Palumbo Ryan J, Hanes Steven D
Department of Biochemistry & Molecular Biology, SUNY Upstate Medical University 750 East Adams Street, 4283 Weiskotten Hall, Syracuse, New York, 13210.
bioRxiv. 2023 Jun 8:2023.06.01.543302. doi: 10.1101/2023.06.01.543302.
Methylphosphate Capping Enzyme (MEPCE) monomethylates the gamma phosphate at the 5' end of the 7SK noncoding RNA, a modification thought to protect 7SK from degradation. 7SK serves as a scaffold for assembly of a snRNP complex that inhibits transcription by sequestering the positive elongation factor P-TEFb. While much is known about the biochemical activity of MEPCE , little is known about its functions , or what roles- if any-there are for regions outside the conserved methyltransferase domain. Here, we investigated the role of Bin3, the ortholog of MEPCE, and its conserved functional domains in development. We found that mutant females had strongly reduced rates of egg-laying, which was rescued by genetic reduction of P-TEFb activity, suggesting that Bin3 promotes fecundity by repressing P-TEFb. mutants also exhibited neuromuscular defects, analogous to a patient with haploinsufficiency. These defects were also rescued by genetic reduction of P-TEFb activity, suggesting that Bin3 and MEPCE have conserved roles in promoting neuromuscular function by repressing P-TEFb. Unexpectedly, we found that a Bin3 catalytic mutant (Bin3) could still bind and stabilize 7SK and rescue all mutant phenotypes, indicating that Bin3 catalytic activity is dispensable for 7SK stability and snRNP function . Finally, we identified a metazoan-specific motif (MSM) outside of the methyltransferase domain and generated mutant flies lacking this motif (Bin3). Bin3 mutant flies exhibited some-but not all- mutant phenotypes, suggesting that the MSM is required for a 7SK-independent, tissue-specific function of Bin3.
甲基磷酸帽化酶（MEPCE）使7SK非编码RNA 5'端的γ磷酸单甲基化，这种修饰被认为可保护7SK不被降解。7SK作为一种小核核糖核蛋白（snRNP）复合体组装的支架，该复合体通过隔离正性延伸因子P-TEFb来抑制转录。虽然人们对MEPCE的生化活性了解很多，但对其功能知之甚少，对于保守甲基转移酶结构域之外的区域是否有作用（如果有的话）也了解甚少。在这里，我们研究了MEPCE的直系同源物Bin3及其保守功能结构域在发育中的作用。我们发现突变雌性的产卵率大幅降低，而通过基因降低P-TEFb的活性可使其恢复，这表明Bin3通过抑制P-TEFb来促进繁殖力。突变体还表现出神经肌肉缺陷，类似于单倍剂量不足的患者。这些缺陷也通过基因降低P-TEFb的活性而得到恢复，这表明Bin3和MEPCE在通过抑制P-TEFb促进神经肌肉功能方面具有保守作用。出乎意料的是，我们发现Bin3催化突变体（Bin3）仍能结合并稳定7SK，并挽救所有突变表型，这表明Bin3催化活性对于7SK稳定性和snRNP功能是可有可无的。最后，我们在甲基转移酶结构域之外鉴定出一个后生动物特异性基序（MSM），并构建了缺失该基序的突变果蝇（Bin3）。Bin3突变果蝇表现出一些但不是所有的突变表型，这表明MSM是Bin3独立于7SK的组织特异性功能所必需的。
