DMS 结构探针在人类细胞中量化 RNA 发夹上的蛋白占据并关联功能
Structural probing of RNA hairpins quantifies protein occupancy on RNA and links it to function in human cells
研究建立了一套基于 dimethyl sulfate 突变谱测序（DMS-MaP）的定量框架，可在人类细胞中以单核苷酸分辨率同时测量 RNA 结构与 RNA 结合蛋白（RBP）的结合。作者将该方法应用于含 1600 个成员的 MS2 发夹文库，发现 RNA 折叠是 MS2 coat protein（MCP）识别的最强决定因素，需要形成稳定发夹和共有环序列，茎长或 GC 含量主要通过影响发夹折叠起作用。
来源：Nucleic Acids Research · doi.org
RNA structures that form inside living cells govern processes ranging from translation to RNA decay, many of which are controlled by RNA-binding proteins (RBPs). Because RBP specificity can depend on both RNA sequence and folding, traditional pulldown-based methods often obscure the structural context of bound RNAs. Here, a quantitative framework based on dimethyl sulfate mutational profiling and sequencing was established to directly measure RNA structure and RBP binding in human cells at single-nucleotide resolution. Applied to a 1600-member MS2 hairpin library, this approach revealed that RNA folding is the strongest determinant of MS2 coat protein (MCP) recognition in the MS2–MCP system, requiring formation of a stable hairpin and a consensus loop sequence, with stem length or GC content contributing primarily through their effect on hairpin folding. An in-cell occupancy model was derived to quantify fractional binding from structural readouts. Complementary systems using degron-tuned MCP and a downregulatory MCP fusion demonstrated binding behavior under subsaturating protein concentrations and showed that inferred occupancy quantitatively predicts RNA degradation efficiency. Together, these results establish a quantitative strategy for measuring RBP–RNA specificity and affinity directly in cells, using the MS2–MCP system to link RNA structure, protein occupancy, and regulatory outcome.