SINE-mediated Regulation of mRNA Epitranscriptome for Pluripotency Maintenance and Differentiation

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PROJECT SUMMARY The transcriptional control of mRNA expression has been extensively investigated; however, less attention has been paid to their internal modifications, the mRNA “epitranscriptome”. Recently, evidence is accumulating that modifications on mRNAs are functionally significant in a variety of molecular processes including pre-mRNA splicing, nuclear export, and stability, and play important biological roles in stem cells differentiation and human development. Transposable elements (TEs), including SINEs, LINEs, and LTRs are the most abundant DNA elements in the mammalian genomes. Unlike the other TEs such as LINEs and ERVs, SINEs are frequently embedded in the non-coding regions inside a gene, such as the introns and UTRs, with functional implications on their host mRNA expression. SINEs have more than a million genomic copies and occupy 8.22% of DNA sequences of the mouse genome and 13.64% of the human genome. TEs, including SINEs, were historically considered as “junk” DNA but now it is widely accepted that this portion of the genome plays a significant role in diverse cellular processes. Our preliminary analysis of RNA-bisulfite sequencing (BS-seq) in embryonic stem cells (ESCs) identified that the 5-methylcytosine (m5C)-enriched regions on mRNA transcripts are significantly associated with the embedded SINE elements at introns or UTRs of the host genes. Despite the well-established metabolism of DNA methylation and demethylation by Dnmt1/3a/3b and Tet1/2/3 for epigenetic regulations, our knowledge on RNA m5C for posttranscriptional gene regulation is quite limited. We hypothesize that self-renewal and differentiation of ESCs may be controlled by m5C-mediated active nuclear export and nuclear retention/ destabilization, respectively, of pluripotency mRNAs bearing SINE elements. We propose two aims to test this hypothesis. Aim 1. We will determine the regulation of pluripotency mRNA with embedded SINE region and m5C for nuclear export in ESC self-renewal. We determined that Alyref is indispensable for ESC self-renewal. We will examine if active nuclear export of pluripotency mRNAs (e.g. Nanog) are mediated by the m5C reader Alyref. Next, we will test if Alyref represses the affinity of m5C-modified mRNAs to the Pspc1/Nono heterodimer. Aim 2. We will dissect the mechanism of SINE non-coding (nc) RNA-mediated pluripotency mRNA degradation in ESC differentiation. Our preliminary work revealed that m5C-modified pluripotency gene transcripts (e.g., Nanog) have impaired degradation in Pspc1KO ESCs upon retinoic acid (RA)-induced differentiation. RA treatment activates SINE ncRNAs, which form double-stranded (ds) RNAs in trans with the host mRNAs and recruit the Nono/Pspc1/ Tet2 complex for mRNA nuclear retention and destabilization. We will explore the Pspc1 RNA targets by eCLIP- seq in ESCs and with RA treatment to examine the interactions between SINE ncRNAs and host mRNAs. Next, we will examine if Tet2 is recruited by Pspc1 for mRNA demethylation and destabilization. In summary, we will establish a novel paradigm of pluripotent cell fate determination via SINE-mediated mRNA metabolism and the functions of mRNA m5C readers, erasers, and effectors in pluripotency maintenance and during differentiation.