Translatome and Transcriptome Profiling of Hypoxic-Induced Rat Cardiomyocytes

Abstract

Adult cardiac hypoxia as a crucial pathogenesis factor can induce detrimental effects on cardiac injury and dysfunction. The global transcriptome and translatome reflecting the cellular response to hypoxia have not yet been extensively studied in myocardium. In this study, we conducted RNA sequencing (RNA-seq) and ribosome profiling technique (polyribo-seq) in rat heart tissues and H9C2 cells exposed to different periods of hypoxia stress in vivo and in vitro. The temporal gene-expression profiling displayed the distinction of transcriptome and translatome, which were mainly concentrated in cell apoptosis, autophagy, DNA repair, angiogenesis, vascular process, and cardiac cell proliferation and differentiation. A large number of genes such as GNAI3, SEPT4, FANCL, BNIP3, TBX3, ESR2, PTGS2, KLF4, and ADRB2, whose transcript and translation levels are closely correlated, were identified to own a common RNA motif "5 0-GAAGCUGCC-3 0 " in 5 0 UTR. NCBP3 was further determined to recognize this RNA motif and facilitate translational process in myocardium under hypoxia stress. Taken together, our data show the close connection between alterations of transcriptome and translatome after hypoxia exposure, emphasizing the significance of translational regulation in related studies. The profiled molecular responses in current study may be valuable resources for advanced understanding of the mechanisms underlying hypoxia-induced effect on heart diseases.

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

“…Moreover, MeRIP‐seq data indicated the four profiles of m6A differentially enriched genes (log2FC >1 or <−1, p < 0.05) that were higher or lower in hypoxia compared with control, and combined with lower or higher compared with hypoxia plus NCBP3 knockdown (Figure 3B). Surprisingly, we notice that the profile C containing 85 genes with highly enriched m6A methylation in hypoxia and reduced in NCBP3 knockdown was 87.6% identical with DEGs of translatome data between normoxic and hypoxic H9C2 cells in our previous study 10 (Figure 3C). In profile C, genes with differential m6A RNA methylation at 5’ UTR were all included in RNA‐seq data (Figure 3D ), indicating that METTL3‐mediated m6A methylation at 5' UTR might play a regulatory role in translational process in hypoxic H9C2 cells.…”

“…Our previous study has unmasked that NCBP3 can specially recognize ‘GAAGCUGCC’ at 5' UTR of mRNAs, and further affects a large number of genes’ translation in hypoxic H9C2 cells 10 . To further understand the role of NCBP3 in translational process, IP was conducted to pull down NCBP3 (Figure ) followed by mass spectrum (Figure ).…”

“…Our previous study has unmasked that NCBP3 can specially recognize ‘GAAGCUGCC’ at 5' UTR of mRNAs, and further affects a large number of genes’ translation in hypoxic H9C2 cells. 10 To further understand the role of NCBP3 in translational process, IP was conducted to pull down NCBP3 (Figure S1A ) followed by mass spectrum (Figure S1B ). The differential binding protein profiling of NCBP3 between normoxic and hypoxic rat H9C2 cardiomyocytes was identified (log2FC>1 or <−1, p < 0.05) (Figure 1A ).…”

“…8 To monitor the localized protein synthesis and explore cotranslational folding and targeting, ribosome profiling technique (polyriboseq) based on sucrose-gradient separation of polysome-associated RNAs is allowed to assess the coding potential of mRNAs. 9 Our previous study 10 has compared the global transcriptome and translatome in hypoxic myocardium in vitro and in vivo via RNA-seq and polyribo-seq. A large number of differential expressed genes between transcriptome and translatome indicate the complicated posttranscriptional and translational reactions responding to hypoxia.…”

Preferential Ribosome Loading on the Stress-Upregulated mRNA Pool Shapes the Selective Translation under Stress Conditions

“…Recent years have also seen a substantial interest in dissecting the relationship between transcriptional and translational regulations in yeasts, plants and mammals under diverse stresses or across different tissues [ 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 ]. However, most of these studies assess the mRNA translation through polysome profiling, which cannot measure the exact number of ribosomes loading on polysome-associated mRNAs for each gene [ 19 , 20 , 21 , 22 , 23 ]. By contrast, a few studies comprehensively investigated the translatome based on ribosome footprints [ 15 , 16 , 17 , 18 , 24 ].…”