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The fidelity of start codon recognition by ribosomes is paramount during protein synthesis. The current knowledge of eukaryotic translation initiation implies unidirectional 5ʹ→3ʹ migration of the pre-initiation complex (PIC) along the 5ʹ UTR. In probing translation initiation from ultra-short 5ʹ UTR, we report that an AUG triplet near the 5ʹ end can be selected via PIC backsliding. Bi-directional ribosome scanning is supported by competitive selection of closely spaced AUG codons and recognition of two initiation sites flanking an internal ribosome entry site. Transcriptome-wide PIC profiling reveals footprints with an oscillation pattern near the 5ʹ end and start codons. Depleting the RNA helicase eIF4A leads to reduced PIC oscillations and impaired selection of 5ʹ end start codons. Enhancing the ATPase activity of eIF4A promotes nonlinear PIC scanning and stimulates upstream translation initiation. The helicase-mediated PIC conformational switch may provide an operational mechanism that unifies ribosome recruitment, scanning, and start codon selection., Start codon selection is commonly thought to occur through the unidirectional scanning of the mRNA by the 40 S ribosome. Here the authors provide evidence that the pre-initiation complex can backslide on the mRNA to initiate translation at upstream AUG codons.

Transcriptional programming plays a key role in determining the cell state. Timely reconfiguration of chromatin structure and attenuation of pluripotent genes are required for efficient embryonic stem cell (ESC) differentiation. Here, we identify METTL3, a core N

Significance SAMD9 and SAMD9L (SAMD9/9L) are important innate immune defenders against viruses and the development of myeloid tumors. They form a crucial host barrier that poxviruses must overcome for successful infection. A myriad of human diseases including many pediatric myelodysplastic syndromes are caused by mutations in SAMD9/9L. However, the molecular functions of SAMD9/9L and how their functions are executed are unknown, hindering progress in developing effective therapies for SAMD9/9L-associated human diseases. Here, we identified the structure and function of a SAMD9/9L effector domain that is essential for their physiological functions as well as the pathogenic effects exerted by patient-derived mutations. Our study revealed a potential therapeutic target for SAMD9/9L-associated human diseases., SAMD9 and SAMD9L (SAMD9/9L) are antiviral factors and tumor suppressors, playing a critical role in innate immune defense against poxviruses and the development of myeloid tumors. SAMD9/9L mutations with a gain-of-function (GoF) in inhibiting cell growth cause multisystem developmental disorders including many pediatric myelodysplastic syndromes. Predicted to be multidomain proteins with an architecture like that of the NOD-like receptors, SAMD9/9L molecular functions and domain structures are largely unknown. Here, we identified a SAMD9/9L effector domain that functions by binding to double-stranded nucleic acids (dsNA) and determined the crystal structure of the domain in complex with DNA. Aided with precise mutations that differentially perturb dsNA binding, we demonstrated that the antiviral and antiproliferative functions of the wild-type and GoF SAMD9/9L variants rely on dsNA binding by the effector domain. Furthermore, we showed that GoF variants inhibit global protein synthesis, reduce translation elongation, and induce proteotoxic stress response, which all require dsNA binding by the effector domain. The identification of the structure and function of a SAMD9/9L effector domain provides a therapeutic target for SAMD9/9L-associated human diseases.

In eukaryotic cells, many messenger RNAs (mRNAs) possess upstream open reading frames (uORFs) in addition to the main coding region. After uORF translation, the ribosome could either recycle at the stop codon or resume scanning for downstream start codons in a process known as reinitiation. Accumulating evidence suggests that some initiation factors, including eukaryotic initiation factor 3 (eIF3), linger on the early elongating ribosome, forming an eIF3-80S complex. Very little is known about how eIF3 is carried along with the 80S during elongation and whether the eIF3-80S association is subject to regulation. Here, we report that eIF3a undergoes dynamic O-linked N-acetylglucosamine (O-GlcNAc) modification in response to nutrient starvation. Stress-induced de-O-GlcNAcylation promotes eIF3 retention on the elongating ribosome and facilitates activating transcription factor 4 (ATF4) reinitiation. Eliminating the modification site from eIF3a via CRISPR genome editing induces ATF4 reinitiation even under the nutrient-rich condition. Our findings illustrate a mechanism in balancing ribosome recycling and reinitiation, thereby linking the nutrient stress response and translational reprogramming.

The integrated stress response (ISR) plays a pivotal role in adaptation of translation machinery to cellular stress. Here, we demonstrate an ISR-independent osmoadaptation mechanism involving reprogramming of translation via coordinated but independent actions of mTOR and plasma membrane amino acid transporter SNAT2. This biphasic response entails reduced global protein synthesis and mTOR signaling followed by translation of SNAT2. Induction of SNAT2 leads to accumulation of amino acids and reactivation of mTOR and global protein synthesis, paralleled by partial reversal of the early-phase, stress-induced translatome. We propose SNAT2 functions as a molecular switch between inhibition of protein synthesis and establishment of an osmoadaptive translation program involving the formation of cytoplasmic condensates of SNAT2-regulated RNA-binding proteins DDX3X and FUS. In summary, we define key roles of SNAT2 in osmotolerance.

RNA modification in the form of N6-methyladenosine (m6A) regulates nearly all the post-transcriptional processes. The asymmetric m6A deposition suggests that regional methylation may have distinct functional consequences. However, current RNA biology tools do not distinguish the contribution of individual m6A modifications. Here we report the development of “m6A editing”, a powerful approach that enables m6A installation and erasure from cellular RNAs without changing the primary sequence. We engineered fusions of CRISPR-Cas9 and a single chain m6A methyltransferase that can be programmed with a guide RNA. The resultant m6A “writers” allow functional comparison of single site methylation in different mRNA regions. We further engineered m6A “erasers” by fusing CRISPR-Cas9 with ALKBH5 or FTO to achieve site-specific demethylation of RNAs. The development of programmable m6A editing not only expands the scope of RNA engineering, but also facilitates mechanistic understanding of epitranscriptome., Graphical Abstract, Reporting Summary Further information on research design is available in the Nature Research Reporting Summary linked to this article.

The structure of mRNA in vivo is unwound to some extent in response to multiple factors involved in the translation process, resulting in significant differences from the structure of the same mRNA in vitro. In this study, we have proposed a novel application of deep neural networks, named DeepDRU, to predict the degree of mRNA structure unwinding in vivo by fitting five quantifiable features that may affect mRNA folding: ribosome density (RD), minimum folding free energy (MFE), GC content, translation initiation ribosome density (INI) and mRNA structure position (POS). mRNA structures with adjustment of the simulated structural features were designed and then fed into the trained DeepDRU model. We found unique effect regions of these five features on mRNA structure in vivo. Strikingly, INI is the most critical factor affecting the structure of mRNA in vivo, and structural sequence features, including MFE and GC content, have relatively smaller effects. DeepDRU provides a new paradigm for predicting the unwinding capability of mRNA structure in vivo. This improved knowledge about the mechanisms of factors influencing the structural capability of mRNA to unwind will facilitate the design and functional analysis of mRNA structure in vivo.

Long noncoding RNAs (lncRNAs) are key regulators of gene expression in diverse cellular contexts and biological processes. Given the surprising range of shapes and sizes, how distinct lncRNAs achieve functional specificity remains incompletely understood. Here, we identified a heat shock–inducible lncRNA, Heat, in mouse cells that acts as a transcriptional brake to restrain stress gene expression. Functional characterization reveals that Heat directly binds to heat shock transcription factor 1 (HSF1), thereby targeting stress genes in a trans-acting manner. Intriguingly, Heat is heavily methylated in the form of m(6)A. Although dispensable for HSF1 binding, Heat methylation is required for silencing stress genes to attenuate heat shock response. Consistently, m(6)A depletion results in prolonged activation of stress genes. Furthermore, Heat mediates these effects via the nuclear m(6)A reader YTHDC1, forming a transcriptional silencing complex for stress genes. Our study reveals a crucial role of nuclear epitranscriptome in the transcriptional regulation of heat shock response.

Long noncoding RNAs (lncRNAs) are key regulators of gene expression in diverse cellular contexts and biological processes. Given the surprising range of shapes and sizes, how distinct lncRNAs achieve functional specificity remains incompletely understood. Here, we identified a heat shock-inducible lncRNA, Heat, in mouse cells that acts as a transcriptional brake to restrain stress gene expression. Functional characterization reveals that Heat directly binds to heat shock transcription factor 1 (HSF1), thereby targeting stress genes in a trans-acting manner. Intriguingly, Heat is heavily methylated in the form of m6A. Although dispensable for HSF1 binding, Heat methylation is required for silencing stress genes to attenuate heat shock response. Consistently, m6A depletion results in prolonged activation of stress genes. Furthermore, Heat mediates these effects via the nuclear m6A reader YTHDC1, forming a transcriptional silencing complex for stress genes. Our study reveals a crucial role of nuclear epitranscriptome in the transcriptional regulation of heat shock response.

Ribosome pausing at start codons limits translational output, which is controlled by altered mRNA methylation in cancer cells., Translation is a crucial process in cancer development and progression. Many oncogenic signaling pathways target the translation initiation stage to satisfy the increased anabolic demands of cancer cells. Using quantitative profiling of initiating ribosomes, we found that ribosomal pausing at the start codon serves as a “brake” to restrain the translational output. In response to oncogenic RAS signaling, the initiation pausing relaxes and contributes to the increased translational flux. Intriguingly, messenger RNA (mRNA) m6A modification in the vicinity of start codons influences the behavior of initiating ribosomes. Under oncogenic RAS signaling, the reduced mRNA methylation leads to relaxed initiation pausing, thereby promoting malignant transformation and tumor growth. Restored initiation pausing by inhibiting m6A demethylases suppresses RAS-mediated oncogenic translation and subsequent tumorigenesis. Our findings unveil a paradigm of translational control that is co-opted by RAS mutant cancer cells to drive malignant phenotypes.

SummaryMammalian cells have to adapt to environmental challenges that range from mild to severe stress. While the cellular response to mild stress has been widely studied, how cells respond to severe stress remains unclear. We show here that under severe stress conditions, cells induce a transient hibernation-like mechanism that anticipates recovery. We demonstrate that this Adaptive Pausing Response (APR) is a coordinated cellular response that limits ATP supply and consumption though mitochondrial fragmentation and widespread pausing of mRNA translation. This pausing is accomplished by ribosome stalling at translation initiation codons, which keeps mRNAs poised to resume translation upon recovery from severe stress. We further show that recovery from severe stress involves adaptive ISR (Integrated Stress Response) signaling that in turn permits cell cycle progression, resumption of growth, and reversal of mitochondria fragmentation. Our findings indicate that cells can respond to severe stress through the APR, a mechanism that preserves vital elements of cellular function under harsh environmental conditions.

Precise control of protein synthesis by engineering sequence elements in 5’ untranslated region (5’UTR) remains a fundamental challenge. To accelerate our understanding of cis-regulatory code embedded in 5’UTR, we devised massively parallel reporter assays from a synthetic mRNA library composed of over one million 5’UTR variants. A completely randomized 10-nucleotide sequence preceding an upstream open reading frame (uORF) and downstream GFP leads to a broad range of mRNA translatability and stability in mammalian cells. While efficient translation protects mRNA from degradation, uORF translation triggers mRNA decay in a UPF1-dependent manner. We also identified translational inhibitory elements in 5’UTR with G-quadruplex as a mark for mRNA decay in the P-body. Unexpectedly, an unstructured A-rich element in 5’UTR, while enabling cap-independent translation, destabilizes mRNAs in the absence of translation. Our results not only expose diverse sequence features of 5’UTR in controlling mRNA translatability, but also reveal ribosome-dependent and -independent mRNA surveillance pathways.

Although amino acids are known regulators of translation, the unique contributions of specific amino acids are not well understood. We compared effects of culturing HEK293T cells in medium lacking either leucine, methionine, histidine, or arginine on eIF2 and 4EBP1 phosphorylation and measures of mRNA translation. Methionine starvation caused the most drastic decrease in translation as assessed by polysome formation, ribosome profiling, and a measure of protein synthesis (puromycin-labeled polypeptides) but had no significant effect on eIF2 phosphorylation, 4EBP1 hyperphosphorylation or 4EBP1 binding to eIF4E. Leucine starvation suppressed polysome formation and was the only tested condition that caused a significant decrease in 4EBP1 phosphorylation or increase in 4EBP1 binding to eIF4E, but effects of leucine starvation were not replicated by overexpressing nonphosphorylatable 4EBP1. This suggests the binding of 4EBP1 to eIF4E may not by itself explain the suppression of mRNA translation under conditions of leucine starvation. Ribosome profiling suggested that leucine deprivation may primarily inhibit ribosome loading, whereas methionine deprivation may primarily impair start site recognition. These data underscore our lack of a full understanding of how mRNA translation is regulated and point to a unique regulatory role of methionine status on translation initiation that is not dependent upon eIF2 phosphorylation.

To survive, mammalian cells must adapt to environmental challenges. While the cellular response to mild stress has been widely studied, how cells respond to severe stress remains unclear. We show here that under severe hyperosmotic stress, cells enter a transient hibernation-like state in anticipation of recovery. We demonstrate this adaptive pausing response (APR) is a coordinated cellular response that limits ATP supply and consumption through mitochondrial fragmentation and widespread pausing of mRNA translation. This pausing is accomplished by ribosome stalling at translation initiation codons, which keeps mRNAs poised to resume translation upon recovery. We further show that recovery from severe stress involves ISR (integrated stress response) signaling that permits cell cycle progression, resumption of growth, and reversal of mitochondria fragmentation. Our findings indicate that cells can respond to severe stress via a hibernation-like mechanism that preserves vital elements of cellular function under harsh environmental conditions.

Zika virus (ZIKV) is a member of the family Flaviviridae and contains a single-stranded RNA genome with positive-polarity. Like Dengue, Zika virus uses Aedes aegypti mosquito as a vector to infect human with a wide range of clinical signs, from asymptomatic to influenza-like syndrome. Despite significant progress in genomic analyses, how a viral relationship with two different hosts affect the overall fitness, constancy, and dodging of hosts immune system are elusive. Here we analyzed Zika virus codon-based evolution using eleven strains from different geographical locations. The overall codon usage was similar and slightly bias among all strains. An occurrence of A-ending in highly-preferred codons and analysis by various approaches strongly suggests that mutational bias is the main force shaping codon usage in this virus. However, natural selection and geographical realities cannot be ignored in marginal influence on codon usage. The viral genomes naturally favor Aedes aegypti over human host for tRNA pool in translation. Such findings will assist researchers in understanding elements contribute to viral adaptation and evolutionary setup with hosts.

Precise control of protein synthesis by engineering sequence elements in 5' untranslated regions (5' UTRs) remains a fundamental challenge. To accelerate our understanding of the cis-regulatory code embedded in 5' UTRs, we devised massively parallel reporter assays from a synthetic messenger RNA library composed of over one million 5' UTR variants. A completely randomized 10-nucleotide sequence preceding an upstream open reading frame (uORF) and downstream GFP drives a broad range of translational outputs and mRNA stability in mammalian cells. While efficient translation protects mRNA from degradation, uORF translation triggers mRNA decay in a UPF1-dependent manner. We also identified translational inhibitory elements with G-quadruplexes as marks for mRNA decay in P-bodies. Unexpectedly, an unstructured A-rich element in 5' UTRs destabilizes mRNAs in the absence of translation, although it enables cap-independent translation. Our results not only identify diverse sequence features of 5' UTRs that control mRNA translatability, but they also reveal ribosome-dependent and ribosome-independent mRNA-surveillance pathways.

Traditional annotation of protein-encoding genes relied on assumptions, such as one open reading frame (ORF) encodes one protein and minimal lengths for translated proteins. With the serendipitous discoveries of translated ORFs encoded upstream and downstream of annotated ORFs, from alternative start sites nested within annotated ORFs and from RNAs previously considered noncoding, it is becoming clear that these initial assumptions are incorrect. The findings have led to the realization that genetic information is more densely coded and that the proteome is more complex than previously anticipated. As such, interest in the identification and characterization of the previously ignored ‘dark proteome’ is increasing, though we note that research in eukaryotes and bacteria has largely progressed in isolation. To bridge this gap and illustrate exciting findings emerging from studies of the dark proteome, we highlight recent advances in both eukaryotic and bacterial cells. We discuss progress in the detection of alternative ORFs as well as in the understanding of functions and the regulation of their expression and posit questions for future work.

It is common wisdom that codon usage bias has evolved in the selection for efficient translation, in which highly expressed genes are encoded predominantly by optimal codons. However, a growing body of evidence suggests regulatory roles for non-optimal codons in translation dynamics. Here we report that in mammalian cells, non-optimal codons play a critical role in promoting selective mRNA translation during amino acid starvation. During starvation, in contrast to genes encoding ribosomal proteins whose translation is highly sensitive to amino acid deprivation, translation of genes involved in the cellular protein degradation pathways remains unaffected. We found that these two gene groups bear different codon composition, with non-optimal codons being highly enriched in genes encoding the ubiquitin–proteasome system. Supporting the selective tRNA charging model originally proposed in Escherichia coli, we demonstrated that tRNA isoacceptors decoding rare codons are maintained in translating ribosomes under amino acid starvation. Finally, using luciferase reporters fused with endogenous gene-derived sequences, we show that codon optimality contributes to differential mRNA translation in response to amino acid starvation. These results highlight the physiological significance of codon usage bias in cellular adaptation to stress.

m6A modification in the 5′ vicinity of the coding sequence of transcripts provides a selective mechanism for triaging mRNAs to stress granules and is mediated by the YTHDF3 “reader” protein., Reversible post-transcriptional modifications on messenger RNA emerge as prevalent phenomena in RNA metabolism. The most abundant among them is N6-methyladenosine (m6A) which is pivotal for RNA metabolism and function; its role in stress response remains elusive. We have discovered that in response to oxidative stress, transcripts are additionally m6A modified in their 5′ vicinity. Distinct from that of the translationally active mRNAs, this methylation pattern provides a selective mechanism for triaging mRNAs from the translatable pool to stress-induced stress granules. These stress-induced newly methylated sites are selectively recognized by the YTH domain family 3 (YTHDF3) “reader” protein, thereby revealing a new role for YTHDF3 in shaping the selectivity of stress response. Our findings describe a previously unappreciated function for RNA m6A modification in oxidative-stress response and expand the breadth of physiological roles of m6A.

The structure of mRNA in vivo is influenced by various factors involved in the translation process, resulting in significant differentiation of mRNA structure from that in vitro. Because multiple factors cause the differentiation of in vivo and in vitro mRNA structures, it was difficult to perform a more accurate analysis of mRNA structures in previous studies. In this study, we have proposed a novel application of a deep neural network (DNN) model to predict the structural stability of mRNA in vivo by fitting six quantifiable features that may affect mRNA folding: ribosome density, minimum folding free energy, GC content, mRNA abundance, ribosomal initial density and position of mRNA structure. Simulated mutations of the mRNA structure were designed and then fed into the trained DNN model to compute their structural stability. We found unique effects of these six features on mRNA structural stability in vivo. Strikingly, the ribosome density of the structural region is the most important factor affecting the structural stability of mRNA in vivo, and the strength of the mRNA structure in vitro should have a relatively small effect on its structural stability in vivo. The recruitment of DNNs provides a new paradigm to decipher the differentiation of mRNA structure in vivo and in vitro. This improved knowledge on the mechanisms of factors influencing mRNA structural stability will facilitate the design and functional analysis of mRNA structure in vivo.

SUMMARYGene expression can be post-transcriptionally regulated via dynamic and reversible RNA modifications. N1-methyladenosine (m1A) is a recently identified mRNA modification; however, little is known about its precise location, regulation and function. Here, we develop a base-resolution m1A profiling method, based on m1A-induced misincorporation during reverse transcription, and report distinct classes of m1A methylome in the human transcriptome. m1A in 5’-UTR, particularly those at the first nucleotide of mRNA, associate with increased translation efficiency. A different subset of m1A exhibit a GUUCRA tRNA-like motif, are evenly distributed in the transcriptome and are dependent on the methyltransferase TRMT6/61A. Additionally, we show for the first time that m1A is prevalent in the mitochondrial-encoded transcripts. Manipulation of m1A level via TRMT61B, a mitochondria-localizing m1A methyltransferase, demonstrates that m1A in mitochondrial mRNA interferes with translation. Collectively, our approaches reveal distinct classes of m1A methylome and provide a resource for functional studies of m1A-mediated epitranscriptomic regulation.

The integrated stress response (ISR) facilitates cellular adaptation to stress conditions via the common target eIF2α. During ISR, the selective translation of stress-related mRNAs often relies on alternative mechanisms, such as leaky scanning or reinitiation, but the underlying mechanism remains incompletely understood. Here we report that, in response to amino acid starvation, the reinitiation of ATF4 is not only governed by the eIF2α signaling pathway, but is also subjected to regulation by mRNA methylation in the form of N

Codon bias at the 5' terminal of coding sequence (CDS) is known to be distinct from the rest of the CDS. A number of events occur in this short region to regulate early translation elongation and co-translational translocation. In the genes encoding secretory proteins, there is a special signal sequence which has a higher occurrence of rare codons. In this study, we analyzed codon bias of secretory genes in several eukaryotes. The results showed that secretory genes in the species except mammals had a higher occurrence of rare codons in the 5' terminal of CDS, and the bias was greater than the same region of non-secretory genes. GO analysis revealed that secretory genes containing rare codon clusters in different regions were responsible for various roles in gene functions. Moreover, codon bias in the region encoding the hydrophobic region of protein is similar in secretory and non-secretory genes, indicating that codon bias in secretory genes was partly influenced by amino acid bias. Rare codon clusters are found more frequently in specific regions, and continuous rare codons are not favoured probably because they will increase the probability of ribosome collision and drop-off. Based on ribosome profiling data, there is no significant difference in the average translation efficiencies between rare and optimal codons. Higher ribosomal density in the 5' terminal may result from ribosome pausing which could be involved in different translation events. These findings collectively provided rich information on codon bias in secretory genes, which may shed light on the co-effect of codon bias, mRNA structure and tRNA abundance in translational regulations.

Amino acids show apparent propensities toward their neighbors. In addition to preferences of amino acids for their neighborhood context, amino acid substitutions are also considered to be context-dependent. However, context-dependence patterns of amino acid substitutions still remain poorly understood. Using relative entropy, we investigated the neighbor preferences of 20 amino acids and the context-dependent effects of amino acid substitutions with protein sequences in human, mouse, and dog. For 20 amino acids, the highest relative entropy was mostly observed at the nearest adjacent site of either N- or C-terminus except C and G. C showed the highest relative entropy at the third flanking site and periodic pattern was detected at G flanking sites. Furthermore, neighbor preference patterns of amino acids varied greatly in different secondary structures. We then comprehensively investigated the context-dependent effects of amino acid substitutions. Our results showed that nearly half of 380 substitution types were evidently context dependent, and the context-dependent patterns relied on protein secondary structures. Among 20 amino acids, P elicited the greatest effect on amino acid substitutions. The underlying mechanisms of context-dependent effects of amino acid substitutions were possibly mutation bias at a DNA level and natural selection. Our findings may improve secondary structure prediction algorithms and protein design, moreover, this study provided useful information to develop empirical models of protein evolution that consider dependence between residues.

Messenger RNA (mRNA) secondary structure decreases the elongation rate, as ribosomes must unwind every structure they encounter during translation. Therefore, the strength of mRNA secondary structure is assumed to be reduced in highly translated mRNAs. However, previous studies in vitro reported a positive correlation between mRNA folding strength and protein abundance. The counterintuitive finding suggests that mRNA secondary structure affects translation efficiency in an undetermined manner. Here, we analyzed the folding behavior of mRNA during translation and its effect on translation efficiency. We simulated translation process based on a novel computational model, taking into account the interactions among ribosomes, codon usage and mRNA secondary structures. We showed that mRNA secondary structure shortens ribosomal distance through the dynamics of folding strength. Notably, when adjacent ribosomes are close, mRNA secondary structures between them disappear, and codon usage determines the elongation rate. More importantly, our results showed that the combined effect of mRNA secondary structure and codon usage in highly translated mRNAs causes a short ribosomal distance in structural regions, which in turn eliminates the structures during translation, leading to a high elongation rate. Together, these findings reveal how the dynamics of mRNA secondary structure coupling with codon usage affect translation efficiency.

Local secondary structures in coding sequences have important functions across various translational processes. To date, however, the local structures and their functions in the early stage of translation elongation remain poorly understood. Here, we surveyed the structural stability in the first 180 nucleotides of the coding sequence of 27 species using computational method. We found that the structural stability in the 30-80 nucleotide interval was significantly higher than that in other regions in eukaryotes and most prokaryotes. No significant correlation between local translation efficiency and structural stability was observed, suggesting that this structural region has undergone selection pressure directly to maintain high stability. Furthermore, ribosome was blocked by this region, providing an opportunity for co-translational regulation. Remarkably, in eukaryotes, we found that mRNAs with higher structural stability in the 30-80 nucleotide interval tended to encode the secreted proteins. Overall, our results revealed a previously unappreciated correlation between structural stability and protein localization.

Summary blurbm6A-modification in the 5’ vicinity of the coding sequence of transcripts provides a selective mechanism for triaging mRNAs to stress granules and is mediated by YTHDF3 ‘reader’ protein.AbstractReversible post-transcriptional modifications on messenger RNA emerge as prevalent phenomena in RNA metabolism. The most abundant among them is N6-methyladenosine (m6A) which is pivotal for RNA metabolism and function, its role in stress response remains elusive. We have discovered that in response to oxidative stress, transcripts are additionally m6A-modified in their 5’ vicinity. Distinct from that of the translationally-active mRNAs, this methylation pattern provides a selective mechanism for triaging mRNAs from the translatable pool to stress-induced stress granules. These stress-induced newly methylated sites are selectively recognized by the YTH domain family 3 (YTHDF3) ‘reader’ protein, thereby revealing a new role for YTHDF3 in shaping the selectivity of stress response. Our findings describe a previously unappreciated function for RNA m6A modification in the oxidative-stress response and expand the breadth of physiological roles of m6A.

The integrated stress response (ISR) facilitates cellular adaptation to stress conditions via the common target eIF2α. During ISR, the selective translation of stress-related mRNAs often relies on alternative mechanisms, such as leaky scanning or reinitiation, but the underlying mechanism remains incompletely understood. Here we report that, in response to amino acid starvation, the reinitiation of ATF4 is not only governed by the eIF2α signaling pathway, but is also subjected to regulation by mRNA methylation in the form of N6-methyladenosine (m6A). While depleting m6A demethylases represses ATF4 reinitiation, knocking down m6A methyltransferases promotes ATF4 translation. We demonstrate that m6A in the 5' UTR controls ribosome scanning and subsequent start codon selection. Global profiling of initiating ribosomes reveals widespread alternative translation events influenced by dynamic mRNA methylation. Consistently, Fto transgenic mice manifest enhanced ATF4 expression, highlighting the critical role of m6A in translational regulation of ISR at cellular and organismal levels.

By introducing synonymous mutations into the coding sequences of GP64sp and FibHsp signal peptides, the influences of mRNA secondary structure and codon usage of signal sequences on protein expression and secretion were investigated using baculovirus/insect cell expression system. The results showed that mRNA structural stability of the signal sequences was not correlated with the protein production and secretion levels, and FibHsp was more tolerable to codon changes than GP64sp. Codon bias analyses revealed that codons for GP64sp were well de-optimized and contained more non-optimal codons than FibHsp. Synonymous mutations in GP64sp sufficiently increased its average codon usage frequency and resulted in dramatic reduction of the activity and secretion of luciferase. Protein degradation inhibition assay with MG-132 showed that higher codon usage frequency in the signal sequence increased the production as well as the degradation of luciferase protein, indicating that the synonymous codon substitutions in the signal sequence caused misfolding of luciferase instead of slowing down the protein production. Meanwhile, we found that introduction of more non-optimal codons into FibHsp could increase the production and secretion levels of luciferase, which suggested a new strategy to improve the production of secretory proteins in insect cells.

In eukaryotic cells, protein synthesis typically begins with the binding of eIF4F to the 7-methylguanylate (m7G) cap found on the 5' end of the majority of mRNAs. Surprisingly, overall translational output remains robust under eIF4F inhibition. The broad spectrum of eIF4F-resistant translatomes is incompatible with cap-independent translation mediated by internal ribosome entry sites (IRESs). Here, we report that N6-methyladenosine (m6A) facilitates mRNA translation that is resistant to eIF4F inactivation. Depletion of the methyltransferase METTL3 selectively inhibits translation of mRNAs bearing 5' UTR methylation, but not mRNAs with 5' terminal oligopyrimidine (TOP) elements. We identify ABCF1 as a critical mediator of m6A-promoted translation under both stress and physiological conditions. Supporting the role of ABCF1 in m6A-facilitated mRNA translation, ABCF1-sensitive transcripts largely overlap with METTL3-dependent mRNA targets. By illustrating the scope and mechanism of eIF4F-independent mRNA translation, these findings reshape our current perceptions of cellular translational pathways.

F-box proteins are substrate adaptors used by the SKP1-CUL1-F-box protein (SCF) complex, a type of E3 ubiquitin ligase complex in the ubiquitin proteasome system (UPS). SCF-mediated ubiquitylation regulates proteolysis of hundreds of cellular proteins involved in key signaling and disease systems. However, our knowledge of the evolution of the F-box gene family in Euarchontoglires is limited. In the present study, 559 F-box genes and nine related pseudogenes were identified in eight genomes. Lineage-specific gene gain and loss events occurred during the evolution of Euarchontoglires, resulting in varying F-box gene numbers ranging from 66 to 81 among the eight species. Both tandem duplication and retrotransposition were found to have contributed to the increase of F-box gene number, whereas mutation in the F-box domain was the main mechanism responsible for reduction in the number of F-box genes, resulting in a balance of expansion and contraction in the F-box gene family. Thus, the Euarchontoglire F-box gene family evolved under a birth-and-death model. Signatures of positive selection were detected in substrate-recognizing domains of multiple F-box proteins, and adaptive changes played a role in evolution of the Euarchontoglire F-box gene family. In addition, single nucleotide polymorphism (SNP) distributions were found to be highly non-random among different regions of F-box genes in 1092 human individuals, with domain regions having a significantly lower number of non-synonymous SNPs.

Various regulatory elements in messenger RNAs (mRNAs) carrying the secondary structure play important roles in a wide range of expression processes. Numerous recent works have focused on the discovery of these functional elements that contain the conserved mRNA structures. However, to date, regions with high structural stability have been largely overlooked. In this study, we defined high stability regions (HSRs) in the coding sequences (CDSs) in bacteria based on the normalized folding free energy. We found that CDSs had high number of HSRs, and these HSRs showed high structural context robustness compared with random sequences, indicating a direct selective constraint imposed on HSRs. A reduced ribosome speed was detected near the start position of HSR, implying a possibility that HSR acted as obstacle to drive translational pausing that coordinated protein synthesis. Interestingly, we found that genes with high HSR density were enriched in the processes of translation, protein folding, and cell division. In addition, essential genes exhibited higher HSR density than nonessential genes. Overall, our study presented the previously unappreciated correlation between the number variation of HSRs and cellular processes.