Dynamic mRNA modification in the form of N6-methyladenosine (m6A) adds considerable richness and sophistication to gene regulation. The m6A mark is asymmetrically distributed along mature mRNAs, with approximately 35% of m6A residues located within the coding region (CDS). It has been suggested that methylation in CDS slows down translation elongation. However, neither the decoding feature of endogenous mRNAs nor the physiological significance of CDS m6A has been clearly defined. Here, we found that CDS m6A leads to ribosome pausing in a codon-specific manner. Unexpectedly, removing CDS m6A from these transcripts results in a further decrease of translation. A systemic analysis of RNA structural datasets revealed that CDS m6A positively regulates translation by resolving mRNA secondary structures. We further demonstrate that the elongation-promoting effect of CDS methylation requires the RNA helicase-containing m6A reader YTHDC2. Our findings established the physiological significance of CDS methylation and uncovered non-overlapping function of m6A reader proteins.

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-methyladenosine (mA). While depleting mA demethylases represses ATF4 reinitiation, knocking down mA methyltransferases promotes ATF4 translation. We demonstrate that mA 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 mA in translational regulation of ISR at cellular and organismal levels.

SUMMARY 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 (IRES). 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.

RNA modification in the form of N 6 -methyladenosine (m 6 A) regulates nearly all the post-transcriptional processes. The asymmetric m 6 A deposition suggests that regional methylation may have distinct functional consequences. However, current RNA biology tools do not distinguish the contribution of individual m 6 A modifications. Here we report the development of “m 6 A editing”, a powerful approach that enables m 6 A installation and erasure from cellular RNAs without changing the primary sequence. We engineered fusions of CRISPR-Cas9 and a single chain m 6 A methyltransferase that can be programmed with a guide RNA. The resultant m 6 A “writers” allow functional comparison of single site methylation in different mRNA regions. We further engineered m 6 A “erasers” by fusing CRISPR-Cas9 with ALKBH5 or FTO to achieve site-specific demethylation of RNAs. The development of programmable m 6 A editing not only expands the scope of RNA engineering, but also facilitates mechanistic understanding of epitranscriptome.

Lung cancer is one of the most common malignant diseases globally, composed of non-small cell lung cancer (NSCLC, 85%) and small cell lung cancer (SCLC, 15%). MicroRNAs (miRNAs) are single-stranded noncoding RNAs having important roles in lung cancer development. miR-411-5p/3p were reported to be increased significantly in human NSCLC tissues and cell lines. Moreover, miR-411-5p/3p overexpression could accelerate cell proliferation and migration, and impede cell apoptosis in NSCLC cell lines. Mechanically, SPRY4 is confirmed a direct target of miR-411-5p/3p. Furthermore, our findings showed that miR-411-5p/3p promoted lung tumor growth in vivo, decreased SPRY4 expression dramatically, and induced EGFR, AKT signaling activation, as well as epithelial–mesenchymal transition (EMT) simultaneously in tumor tissues. In addition, we showed that miR-411-5p also targeted tumor suppressor TXNIP, involved in regulating positively cell cycle progress in SPC-A1 cells rather than in H1299. Whether cell specificity of low TXNIP mRNA level in H1299 is responsible for the different response to cell cycle between H1299 and SPC-A1 would need further explorations. Collectively, these results suggest that miR-411-5p/3p are required for NSCLC development by suppressing SPRY4 and TXNIP; thus, the miR-411-SPRY4-AKT axis might act as a promising target for lung cancer therapy clinically.

The formation of a series of side products causes low yield and difficult separation in 5-hydroxymethylfurfural (HMF) preparation. To overcome these problems, a novel switchable biphasic reaction/separation coupling system is proposed for the efficient preparation of HMF from fructose. The miscible ionic liquid (IL)/organic solvent is adjusted to two phases with CO2 as the “phase separation switch”. Reactants and catalyst are mainly enriched in the IL phase and the produced HMF is continuously extracted into the organic solvent phase. This leads to 84% yield of HMF and the mass ratio of HMF in the organic solvent phase to the IL phase of 1.3:1 in a biphasic system. Comparing to several commonly used systems and a biphasic system, the switchable biphasic reaction/separation coupling system is more efficient for HMF preparation. On the basis of the results, a biphasic consecutive process for HMF preparation is also proposed, which is an effective production process through recycling of the inexpensive and abundantly available solvents and CO2. It is expected that such a system may provide more applications for efficient reactions and separations in ILs.

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