Junho Choe

The m(6)A Methyltransferase METTL3 Promotes Translation in Human Cancer Cells.

METTL3 is an RNA methyltransferase implicated in mRNA biogenesis, decay, and translation control through N(6)-methyladenosine (m(6)A) modification. Here we find that METTL3 promotes translation of certain mRNAs including epidermal growth factor receptor (EGFR) and the Hippo pathway effector TAZ in human cancer cells. In contrast to current models that invoke m(6)A reader proteins downstream of nuclear METTL3, we find METTL3 associates with ribosomes and promotes translation in the cytoplasm. METTL3 depletion inhibits translation, and both wild-type and catalytically inactive METTL3 promote translation when tethered to a reporter mRNA. Mechanistically, METTL3 enhances mRNA translation through an interaction with the translation initiation machinery. METTL3 expression is elevated in lung adenocarcinoma and using both loss- and gain-of-function studies, we find that METTL3 promotes growth, survival, and invasion of human lung cancer cells. Our results uncover an important role of METTL3 in promoting translation of oncogenes in human lung cancer.

microRNA/Argonaute 2 regulates nonsense-mediated messenger RNA decay

Imperfect base‐pairing between microRNA (miRNA) and the 3′‐untranslated region of target messenger RNA (mRNA) triggers translational repression of the target mRNA. Here, we provide evidence that human Argonaute 2 targets cap‐binding protein (CBP)80/20‐bound mRNAs and exon junction complex‐bound mRNAs and inhibits nonsense‐mediated mRNA decay (NMD), which is restricted tightly to CBP80/20‐bound mRNAs. Furthermore, microarray analyses reveal that a subset of cellular transcripts, which are expected to be targeted for NMD, is stabilized by miRNA‐mediated gene silencing. The regulation of NMD by miRNAs will shed light on a new post‐transcriptional regulation mechanism of gene expression in mammalian cells.

Translation initiation on mRNAs bound by nuclear cap-binding protein complex CBP80/20 requires interaction between CBP80/20-dependent translation initiation factor and eukaryotic translation initiation factor 3g.

Background: How the eIF3 complex and ribosomes are recruited during translation on CBP80/20-bound mRNAs remains obscure. Results: CTIF interacts with eIF3g to recruit the eIF3 complex. Conclusion: Translation on CBP80/20-bound mRNAs requires CTIF-eIF3g interaction. Significance: The use of different eIF3 subunits for recruiting eIF3 complex implies that translation on CBP80/20-bound mRNAs differs mechanistically from translation on eIF4E-bound mRNAs. In the cytoplasm of mammalian cells, either cap-binding proteins 80 and 20 (CBP80/20) or eukaryotic translation initiation factor (eIF) 4E can direct the initiation of translation. Although the recruitment of ribosomes to mRNAs during eIF4E-dependent translation (ET) is well characterized, the molecular mechanism for CBP80/20-dependent translation (CT) remains obscure. Here, we show that CBP80/20-dependent translation initiation factor (CTIF), which has been shown to be preferentially involved in CT but not ET, specifically interacts with eIF3g, a component of the eIF3 complex involved in ribosome recruitment. By interacting with eIF3g, CTIF serves as an adaptor protein to bridge the CBP80/20 and the eIF3 complex, leading to efficient ribosome recruitment during CT. Accordingly, down-regulation of CTIF using a small interfering RNA causes a redistribution of CBP80 from polysome fractions to subpolysome fractions, without significant consequence to eIF4E distribution. In addition, down-regulation of eIF3g inhibits the efficiency of nonsense-mediated mRNA decay, which is tightly coupled to CT but not to ET. Moreover, the artificial tethering of CTIF to an intercistronic region of dicistronic mRNA results in translation of the downstream cistron in an eIF3-dependent manner. These findings support the idea that CT mechanistically differs from ET.

Exon junction complex enhances translation of spliced mRNAs at multiple steps

Translation of spliced mRNAs is enhanced by exon junction complex (EJC), which is deposited on mRNAs as a result of splicing. Although this phenomenon itself is well known, the underlying molecular mechanism remains poorly understood. Here we show, using siRNAs against Y14 and eIF4AIII and spliced or intronless constructs that contain different types of internal ribosome entry sites (IRESes), that Y14 and eIF4AIII increase translation of spliced mRNAs before and after formation of the 80S ribosome complex, respectively. These results suggest that EJC modulates translation of spliced mRNA at multiple steps.

eIF4AIII enhances translation of nuclear cap-binding complex–bound mRNAs by promoting disruption of secondary structures in 5′UTR

Significance Two major cap-binding components can drive mammalian translation initiation: the nuclear cap-binding complex (CBC) and eukaryotic translation initiation factor 4E (eIF4E). Although eIF4E-dependent translation has been well characterized, the mechanism of CBC-dependent translation remains unclear. Here, we demonstrate the previously unappreciated role of eIF4AIII in CBC-dependent translation. eIF4AIII is traditionally considered a component of the exon junction complex loaded onto mRNAs after splicing. In addition, we found that eIF4AIII can be recruited to the 5′-end of CBC-associated mRNA and promotes the translation of CBC-associated mRNA by helping to unwind secondary structures in 5′UTR. Therefore, our data provide evidence that eIF4AIII is a translation initiation factor specifically required for translation of CBC-associated mRNAs. It has long been considered that intron-containing (spliced) mRNAs are translationally more active than intronless mRNAs (identical mRNA not produced by splicing). The splicing-dependent translational enhancement is mediated, in part, by the exon junction complex (EJC). Nonetheless, the molecular mechanism by which each EJC component contributes to the translational enhancement remains unclear. Here, we demonstrate the previously unappreciated role of eukaryotic translation initiation factor 4AIII (eIF4AIII), a component of EJC, in the translation of mRNAs bound by the nuclear cap-binding complex (CBC), a heterodimer of cap-binding protein 80 (CBP80) and CBP20. eIF4AIII is recruited to the 5′-end of mRNAs bound by the CBC by direct interaction with the CBC-dependent translation initiation factor (CTIF); this recruitment of eIF4AIII is independent of the presence of introns (deposited EJCs after splicing). Polysome fractionation, tethering experiments, and in vitro reconstitution experiments using recombinant proteins show that eIF4AIII promotes efficient unwinding of secondary structures in 5′UTR, and consequently enhances CBC-dependent translation in vivo and in vitro. Therefore, our data provide evidence that eIF4AIII is a specific translation initiation factor for CBC-dependent translation.

Ago2/miRISC-mediated inhibition of CBP80/20-dependent translation and thereby abrogation of nonsense-mediated mRNA decay require the cap-associating activity of Ago2

AGO2 physically interacts with GW182 by anti tag coimmunoprecipitation (View interaction)

Rapid degradation of replication-dependent histone mRNAs largely occurs on mRNAs bound by nuclear cap-binding proteins 80 and 20

The translation of mammalian messenger RNAs (mRNAs) can be driven by either cap-binding proteins 80 and 20 (CBP80/20) or eukaryotic translation initiation factor (eIF)4E. Although CBP80/20-dependent translation (CT) is known to be coupled to an mRNA surveillance mechanism termed nonsense-mediated mRNA decay (NMD), its molecular mechanism and biological role remain obscure. Here, using a yeast two-hybrid screening system, we identify a stem-loop binding protein (SLBP) that binds to a stem-loop structure at the 3′-end of the replication-dependent histone mRNA as a CT initiation factor (CTIF)-interacting protein. SLBP preferentially associates with the CT complex of histone mRNAs, but not with the eIF4E-depedent translation (ET) complex. Several lines of evidence indicate that rapid degradation of histone mRNA on the inhibition of DNA replication largely takes place during CT and not ET, which has been previously unappreciated. Furthermore, the ratio of CBP80/20-bound histone mRNA to eIF4E-bound histone mRNA is larger than the ratio of CBP80/20-bound polyadenylated β-actin or eEF2 mRNA to eIF4E-bound polyadenylated β-actin or eEF2 mRNA, respectively. The collective findings suggest that mRNAs harboring a different 3′-end use a different mechanism of translation initiation, expanding the repertoire of CT as a step for determining the fate of histone mRNAs.

Pioneer round of translation mediated by nuclear cap-binding proteins CBP80/20 occurs during prolonged hypoxia.

Nonsense‐mediated mRNA decay (NMD) is one of the mRNA surveillance mechanisms, which eliminates aberrant mRNAs harboring premature termination codons. NMD targets only mRNAs bound by the nuclear cap‐binding protein complex CBP80/20 which directs the pioneer round of translation. Here we demonstrate that NMD occurs efficiently during prolonged hypoxia in which steady‐state translation is drastically inhibited. Accordingly, CBP80 remains in the nucleus, and processing bodies are unaffected with regard to their abundance and number under prolonged hypoxic conditions. These results indicate that mRNAs enter the pioneer round of translation during prolonged hypoxia.

The mRNP remodeling mediated by UPF1 promotes rapid degradation of replication-dependent histone mRNA

Histone biogenesis is tightly controlled at multiple steps to maintain the balance between the amounts of DNA and histone protein during the cell cycle. In particular, translation and degradation of replication-dependent histone mRNAs are coordinately regulated. However, the underlying molecular mechanisms remain elusive. Here, we investigate remodeling of stem-loop binding protein (SLBP)-containing histone mRNPs occurring during the switch from the actively translating mode to the degradation mode. The interaction between a CBP80/20-dependent translation initiation factor (CTIF) and SLBP, which is important for efficient histone mRNA translation, is disrupted upon the inhibition of DNA replication or at the end of S phase. This disruption is mediated by competition between CTIF and UPF1 for SLBP binding. Further characterizations reveal hyperphosphorylation of UPF1 by activated ATR and DNA-dependent protein kinase upon the inhibition of DNA replication interacts with SLBP more strongly, promoting the release of CTIF and eIF3 from SLBP-containing histone mRNP. In addition, hyperphosphorylated UPF1 recruits PNRC2 and SMG5, triggering decapping followed by 5′-to-3′ degradation of histone mRNAs. The collective observations suggest that both inhibition of translation and recruitment of mRNA degradation machinery during histone mRNA degradation are tightly coupled and coordinately regulated by UPF1 phosphorylation.

Nonsense‐mediated translational repression involves exon junction complex downstream of premature translation termination codon

Human transforming growth factor‐β receptor type 2 (TGFβR2) mRNA harboring a premature translation termination codon (PTC) generated by frameshift mutation is targeted for nonsense‐mediated translational repression (NMTR), rather than nonsense‐mediated mRNA decay (NMD). Here we show that exon junction complex (EJC) downstream of a PTC plays an inhibitory role in translation of TGFβR2 mRNA. Translational repression by core EJC components occurs after formation of 80S ribosome complex, which is demonstrated using different types of internal ribosome entry sites (IRESes). Our findings implicate EJCs or core EJC components as negative regulators of translation.