Extracellular arginine availability modulates eIF2α O-GlcNAcylation and heme oxygenase 1 translation for cellular homeostasis.

BACKGROUND: Nutrient limitations often lead to metabolic stress during cancer initiation and progression. To combat this stress, the enzyme heme oxygenase 1 (HMOX1, commonly known as HO-1) is thought to play a key role as an antioxidant. However, there is a discrepancy between the level of HO-1 mRNA and its protein, particularly in cells under stress. O-linked ß-N-acetylglucosamine (O-GlcNAc) modification of proteins (O-GlcNAcylation) is a recently discovered cellular signaling mechanism that rivals phosphorylation in many proteins, including eukaryote translation initiation factors (eIFs). The mechanism by which eIF2α O-GlcNAcylation regulates translation of HO-1 during extracellular arginine shortage (ArgS) remains unclear. METHODS: We used mass spectrometry to study the relationship between O-GlcNAcylation and Arg availability in breast cancer BT-549 cells. We validated eIF2α O-GlcNAcylation through site-specific mutagenesis and azido sugar N-azidoacetylglucosamine-tetraacylated labeling. We then evaluated the effect of eIF2α O-GlcNAcylation on cell recovery, migration, accumulation of reactive oxygen species (ROS), and metabolic labeling during protein synthesis under different Arg conditions. RESULTS: Our research identified eIF2α, eIF2ß, and eIF2γ, as key O-GlcNAcylation targets in the absence of Arg. We found that O-GlcNAcylation of eIF2α plays a crucial role in regulating antioxidant defense by suppressing the translation of the enzyme HO-1 during Arg limitation. Our study showed that O-GlcNAcylation of eIF2α at specific sites suppresses HO-1 translation despite high levels of HMOX1 transcription. We also found that eliminating eIF2α O-GlcNAcylation through site-specific mutagenesis improves cell recovery, migration, and reduces ROS accumulation by restoring HO-1 translation. However, the level of the metabolic stress effector ATF4 is not affected by eIF2α O-GlcNAcylation under these conditions. CONCLUSIONS: Overall, this study provides new insights into how ArgS fine-tunes the control of translation initiation and antioxidant defense through eIF2α O-GlcNAcylation, which has potential biological and clinical implications.

O-GlcNAc transferase is important for homology-directed repair.

O-Linked ß-N-acetylglucosamine glycosylation (O-GlcNAcylation) to serine or threonine residues is a reversible and dynamic post-translational modification. O-GlcNAc transferase (OGT) is the only enzyme for O-GlcNAcylation, and is a potential cancer therapeutic target in combination with clastogenic (i.e., chromosomal breaking) therapeutics. Thus, we sought to examine the influence of O-GlcNAcylation on chromosomal break repair. Using a set of DNA double strand break (DSB) reporter assays, we found that the depletion of OGT, and its inhibition with a small molecule each caused a reduction in repair pathways that involve use of homology: RAD51-dependent homology-directed repair (HDR), and single strand annealing. In contrast, such OGT disruption did not obviously affect chromosomal break end joining, and furthermore caused an increase in homology-directed gene targeting. Such disruption in OGT also caused a reduction in clonogenic survival, as well as modifications to cell cycle profiles, particularly an increase in G1-phase cells. We also examined intermediate steps of HDR, finding no obvious effects on an assay for DSB end resection, nor for RAD51 recruitment into ionizing radiation induced foci (IRIF) in proliferating cells. However, we also found that the influence of OGT on HDR and homology-directed gene targeting were dependent on RAD52, and that OGT is important for RAD52 IRIF in proliferating cells. Thus, we suggest that OGT is important for regulation of HDR that is partially linked to RAD52 function.

To indel or not to indel: Factors influencing mutagenesis during chromosomal break end joining.

Chromosomal DNA double-strand breaks (DSBs) are the effective lesion of radiotherapy and other clastogenic cancer therapeutics, and are also the initiating event of many approaches to gene editing. Ligation of the DSBs by end joining (EJ) pathways can restore the broken chromosome, but the repair junctions can have insertion/deletion (indel) mutations. The indel patterns resulting from DSB EJ are likely defined by the initial structure of the DNA ends, how the ends are processed and synapsed prior to ligation, and the factors that mediate the ligation step. In this review, we describe key factors that influence these steps of DSB EJ in mammalian cells, which is significant both for understanding mutagenesis resulting from clastogenic cancer therapeutics, and for developing approaches to manipulating gene editing outcomes.

Early echocardiographic predictors of outcomes in the mouse transverse aortic constriction heart failure model.

INTRODUCTION: Mouse transverse aortic constriction (TAC) is a widely-used model of pressure overload-induced heart failure. An intrinsic limitation of the model is variability in the response to pressure overload even when employing a standard severity of stenosis. Few literature studies have explicitly reported the use of entry criteria or early predictors to mitigate variability and enrich outcomes in this model. METHODS: Eleven-week-old male C57BL/6J mice underwent TAC or sham surgery. Left ventricular (LV) function and dimensions were assessed by M-mode echocardiography at baseline (pre) and 3, 9 and 12weeks post-procedure (end-study). At 24h post-procedure, transverse aortic flow velocities were obtained for estimating trans-TAC pressure gradients. Invasive LV hemodynamic assessments were performed and terminal heart and lung weights obtained at end-study. RESULTS: TAC mice displayed early development of LV hypertrophy and wall thickening followed by the later development of LV chamber dilation, and progressive development of LV systolic and diastolic dysfunction. The use of a pre-defined trans-TAC pressure gradient criterion of 45-60mmHg did not affect end-study organ weight, echocardiographic and invasive hemodynamic outcomes. A post-hoc receiver operator characteristic (ROC) analysis identified early 3week echocardiographic measures of LVmass(echo) and ejection fraction, with threshold changes of ~+30% and -10% normalized to baseline respectively, as good predictors for multiple end-study organ weight, echocardiographic and invasive hemodynamic outcomes. DISCUSSION: This ROC analysis has identified early predictive threshold changes which may serve, alone or in combination, as entry criteria to enrich outcome in this model.

Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing.

The regulatory role of N(6)-methyladenosine (m(6)A) and its nuclear binding protein YTHDC1 in pre-mRNA splicing remains an enigma. Here we show that YTHDC1 promotes exon inclusion in targeted mRNAs through recruiting pre-mRNA splicing factor SRSF3 (SRp20) while blocking SRSF10 (SRp38) mRNA binding. Transcriptome assay with PAR-CLIP-seq analysis revealed that YTHDC1-regulated exon-inclusion patterns were similar to those of SRSF3 but opposite of SRSF10. In vitro pull-down assay illustrated a competitive binding of SRSF3 and SRSF10 to YTHDC1. Moreover, YTHDC1 facilitates SRSF3 but represses SRSF10 in their nuclear speckle localization, RNA-binding affinity, and associated splicing events, dysregulation of which, as the result of YTHDC1 depletion, can be restored by reconstitution with wild-type, but not m(6)A-binding-defective, YTHDC1. Our findings provide the direct evidence that m(6)A reader YTHDC1 regulates mRNA splicing through recruiting and modulating pre-mRNA splicing factors for their access to the binding regions of targeted mRNAs.

FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis.

The role of Fat Mass and Obesity-associated protein (FTO) and its substrate N6-methyladenosine (m6A) in mRNA processing and adipogenesis remains largely unknown. We show that FTO expression and m6A levels are inversely correlated during adipogenesis. FTO depletion blocks differentiation and only catalytically active FTO restores adipogenesis. Transcriptome analyses in combination with m6A-seq revealed that gene expression and mRNA splicing of grouped genes are regulated by FTO. M6A is enriched in exonic regions flanking 5'- and 3'-splice sites, spatially overlapping with mRNA splicing regulatory serine/arginine-rich (SR) protein exonic splicing enhancer binding regions. Enhanced levels of m6A in response to FTO depletion promotes the RNA binding ability of SRSF2 protein, leading to increased inclusion of target exons. FTO controls exonic splicing of adipogenic regulatory factor RUNX1T1 by regulating m6A levels around splice sites and thereby modulates differentiation. These findings provide compelling evidence that FTO-dependent m6A demethylation functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis.

Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase.

The methyltransferase like 3 (METTL3)-containing methyltransferase complex catalyzes the N6-methyladenosine (m6A) formation, a novel epitranscriptomic marker; however, the nature of this complex remains largely unknown. Here we report two new components of the human m6A methyltransferase complex, Wilms' tumor 1-associating protein (WTAP) and methyltransferase like 14 (METTL14). WTAP interacts with METTL3 and METTL14, and is required for their localization into nuclear speckles enriched with pre-mRNA processing factors and for catalytic activity of the m6A methyltransferase in vivo. The majority of RNAs bound by WTAP and METTL3 in vivo represent mRNAs containing the consensus m6A motif. In the absence of WTAP, the RNA-binding capability of METTL3 is strongly reduced, suggesting that WTAP may function to regulate recruitment of the m6A methyltransferase complex to mRNA targets. Furthermore, transcriptomic analyses in combination with photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) illustrate that WTAP and METTL3 regulate expression and alternative splicing of genes involved in transcription and RNA processing. Morpholino-mediated knockdown targeting WTAP and/or METTL3 in zebrafish embryos caused tissue differentiation defects and increased apoptosis. These findings provide strong evidence that WTAP may function as a regulatory subunit in the m6A methyltransferase complex and play a critical role in epitranscriptomic regulation of RNA metabolism.