetic study, we establish a model for the secondary structure of the 5′UTR and Gag coding region. This work highlights properties common to all lentiviruses, like the primer binding site structure and the presence of a stable stem-loop at the 5′ extremity. We find that FIV has also evolved specific features, including a long stem loop overlapping the end of the 5′UTR and the beginning of the coding region. In addition, we observed footprints of Gag protein on each side of the initiation codon, this sheds light on the role of the sequences required for encapsidation.

Upf1/Upf2 Regulation of 3′ Untranslated Region Splice Variants of AUF1 Links Nonsense-Mediated and A+U-Rich Element-Mediated mRNA Decay

AUF1 is an RNA-binding protein that targets mRNAs containing A+U-rich elements (AREs) for rapid cytoplasmic turnover. Alternative pre-mRNA splicing produces five variants of AUF1 mRNA that differ in the composition of their 3'-untranslated regions (3'-UTRs). Previous work suggested that this heterogeneity in 3'-UTR sequence could regulate AUF1 expression by two potential mechanisms. First, AUF1 may regulate its own expression by binding to AREs in 3'-UTR splice variants that retain intron 9. The second potential mechanism, and the focus of this report, is regulation of a subset of 3'-UTR splice variants by the nonsense-mediated mRNA decay (NMD) pathway. Two of the five AUF1 mRNA 3'-UTR variants position the translational termination codon more than 50 nucleotides upstream of an exon-exon junction, creating a potential triggering signal for NMD in mammalian cells. Disruption of cellular NMD pathways by RNA interference-mediated knockdown of Upf1/Rent1 or Upf2/Rent2 or transfection of a dominant-negative Upf1 mutant specifically enhanced expression of these two candidate NMD substrate mRNAs in cells, involving stabilization of each transcript. Ribonucleoprotein immunoprecipitation experiments revealed that both Upf1 and Upf2 can associate with an NMD-sensitive AUF1 mRNA 3'-UTR variant in cells. Finally, quantitation of AUF1 mRNA 3'-UTR splice variants during murine embryonic development showed that the expression of NMD-sensitive AUF1 mRNAs is specifically enhanced as development proceeds, contributing to dynamic changes in AUF1 3'-UTR structures during embryogenesis. Together, these studies provide the first evidence of linkage between the nonsense- and ARE-mediated mRNA decay pathways, which may constitute a new mechanism regulating the expression of ARE-containing mRNAs.

Molecular, genetic, and morphological markers during persistence of RNA-containing hepatitis C virus in the body.

A comparison of 5'-UTR structures in Siberian isolates of hepatitis C virus showed that the majority of genotyped samples had 1b genotype. A correlation was found between HLA antigens and their stable complexes and clinical manifestations of hepatitis C. We revealed immunogenetic criteria of organism's resistance or susceptibility to hepatitis C virus. The presence of hepatitis C virus RNA in the serum determined by the reverse transcription polymerase chain reaction was accompanied by the appearance of pathomorphological signs characteristic of liver tissue infection, but did not correspond to the severity of fibrosis.