The effects of NCBP3 on METTL3‐mediated m6A RNA methylation to enhance translation process in hypoxic cardiomyocytes

Abstract

Hypoxia as a crucial pathogenesis factor usually results in huge harmful effects on cardiac injury and dysfunction. Our previous study has uncovered the global transcriptome and translatome profiles of cardiomyocytes in vitro and in vivo to response to hypoxia by RNA sequencing and ribosome profiling sequencing. We observe a series of differential expressed genes between transcription and translation, which may be attributed to the hypoxia‐specific binding affinity of nuclear cap‐binding subunit 3 (NCBP3) at 5' untranslation region of target genes. Although we observe that NCBP3 can facilitate translational process in myocardium under hypoxia stress, the underlying molecular mechanism of NCBP3 for gene translation modulation remains unclear. In this study, we performed NCBP3 immunoprecipitation for mass spectrum and found that METTL3 and eIF4A2 particularly interacted with NCBP3 in hypoxic rat H9C2 cardiomyocytes. Furthermore, we observed that METTL3‐mediated N6‐methyladenosine (m6A) methylation was elevated in hypoxia, but compromised by NCBP3 or METTL3 knockdown. Finally, we also demonstrated that NCBP3/METTL3/eIF4A2 regulatory axis plays a specific role in cardiomyocytes undergoing hypoxic stress. Taken together, we unmasked NCBP3, a novel hypoxia‐specific response protein functions as a scaffold to coordinate METTL3 and eIF4A2 for enhancing gene translation by m6A RNA methylation in cardiomyocytes upon hypoxic stress.

Emerging roles of the RNA modifications N6-methyladenosine and adenosine-to-inosine in cardiovascular diseases

“…Hypoxia-inducible m 6 A deposition by METTL3 was recently described to operate specifically in cardiomyocytes, which could possibly provide an operating rationale for some of the above speculations. 144 Namely, a hypoxia-inducible, cardiomyocyte-enriched, and mesoderm-restricted upregulation of a nuclear cap-binding subunit 3 (NCBP3) protein was identified to occupy the 5′ UTRs of 85 distinct mRNAs in hypoxic cardiomyocytes with a striking 87.6% congruency to a previously published hypoxic cardiomyocyte dataset of transcripts with incongruent translation activity to their transcriptomic expression. 346 NCBP3 was shown to recruit METTL3, promoting the bound mRNA m 6 A methylation and eukaryotic translation initiation factor 4A2 (eIF4A2) to initiate their translation.…”

“… 346 NCBP3 was shown to recruit METTL3, promoting the bound mRNA m 6 A methylation and eukaryotic translation initiation factor 4A2 (eIF4A2) to initiate their translation. 144 …”

“…346 NCBP3 was shown to recruit METTL3, promoting the bound mRNA m 6 A methylation and eukaryotic translation initiation factor 4A2 (eIF4A2) to initiate their translation. 144 Furthermore, ALKBH5 overexpression has also been shown beneficial by enhancing cardiac regeneration and salvage myocardial function after MI in both neonatal and adult mice (see section "cardiogenesis and cardiac regeneration") via m 6 A demethylationdependent increase in Ythdf1 translation and consequent YTHDF1dependently enhanced Yap1 translation to YAP1. 54 This YTHDF1m 6 A-Yap1 interaction was confirmed operative irrespective of ALKBH5 activity when YTHDF1 was overexpressed, suggesting incapability of ALKBH5 to demethylate Yap1.…”

m6A Methylation in Cardiovascular Diseases: From Mechanisms to Therapeutic Potential

“…Rapid gene expression after hypoxia stress is required to prevent cardiomyocytes from the hypoxia-induced injury. As the most abundant RNA modification, m6A methylation is dramatically up-regulated in cardiomyocytes after hypoxia exposure ( Fry et al, 2017 ; Chokkalla et al, 2019 ; Ye et al, 2021 ). Ye et al reported that m6A methylation at the 5′-UTR of hypoxia-related mRNA is upregulated via overexpression of METTL3 induced by hypoxia; and this promote the localization of eIF4A2 to target genes and enhance the translation of these genes in hypoxic cardiomyocytes, which can alleviate hypoxia-induced injury ( Ye et al, 2021 ; Table 3 and Figure 2 ).…”

N6-methyladenosine (m6A) RNA modification in the pathophysiology of heart failure: a narrative review

“…In addition to the YTH family, recent study found that the m6A writer can also interact with eIF4A2 to facilitate gene expression in cardiac cells under hypoxic conditions via the scaffold function of nuclear cap-binding subunit 3 (NCBP3) ( 31 ). Besides, the METTL3 was also found to be involved in the RNA splicing process.…”