Mechanism Of Translation Initiation Research Articles

The new function of circRNA: translation.

Circular RNAs (circRNAs) have been considered a special class of non-coding RNAs without 5' caps and 3' tails which are covalently closed RNA molecules generated by back splicing of mRNA. For a long time, circRNAs have been considered to be directly involved in various biological processes as functional RNA. In recent years, a variety of circRNAs have been found to have translational functions, and the resultant peptides also play biological roles in the emergence and progression of human disease. The discovery of these circRNAs and their encoded peptides has enriched genomics, helped us to study the causes of diseases, and promoted the development of biotechnology. The purpose of this review is to summarize the research progress of the detection methods, translation initiation mechanism, as well as functional mechanism of peptides encoded by circRNAs, with the goal of providing the directions for the discovery of biomarkers for diagnosis, prognosis, and therapeutic targets for human disease.

A Spontaneous RAG1 Nonsense Mutation Unveils Naturally Occurring N-Terminal Truncated RAG1 Isoforms.

The RAG1 and RAG2 proteins are essential for the assembly of Ag receptor genes in the process known as VDJ recombination, allowing for an immense diversity of lymphocyte Ag receptors. Congruent with their importance, RAG1 and RAG2 have been a focus of intense study for decades. To date, RAG1 has been studied as a single isoform; however, our identification of a spontaneous nonsense mutation in the 5' region of the mouse Rag1 gene lead us to discover N-truncated RAG1 isoforms made from internal translation initiation. Mice homozygous for the RAG1 nonsense mutation only express N-truncated RAG1 isoforms and have defects in Ag receptor rearrangement similar to human Omenn syndrome patients with truncating 5' RAG1 frameshift mutations. We show that the N-truncated RAG1 isoforms are derived from internal translation initiation start sites. Given the seemingly inactivating Rag1 mutation, it is striking that homozygous mutant mice do not have the expected SCID. We propose that evolution has garnered RAG1 and other important genes with the ability to form truncated proteins via internal translation to minimize the deleterious effects of 5' nonsense mutations. This mechanism of internal translation initiation is particularly important to consider when interpreting nonsense or frameshift mutations in whole-genome sequencing, as such mutations may not lead to loss of protein.

Circular code motifs in the ribosome: a missing link in the evolution of translation?

The origin of the genetic code remains enigmatic five decades after it was elucidated, although there is growing evidence that the code coevolved progressively with the ribosome. A number of primordial codes were proposed as ancestors of the modern genetic code, including comma-free codes such as the RRY, RNY, or GNC codes (R = G or A, Y = C or T, N = any nucleotide), and the X circular code, an error-correcting code that also allows identification and maintenance of the reading frame. It was demonstrated previously that motifs of the X circular code are significantly enriched in the protein-coding genes of most organisms, from bacteria to eukaryotes. Here, we show that imprints of this code also exist in the ribosomal RNA (rRNA). In a large-scale study involving 133 organisms representative of the three domains of life, we identified 32 universal X motifs that are conserved in the rRNA of >90% of the organisms. Intriguingly, most of the universal X motifs are located in rRNA regions involved in important ribosome functions, notably in the peptidyl transferase center and the decoding center that form the original “proto-ribosome.” Building on the existing accretion models for ribosome evolution, we propose that error-correcting circular codes represented an important step in the emergence of the modern genetic code. Thus, circular codes would have allowed the simultaneous coding of amino acids and synchronization of the reading frame in primitive translation systems, prior to the emergence of more sophisticated start codon recognition and translation initiation mechanisms.

IRES-mediated cap-independent translation, a path leading to hidden proteome.

Most eukaryotic mRNAs are translated in a cap-dependent fashion; however, under stress conditions, the cap-independent translation driven by internal ribosomal entry sites (IRESs) can serve as an alternative mechanism for protein production. Many IRESs have been discovered from viral or cellular mRNAs to promote ribosome assembly and initiate translation by recruiting different trans-acting factors. Although the mechanisms of translation initiation driven by viral IRESs are relatively well understood, the existence of cellular IRESs is still under debate due to the limitations of translation reporter systems used to assay IRES activities. A recent screen identified > 1000 putative IRESs from viral and human mRNAs, expanding the scope and mechanism for cap-independent translation. Additionally, a large number of circular RNAs lacking free ends were identified in eukaryotic cells, many of which are found to be translated through IRESs. These findings suggest that IRESs may play a previously unappreciated role in driving translation of the new type of mRNA, implying a hidden proteome produced from cap-independent translation.

IRESpy: an XGBoost model for prediction of internal ribosome entry sites

BackgroundInternal ribosome entry sites (IRES) are segments of mRNA found in untranslated regions that can recruit the ribosome and initiate translation independently of the 5′ cap-dependent translation initiation mechanism. IRES usually function when 5′ cap-dependent translation initiation has been blocked or repressed. They have been widely found to play important roles in viral infections and cellular processes. However, a limited number of confirmed IRES have been reported due to the requirement for highly labor intensive, slow, and low efficiency laboratory experiments. Bioinformatics tools have been developed, but there is no reliable online tool.ResultsThis paper systematically examines the features that can distinguish IRES from non-IRES sequences. Sequence features such as kmer words, structural features such as QMFE, and sequence/structure hybrid features are evaluated as possible discriminators. They are incorporated into an IRES classifier based on XGBoost. The XGBoost model performs better than previous classifiers, with higher accuracy and much shorter computational time. The number of features in the model has been greatly reduced, compared to previous predictors, by including global kmer and structural features. The contributions of model features are well explained by LIME and SHapley Additive exPlanations. The trained XGBoost model has been implemented as a bioinformatics tool for IRES prediction, IRESpy (https://irespy.shinyapps.io/IRESpy/), which has been applied to scan the human 5′ UTR and find novel IRES segments.ConclusionsIRESpy is a fast, reliable, high-throughput IRES online prediction tool. It provides a publicly available tool for all IRES researchers, and can be used in other genomics applications such as gene annotation and analysis of differential gene expression.

The Role of miRNA in Differentiation, Cell Proliferation, and Pathogenesis of Poultry Diseases

To date, several noncoding RNAs are known that play a prominent role in the processes of transcription, translation, and structural conformation of RNA. By binding miRNA to 3'-untranslated regions, mRNA regulates gene expression in animals through inhibition of translation initiation, elongation, and other mechanisms. There is evidence for the differential expression of miRNA that regulates transcription with the inclusion of several stages of myoblast proliferation, differentiation, and apoptosis. These molecules play a significant role in cell differentiation, proliferation, apoptosis, and tumorigenesis. Some miRNAs are known to target 100–200 genes. Polymorphisms of miRNA genes, especially in the competent regions of the genome, can be biomarkers for phenotypic traits important for breeding birds. The findings confirm the key role of miRNA in controlling the metabolic switch that occurs between the embryo development and the chick hatching. Differentially expressed miRNAs and their possible target genes are involved in egg production functions. The available miRNA data complement our understanding of the molecular genetic control underlying abdominal fat accumulation and myogenesis in chickens. Hundreds of differentially expressed miRNAs from individuals differing in body weight were found. A new understanding into the functions of miRNA during chicken gonads’ rapid development is being formed. Hundreds of miRNAs expressed by hypothalamic genes and involved in the initial phase of rapid gonad growth have been identified. There is increasingly clear evidence that miRNA play a major role in regulating the innate immune response and are important effectors in complex host–pathogen interaction networks for salmonellosis, Marek’s disease, carcinogenic, and other diseases. Many of the miRNAs are associated with cell proliferation, apoptosis, and tumorigenesis. Bacterial pathogens are able to modulate the expression of the host miRNAs and affect the regulation of miRNAs and the outcome of infection. Several miRNAs are induced by TLR activation in innate immune cells and target the 3′-untranslated regions of the mRNA encoding the components of the TLR signaling system. Modern genome-editing tools suggest an artificial increase in the diversity of miRNAs and an increased use of miRNAs for directional action. Structure determination of RNA, RNA–RNA, RNA–DNA, and ribonucleoprotein complexes is becoming a rapidly developing area requiring the development of new technologies.

Molecular Mechanisms of Cue‐Induced Translation Regulation

Precise tuning of local gene expression is essential for proper neural development. In particular, environmental cues spatially and temporally direct neuron growth by stimulating or repressing protein synthesis. The integral membrane protein, deleted in colorectal cancer, or DCC, detects the environmental growth cue netrin‐1, and transduces the growth signal to a variety of intracellular responses, including activating protein synthesis. DCC is primed to function in this way; in the absence of netrin‐1, translation machinery is tethered to its C‐terminal tail (C‐tail) and silenced. Once netrin‐1 binds DCC, the receptor dimerizes, translation machinery is released, and protein synthesis commences. How DCC's C‐tail binds to the protein synthesis machinery and prevents translation in the absence of netrin‐1 is unknown. The C‐tail is a 35 kDa protein that is composed of three domains (P1–3) and is predicted 91% unstructured. Dimerization is facilitated by the P3 domain, while the P1 domain binds to the 60S subunit protein, ribosomal protein L5 (rpL5). Titrating the full‐length C‐tail into translation‐competent lysates, we found that the tail specifically inhibits eukaryotic translation, and inhibits protein synthesis of messages that use both canonical and non‐canonical translation initiation mechanisms. Hence, we propose that the C‐tail functions primarily through binding the ribosome, not eukaryotic initiation factors. We have evidence from cosedimentation and in vitro binding studies to show that the full‐length tail binds to the small and large subunits, as well as the 80S ribosome in the absence of mRNA. Binding and luciferase reporter assays show that the P3 dimerization domain is dispensable for translation inhibition, and the P1 domain is necessary but not sufficient to bind the ribosome and decrease translation. Thus the P2 domain and/or linker sequence(s) are also important for function. Collectively, our results suggest that DCC's C‐tail inhibits translation by effectively preventing ribosomes from binding to messages. We hypothesize the unstructured C‐tail precludes mRNA binding by interacting with the decoding groove of the ribosome until receptor dimerization releases 80S ribosomes. Thus chemoattraction via netrin‐induced DCC receptor dimerization might lead to a global (not message‐specific) increase in localized protein synthesis at axonal growth cones.Support or Funding InformationMSU Denver Provost Seed GrantMSU Denver College of Letters, Arts and Sciences Dean's Seed GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Eukaryotic initiation factor 5B (eIF5B) provides a critical cell survival switch to glioblastoma cells via regulation of apoptosis

Physiological stress conditions attenuate global mRNA translation via modifications of key eukaryotic initiation factors. However, non-canonical translation initiation mechanisms allow cap-independent translation of certain mRNAs. We have previously demonstrated that eIF5B promotes cap-independent translation of the mRNA encoding the antiapoptotic factor, XIAP, during cellular stress. Here, we show that depletion of eIF5B sensitizes glioblastoma multiforme cells to TRAIL-induced apoptosis by a pathway involving caspases-8, −9, and −7, with no significant effect on cell cycle progression. eIF5B promotes evasion of apoptosis by promoting the translation of several IRES-containing mRNAs, encoding the antiapoptotic proteins XIAP, Bcl-xL, cIAP1, and c-FLIPS. We also show that eIF5B promotes translation of nuclear factor erythroid 2-related factor 2 and suggest that reactive oxygen species contribute to increased apoptosis under conditions of eIF5B depletion. Finally, eIF5B depletion leads to decreased activation of the canonical NF-κB pathway. Taken together, our data suggest that eIF5B represents a regulatory node, allowing cancer cells to evade apoptosis by promoting the translation of pro-survival proteins from IRES-containing mRNAs.

Alternative Mechanisms of mRNA Translation Initiation in Cellular Stress Response and Cancer.

Throughout evolution, eukaryotic cells have devised different mechanisms to cope with stressful environments. When eukaryotic cells are exposed to stress stimuli, they activate adaptive pathways that allow them to restore cellular homeostasis. Most types of stress stimuli have been reported to induce a decrease in overall protein synthesis accompanied by induction of alternative mechanisms of mRNA translation initiation. Here, we present well-studied and recent examples of such stress responses and the alternative translation initiation mechanisms they induce, and discuss the consequences of such regulation for cell homeostasis and oncogenic transformation.

IRES-dependent ribosome repositioning directs translation of a +1 overlapping ORF that enhances viral infection.

RNA structures can interact with the ribosome to alter translational reading frame maintenance and promote recoding that result in alternative protein products. Here, we show that the internal ribosome entry site (IRES) from the dicistrovirus Cricket paralysis virus drives translation of the 0-frame viral polyprotein and an overlapping +1 open reading frame, called ORFx, via a novel mechanism whereby a subset of ribosomes recruited to the IRES bypasses 37 nucleotides downstream to resume translation at the +1-frame 13th non-AUG codon. A mutant of CrPV containing a stop codon in the +1 frame ORFx sequence, yet synonymous in the 0-frame, is attenuated compared to wild-type virus in a Drosophila infection model, indicating the importance of +1 ORFx expression in promoting viral pathogenesis. This work demonstrates a novel programmed IRES-mediated recoding strategy to increase viral coding capacity and impact virus infection, highlighting the diversity of RNA-driven translation initiation mechanisms in eukaryotes.

Exploring the Role of AUG Triplets in Human Cap-Independent Translation Enhancing Elements.

Cap-independent translation is believed to play an important role in eukaryotic protein synthesis, but the mechanisms of ribosomal recruitment and translation initiation remain largely unknown. Messenger RNA display was previously used to profile the human genome for RNA leader sequences that can enhance cap-independent translation. Surprisingly, many of the isolated sequences contain AUG triplets, suggesting a possible functional role for these motifs during translation initiation. Herein, we examine the sequence determinants of AUG triplets within a set of human translation enhancing elements (TEEs). Functional analyses performed in vitro and in cultured cells indicate that AUGs have the capacity to modulate mRNA translation either by serving as part of a larger ribosomal recruitment site or by directing the ribosome to defined initiation sites. These observations help constrain the functional role of AUG triplets in human TEEs and advance our understanding of this specific mechanism of cap-independent translation initiation.

MNK1 inhibitor as an antiviral agent suppresses buffalopox virus protein synthesis

A small molecule chemical inhibitor CGP57380 that blocks activation of MAPK interacting kinase 1 (MNK1) was found to significantly suppress buffalopox virus (BPXV) replication. BPXV infection was shown to induce MNK1 activation. Depletion of MNK1 by small interfering RNA (siRNA), blocking activation of extracellular regulated kinase (ERK, an upstream activator of MNK1) and disruption of eIF4E/eIF4G interaction (downstream substrate of MNK1 which plays a central role in cap-dependent translation initiation), resulted in reduced BPXV replication, suggesting that ERK/MNK1/eIF4E signaling is a prerequisite for BPXV replication. With the help of time-of-addition and virus step-specific assays, CGP57380 treatment was shown to decrease synthesis of viral genome (DNA). Disruption of ERK/MNK1/eIF4E signaling resulted in reduced synthesis of viral proteins, suggesting that BPXV utilizes cap-dependent mechanism of translation initiation. Therefore, we concluded that decreased synthesis of viral genome in presence of MNK1 inhibitor is the result of reduced synthesis of viral proteins. Furthermore, BPXV was sequentially passaged (P = 40) in presence of CGP57380 or vehicle control (DMSO). As compared to P0 and P40-control viruses, P40-CGP57380 virus replicated at significantly higher (∼10-fold) titers in presence of CGP57380, although a complete resistance could not be achieved. In a BPXV egg infection model, CGP57380 was found to prevent development of pock lesions on chorioallantoic membrane (CAM) as well as associated mortality of the embryonated chicken eggs. We for the first time demonstrated in vitro and in ovo antiviral efficacy of CGP57380 against BPXV and identified that ERK/MNK1 signaling is a prerequisite for synthesis of viral proteins. Our study also describes a rare report about generation of drug-resistant viral variants against a host-targeting antiviral agent.

Ribosome Profiling of Synechocystis Reveals Altered Ribosome Allocation at Carbon Starvation

Ribosome profiling accesses the translational step of gene expression via deep sequencing of ribosome-protected mRNA footprints. Pairing of ribosome profiling and transcriptomics data provides a translational efficiency for each gene. Here, the translatome and transcriptome of the model cyanobacterium Synechocystis were compared under carbon-replete and carbon starvation conditions. The latter may be experienced when cyanobacteria are cultivated in poorly mixed bioreactors or engineered to be product-secreting cell factories. A small fraction of genes (<200), including stress response genes, showed changes in translational efficiency during carbon starvation, indicating condition-dependent translation-level regulation. We observed ribosome occupancy in untranslated regions, possibly due to an alternative translation initiation mechanism in Synechocystis. The higher proportion of ribosomes residing in untranslated regions during carbon starvation may be a mechanism to quickly inactivate superfluous ribosomes. This work provides the first ribosome profiling data for cyanobacteria and reveals new regulation strategies for coping with nutrient limitation.

Translation acrobatics: how cancer cells exploit alternate modes of translational initiation.

Recent work has brought to light many different mechanisms of translation initiation that function in cells in parallel to canonical cap-dependent initiation. This has important implications for cancer. Canonical cap-dependent translation initiation is inhibited by many stresses such as hypoxia, nutrient limitation, proteotoxic stress, or genotoxic stress. Since cancer cells are often exposed to these stresses, they rely on alternate modes of translation initiation for protein synthesis and cell growth. Cancer mutations are now being identified in components of the translation machinery and in cis-regulatory elements of mRNAs, which both control translation of cancer-relevant genes. In this review, we provide an overview on the various modes of non-canonical translation initiation, such as leaky scanning, translation re-initiation, ribosome shunting, IRES-dependent translation, and m6A-dependent translation, and then discuss the influence of stress on these different modes of translation. Finally, we present examples of how these modes of translation are dysregulated in cancer cells, allowing them to grow, to proliferate, and to survive, thereby highlighting the importance of translational control in cancer.

Research progress in the mechanism of translation initiation of eukaryotic mRNAs

The translation of mRNA is a complicated multi-step process, including initiation, elongation and termination. Among them, the regulation of the initial stage plays the key role. There are many ways to initiate mRNA translation, and the most classical way is the m 7G cap-dependent scanning mechanism that was also the first mechanism identified. When cells encounter adversity and the classical mechanism is inhibited, other types of translation initiation mechanisms will be activated. In this review, we summarize the translation initiation mechanisms of eukaryotic mRNAs, especially some alternative mechanisms. It will provide a reference for further understanding of the expression and regulation of eukaryotic genes at the translation levels.

Translation of Hepatitis A Virus IRES Is Upregulated by a Hepatic Cell-Specific Factor.

Many viruses strongly prefer to infect certain cell types, a phenomenon known as “tropism.” Understanding tropism’s molecular basis is important for the design of vaccines and antiviral therapy. A common mechanism involves viral protein interactions with cell-specific surface receptors, but intracellular mechanisms involving translation have also been described. In this report, we focus on Hepatitis A Virus (HAV) tissue tropism from the standpoint of the translational machinery. HAV genomic RNA, like other positive stranded RNA viruses, is devoid of a cap structure and its translation is driven by highly structured RNA sequences termed internal ribosome entry site (IRES) in the 5′ untranslated region (UTR). Unlike most viral IRESs, HAV IRES-mediated translation requires eIF4E and the 3′ end of HAV RNA is polyadenylated. However, the molecular mechanism of HAV IRES-mediated translation initiation remains poorly understood. We analyzed HAV-IRES-mediated translation in a cell-free system derived from either non-hepatic cells (HeLa) or hepatoma cells (Huh-7) that enables investigation of the contribution of the cap and the poly(A) tail. This revealed that HAV IRES-mediated translation activity in hepatoma cell extracts is higher as compared to extracts derived from a non-hepatic line. Our data suggest that HAV IRES-mediated translation is upregulated by a hepatic cell-specific activator in a poly(A) tail-independent manner.

Differential Requirement for Translation Initiation Factor Pathways during Ecdysone-Dependent Neuronal Remodeling in Drosophila

Dendrite pruning of Drosophila sensory neurons during metamorphosis is induced by the steroid hormone ecdysone through a transcriptional program. In addition, ecdysone activates the eukaryotic initiation factor 4E-binding protein (4E-BP) to inhibit cap-dependent translation initiation. To uncover how efficient translation of ecdysone targets is achieved under these conditions, we assessed the requirements for translation initiation factors during dendrite pruning. We found that the canonical cap-binding complex eIF4F is dispensable for dendrite pruning, but the eIF3 complex and the helicase eIF4A are required, indicating that differential translation initiation mechanisms are operating during dendrite pruning. eIF4A and eIF3 are stringently required for translation of the ecdysone target Mical, and this depends on the 5' UTR of Mical mRNA. Functional analyses indicate that eIF4A regulates eIF3-mRNA interactions in a helicase-dependent manner. We propose that an eIF3-eIF4A-dependent alternative initiation pathway bypasses 4E-BP to ensure adequate translation of ecdysone-induced genes.

Noncanonical Translation Initiation in Eukaryotes

The vast majority of eukaryotic messenger RNAs (mRNAs) initiate translation through a canonical, cap-dependent mechanism requiring a free 5' end and 5' cap and several initiation factors to form a translationally active ribosome. Stresses such as hypoxia, apoptosis, starvation, and viral infection down-regulate cap-dependent translation during which alternative mechanisms of translation initiation prevail to express proteins required to cope with the stress, or to produce viral proteins. The diversity of noncanonical initiation mechanisms encompasses a broad range of strategies and cellular cofactors. Herein, we provide an overview and, whenever possible, a mechanistic understanding of the various noncanonical mechanisms of initiation used by cells and viruses. Despite many unanswered questions, recent advances have propelled our understanding of the scope, diversity, and mechanisms of alternative initiation.

Leaderless mRNAs are circularized in Chlamydomonas reinhardtii mitochondria.

The mitochondrial genome of Chlamydomonas reinhardtii encodes eight protein coding genes transcribed on two polycistronic primary transcripts. The mRNAs are endonucleolytically cleaved from these transcripts directly upstream of their AUG start codons, creating leaderless mRNAs with 3' untranslated regions (UTR) comprised of most or all of their downstream intergenic regions. In this report, we provide evidence that these processed linear mRNAs are circularized, which places the 3' UTR upstream of the 5' start codon, creating a leader sequence ex post facto. The circular mRNAs were found to be ribosome associate by polysome profiling experiments suggesting they are translated. Sequencing of the 3'-5' junctions of the circularized mRNAs found the intra-molecular ligations occurred between fully processed 5' ends (the start AUG) and a variable 3' terminus. For five genes (cob, cox, nd2, nd4, and nd6), some of the 3' ends maintained an oligonucleotide addition during ligation, and for two of them, cob and nd6, these 3' termini were the most commonly recovered sequence. Previous reports have shown that after cleavage, three untemplated oligonucleotide additions may occur on the 3' termini of these mRNAs-adenylation, uridylylation, or cytidylation. These results suggest oligo(U) and oligo(C) additions may be part of the maturation process since they are maintained in the circular mRNAs. Circular RNAs occur in organisms across the biological spectrum, but their purpose in some systems, such as organelles (mitochondria and chloroplasts) is unclear. We hypothesize, that in C. reinhardtii mitochondria it may create a leader sequence to facilitate translation initiation, which may negate the need for an alternative translation initiation mechanism in this system, as previously speculated. In addition, circularization may play a protective role against exonucleases, and/or increase translational productivity.

Translation initiation by cap‐dependent ribosome recruitment: Recent insights and open questions

Gene expression universally relies on protein synthesis, where ribosomes recognize and decode the messenger RNA template by cycling through translation initiation, elongation, and termination phases. All aspects of translation have been studied for decades using the tools of biochemistry and molecular biology available at the time. Here, we focus on the mechanism of translation initiation in eukaryotes, which is remarkably more complex than prokaryotic initiation and is the target of multiple types of regulatory intervention. The "consensus" model, featuring cap-dependent ribosome entry and scanning of mRNA leader sequences, represents the predominantly utilized initiation pathway across eukaryotes, although several variations of the model and alternative initiation mechanisms are also known. Recent advances in structural biology techniques have enabled remarkable molecular-level insights into the functional states of eukaryotic ribosomes, including a range of ribosomal complexes with different combinations of translation initiation factors that are thought to represent bona fide intermediates of the initiation process. Similarly, high-throughput sequencing-based ribosome profiling or "footprinting" approaches have allowed much progress in understanding the elongation phase of translation, and variants of them are beginning to reveal the remaining mysteries of initiation, as well as aspects of translation termination and ribosomal recycling. A current view on the eukaryotic initiation mechanism is presented here with an emphasis on how recent structural and footprinting results underpin axioms of the consensus model. Along the way, we further outline some contested mechanistic issues and major open questions still to be addressed. This article is categorized under: Translation > Translation Mechanisms Translation > Translation Regulation RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.