Nuclear M~6A Reader YTHDC1 Regulates MRNA Splicing

N6-methyl-adenosine (m6A) is one of the most common and abundant modifications on mRNA molecules present in eukaryotes. Early studies described that m6A occurs at a level of three to five sites per mRNA on average in mammals and occurs in highly-conserved regions with a consensus sequence identified as:RRACH (R= G or A, H= A, C or U). m6A methylation is a reversible modification catalyzed by RNA methyltransferase versus demethylases, where the former one is a multicomponent complex composed of at least three subunits, including methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and Wilms tumor 1-associated protein (WTAP). and the latter ones that have been discovered so far include alkylated DNA repair protein A1kB homolog 5 (ALKBH5) and fat mass and obesity-associated protein (FTO). Additionally, several m6A-binding proteins with YTH domain located in cytoplasmic (YTHDF1, YTHDF2, YTHDF3) and nuclear (YTHDC1) compartments have been identified. Cytoplasmic YTHDF2 and YTHDF1 have been reported to modulate mRNA stability and translation, respectively. However, the functions of nuclear m6A binding protein YTHDC1 are largely unknown. YTHDC1 was reported to localize in YT-body ajacent to speckles which is the RNA processing organelle. These suggest that YTHDC1 may interact with other RNA processing factors to involve in splicing process.To verify these hypothesis, we identified five YTHDC1 interacting SR family members including SRSF1, SRSF3, SRSF7, SRSF9, SRSF10 by using immunoprecipitation in combination with MS/MS technology. With splicing pattern analysis on transcriptome, we found that YTHDC1, SRSF3 and METTL3 promote cassette exon inclusion, while SRSF10 promtes cassette exon skipping. In order to investigate the splicing patterns of their targeting exon, we combined PAR-CLIP-seq with RNA-seq analyses and found YTHDC1 and SRSF3 promote their targeting exons inclusion while SRSF10 inhibits its targeting exons inclusion. However, for the other three YTHDC1 interacting SR proteins, we did not find significant splicing pattern change of their individual targeting exons. So we focused on SRSF3 and SRSF10. The RT-PCR verification of splicing change of randomly picked two genes were consistent with our sequencing splicing analysis. We men found the average binding sites distance between SRSR3 and YTHDC1 is closer than that of SRSF10 and YTHDC1. In order to investigate the spacial relationship between these three proteins, we did in vitro immunoprecipitation and found that YTHDC1, SRSF3 and SRSF10 can directly binds with each other. We also found SRSF3 and SRSF10 can competitively interact with YTHDC1 which may account for the opposite alternative splicing pattern of knockdown of SRSF3 and SRSF10.We speculated that the opposite splicing patterns of SRSF3 and SRSF10 is due to their RNA binding sites preference. By using PAR-CLIP method in combination with 3’biotin-labelling assay, we found RNA binding affinity of SRSF3 decreased, while the RNA binding affinity of SRSF10 increased with YTHDC1 knockdown.These results suggest that YTHDC1 can recruit SRSF3 for its RNA binding while inhibits SRSF10 binding with RNA to induce exon inclusion. Interestingly, we found that 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 m6A-binding-defective, YTHDC1. These results robustly desmonstrate the RNA binding affinity and associated splicing events of SRSF3 and SRSF10 are m6A dependent.We next combined purified protein quantification and western blotting techniqes to detect the individual endogenous amount of YTHDC1, SRSF3 and SRSF10. We found that the endogenous amount of SRSF3 was much higher than that of SRSF10. The much higher endogenous amount of SRSF3 compared SRSF10 suggest the protective mechanism by which most of YTHDC1 occupied by SRSF3 makes their targeting exons a very high inclusion level in physiological conditions.Our discoveries provide direct evidence for biological functions of m6A and RNA epigenetics, and also provide the molecular mechanism of epigenetical research direction for nomal physiology (like stem cell pluripotency maintianing and differentiation) and abnormal pathology (like cancer).