SUMMARY A hallmark of both embryonic and adult stem cells is their dependence on low translation rates to maintain an active self-renewal and undifferentiated state independent of the cell cycle. As initiation is the rate-limiting step of translation, currently, it is widely accepted that the accumulation of tRNA fragments (tRFs) negatively regulates translation initiation leading to global translation attenuation. Notably, NSUN2, an RNA methyl-transferase that converts cytosine to 5-methylcytosine (m5C), is required for decoding activity and stability of tRNAs and coding/noncoding mRNAs. NSUN2 mutations have been broadly involved in developmental defects, including male infertility, Dubowitz-like syndrome, and intellectual disability, as well as in cancer. Hypo-m5C-methylated tRNAs accumulated in NSUN2 knockout (KO) or mutant somatic cells are cleaved by endonuclease angiogenin, and the resulting tRFs inhibit translation initiation globally. Lower protein synthesis correlates well with lower NSUN2 expression in somatic/cancer stem cells than committed or differentiated cells under homeostasis and tumorigenesis. However, this cannot explain the translation attenuation in pluripotent stem cells, the versatile cellular system for modeling diseases and early development, because NSUN2 is highly enriched under the self- renewal and undifferentiated state of these cells. Moreover, our preliminary data show that NSUN2 KO ESCs are well maintained without global translation reduction but with a hyper-pluripotent state concomitant with the upregulation of the naïve marker NANOG protein expression. This proposal aims to study m5C RNA targets of NSUN2 and understand how they are regulated at the translation level to dissect unique functions of NSUN2- regulated epitranscriptome in the translation control of stem cell pluripotency, which is fundamental to developmental biology and disease modeling. This proposal will explore the liquid-liquid phase separation (LLPS) potential of m5C mRNAs and their readers for translation control. We will test the hypothesis that NSUN2- modified m5C mRNAs are sequestered into P-bodies (PBs) through the RNA-binding protein YBX1-mediated LLPS process, leading to translation attenuation of NSUN2 targets in pluripotent stem cells. Two aims are proposed to test the hypothesis. 1) Identify m5C RNA targets of NSUN2 and YBX1 for translational control of stem cell pluripotency. 2) Establish LLPS-mediated translation repression of m5C RNAs in pluripotent stem cells. The proposed work will fill a significant knowledge gap in our understanding of NSUN2-mediated RNA methylation for specialized translation control in pluripotent stem cells. It will also establish a novel paradigm for translational attenuation through the phase separation potential of m5C-modified RNAs and their reader proteins.

RELEVANCE NSUN2 is an RNA methyltransferase that converts cytosine to 5-methylcytosine (m5C) and is required for the decoding activity and stability of tRNAs and coding/noncoding mRNAs. NSUN2 mutations have been widely involved in developmental defects, including male infertility, Dubowitz-like syndrome, and intellectual disability, as well as in cancer. Dissecting the unique functions of the NSUN2-regulated epitranscriptome through liquid- liquid phase separation in the translational control of pluripotent stem cells is fundamental to developmental biology and disease modeling.