Data_Sheet_1_SUMOylation of Arginyl tRNA Synthetase Modulates the Drosophila Innate Immune Response.pdf

SUMO conjugation of a substrate protein can modify its activity, localization, interaction or function. A large number of SUMO targets in cells have been identified by Proteomics, but biological roles for SUMO conjugation for most targets remains elusive. The multi-aminoacyl tRNA synthetase complex (MARS) is a sensor and regulator of immune signaling. The proteins of this 1.2 MDa complex are targets of SUMO conjugation, in response to infection. Arginyl tRNA Synthetase (RRS), a member of the sub-complex II of MARS, is one such SUMO conjugation target. The sites for SUMO conjugation are Lys 147 and 383. Replacement of these residues by Arg (RRSK147R,K383R), creates a SUMO conjugation resistant variant (RRSSCR). Transgenic Drosophila lines for RRSWT and RRSSCR were generated by expressing these variants in a RRS loss of function (lof) animal, using the UAS-Gal4 system. The RRS-lof line was itself generated using CRISPR/Cas9 genome editing. Expression of both RRSWT and RRSSCR rescue the RRS-lof lethality. Adult animals expressing RRSWT and RRSSCR are compared and contrasted for their response to bacterial infection by gram positive M. luteus and gram negative Ecc15. We find that RRSSCR, when compared to RRSWT, shows modulation of the transcriptional response, as measured by quantitative 3′ mRNA sequencing. Our study uncovers a possible non-canonical role for SUMOylation of RRS, a member of the MARS complex, in host-defense.

底物蛋白的SUMO化修饰（SUMO conjugation）可改变其活性、定位、相互作用或生物学功能。蛋白质组学已在细胞中鉴定出大量SUMO化靶标，但绝大多数靶标的SUMO化修饰生物学功能仍有待阐明。多氨酰-tRNA合成酶复合物（multi-aminoacyl tRNA synthetase complex, MARS）是免疫信号的感受器与调控因子，该分子量1.2 MDa的复合物中的蛋白可在感染应答过程中发生SUMO化修饰。精氨酰-tRNA合成酶（Arginyl tRNA Synthetase, RRS）作为MARS亚复合物II的成员，正是这类SUMO化靶标之一，其SUMO化修饰位点为赖氨酸147与赖氨酸383。将上述残基替换为精氨酸（即RRSK147R、K383R突变体），可获得SUMO化抗性变体（RRSSCR）。本研究通过UAS-Gal4系统，在RRS功能缺失（loss of function, lof）果蝇中分别表达野生型RRS（RRSWT）与SUMO化抗性变体RRSSCR，成功构建了对应的转基因果蝇品系；其中RRS功能缺失品系是通过CRISPR/Cas9基因组编辑技术获得的。RRSWT与RRSSCR均可挽救RRS功能缺失果蝇的致死表型。本研究对表达RRSWT与RRSSCR的成年果蝇分别开展革兰氏阳性菌藤黄微球菌（M. luteus）与革兰氏阴性菌Ecc15感染后的应答分析，并通过定量3'端mRNA测序比较两者的转录组差异。结果显示，与RRSWT相比，RRSSCR可调控宿主的转录应答过程。本研究揭示了MARS复合物成员RRS的SUMO化修饰在宿主防御中可能存在的非经典生物学功能。