Molecular Mechanisms Driving mRNA Degradation by m6A Modification.

N6-Methyladenosine (m6A), the most prevalent internal modification associated with eukaryotic mRNAs, influences many steps of mRNA metabolism, including splicing, export, and translation, as well as stability. Recent studies have revealed that m6A-containing mRNAs undergo one of two distinct pathways of rapid degradation: deadenylation via the YT521-B homology (YTH) domain-containing family protein 2 (YTHDF2; an m6A reader protein)-CCR4/NOT (deadenylase) complex or endoribonucleolytic cleavage by the YTHDF2-HRSP12-ribonuclease (RNase) P/mitochondrial RNA-processing (MRP) (endoribonuclease) complex. Some m6A-containing circular RNAs (circRNAs) are also subject to endoribonucleolytic cleavage by YTHDF2-HRSP12-RNase P/MRP. Here, we highlight recent progress on the molecular mechanisms underlying rapid mRNA degradation via m6A and describe our current understanding of the dynamic regulation of m6A-mediated mRNA decay through the crosstalk between m6A (or YTHDF2) and other cellular factors.

Endoribonucleolytic Cleavage of m6A-Containing RNAs by RNase P/MRP Complex.

N6-methyladenosine (m6A) is the most abundant internal modification in RNAs and plays regulatory roles in a variety of biological and physiological processes. Despite its important roles, the molecular mechanism underlying m6A-mediated gene regulation is poorly understood. Here, we show that m6A-containing RNAs are subject to endoribonucleolytic cleavage via YTHDF2 (m6A reader protein), HRSP12 (adaptor protein), and RNase P/MRP (endoribonucleases). We demonstrate that HRSP12 functions as an adaptor to bridge YTHDF2 and RNase P/MRP, eliciting rapid degradation of YTHDF2-bound RNAs. Transcriptome-wide analyses show that m6A RNAs that are preferentially targeted for endoribonucleolytic cleavage have an HRSP12-binding site and a RNase P/MRP-directed cleavage site upstream and downstream of the YTHDF2-binding site, respectively. We also find that a subset of m6A-containing circular RNAs associates with YTHDF2 in an HRSP12-dependent manner and is selectively downregulated by RNase P/MRP. Thus, our data expand the known functions of RNase P/MRP to endoribonucleolytic cleavage of m6A RNAs.

HuR stabilizes a polyadenylated form of replication-dependent histone mRNAs under stress conditions.

All metazoan mRNAs have a poly(A) tail at the 3' end with the exception of replication-dependent histone (RDH) mRNAs, which end in a highly conserved stem-loop (SL) structure. However, a subset of RDH mRNAs are reported to be polyadenylated under physiologic conditions. The molecular details of the biogenesis of polyadenylated RDH [poly(A)+ RDH] mRNAs remain unknown. In this study, our genome-wide analyses reveal that puromycin treatment or UVC irradiation stabilizes poly(A)+ RDH mRNAs, relative to canonical RDH mRNAs, which end in an SL structure. We demonstrate that the stabilization of poly(A)+ RDH mRNAs occurs in a translation-independent manner and is regulated via human antigen R (HuR) binding to the extended 3' UTR under stress conditions. Our data suggest that HuR regulates the expression of poly(A)+ RDH mRNAs.-Ryu, I., Park, Y., Seo, J.-W., Park, O. H., Ha, H., Nam, J.-W., Kim, Y. K. HuR stabilizes a polyadenylated form of replication-dependent histone mRNAs under stress conditions.

Insights into degradation mechanism of N-end rule substrates by p62/SQSTM1 autophagy adapter.

p62/SQSTM1 is the key autophagy adapter protein and the hub of multi-cellular signaling. It was recently reported that autophagy and N-end rule pathways are linked via p62. However, the exact recognition mode of degrading substrates and regulation of p62 in the autophagic pathway remain unknown. Here, we present the complex structures between the ZZ-domain of p62 and various type-1 and type-2 N-degrons. The binding mode employed in the interaction of the ZZ-domain with N-degrons differs from that employed by classic N-recognins. It was also determined that oligomerization via the PB1 domain can control functional affinity to the R-BiP substrate. Unexpectedly, we found that self-oligomerization and disassembly of p62 are pH-dependent. These findings broaden our understanding of the functional repertoire of the N-end rule pathway and provide an insight into the regulation of p62 during the autophagic pathway.

Identification and molecular characterization of cellular factors required for glucocorticoid receptor-mediated mRNA decay.

Glucocorticoid (GC) receptor (GR) has been shown recently to bind a subset of mRNAs and elicit rapid mRNA degradation. However, the molecular details of GR-mediated mRNA decay (GMD) remain unclear. Here, we demonstrate that GMD triggers rapid degradation of target mRNAs in a translation-independent and exon junction complex-independent manner, confirming that GMD is mechanistically distinct from nonsense-mediated mRNA decay (NMD). Efficient GMD requires PNRC2 (proline-rich nuclear receptor coregulatory protein 2) binding, helicase ability, and ATM-mediated phosphorylation of UPF1 (upstream frameshift 1). We also identify two GMD-specific factors: an RNA-binding protein, YBX1 (Y-box-binding protein 1), and an endoribonuclease, HRSP12 (heat-responsive protein 12). In particular, using HRSP12 variants, which are known to disrupt trimerization of HRSP12, we show that HRSP12 plays an essential role in the formation of a functionally active GMD complex. Moreover, we determine the hierarchical recruitment of GMD factors to target mRNAs. Finally, our genome-wide analysis shows that GMD targets a variety of transcripts, implicating roles in a wide range of cellular processes, including immune responses.

A new function of glucocorticoid receptor: regulation of mRNA stability.

It has long been thought that glucocorticoid receptor (GR) functions as a DNA-binding transcription factor in response to its ligand (a glucocorticoid) and thus regulates various cellular and physiological processes. It is also known that GR can bind not only to DNA but also to mRNA; this observation points to the possible role of GR in mRNA metabolism. Recent data revealed a molecular mechanism by which binding of GR to target mRNA elicits rapid mRNA degradation. GR binds to specific RNA sequences regardless of the presence of a ligand. In the presence of a ligand, however, the mRNA-associated GR can recruit PNRC2 and UPF1, both of which are specific factors involved in nonsense-mediated mRNA decay (NMD). PNRC2 then recruits the decapping complex, consequently promoting mRNA degradation. This mode of mRNA decay is termed "GR-mediated mRNA decay" (GMD). Further research demonstrated that GMD plays a critical role in chemotaxis of immune cells by targeting CCL2 mRNA. All these observations provide molecular insights into a previously unappreciated function of GR in posttranscriptional regulation of gene expression.

Glucocorticoid receptor interacts with PNRC2 in a ligand-dependent manner to recruit UPF1 for rapid mRNA degradation.

Glucocorticoid receptor (GR), which was originally known to function as a nuclear receptor, plays a role in rapid mRNA degradation by acting as an RNA-binding protein. The mechanism by which this process occurs remains unknown. Here, we demonstrate that GR, preloaded onto the 5'UTR of a target mRNA, recruits UPF1 through proline-rich nuclear receptor coregulatory protein 2 (PNRC2) in a ligand-dependent manner, so as to elicit rapid mRNA degradation. We call this process GR-mediated mRNA decay (GMD). Although GMD, nonsense-mediated mRNA decay (NMD), and staufen-mediated mRNA decay (SMD) share upstream frameshift 1 (UPF1) and PNRC2, we find that GMD is mechanistically distinct from NMD and SMD. We also identify de novo cellular GMD substrates using microarray analysis. Intriguingly, GMD functions in the chemotaxis of human monocytes by targeting chemokine (C-C motif) ligand 2 (CCL2) mRNA. Thus, our data provide molecular evidence of a posttranscriptional role of the well-studied nuclear hormone receptor, GR, which is traditionally considered a transcription factor.

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

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.

SMG1 regulates adipogenesis via targeting of staufen1-mediated mRNA decay.

Suppressor of morphogenesis in genitalia 1 (SMG1), a member of the phosphatidylinositol 3-kinase-related kinase family, is involved in nonsense-mediated mRNA decay (NMD). SMG1 phosphorylates Upf1, a key NMD factor. Subsequently, hyperphosphorylated Upf1 associates with SMG5-7 or proline-rich nuclear receptor coregulatory protein (PNRC2) to elicit rapid mRNA degradation. Upf1 is also known to be involved in staufen 1 (Stau1)-mediated mRNA decay (SMD), which is closely related to NMD. However, the biological and molecular roles of SMG1 in SMD remain unknown. Here, we provide evidence that SMG1 is involved in SMD. The immunoprecipitation results show that SMG1 is complexed with Stau1, Upf1, and Dcp1a. Downregulation of SMG1 or overexpression of a kinase-inactive mutant of SMG1 inhibits SMD efficiency. In addition, downregulation of SMG1 inhibits rapid degradation elicited by artificially tethered Stau1 or Upf1 downstream of the normal termination codon. Furthermore, Stau1 and Upf1 colocalize in processing bodies in an SMG1-dependent manner. We also find that the level of SMG1 increases during adipogenesis. Accordingly, downregulation of SMG1 causes the reduction in the level of Upf1 phosphorylation and delays adipogenesis, suggesting the functional involvement of SMG1 in adipogenesis via SMD.

Enhancement of cometabolic biodegradation of trichloroethylene (TCE) gas in biofiltration.

A biofilter column inoculated with Pseudomonas putida F1 was operated to study cometabolic biodegradation of trichloroethylene (TCE) gas using toluene as a primary substrate. Variations in the efficiency and capacity of TCE elimination with different inlet concentrations of toluene and TCE were investigated in order to understand the competitive inhibition between toluene and TCE. Two toluene feeding methods, stage feeding along the column and cyclic feeding, were examined as strategies to enhance TCE cometabolic biodegradation by avoiding the toluene inhibition of TCE biodegradation and the toxic effect of TCE on cells and toluene dioxygenase enzymes. It was concluded that both methods are promising and that the determination of a suitable feeding frequency, recovery period, and inlet toluene concentration was required to optimize cyclic feeding in the cometabolic biodegradation of TCE.