LIN28A is a suppressor of ER-associated translation in embryonic stem cells.

LIN28 plays a critical role in developmental transition, glucose metabolism, and tumorigenesis. At the molecular level, LIN28 is known to repress maturation of let-7 microRNAs and enhance translation of certain mRNAs. In this study, we obtain a genome-wide view of the molecular function of LIN28A in mouse embryonic stem cells by carrying out RNA crosslinking-immunoprecipitation-sequencing (CLIP-seq) and ribosome footprinting. We find that, in addition to let-7 precursors, LIN28A binds to a large number of spliced mRNAs. LIN28A recognizes AAGNNG, AAGNG, and less frequently UGUG, which are located in the terminal loop of a small hairpin. LIN28A is localized to the periendoplasmic reticulum (ER) area and inhibits translation of mRNAs that are destined for the ER, reducing the synthesis of transmembrane proteins, ER or Golgi lumen proteins, and secretory proteins. Our study suggests a selective regulatory mechanism for ER-associated translation and reveals an unexpected role of LIN28A as a global suppressor of genes in the secretory pathway.

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.

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.

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.

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.

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.

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.

LSM12-EPAC1 defines a neuroprotective pathway that sustains the nucleocytoplasmic RAN gradient.

Nucleocytoplasmic transport (NCT) defects have been implicated in neurodegenerative diseases such as C9ORF72-associated amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). Here, we identify a neuroprotective pathway of like-Sm protein 12 (LSM12) and exchange protein directly activated by cyclic AMP 1 (EPAC1) that sustains the nucleocytoplasmic RAN gradient and thereby suppresses NCT dysfunction by the C9ORF72-derived poly(glycine-arginine) protein. LSM12 depletion in human neuroblastoma cells aggravated poly(GR)-induced impairment of NCT and nuclear integrity while promoting the nuclear accumulation of poly(GR) granules. In fact, LSM12 posttranscriptionally up-regulated EPAC1 expression, whereas EPAC1 overexpression rescued the RAN gradient and NCT defects in LSM12-deleted cells. C9-ALS patient-derived neurons differentiated from induced pluripotent stem cells (C9-ALS iPSNs) displayed low expression of LSM12 and EPAC1. Lentiviral overexpression of LSM12 or EPAC1 indeed restored the RAN gradient, mitigated the pathogenic mislocalization of TDP-43, and suppressed caspase-3 activation for apoptosis in C9-ALS iPSNs. EPAC1 depletion biochemically dissociated RAN-importin ß1 from the cytoplasmic nuclear pore complex, thereby dissipating the nucleocytoplasmic RAN gradient essential for NCT. These findings define the LSM12-EPAC1 pathway as an important suppressor of the NCT-related pathologies in C9-ALS/FTD.

Upf1 phosphorylation triggers translational repression during nonsense-mediated mRNA decay.

In mammalian cells, nonsense-mediated mRNA decay (NMD) generally requires that translation terminates sufficiently upstream of a post-splicing exon junction complex (EJC) during a pioneer round of translation. The subsequent binding of Upf1 to the EJC triggers Upf1 phosphorylation. We provide evidence that phospho-Upf1 functions after nonsense codon recognition during steps that involve the translation initiation factor eIF3 and mRNA decay factors. Phospho-Upf1 interacts directly with eIF3 and inhibits the eIF3-dependent conversion of 40S/Met-tRNA(i)(Met)/mRNA to translationally competent 80S/Met-tRNA(i)(Met)/mRNA initiation complexes to repress continued translation initiation. Consistent with phospho-Upf1 impairing eIF3 function, NMD fails to detectably target nonsense-containing transcripts that initiate translation independently of eIF3 from the CrPV IRES. There is growing evidence that translational repression is a key transition that precedes mRNA delivery to the degradation machinery. Our results uncover a critical step during NMD that converts a pioneer translation initiation complex to a translationally compromised mRNP.

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.

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.

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.

UPFront and center in RNA decay: UPF1 in nonsense-mediated mRNA decay and beyond.

Nonsense-mediated mRNA decay (NMD), which is arguably the best-characterized translation-dependent regulatory pathway in mammals, selectively degrades mRNAs as a means of post-transcriptional gene control. Control can be for the purpose of ensuring the quality of gene expression. Alternatively, control can facilitate the adaptation of cells to changes in their environment. The key to NMD, no matter what its purpose, is the ATP-dependent RNA helicase upstream frameshift 1 (UPF1), without which NMD fails to occur. However, UPF1 does much more than regulate NMD. As examples, UPF1 is engaged in functionally diverse mRNA decay pathways mediated by a variety of RNA-binding proteins that include staufen, stem-loop-binding protein, glucocorticoid receptor, and regnase 1. Moreover, UPF1 promotes tudor-staphylococcal/micrococcal-like nuclease-mediated microRNA decay. In this review, we first focus on how the NMD machinery recognizes an NMD target and triggers mRNA degradation. Next, we compare and contrast the mechanisms by which UPF1 functions in the decay of other mRNAs and also in microRNA decay. UPF1, as a protein polymath, engenders cells with the ability to shape their transcriptome in response to diverse biological and physiological needs.

Staufen1 and UPF1 exert opposite actions on the replacement of the nuclear cap-binding complex by eIF4E at the 5' end of mRNAs.

Newly synthesized mRNAs are exported from the nucleus to cytoplasm with a 5'-cap structure bound by the nuclear cap-binding complex (CBC). During or after export, the CBC should be properly replaced by cytoplasmic cap-binding protein eIF4E for efficient protein synthesis. Nonetheless, little is known about how the replacement takes place. Here, we show that double-stranded RNA-binding protein staufen1 (STAU1) promotes efficient replacement by facilitating an association between the CBC-importin α complex and importin ß. Our transcriptome-wide analyses and artificial tethering experiments also reveal that the replacement occurs more efficiently when an mRNA associates with STAU1. This event is inhibited by a key nonsense-mediated mRNA decay factor, UPF1, which directly interacts with STAU1. Furthermore, we find that cellular apoptosis that is induced by ionizing radiation is accompanied by inhibition of the replacement via increased association between STAU1 and hyperphosphorylated UPF1. Altogether, our data highlight the functional importance of STAU1 and UPF1 in the course of the replacement of the CBC by eIF4E, adding a previously unappreciated layer of post-transcriptional gene regulation.

AU-rich element-mediated mRNA decay via the butyrate response factor 1 controls cellular levels of polyadenylated replication-dependent histone mRNAs.

Replication-dependent histone (RDH) mRNAs have a nonpolyadenylated 3'-UTR that ends in a highly conserved stem-loop structure. Nonetheless, a subset of RDH mRNAs has a poly(A) tail under physiological conditions. The biological meaning of poly(A)-containing (+) RDH mRNAs and details of their biosynthesis remain elusive. Here, using HeLa cells and Western blotting, qRT-PCR, and biotinylated RNA pulldown assays, we show that poly(A)+ RDH mRNAs are post-transcriptionally regulated via adenylate- and uridylate-rich element-mediated mRNA decay (AMD). We observed that the rapid degradation of poly(A)+ RDH mRNA is driven by butyrate response factor 1 (BRF1; also known as ZFP36 ring finger protein-like 1) under normal conditions. Conversely, cellular stresses such as UV C irradiation promoted BRF1 degradation, increased the association of Hu antigen R (HuR; also known as ELAV-like RNA-binding protein 1) with the 3'-UTR of poly(A)+ RDH mRNAs, and eventually stabilized the poly(A)+ RDH mRNAs. Collectively, our results provide evidence that AMD surveils poly(A)+ RDH mRNAs via BRF1-mediated degradation under physiological conditions.

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.

Staufen1-mediated mRNA decay functions in adipogenesis.

The double-stranded RNA binding protein Staufen1 (Stau1) is involved in diverse gene expression pathways. For Stau1-mediated mRNA decay (SMD) in mammals, Stau1 binds to the 3' untranslated region of target mRNA and recruits Upf1 to elicit rapid mRNA degradation. However, the events downstream of Upf1 recruitment and the biological importance of SMD remain unclear. Here we show that SMD involves PNRC2, decapping activity, and 5'-to-3' exonucleolytic activity. In particular, Upf1 serves as an adaptor protein for the association of PNRC2 and Stau1. During adipogenesis, Stau1 and PNRC2 increase in abundance, Upf1 becomes hyperphosphorylated, and consequently SMD efficiency is enhanced. Intriguingly, downregulation of SMD components attenuates adipogenesis in a way that is rescued by downregulation of an antiadipogenic factor, Krüppel-like factor 2 (KLF2), the mRNA of which is identified as a substrate of SMD. Our data thus identify a biological role for SMD in adipogenesis.

DLK regulates a distinctive transcriptional regeneration program after peripheral nerve injury.

Following damage to a peripheral nerve, injury signaling pathways converge in the cell body to generate transcriptional changes that support axon regeneration. Here, we demonstrate that dual leucine zipper kinase (DLK), a central regulator of injury responses including axon regeneration and neuronal apoptosis, is required for the induction of the pro-regenerative transcriptional program in response to peripheral nerve injury. Using a sensory neuron-conditional DLK knockout mouse model, we show a time course for the dependency of gene expression changes on the DLK pathway after sciatic nerve injury. Gene ontology analysis reveals that DLK-dependent gene sets are enriched for specific functional annotations such as ion transport and immune response. A series of comparative analyses shows that the DLK-dependent transcriptional program is distinct from that promoted by the importin-dependent retrograde signaling pathway, while it is partially shared between PNS and CNS injury responses. We suggest that DLK-dependency might provide a selective filter for regeneration-associated genes among the injury-responsive transcriptome.

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.

Human proline-rich nuclear receptor coregulatory protein 2 mediates an interaction between mRNA surveillance machinery and decapping complex.

Nonsense-mediated mRNA decay (NMD) is the best-characterized mRNA surveillance mechanism by which aberrant mRNAs harboring premature termination codons are degraded before translation. However, to date, how NMD machinery recruits the general decay complex to faulty mRNAs and degrades those mRNAs remains unclear. Here we identify human proline-rich nuclear receptor coregulatory protein 2 (PNRC2) as a Upf1- and Dcp1a-interacting protein. Downregulation of PNRC2 abrogates NMD, and artificially tethering PNRC2 downstream of a normal termination codon reduces mRNA abundance. Accordingly, PNRC2 preferentially interacts with hyperphosphorylated Upf1 compared with wild-type Upf1 and triggers movement of hyperphosphorylated Upf1 into processing bodies (P bodies). Our observations suggest that PNRC2 plays an essential role in mammalian NMD, mediating the interaction between the NMD machinery and the decapping complex, so as to target the aberrant mRNA-containing RNPs into P bodies.