LncRNA HOTAIRM1 functions in DNA double-strand break repair via its association with DNA repair and mRNA surveillance factors.

The eukaryotic exon junction complex component Y14 participates in double-strand break (DSB) repair via its RNA-dependent interaction with the non-homologous end-joining (NHEJ) complex. Using immunoprecipitation-RNA-seq, we identified a set of Y14-associated long non-coding RNAs (lncRNAs). The lncRNA HOTAIRM1 serves as a strong candidate that mediates the interaction between Y14 and the NHEJ complex. HOTAIRM1 localized to near ultraviolet laser-induced DNA damage sites. Depletion of HOTAIRM1 delayed the recruitment of DNA damage response and repair factors to DNA lesions and compromised the efficiency of NHEJ-mediated DSB repair. Identification of the HOTAIRM1 interactome revealed a large set of RNA processing factors including mRNA surveillance factors. The surveillance factors Upf1 and SMG6 localized to DNA damage sites in a HOTAIRM1-dependent manner. Depletion of Upf1 or SMG6 increased the level of DSB-induced non-coding transcripts at damaged sites, indicating a pivotal role for Upf1/SMG6-mediated RNA degradation in DNA repair. We conclude that HOTAIRM1 serves as an assembly scaffold for both DNA repair and mRNA surveillance factors that act in concert to repair DSBs.

Co-phase separation of Y14 and RNA in vitro and its implication for DNA repair.

The multifunctional RNA recognition motif-containing protein Y14/RBM8A participates in mRNA metabolism and is essential for the efficient repair of DNA double-strand breaks (DSBs). Y14 contains highly charged, low-complexity sequences in both the amino- and carboxy-terminal domains. The feature of charge segregation suggests that Y14 may undergo liquid-liquid phase separation (LLPS). Recombinant Y14 formed phase-separated droplets, which were sensitive to pH and salt concentration. Domain mapping suggested that LLPS of Y14 involves multivalent electrostatic interactions and is partly determined by the net charge of its low-complexity regions. Phospho-mimicry of the carboxy-terminal arginine-serine dipeptides of Y14 suppressed phase separation. Moreover, RNA could phase separate into Y14 droplets and modulate Y14 LLPS in a concentration-dependent manner. Finally, the capacity of Y14 in LLPS and coacervation with RNA in vitro correlated with its activity in DSB repair. These results reveal a molecular rule for LLPS of Y14 in vitro and an implication for its co-condensation with RNA in genome stability.

Effect of heat stress on oxidative damage and antioxidant defense system in white clover (Trifolium repens L.).

MAIN CONCLUSION: This study leads to advances in the field of heat tolerance among different plant species. We concluded that a coordinated, increased antioxidant defense system enabled white clover to reduce heat-induced oxidative damage. The rise in global ambient temperature has a wide range of effects on plant growth, and, therefore, on the activation of various molecular defenses before the appearance of heat damage. Elevated temperatures result in accelerated generation of reactive oxygen species (ROS), causing an imbalance between ROS production and the ability of scavenging systems to detoxify and remove the reactive intermediates. The aim of this study was to determine the role of antioxidant defense systems in the alleviation of heat stress (HS) consequences in white clover (Trifolium repens L.), which is cultivated worldwide. We evaluated how temperature and time parameters contribute to the thermotolerance of white clover at different growth stages. We revealed HS protection in white clover from 37 to 40 °C, with 40 °C providing the greatest protection of 3-day-old seedlings and 28-day-old adult plants. Heat-provoked oxidative stress in white clover was confirmed by substantial changes in electrolyte leakage, malondialdehyde (MDA), and chlorophyll content, as well as superoxide anion (O2·-) and hydrogen peroxide (H2O2) production. Furthermore, superoxide dismutase (SOD) and ascorbate peroxidase (APX) as well as a high level of GSH non-enzymatic antioxidant were the most responsive, and were associated with acquired thermotolerance through the regulation of ROS generation. We demonstrated, by studying protoplast transient gene expression, direct genetic evidence of endogenous antioxidant-related genes that confer HS tolerance in white clover. Our present study clearly establishes that oxidative stress ensues from HS, which triggers the induction of antioxidant defense systems for ROS scavenging in white clover.

The RNA Processing Factor Y14 Participates in DNA Damage Response and Repair.

DNA repair deficiency leads to genome instability and hence human disease. Depletion of the RNA processing factor Y14/RBM8A in cultured cells or Rbm8a haplodeficiency in the developing mouse cortex results in the accumulation of DNA damage. Y14 depletion differentially affected the expression of DNA damage response (DDR) factors and induced R-loops, both of which threaten genomic stability. Immunoprecipitation coupled with mass spectrometry revealed DDR factors as potential Y14-interacting partners. Further results confirmed that Y14 interacts with Ku and several DDR factors in an ATM-dependent manner. Y14 co-fractionated with Ku in chromatin-enriched fractions and further accumulated on chromatin upon DNA damage. Y14 knockdown delayed recruitment of DDR factors to DNA damage sites and formation of γH2AX foci and also led to Ku retention on chromatin. Accordingly, Y14 depletion compromised the efficiency of DNA end joining. Therefore Y14 likely plays a direct role in DNA damage repair via its interaction with DDR factors.

A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement.

Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis.

Function and pathological implications of exon junction complex factor Y14.

Eukaryotic mRNA biogenesis involves a series of interconnected steps, including nuclear pre-mRNA processing, mRNA export, and surveillance. The exon-junction complex (EJC) is deposited on newly spliced mRNAs and coordinates several downstream steps of mRNA biogenesis. The EJC core protein, Y14, functions with its partners in nonsense-mediated mRNA decay and translational enhancement. Y14 plays additional roles in mRNA metabolism, some of which are independent of the EJC, and it is also involved in other cellular processes. Genetic mutations or aberrant expression of Y14 results in physiological abnormality and may cause disease. Therefore, it is important to understand the various functions of Y14 and its physiological and pathological roles.

The RNA-binding protein Y14 inhibits mRNA decapping and modulates processing body formation.

The exon-junction complex (EJC) deposited on a newly spliced mRNA plays an important role in subsequent mRNA metabolic events. Here we show that an EJC core heterodimer, Y14/Magoh, specifically associates with mRNA-degradation factors, including the mRNA-decapping complex and exoribonucleases, whereas another core factor, eIF4AIII/MLN51, does not. We also demonstrate that Y14 interacts directly with the decapping factor Dcp2 and the 5' cap structure of mRNAs via different but overlapping domains and that Y14 inhibits the mRNA-decapping activity of Dcp2 in vitro. Accordingly, overexpression of Y14 prolongs the half-life of a reporter mRNA. Therefore Y14 may function independently of the EJC in preventing mRNA decapping and decay. Furthermore, we observe that depletion of Y14 disrupts the formation of processing bodies, whereas overexpression of a phosphomimetic Y14 considerably increases the number of processing bodies, perhaps by sequestering the mRNA-degradation factors. In conclusion, this report provides unprecedented evidence for a role of Y14 in regulating mRNA degradation and processing body formation and reinforces the influence of phosphorylation of Y14 on its activity in postsplicing mRNA metabolism.

The exon junction complex component Y14 modulates the activity of the methylosome in biogenesis of spliceosomal small nuclear ribonucleoproteins.

The RNA-binding protein Y14 heterodimerizes with Mago as the core of the exon junction complex during precursor mRNA splicing and plays a role in mRNA surveillance in the cytoplasm. Using the Y14/Magoh heterodimer as bait in a screening for its interacting partners, we identified the protein-arginine methyltransferase PRMT5 as a candidate. We show that Y14 and Magoh, but not other factors of the exon junction complex, interact with the cytoplasmic PRMT5-containing methylosome. We further provide evidence that Y14 promoted the activity of PRMT5 in methylation of Sm proteins of the small nuclear ribonucleoprotein core, whereas knockdown of Y14 reduced their methylation level. Moreover, Y14 overexpression induced the formation of a large, active, and small nuclear ribonucleoprotein (snRNP)-associated methylosome complex. However, Y14 may only transiently associate with the snRNP assembly complex in the cytoplasm. Together, our results suggest that Y14 facilitates Sm protein methylation probably by its activity in promoting the formation or stability of the methylosome-containing complex. We hypothesize that Y14 provides a regulatory link between pre-mRNA splicing and snRNP biogenesis.

Phosphorylation of Y14 modulates its interaction with proteins involved in mRNA metabolism and influences its methylation.

The multicomponent exon junction complex (EJC) is deposited on the spliced mRNA during pre-mRNA splicing and is implicated in several post-splicing events, including mRNA export, nonsense-mediated mRNA decay (NMD), and translation control. This report is the first to identify potential post-translational modifications of the EJC core component Y14. We demonstrate that Y14 is phosphorylated at its repeated arginine/serine (RS) dipeptides, likely by SR protein-specific kinases. Phosphorylation of Y14 abolished its interaction with EJC components as well as factors that function downstream of the EJC. A non-phosphorylatable Y14 mutant was equivalent to the wild-type protein with respect to its association with spliced mRNA and its ability in NMD activation, but the mutant sequestered EJC and NMD factors on ribosome-containing mRNA ribonucleoproteins (mRNPs). We therefore hypothesize that phosphorylation of Y14 occurs upon completion of mRNA surveillance, leading to dissociation of Y14 from ribosome-containing mRNPs. Moreover, we found that Y14 is possibly methylated at multiple arginine residues in the carboxyl-terminal domain and that methylation of Y14 was antagonized by phosphorylation of RS dipeptides. This study reveals antagonistic post-translational modifications of Y14 that may be involved in the remodeling of Y14-containing mRNPs.