Longitudinal epi-transcriptome profiling reveals the crucial role of m⁶A in prenatal skeletal muscle development of pigs

Abstract

18Background: N6-methyladenosine (m 6 A) is the most abundant RNA modification and essentially 19 participates in the regulation of skeletal muscle development. However, the status and function of 20 m 6 A methylation in prenatal myogenesis remains unclear now. 21Results: In our present study, we first demonstrate that chemical suppression of m 6 A and 22 knockdown METTL14 significantly inhibit the differentiation and promote the proliferation of 23 C2C12 myoblast cells. The mRNA expression of m 6 A reader protein IGF2BP1, which functions to 24 promote the stability of target mRNA, continually decreases during the prenatal skeletal muscle 25 development. Thereafter, profiling transcriptome-wide m 6 A for six developmental stage of prenatal 26 skeletal muscle, which spanning two important waves of pig myogenesis, were performed using a 27 refined MeRIP sequencing technology that is optimal for small-amount of RNA samples. Highly 28 dynamic m 6 A methylomes across different development stages were then revealed, with majority 29 of the affected genes enriched in pathways of skeletal muscle development. In association with the 30 transcriptome-wide alterations, transcriptional regulatory factors (MyoD) and differentiated 31 markers (MyHC, MYH1) of muscle development are simultaneously regulated with m 6 A and 32 IGF2BP1. Knockdown of IGF2BP1 also suppresses myotube formation and promotes cell 33 proliferation. 34 Conclusions: The present study clarifies the dynamics of RNA m 6 A methylation in the regulation 35 of prenatal skeletal muscle development, providing a data baseline for future developmental as well 36 as biomedical studies of m 6 A functions in muscle development and disease. 37 Keywords: N6-methyladenosine; IGF2BP1; myoblast; skeletal muscle 38 39 Introduction 40Understanding the development of skeletal muscle is crucial to unravel the molecular basis 41 of formation and diseases of skeletal muscle [1, 2]. The formation of skeletal muscle is regulated 42 not only by genetic factors but also by epigenetic factors [3, 4]. Among these factors, N6-43 methyladenosine (m 6 A) is an epigenetic factor newly discovered in recent years [5, 6]. A dynamic 44 regulation of m 6 A during development is achieved through interplay among m 6 A methyltransferases 45 (METTL3/METTL14/WTAP) and demethylase (FTO/ALKBH5) [7][8][9]. Despite that a previous 46 study revealed the transcriptome-wide m 6 A profile of porcine muscle tissue, to date, very limited numbers of m 6 A peaks and the methylated genes were revealed among different stages ( Fig. 2a). 111Gene Ontology (GO) term enrichment analyses revealed that the m 6 A genes were commonly 112 associated with RNA binding, nucleoplasm and macromolecule metabolic process, but relatively 113 more divergent for functions of regulation of gene expression and biosynthesis processes (Fig. 2b).all the DEGs identified across these stages of skeletal muscle development, with considering their 130 correlation with body weight, body length and developmental stages [19]. We identified seve...