ABSTRACT

RNautophagy and DNautophagy (RDA) are unconventional autophagic pathways where nucleic acids are directly transported through the lysosomal membrane, then degraded inside lysosomes. We have previously shown that bitopic protein LAMP2C and putative RNA transporter SIDT2, both lysosomal membrane proteins, mediate the direct transport of nucleic acids into lysosomes and that LAMP2C interacts with the nucleic acids and functions as a receptor during RDA. Because SIDT2-mediated RDA occurs in isolated lysosomes that lack LAMP2C, in this study, we tested the hypothesis that SIDT2 itself could also interact with the nucleic acids. Our results show that SIDT2 directly binds RNA and DNA through an arginine-rich motif (ARM) located within its main cytosolic domain, and disruption of this motif dramatically impairs SIDT2-mediated RNautophagic activity. We also found that SIDT2 interacts with exon 1 of HTT (huntingtin) transcript through the ARM in a CAG-dependent manner. Moreover, overexpression of SIDT2 promoted degradation of HTT mRNA and reduced the levels of polyglutamine-expanded HTT aggregates, hallmarks of Huntington disease. In addition, a comparative analysis of LAMP2C and SIDT2 functions at the cellular level revealed that the two proteins exert a synergistic effect on RNautophagic activity and that the ARMs which mediate the interactions of SIDT2 and LAMP2C with RNA are essential for the synergy. Together, our results point out the importance of nucleic acid-binding capacity of SIDT2 for its function in translocating nucleic acids through the lipid bilayer and suggests a potential application of RNautophagy activation to reduce the expression levels of disease-causing toxic proteins.

Abbreviations: ACTB/β-actin: actin beta; ARM: arginine-rich motif; CBB: Coomassie Brilliant Blue; CD: cytosolic domain; COX4I1/COX4: cytochrome c oxidase subunit 4I1; E. coli: Escherichia coli; EGFP: enhanced green fluorescent protein; EtBr: ethidium bromide; FITC: fluorescein isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GOLGA2/GM130: golgin A2; GST: glutathione S-transferase; HRP: horseradish peroxidase; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; HTT: huntingtin; HTTex1: exon 1 of the HTT gene; LAMP2: lysosomal associated membrane protein 2; LMNA: lamin A/C; PAGE: polyacrylamide gel electrophoresis; PBS: phosphate-buffered saline; PEI: polyethyleneimine; polyQ: polyglutamine; qPCR: quantitative PCR; RAB5A: RAB5A, member RAS oncogene family; RDA: RNautophagy and DNautophagy; SCARB2/LIMP2: scavenger receptor class B member 2; SDS: sodium dodecyl sulfate; SID-1: systemic RNA interference deficient-1; SIDT2: SID1 transmembrane family member 2; WT: wild type.

 抽象的

RN自噬和DN自噬（RDA）是非常规的自噬途径，其中核酸直接通过溶酶体膜转运，然后在溶酶体内降解。我们之前已经证明，双位蛋白 LAMP2C 和推定的 RNA 转运蛋白 SIDT2 都是溶酶体膜蛋白，介导核酸直接转运到溶酶体中，并且 LAMP2C 与核酸相互作用并在 RDA 期间充当受体。由于 SIDT2 介导的 RDA 发生在缺乏 LAMP2C 的分离溶酶体中，因此在本研究中，我们测试了 SIDT2 本身也可以与核酸相互作用的假设。我们的结果表明，SIDT2 通过位于其主要胞质结构域内的富含精氨酸的基序 (ARM) 直接结合 RNA 和 DNA，并且该基序的破坏会显着损害 SIDT2 介导的 RN 自噬活性。我们还发现 SIDT2 通过 ARM 以 CAG 依赖性方式与HTT （亨廷顿蛋白）转录物的外显子 1 相互作用。此外，SIDT2 的过度表达促进了HTT mRNA 的降解，并降低了多聚谷氨酰胺扩增的 HTT 聚集体的水平，这是亨廷顿病的标志。此外，在细胞水平上对LAMP2C和SIDT2功能的比较分析表明，这两种蛋白对RN自噬活性发挥协同作用，并且介导SIDT2和LAMP2C与RNA相互作用的ARM对于协同作用至关重要。总之，我们的结果指出了 SIDT2 的核酸结合能力对于其通过脂质双层转运核酸的功能的重要性，并表明了 RN 自噬激活的潜在应用，以降低致病有毒蛋白的表达水平。

缩写： ACTB/β-肌动蛋白：肌动蛋白β； ARM：富含精氨酸的基序； CBB：考马斯亮蓝； CD：胞质结构域； COX4I1/COX4：细胞色素c氧化酶亚基4I1；大肠杆菌：大肠杆菌； EGFP：增强型绿色荧光蛋白； EtBr：溴化乙锭； FITC：异硫氰酸荧光素； GAPDH：3-磷酸甘油醛脱氢酶； GOLGA2/GM130：高尔金A2； GST：谷胱甘肽S-转移酶； HRP：辣根过氧化物酶； HSPA5/GRP78：热休克蛋白家族 A (Hsp70) 成员 5； HTT：亨廷顿蛋白； HTTex1 ： HTT基因的外显子1； LAMP2：溶酶体相关膜蛋白2； LMNA：核纤层 A/C； PAGE：聚丙烯酰胺凝胶电泳； PBS：磷酸盐缓冲盐水； PEI：聚乙烯亚胺； PolyQ：聚谷氨酰胺； qPCR：定量PCR； RAB5A：RAB5A，RAS癌基因家族成员； RDA：RN自噬和DN自噬； SCARB2/LIMP2：B类清道夫受体成员2； SDS：十二烷基硫酸钠； SID-1：系统性RNA干扰缺陷-1； SIDT2：SID1跨膜家族成员2； WT：野生型。