Characterization of the Dnmt2 metyltransferase homolog pmt1 from Schizosaccharomyces pombe

胞嘧啶残基上的DNA甲基化是许多生物体用来调节基因表达的一种修饰。分裂酵母Schizosaccharomyces pombe携带Dnmt2家族的同源甲基转移酶，称为Pmt1，但没有可检测到的DNA甲基化。我们的工作表明，Pmt1在体外和体内都具有tRNA甲基化活性。令人惊讶的是，它的体内活性受到营养条件的强烈调节，这种调节需要丝氨酸/苏氨酸激酶Sck2，并对S. pombe的时间衰老有影响。在这个项目中，我们将扩展这项工作，并将进一步定义Pmt1依赖性tRNA甲基化的营养需求和动力学，我们将寻求确定Pmt1诱导的其他条件。我们将评估Sck2以及其他激酶信号通路在Pmt1激活中的作用，并将询问它是通过Pmt1的直接或间接磷酸化还是Pmt1与其他蛋白质的相互作用发生的。此外，我们将在已知衰老途径的背景下放置Pmt1对衰老的影响。基于在第一个资助期获得的知识，我们将寻找与pmt1Δ有合成致死相互作用的基因，这将为Pmt1的功能作用提供指示。最后，考虑到Pmt1激活的营养环境，我们将重新评估Pmt1在表观遗传基因沉默中的作用。

DNA methylation on cytosine residues is a modification used by many organisms to regulate gene expression. The fission yeast Schizosaccharomyces pombe carries a homolog of the Dnmt2 family of methyltransferases called Pmt1, but possesses no detectable DNA methylation. Our work has shown that Pmt1 has tRNA methylation activity, both in vitro and in vivo. Surprisingly, its in vivo activity is strongly regulated by nutritional conditions, and this regulation requires the serine/ threonine kinase Sck2 and has an impact on chronological aging in S. pombe.In this project, we will expand on this work and will further define the nutritional requirements and dynamics of Pmt1-dependent tRNA methylation, and we will seek to identify other conditions for Pmt1 induction. We will evaluate Sck2 as well as other kinase signaling pathways for their role in Pmt1 activation and will ask whether it occurs via a direct or indirect phosphorylation of Pmt1 or by interaction of Pmt1 with other proteins. Furthermore, we will place the effect of Pmt1 on aging in the context of known aging pathways. Based on the knowledge gained in the first grant period, we will search for genes that show a synthetic lethal interaction with pmt1Δ, which will give indications as to functional roles of Pmt1. Finally, we will reevaluate the role of Pmt1 on epigenetic gene silencing, taking into consideration the nutritional circumstances of Pmt1 activation.