Molecular mechanisms of circular RNA translation.

Circular RNAs (circRNAs) are covalently closed single-stranded RNAs without a 5' cap structure and a 3' poly(A) tail typically present in linear mRNAs of eukaryotic cells. CircRNAs are predominantly generated through a back-splicing process within the nucleus. CircRNAs have long been considered non-coding RNAs seemingly devoid of protein-coding potential. However, many recent studies have challenged this idea and have provided substantial evidence that a subset of circRNAs can associate with polysomes and indeed be translated. Therefore, in this review, we primarily highlight the 5' cap-independent internal initiation of translation that occurs on circular RNAs. Several molecular features of circRNAs, including the internal ribosome entry site, N6-methyladenosine modification, and the exon junction complex deposited around the back-splicing junction after back-splicing event, play pivotal roles in their efficient internal translation. We also propose a possible relationship between the translatability of circRNAs and their stability, with a focus on nonsense-mediated mRNA decay and nonstop decay, both of which are well-characterized mRNA surveillance mechanisms. An in-depth understanding of circRNA translation will reshape and expand our current knowledge of proteomics.

Nonsense-mediated mRNA decay of mRNAs encoding a signal peptide occurs primarily after mRNA targeting to the endoplasmic reticulum.

Translation of messenger ribonucleic acids (mRNAs) encoding integral membrane proteins or secreted proteins occurs on the surface of the endoplasmic reticulum (ER). When a nascent signal peptide is synthesized from the mRNAs, the ribosome-nascent chain complex (RNC) is recognized by the signal recognition particle (SRP) and then transported to the surface of the ER. The appropriate targeting of the RNC-SRP complex to the ER is monitored by a quality control pathway, a nuclear cap-binding complex (CBC)-ensured translational repression of RNC-SRP (CENTRE). In this study, using ribosome profiling of CBC-associated and eukaryotic translation initiation factor 4E-associated mRNAs, we reveal that, at the transcriptomic level, CENTRE is in charge of the translational repression of the CBC-RNC-SRP until the complex is specifically transported to the ER. We also find that CENTRE inhibits the nonsense-mediated mRNA decay (NMD) of mRNAs within the CBC-RNC-SRP. The NMD occurs only after the CBC-RNC-SRP is targeted to the ER and after eukaryotic translation initiation factor 4E replaces CBC. Our data indicate dual surveillance for properly targeting mRNAs encoding integral membrane or secretory proteins to the ER. CENTRE blocks gene expression at the translation level before the CBC-RNC-SRP delivery to the ER, and NMD monitors mRNA quality after its delivery to the ER.

YTHDF2 facilitates aggresome formation via UPF1 in an m6A-independent manner.

YTHDF2 has been extensively studied and typified as an RNA-binding protein that specifically recognizes and destabilizes RNAs harboring N6-methyladenosine (m6A), the most prevalent internal modification found in eukaryotic RNAs. In this study, we unravel the m6A-independent role of YTHDF2 in the formation of an aggresome, where cytoplasmic protein aggregates are selectively sequestered upon failure of protein homeostasis mediated by the ubiquitin-proteasome system. Downregulation of YTHDF2 in HeLa cells reduces the circularity of aggresomes and the rate of movement of misfolded polypeptides, inhibits aggresome formation, and thereby promotes cellular apoptosis. Mechanistically, YTHDF2 is recruited to a misfolded polypeptide-associated complex composed of UPF1, CTIF, eEF1A1, and DCTN1 through its interaction with UPF1. Subsequently, YTHDF2 increases the interaction between the dynein motor protein and the misfolded polypeptide-associated complex, facilitating the diffusion dynamics of the movement of misfolded polypeptides toward aggresomes. Therefore, our data reveal that YTHDF2 is a cellular factor involved in protein quality control.

An interaction between eIF4A3 and eIF3g drives the internal initiation of translation.

An RNA structure or modified RNA sequences can provide a platform for ribosome loading and internal translation initiation. The functional significance of internal translation has recently been highlighted by the discovery that a subset of circular RNAs (circRNAs) is internally translated. However, the molecular mechanisms underlying the internal initiation of translation in circRNAs remain unclear. Here, we identify eIF3g (a subunit of eIF3 complex) as a binding partner of eIF4A3, a core component of the exon-junction complex (EJC) that is deposited onto spliced mRNAs and plays multiple roles in the regulation of gene expression. The direct interaction between eIF4A3-eIF3g serves as a molecular linker between the eIF4A3 and eIF3 complex, thereby facilitating internal ribosomal entry. Protein synthesis from in vitro-synthesized circRNA demonstrates eIF4A3-driven internal translation, which relies on the eIF4A3-eIF3g interaction. Furthermore, our transcriptome-wide analysis shows that efficient polysomal association of endogenous circRNAs requires eIF4A3. Notably, a subset of endogenous circRNAs can express a full-length intact protein, such as ß-catenin, in an eIF4A3-dependent manner. Collectively, our results expand the understanding of the protein-coding potential of the human transcriptome, including circRNAs.

Freestanding Oxide Membranes for Epitaxial Ferroelectric Heterojunctions.

Since facile routes to fabricate freestanding oxide membranes were previously established, tremendous efforts have been made to further improve their crystallinity, and fascinating physical properties have been also reported in heterointegrated freestanding membranes. Here, we demonstrate our synthetic recipe to manufacture highly crystalline perovskite SrRuO3 freestanding membranes using new infinite-layer perovskite SrCuO2 sacrificial layers. To accomplish this, SrRuO3/SrCuO2 bilayer thin films are epitaxially grown on SrTiO3 (001) substrates, and the topmost SrRuO3 layer is chemically exfoliated by etching the SrCuO2 template layer. The as-exfoliated SrRuO3 membranes are mechanically transferred to various nonoxide substrates for the subsequent BaTiO3 film growth. Finally, freestanding heteroepitaxial junctions of ferroelectric BaTiO3 and metallic SrRuO3 are realized, exhibiting robust ferroelectricity. Intriguingly, the enhancement of piezoelectric responses is identified in freestanding BaTiO3/SrRuO3 heterojunctions with mixed ferroelectric domain states. Our approaches will offer more opportunities to develop heteroepitaxial freestanding oxide membranes with high crystallinity and enhanced functionality.

Single-molecule visualization of mRNA circularization during translation.

Translation is mediated by precisely orchestrated sequential interactions among translation initiation components, mRNA, and ribosomes. Biochemical, structural, and genetic techniques have revealed the fundamental mechanism that determines what occurs and when, where and in what order. Most mRNAs are circularized via the eIF4E-eIF4G-PABP interaction, which stabilizes mRNAs and enhances translation by recycling ribosomes. However, studies using single-molecule fluorescence imaging have allowed for the visualization of complex data that opposes the traditional "functional circularization" theory. Here, we briefly introduce single-molecule techniques applied to studies on mRNA circularization and describe the results of in vitro and live-cell imaging. Finally, we discuss relevant insights and questions gained from single-molecule research related to translation.

The role of LC3B in autophagy as an RNA-binding protein.

The hallmark of cellular events observed upon macroautophagic/autophagic induction is the conjugation of LC3B, one of the mammalian Atg8 homologs, with phosphatidylethanolamine. This conversion from LC3B-I (an unconjugated form) to LC3B-II (a conjugated form) is essential for phagophore expansion and formation of autophagosomes. Our recent study revealed that LC3B binds to RNAs with a preference for the consensus AAUAAA motif and recruits the CCR4-NOT deadenylase complex. Consequently, LC3B elicits rapid degradation of mRNAs, which we have termed as LC3B-mediated mRNA decay (LMD). LMD requires the conversion of LC3B-I to LC3B-II and occurs before the formation of autolysosomes. Furthermore, we identified PRMT1 mRNA, which encodes a protein that functions as a negative regulator of autophagy, as an LMD substrate. A failure of rapid degradation of PRMT1 mRNA via LMD results in inefficient autophagy. Thus, our study unravels an important role of LC3B in autophagy as an RNA-binding protein for efficient mRNA decay.

m1A and m6A modifications function cooperatively to facilitate rapid mRNA degradation.

N6-Methyladenosine (m6A), the most abundant internal mRNA modification, affects multiple steps in gene expression. Mechanistically, the binding of YTHDF2 to m6A on mRNAs elicits rapid mRNA degradation by recruiting several RNA degrading enzymes. Here, we show that N1-methyladenosine (m1A), another type of RNA modification, accelerates rapid m6A RNA degradation. We identify HRSP12 as an RNA-binding protein that recognizes m1A. The binding of HRSP12 to m1A promotes efficient interaction of YTHDF2 with m6A, consequently facilitating endoribonucleolytic cleavage via the RNase P/MRP complex. Transcriptome-wide analyses also reveal that mRNAs harboring both m1A and m6A are downregulated in an HRSP12-dependent manner compared with mRNAs harboring m6A only. Accordingly, a subset of endogenous circular RNAs that harbor m6A and associate with YTHDF2 in an HRSP12-dependent manner is also subjected to m1A-facilitated rapid degradation. Together, our observations provide compelling evidence for crosstalk between different RNA modifications.

Resolving m6A epitranscriptome with stoichiometry.

A recent study by Hu et al. describes N6-methyladenosine (m6A)-selective allyl chemical labeling and sequencing (m6A-SAC-seq), which allows for quantitative, stoichiometric, and positional analyses of m6A at single-nucleotide resolution across the whole transcriptome level. Information on the m6A stoichiometry will provide additional layers of gene regulatory pathways mediated by m6A modification during diverse molecular, cellular, and physiological events.

Single polysome analysis of mRNP.

Eukaryotic translation is a complex process that involves the interplay of various translation factors to convert genetic information into a specific amino acid chain. According to an elegant model of eukaryotic translation initiation, the 3' poly(A) tail of an mRNA, which is occupied by poly(A)-binding proteins (PABPs), communicates with the 5'-cap bound by eIF4E to enhance translation. Although the circularization of mRNA resulting from the communication is widely understood, it has yet to be directly observed. To explore mRNA circularization in translation, we analyzed the level of colocalization of eIF4E, eIF4G, and PABP on individual mRNAs in polysomal and subpolysomal fractions using single polysome analysis. Our results show that the three tested proteins barely coexist in mRNA in either polysomal or subpolysomal fractions, implying that the closed-loop structure generated by the communication between eIF4E, eIF4G, and PAPB may be transient during translation.

UPF1 promotes rapid degradation of m6A-containing RNAs.

N6-methyladenosine (m6A) is the most prevalent internal modification in eukaryotic mRNAs and affects RNA processing and metabolism. When YTHDF2, an m6A-recognizing protein, binds to m6A, it facilitates the destabilization of m6A-containing RNAs (m6A RNAs). Here, we demonstrate that upstream frameshift 1 (UPF1), a key factor for nonsense-mediated mRNA decay, interacts with YTHDF2, thereby triggering rapid degradation of m6A RNAs. The UPF1-mediated m6A RNA degradation depends on a specific interaction between UPF1 and N-terminal residues 101-168 of YTHDF2, UPF1 ATPase/helicase activities, and UPF1 interaction with proline-rich nuclear receptor coactivator 2 (PNRC2), a decapping-promoting factor preferentially involved in nonsense-mediated mRNA decay. Furthermore, transcriptome-wide analyses show that YTHDF2-bound mRNAs that are not substrates for HRSP12-RNase P/MRP-mediated endoribonucleolytic cleavage are destabilized with a higher dependency on UPF1. Collectively, our data indicate dynamic and multilayered regulation of the stability of m6A RNAs and highlight the multifaceted role of UPF1 in mRNA decay.

LC3B is an RNA-binding protein to trigger rapid mRNA degradation during autophagy.

LC3/ATG8 has long been appreciated to play a central role in autophagy, by which a variety of cytoplasmic materials are delivered to lysosomes and eventually degraded. However, information on the molecular functions of LC3 in RNA biology is very limited. Here, we show that LC3B is an RNA-binding protein that directly binds to mRNAs with a preference for a consensus AAUAAA motif corresponding to a polyadenylation sequence. Autophagic activation promotes an association between LC3B and target mRNAs and triggers rapid degradation of target mRNAs in a CCR4-NOT-dependent manner before autolysosome formation. Furthermore, our transcriptome-wide analysis reveals that PRMT1 mRNA, which encodes a negative regulator of autophagy, is one of the major substrates. Rapid degradation of PRMT1 mRNA by LC3B facilitates autophagy. Collectively, we demonstrate that LC3B acts as an RNA-binding protein and an mRNA decay factor necessary for efficient autophagy.

TAZ links exercise to mitochondrial biogenesis via mitochondrial transcription factor A.

Mitochondria are energy-generating organelles and mitochondrial biogenesis is stimulated to meet energy requirements in response to extracellular stimuli, including exercise. However, the mechanisms underlying mitochondrial biogenesis remain unknown. Here, we demonstrate that transcriptional coactivator with PDZ-binding motif (TAZ) stimulates mitochondrial biogenesis in skeletal muscle. In muscle-specific TAZ-knockout (mKO) mice, mitochondrial biogenesis, respiratory metabolism, and exercise ability were decreased compared to wild-type mice. Mechanistically, TAZ stimulates the translation of mitochondrial transcription factor A via Ras homolog enriched in brain (Rheb)/Rheb like 1 (Rhebl1)-mTOR axis. TAZ stimulates Rhebl1 expression via TEA domain family transcription factor. Rhebl1 introduction by adeno-associated virus or mTOR activation recovered mitochondrial biogenesis in mKO muscle. Physiologically, mKO mice did not stimulate exercise-induced mitochondrial biogenesis. Collectively, our results suggested that TAZ is a novel stimulator for mitochondrial biogenesis and exercise-induced muscle adaptation.

The pioneer round of translation ensures proper targeting of ER and mitochondrial proteins.

The pioneer (or first) round of translation of newly synthesized mRNAs is largely mediated by a nuclear cap-binding complex (CBC). In a transcriptome-wide analysis of polysome-associated and CBC-bound transcripts, we identify RN7SL1, a noncoding RNA component of a signal recognition particle (SRP), as an interaction partner of the CBC. The direct CBC-SRP interaction safeguards against abnormal expression of polypeptides from a ribosome-nascent chain complex (RNC)-SRP complex until the latter is properly delivered to the endoplasmic reticulum. Failure of this surveillance causes abnormal expression of misfolded proteins at inappropriate intracellular locations, leading to a cytosolic stress response. This surveillance pathway also blocks protein synthesis through RNC-SRP misassembled on an mRNA encoding a mitochondrial protein. Thus, our results reveal a surveillance pathway in which pioneer translation ensures proper targeting of endoplasmic reticulum and mitochondrial proteins.

A high-resolution temporal atlas of the SARS-CoV-2 translatome and transcriptome.

COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which infected >200 million people resulting in >4 million deaths. However, temporal landscape of the SARS-CoV-2 translatome and its impact on the human genome remain unexplored. Here, we report a high-resolution atlas of the translatome and transcriptome of SARS-CoV-2 for various time points after infecting human cells. Intriguingly, substantial amount of SARS-CoV-2 translation initiates at a novel translation initiation site (TIS) located in the leader sequence, termed TIS-L. Since TIS-L is included in all the genomic and subgenomic RNAs, the SARS-CoV-2 translatome may be regulated by a sophisticated interplay between TIS-L and downstream TISs. TIS-L functions as a strong translation enhancer for ORF S, and as translation suppressors for most of the other ORFs. Our global temporal atlas provides compelling insight into unique regulation of the SARS-CoV-2 translatome and helps comprehensively evaluate its impact on the human genome.

UPF1: From mRNA Surveillance to Protein Quality Control.

Selective recognition and removal of faulty transcripts and misfolded polypeptides are crucial for cell viability. In eukaryotic cells, nonsense-mediated mRNA decay (NMD) constitutes an mRNA surveillance pathway for sensing and degrading aberrant transcripts harboring premature termination codons (PTCs). NMD functions also as a post-transcriptional gene regulatory mechanism by downregulating naturally occurring mRNAs. As NMD is activated only after a ribosome reaches a PTC, PTC-containing mRNAs inevitably produce truncated and potentially misfolded polypeptides as byproducts. To cope with the emergence of misfolded polypeptides, eukaryotic cells have evolved sophisticated mechanisms such as chaperone-mediated protein refolding, rapid degradation of misfolded polypeptides through the ubiquitin-proteasome system, and sequestration of misfolded polypeptides to the aggresome for autophagy-mediated degradation. In this review, we discuss how UPF1, a key NMD factor, contributes to the selective removal of faulty transcripts via NMD at the molecular level. We then highlight recent advances on UPF1-mediated communication between mRNA surveillance and protein quality control.

The regulatory impact of RNA-binding proteins on microRNA targeting.

Argonaute is the primary mediator of metazoan miRNA targeting (MT). Among the currently identified >1,500 human RNA-binding proteins (RBPs), there are only a handful of RBPs known to enhance MT and several others reported to suppress MT, leaving the global impact of RBPs on MT elusive. In this study, we have systematically analyzed transcriptome-wide binding sites for 150 human RBPs and evaluated the quantitative effect of individual RBPs on MT efficacy. In contrast to previous studies, we show that most RBPs significantly affect MT and that all of those MT-regulating RBPs function as MT enhancers rather than suppressors, by making the local secondary structure of the target site accessible to Argonaute. Our findings illuminate the unappreciated regulatory impact of human RBPs on MT, and as these RBPs may play key roles in the gene regulatory network governed by metazoan miRNAs, MT should be understood in the context of co-regulating RBPs.

Translation mediated by the nuclear cap-binding complex is confined to the perinuclear region via a CTIF-DDX19B interaction.

Newly synthesized mRNA is translated during its export through the nuclear pore complex, when its 5'-cap structure is still bound by the nuclear cap-binding complex (CBC), a heterodimer of cap-binding protein (CBP) 80 and CBP20. Despite its critical role in mRNA surveillance, the mechanism by which CBC-dependent translation (CT) is regulated remains unknown. Here, we demonstrate that the CT initiation factor (CTIF) is tethered in a translationally incompetent manner to the perinuclear region by the DEAD-box helicase 19B (DDX19B). DDX19B hands over CTIF to CBP80, which is associated with the 5'-cap of a newly exported mRNA. The resulting CBP80-CTIF complex then initiates CT in the perinuclear region. We also show that impeding the interaction between CTIF and DDX19B leads to uncontrolled CT throughout the cytosol, consequently dysregulating nonsense-mediated mRNA decay. Altogether, our data provide molecular evidence supporting the importance of tight control of local translation in the perinuclear region.

Emerging functions of circular RNA in aging.

Circular RNA (circRNA) is a closed, single-stranded transcript widely detected in eukaryotes. Recent studies indicate that the levels of circRNAs change with age in various tissues in multiple species, ranging from nematodes to mammals. Here we discuss the functional roles of circRNAs in animal aging and longevity. We review studies regarding the differential expression of circRNAs that contributes to cellular senescence and the pathogenesis of aging-associated diseases. We explore the features of aging-associated circRNAs by discussing their potential as biomarkers of aging, tissue specificity, physiological roles, action mechanisms, and evolutionarily conserved characteristics. Our review provides insights into current progress in circRNA research and their significant functions in the aging process.