Many unannotated microproteins and alternative proteins (alt-proteins) are coencoded with canonical proteins, but few of their functions are known. Motivated by the hypothesis that alt-proteins undergoing regulated synthesis could play important cellular roles, we developed a chemoproteomic pipeline to identify nascent alt-proteins in human cells. We identified 22 actively translated alt-proteins or N-terminal extensions, one of which is post-transcriptionally upregulated by DNA damage stress. We further defined a nucleolar, cell-cycle-regulated alt-protein that negatively regulates assembly of the pre-60S ribosomal subunit (MINAS-60). Depletion of MINAS-60 increases the amount of cytoplasmic 60S ribosomal subunit, upregulating global protein synthesis and cell proliferation. Mechanistically, MINAS-60 represses the rate of late-stage pre-60S assembly and export to the cytoplasm. Together, these results implicate MINAS-60 as a potential checkpoint inhibitor of pre-60S assembly and demonstrate that chemoproteomics enables hypothesis generation for uncharacterized alt-proteins.

许多未注释的微生物蛋白和替代蛋白（替代蛋白）与规范蛋白共编码，但它们的功能很少为人所知。受到接受调节合成的 alt 蛋白可以发挥重要细胞作用的假设的启发，我们开发了一种化学蛋白质组学管道来识别人类细胞中的新生 alt 蛋白。我们鉴定了 22 种主动翻译的 alt 蛋白或 N 末端延伸，其中之一在转录后被 DNA 损伤应激上调。我们进一步定义了一种核仁、细胞周期调节的 alt 蛋白，它负向调节前 60S 核糖体亚基 (MINAS-60) 的组装。MINAS-60 的消耗增加了细胞质 60S 核糖体亚基的数量，上调了整体蛋白质合成和细胞增殖。从机械上讲，MINAS-60 抑制晚期 60 年代前组装和输出到细胞质的速率。总之，这些结果表明 MINAS-60 是 60 年代前组装的潜在检查点抑制剂，并证明化学蛋白质组学能够为未表征的 alt 蛋白生成假设。