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PubMed2025

Transcriptome-Wide Analysis of the 5' Cap Status of RNA Using 5' Monophosphate-Dependent Exonuclease Digestion and RNA Sequencing.

Eukaryotic mRNAs carry an N7-methylguanosine (m7G) cap structure at their 5' extremity, which protects them from the degradation by 5'-3' exoribonucleases and plays a pivotal role in mRNA metabolism, promoting splicing, nuclear export, and translation. Decapping, the enzymatic process that removes this structure, is a key event during cytoplasmic mRNA 5'-3' decay, leading to the degradation of the transcript body by Xrn1. In this chapter, we describe a procedure to assess the cap status of RNA at the transcriptome level. It is based on a treatment of total RNA extracts with a 5' monophosphate-dependent exonuclease, which like Xrn1 specifically degrades decapped RNAs harboring 5' monophosphate extremities, but not RNAs with intact m7G cap. The digested RNAs are then analyzed by RNA sequencing.

Exoribonucleases↗

PubMed2025

Establishment of reverse genetics systems for Colorado tick fever virus.

The Colorado tick fever virus (CTFV), which has 12-segmented double-stranded RNA genomes, is a pathogenic arbovirus that causes severe diseases in humans. However, little progress has been made in the analysis of replication mechanisms and pathogenicity. This virological constraint is due to the absence of a reverse genetics system for CTFV; therefore, we aimed to establish the system. Initially, the efficacy of CTFV replication was investigated in various cell lines. CTFV was found to grow in many cell types derived from different hosts and organs. Subsequently, BHK-T7 cells stably expressing T7 RNA polymerase were transfected with plasmids encoding each of the 12 CTFV gene segments, expression plasmids encoding all CTFV proteins, and a vaccinia virus RNA-capping enzyme. Following transfection, the cells were co-cultured with Vero or HeLa cells. Using this system, we rescued monoreassortants and recombinant viruses harboring peptide-tagged viral proteins. Furthermore, an improved system using Expi293F cells expressing T7 RNA polymerase was established, which enabled the generation of recombinant reporter CTFVs. In conclusion, these reverse genetics systems for CTFV will greatly contribute to the understanding of viral replication mechanisms, pathogenesis, and transmission, ultimately facilitating the development of rational treatments and candidate vaccines.

Animals↗

PubMed2022

CMTr mediated 2'-O-ribose methylation status of cap-adjacent nucleotides across animals.

Cap methyltransferases (CMTrs) O methylate the 2' position of the ribose (cOMe) of cap-adjacent nucleotides of animal, protist, and viral mRNAs. Animals generally have two CMTrs, whereas trypanosomes have three, and many viruses encode one in their genome. In the splice leader of mRNAs in trypanosomes, the first four nucleotides contain cOMe, but little is known about the status of cOMe in animals. Here, we show that cOMe is prominently present on the first two cap-adjacent nucleotides with species- and tissue-specific variations in Caenorhabditis elegans, honeybees, zebrafish, mouse, and human cell lines. In contrast, Drosophila contains cOMe primarily on the first cap-adjacent nucleotide. De novo RoseTTA modeling of CMTrs reveals close similarities of the overall structure and near identity for the catalytic tetrad, and for cap and cofactor binding for human, Drosophila and C. elegans CMTrs. Although viral CMTrs maintain the overall structure and catalytic tetrad, they have diverged in cap and cofactor binding. Consistent with the structural similarity, both CMTrs from Drosophila and humans methylate the first cap-adjacent nucleotide of an AGU consensus start. Because the second nucleotide is also methylated upon heat stress in Drosophila, these findings argue for regulated cOMe important for gene expression regulation.

Animals↗

PubMed2026

Altered neuronal start codon stringency favors cap-independent repeat-associated non-AUG translation.

Intronic GGGGCC repeat expansions in C9orf72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This expansion supports a non-canonical form of translational initiation known as repeat-associated non-AUG (RAN) translation to produce toxic dipeptide repeat proteins that contribute to neurodegeneration. Here, we find that the efficiency of RAN translation and its dependency on the 5' 7-methylguanosine mRNA cap are variable across cell types, with both rodent neurons and human iNeurons favoring cap-independent RAN translation from two distinct repeats (CGG and GGGGCC) across multiple reading frames. Treatment with an eIF4E inhibitor that blocks cap-dependent translation enhances RAN translation specifically in neurons. Intriguingly, cap-independent RAN translation exhibits less reliance on near-cognate codons for initiation than cap-dependent RAN translation. This finding led us to identify a surprising global alteration in neuronal start codon stringency as a contributor to the relatively higher cap-independent RAN translation in this cell type. This effect correlates with cytoplasmic redistribution of eIF1 in neurons and is reversed with overexpression of the eukaryotic initiation factor eIF5, which relaxes start codon stringency and preferentially enhances cap-dependent RAN translation. Together, these findings reveal several neuron-specific features of translational regulation that favor cap-independent RAN translation with implications for nucleotide repeat expansion disorder pathogenesis.

Neurons↗

PubMed2025

RNase III cleavage sites spread across splice junctions enforce sequential snoRNA processing.

Small nucleolar RNAs (snoRNAs) are a class of eukaryotic non-coding RNA molecules whose precursor transcripts are capped and polyadenylated. However, these end modifications are detrimental to snoRNA function and must be removed, a process typically involving excision from introns and/or endonucleolytic cleavage. For RNA precursors that host multiple snoRNAs, the sequence of maturation events is potentially important, but not well understood. Here, we report a new mode of maturation concerning snoRNA pairs that are co-hosted in the intron and the adjacent 3' exon of a precursor transcript. For a snoRNA pair with this arrangement in Schizosaccharomyces pombe, we found that the sequence surrounding an exon-exon junction within their precursor transcript folds into a hairpin after splicing of the intron. This hairpin recruits the RNase III ortholog Pac1, which participates in the maturation of the downstream snoRNA by cleaving the precursor. Our findings suggest that conditional RNase III cleavage signals hidden in an exon-exon junction evolved to enforce sequential snoRNA processing. Sequence analysis suggests that this mechanism is conserved in animals and plants.

RNA, Small Nucleolar↗

PubMed2003

Inhibition of the protein kinase PKR by the internal ribosome entry site of hepatitis C virus genomic RNA.

Translation of the hepatitis C genome is mediated by internal ribosome entry on the structurally complex 5' untranslated region of the large viral RNA. Initiation of protein synthesis by this mechanism is independent of the cap-binding factor eIF4E, but activity of the initiator Met-tRNA(f)-binding factor eIF2 is still required. HCV protein synthesis is thus potentially sensitive to the inhibition of eIF2 activity that can result from the phosphorylation of the latter by the interferon-inducible, double-stranded RNA-activated protein kinase PKR. Two virally encoded proteins, NS5A and E2, have been shown to reduce this inhibitory effect of PKR by impairing the activation of the kinase. Here we present evidence for a third viral strategy for PKR inhibition. A region of the viral RNA comprising part of the internal ribosome entry site (IRES) is able to bind to PKR in competition with double-stranded RNA and can prevent autophosphorylation and activation of the kinase in vitro. The HCV IRES itself has no PKR-activating ability. Consistent with these findings, cotransfection experiments employing a bicistronic reporter construct and wild-type PKR indicate that expression of the protein kinase is less inhibitory towards HCV IRES-driven protein synthesis than towards cap-dependent protein synthesis. These data suggest a dual function for the viral IRES, with both a structural role in promoting initiation complex formation and a regulatory role in preventing inhibition of initiation by PKR.

Animals↗

PubMed2026

Host ESCRT machinery orchestrates the assembly of tomato spotted wilt virus ribonucleoproteins.

The genomic RNA of negative-strand RNA viruses is encapsidated by nucleocapsid proteins and associates with RNA polymerase to form a ribonucleoprotein (RNP) complex. Lacking both a 5' cap and a 3' poly (A) tail, viral RNAs are highly unstable and prone to degradation by cellular nucleases. Therefore, newly synthesized genomic and complementary-strand RNAs must be rapidly protected through RNP formation. However, the molecular mechanisms governing RNP assembly in cytoplasm-replicating negative-strand RNA viruses remain largely unknown. Here, we screened a yeast knockout library and isolated mutants in several components of the endosomal sorting complexes required for transport (ESCRT) genes that affected RNA replication of tomato spotted wilt virus (TSWV). In wild-type (WT) yeast cells, TSWV nucleocapsid (N) and RNA polymerase (L) proteins colocalize at the trans-Golgi network (TGN) in a replicon-RNA-dependent manner, suggesting that TSWV RNPs accumulate at the TGN. However, in the snf7Δ, bro1Δ, and doa4Δ mutant cells, N localization to TGN and RNP formation were impaired. Another RNA replication-defective mutant, vps36Δ, showed normal N localization, and SNF7, BRO1, and DOA4 were recruited to the TGN by TSWV N or L proteins, implying that the ESCRT components have additional roles in TSWV RNA replication beyond facilitating N transport. These findings suggest that ESCRT components play multifaceted roles in TSWV RNA replication, including the intracellular transport of N to the TGN-where RNA replication takes place-thereby ensuring accurate and efficient RNP assembly.

Tospovirus↗

PubMed2026

Single-nucleotide transcription start sites profiling via Nascent Strand-Specific RNA sequencing uncovers IFN-γ-induced promoter dynamics.

Transcriptional regulation is a highly dynamic process in which nascent RNAs provide the most immediate readout of transcriptional activity. Precise mapping of transcription start sites (TSSs) is therefore critical for understanding promoter architecture and gene regulation, yet remains technically challenging. Here, we introduce Nascent Strand-Specific RNA sequencing (NSS-seq), a robust and streamlined method for genome-wide profiling of the capped 5' ends of nascent RNAs. By directly capturing transcription initiation events, NSS-seq overcomes the temporal delay inherent to conventional RNA-seq and enables time-resolved interrogation of transcriptional dynamics. Applied to interferon-γ (IFN-γ)-stimulation, NSS-seq uncovers previously unrecognized IFN-γ-responsive genes and transient transcription factor activation patterns underlying interferon-mediated tumor-suppressive functions. Together, NSS-seq provides a cost-effective and technically accessible platform for dissecting promoter-level regulatory dynamics during cellular responses.

Promoter Regions, Genetic↗

PubMed2026

Impaired Glycolysis Leads to Defective Efferocytosis and Impaired Plaque Resolution in Tet2 Clonal Hematopoiesis.

BACKGROUND: Clonal hematopoiesis (CH) arising from mutations in hematopoietic genes has been identified as an important risk factor for atherosclerotic cardiovascular disease. Despite the established role of some CH mutations in promoting atherosclerosis progression, their role in clinically relevant LDL (low-density lipoprotein) lowering-induced plaque remodeling or regression has not been extensively studied. METHODS: To assess the effects of TET2 (tet methylcytosine dioxygenase 2) CH on plaque resolution, we prepared control or chimeric Tet2+/- CH mice with conditional deletion of Tet2 in hematopoietic stem cells during LDL lowering-induced plaque remodeling. After establishing atherosclerosis by Western diet feeding for 12 weeks in Ldlr-/- mice, Tet2 was deleted by tamoxifen injection, and hypercholesterolemia was either normalized to simulate clinical lipid management, or mice were continued on the Western diet. RESULTS: Unlike control mice, Tet2+/- CH mice failed to significantly reduce necrotic core area or increase fibrous cap thickness and showed impaired macrophage efferocytosis during LDL lowering. Single-cell RNA sequencing and gene set enrichment analysis of aortic cell populations revealed that Tet2 deficient monocyte/macrophage populations were defective in glycolysis, phagocytosis, and actin polymerization. Tet2-deficient bone marrow-derived macrophages and Tet2+/- induced pluripotent stem cell-derived human macrophages showed defective ability to sustain continuing rounds of efferocytosis. Bone marrow-derived macrophages displayed reduced apoptotic cell binding and internalization and impaired activity of Wiskott-Aldrich syndrome protein and SCAR (suppressor of cyclic AMP receptor) homolog complex mediated actin polymerization. We linked these defects to reduced anaerobic glycolysis and lactate levels and rescued them by lactate supplementation or by treatment with the HIF-1α (hypoxia-inducible factor 1α) activator molidustat. Molidustat treatment reversed the defects in necrotic core and fibrous cap formation during LDL lowering-induced plaque remodeling in Tet2+/- CH mice. Reduced plasma lactate levels were also shown in TET2 clonal hematopoiesis of indeterminate potential carriers in the UK Biobank. CONCLUSIONS: Our data identify impaired efferocytosis and glycolysis-lactate-actin polymerization pathways in advanced atherosclerosis as potential therapeutic targets to induce proresolving restructuring of the plaque immune cells and to promote beneficial atherosclerosis remodeling in subjects with TET2 CH.

LDL lowering↗

PubMed2024

An Intrinsically Disordered RNA Binding Protein Modulates mRNA Translation and Storage.

Proteins with intrinsically disordered regions (IDR) play diverse functions in regulating gene expression in the cell. Many of these proteins interact with cytoplasmic ribosomes. However, the molecular functions related to the interactions are largely unclear. In this study, using an abundant RNA-binding protein, Sbp1, with a structurally well-defined RNA recognition motif and an intrinsically disordered RGG domain as a model system, we investigated how an RNA binding protein with IDR modulates mRNA storage and translation. Using genomic and molecular approaches, we show that Sbp1 slows ribosome movement on cellular mRNAs and promotes polysome stacking or aggregation. Sbp1-associated polysomes display a ring-shaped structure in addition to a beads-on-string morphology visualized under the electron microscope, likely to be an intermediate slow translation state between actively translating polysomes and the translation-sequestered RNA granule. Moreover, the binding of Sbp1 to the 5'UTRs of mRNAs represses both cap-dependent and cap-independent translation initiation of proteins, many are functionally important for general protein synthesis in the cell. Finally, post-translational modifications at the arginine in the RGG motif change the Sbp1 protein interactome and play important roles in directing cellular mRNAs to either translation or storage. Taken together, our study demonstrates that under physiological conditions, intrinsically disordered RNA binding proteins promote polysome aggregation and regulate mRNA translation and storage using multiple distinctive mechanisms. This research also establishes a framework with which functions of other IDR-containing proteins can be investigated and defined.

RNA-Binding Proteins↗

PubMed2026

RAD54L coordinates the nucleolar DNA damage response to maintain rDNA stability.

The nucleolus is organized around actively transcribed ribosomal RNA genes (rDNA), where high RNA polymerase I (Pol I) activity creates intrinsic susceptibility to replication stress and DNA damage. Here, we identify the DNA translocase RAD54L as a critical regulator of the nucleolar DNA damage response (nDDR) to rDNA double-strand breaks (DSBs) and replication stress. We show that RAD54L localizes to the nucleolus under basal conditions and is recruited to nucleolar caps following CRISPR-Cas9-induced rDNA-DSBs to promote repair. RAD54L loss results in persistent RAD51 foci, increased nucleolar γH2AX, and micronuclei formation, indicating defective resolution of rDNA lesions and genome instability. Under baseline conditions and replication stress induced by the Pol I transcription inhibitor CX-5461, RAD54L limits the accumulation of ssDNA and coordinates nDDR signaling. We further show that rDNA-DSBs induce RNA polymerase II-dependent RNA-DNA hybrids (R-loops) at intergenic rDNA regions, which facilitate nucleolar reorganization and cap formation and repair factor recruitment. Together, these findings establish RAD54L as a key regulator that coordinates replication stress response and rDNA repair, maintaining rDNA stability and genome integrity.

DNA, Ribosomal↗

PubMed2025

Phase separation of hnRNPA1 and TERRA regulates telomeric stability.

Telomeres are the complexes composed of repetitive DNA sequences and associated proteins located at the end of chromatin. As a result of the DNA replication ending issue, telomeric DNA shortens during each cell cycle. The shelterin protein complex caps telomeric ends and forms a high-order protein-DNA structure to protect telomeric DNA. The stability of telomeres is critical for cellular function and related to the progression of many human diseases. Telomeric repeat-containing RNA (TERRA) is a noncoding RNA transcribed from telomeric DNA regions. TERRA plays an essential role in regulating and maintaining the stability of telomeres. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are RNA-binding proteins associated with complex and diverse biological processes. hnRNPA1 can recognize both TERRA and telomeric DNA. Previous research reported that hnRNPA1, TERRA, and POT1, a component of the shelterin complex, work coordinately and displace replication protein A from telomeric single-stranded DNA after DNA replication, promoting telomere capping to preserve genomic integrity. However, the detailed molecular mechanism has remained unclear for >20 years. Here, our study revealed the molecular structure through which the hnRNPA1 UP1 domain interacts with TERRA and identified critical residues on the interacting surface between UP1 and TERRA. Furthermore, we proved that nucleic acids significantly increase the phase-separating ability of hnRNPA1, while disrupting the UP1-TERRA interaction extraordinarily affects hnRNPA1 droplet formation both in vitro and in vivo. Taken together, these data reveal the molecular mechanism of the phase separation of hnRNPA1 and TERRA and the potential contribution of the droplets to maintaining genomic stability.

Heterogeneous Nuclear Ribonucleoprotein A1↗

PubMed2026

Fluorescence-Based mRNA Translation Reporter.

Herein, we describe a fluorescence reporter assay for investigating regulatory elements in the 5'-UTR of mRNA that modulate translational initiation. The canonical translation initiation process generally begins with the assembly of the eIF4F complex at the cap structure, the formation of the ternary complex, which is loaded with the initiator tRNA, and finally, the preinitiation complex. The preinitiation complex then scans the 5'-UTR to identify the authentic translation start site, which is typically characterized by an AUG codon within a sequence context known as the Kozak sequence. It is well established that the 5'-UTR varies extensively in length and contains regulatory, structural, and sequence elements. To investigate these elements, we use a green fluorescence protein (GFP)- and a luciferase-based mRNA reporter. These reporters can be quickly generated by ordering gBlocks or by PCR-based template generation for in vitro transcription, capping and polyadenylation, to produce synthetic mRNAs. These mRNAs can be transfected into cells, and expression can be monitored via GFP fluorescence or by measuring luciferase activity. In this chapter, we provide a step-by-step protocol for designing gBlocks, generating the reporter mRNA, transfecting the cells, and analyzing the results.

Genes, Reporter↗

PubMed2026

Evaluation of methacrylic resin-modified calcium silicate cements for pulpal healing using an experimental pulpitis model.

Recently, vital pulp treatment (VPT), including direct pulp capping, which preserves pulp vitality and extends the functional lifespan of teeth, has garnered significant attention. The purpose of this study was to evaluate the performance of calcium silicate cement, the gold standard for VPT, alongside hydraulic calcium silicate cement (Pro-MTA), a material with extensive evidence of effectiveness, Bis-GMA resin-modified calcium silicate cement (TH), and a newly developed material, methacrylate resin-modified calcium silicate cement (RM-MTA), in a model of pulpitis induced by caries progression. Additionally, the calcium ion (Ca2+) release capacity of these materials and their comprehensive effects on pulpal wound healing were assessed using RNA sequencing (RNA-seq). In both sound pulp models and caries-induced pulpitis models, RM-MTA and Pro-MTA exhibited similar performances. Unlike TH, they induced significant tertiary dentin formation within the dental pulp beneath the material, without any residual inflammatory cells. Inflammation was specifically assessed with a focus on M1/M2 macrophages. While the timing of Ca2+ ion release differed among the materials, the total amount released was comparable, although the release of calcium ions (Ca2+) from TH was significantly lower compared to that observed for the above materials. Moreover, comprehensive genetic analysis revealed that the expression of cell proliferation-related genes was selectively reduced in TH, suggesting that differences in resin composition may account for these variations in behavior. These findings suggest that RM-MTA induces tertiary dentin and demonstrates biocompatibility comparable to that of Pro-MTA. This makes it suitable for the treatment of both sound and mildly inflamed pulp tissues. Additionally, its resin properties are expected to enhance both mechanical performance and clinical handling.

Journal Article↗

PubMed2026

Atherosclerotic plaque fibroblasts derive from adventitial and medial Pdgfra-lineage-positive cells and predominantly maintain fibroblast identity.

AIMS: Fibroblasts are mesenchymal cells in the healthy vascular adventitia. In atherosclerosis, single-cell sequencing datasets suggest fibroblasts are abundant in plaques. However, their identity, origin, and fate during plaque progression remain unclear, which we aim to unravel here. APPROACH AND RESULTS: To robustly define fibroblast identity, origin, and fate, we employed meta-analyses of 54 single-cell RNA sequencing libraries, including murine smooth muscle cell (Myh11) and endothelial cell (EC) (Cdh5) lineage reporter mice with and without atherosclerosis; human control and atherosclerotic arteries; and murine adventitia and atherosclerotic plaques processed separately from low-density lipoprotein (LDL) receptor knockout (Ldlr-/-) mice. These meta-analyses showed that murine and human plaque fibroblast identity was robustly defined by Pdgfra, Pi16, Cygb, and Serpinf1 mRNA. Ninety-five percent of plaque fibroblasts do not derive from the Myh11 lineage, while no Cdh5-lineage-positive cells were present in the fibroblast cluster. We identified five murine arterial fibroblast subsets in atherosclerotic murine aorta: progenitor fibroblasts, matrix fibroblasts, inflammatory fibroblasts, an EC-like fibroblast subset, detected in both adventitia and plaques, and Col5a3+ fibroblasts, unique to the adventitia. We next studied fibroblast identity, origin, and fate using pseudotime analysis and Pdgfra-CreERT2/tdTomato lineage reporter mice (Pdgfra Lin+). Healthy Pdgfra Lin+ reporter mice showed predominant adventitial tdTomato expression, and infrequent medial and intimal Pdgfra Lin+ cells co-expressing MYH11 and PECAM1, respectively. The Pdgfra Lin+ plaque area increased with diet duration. Pdgfra Lin+ cells largely maintain fibroblast identity in the plaque, while <10% co-express SMC markers (MYH11, SM22α), or contribute to ACTA2+ cap cells. ECs gaining mesenchymal markers are transcriptionally distinct from Cdh5-lineage-negative fibroblasts gaining EC markers. Plaque-resident EC-like fibroblasts displayed a mesenchymal-to-endothelial transition transcriptome, which was induced in human primary fibroblasts in vitro by starvation, and dampened or reversed by IL1B, TGFB1, TGFB3, and oxidized LDL. Cross-species integration showed that all murine plaque fibroblasts were conserved in human atherosclerosis, with one additional subset partially resembling murine subsets, and three human-specific subsets. Importantly, human fibroblast subsets differentially correlated to human plaque traits, with EC-like fibroblasts correlating to plaque instability. CONCLUSION: Our results indicate that 95% of plaque-residing fibroblasts are Myh11 Lin- Plaque fibroblasts have a dual origin, predominantly adventitial Pdgfra Lin+ progenitor fibroblasts, with a minor contribution from medial Pdgfra Lin+  Myh11+ SMCs. Most plaque fibroblasts maintain fibroblast identity. Murine plaque fibroblast subsets were conserved in human atherosclerosis. EC-like fibroblasts are linked to human plaque instability. Intervening in progenitor-to-specific fibroblast transitions could present a new avenue to promote plaque stability in atherosclerosis.

Atherosclerosis↗

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

Unbiased screen of human transcriptome reveals an unexpected role of 3'UTRs in translation initiation.

Although most eukaryotic mRNAs require a 5'-cap for translation initiation, some can also be translated through a poorly studied cap-independent pathway. Here we develop a circRNA-based system and unbiasedly identify more than 10,000 sequences in the human transcriptome that contain Cap-independent Translation Initiators (CiTIs). Surprisingly, most of the identified CiTIs are located in 3'UTRs, which mainly promote translation initiation in mRNAs bearing highly structured 5'UTR. Mechanistically, CiTI recruits several translation initiation factors including eIF3 and DHX29, which in turn unwind 5'UTR structures and facilitate ribosome scanning. Functionally, we show that the translation of HIF1A mRNA, an endogenous DHX29 target, is antagonistically regulated by its 5'UTR structure and a new 3'-CiTI in response to hypoxia. Consistently, deletion of 3'-CiTI suppresses cell growth in hypoxia and tumor progression in vivo. Collectively, our study uncovers a new regulatory mode for translation where the 3'UTR actively participate in the translation initiation.

Humans↗