In 2006, Andrew Fire and Craig Mello were awarded the Nobel Prize in Physiology or Medicine for their work on RNA interference (RNAi)，which opened a new chapter in RNAi. Nowadays, RNA interference is increasingly being used to regulate the expression of human genes. In plants and animals, a class of non-coding single-stranded RNA molecules with a length of about 22 nt (miRNAs) exist, which can participate in the expression regulation of post-transcriptional genes through RNAi. In the study of nematode systemic RNAi, it was found that the transmembrane protein SID-1 (SID-1) plays a key role in mediating the absorption of double-stranded RNA (dsRNA) and is considered to be an important nucleic acid transport channel. In mammalian cells, there are two homologous transmembrane proteins that have similar nucleic acid transport functions to SID-1, namely SIDT1 and SIDT2 (SID1 transmembrane family member 1/2). SIDT2 is involved in the process of intracellular DNA/RNA autophagy, and transports lysosome-internalized nucleic acids to the cytoplasm, which triggers innate immune responses. While SIDT1 directly mediates the absorption of food-derived miRNAs. Studies have found that the stomach is the main site for the absorption of small RNA from food, and SIDT1 expressed on the parietal cells of the gastric mucosa (pit cell) of mammals is a key protein for the absorption of exogenous small RNAs. It is worth noting that this process is significantly facilitated by very low pH gastric acid, and acid stimulation greatly promotes miRNA absorption. This subversive discovery provides further evidence for the "cross-border regulation" of small RNAs, and provides a new strategy for small RNA-based therapeutics, which also provides a potential direction for RNA-based drug development. Although more and more studies support the mediation of nucleic acid transport by mammalian SID-1 transmembrane family proteins SIDT1 and SIDT2, the molecular mechanism of SID-1 transmembrane family proteins SIDT1 and SIDT2 mediating heterologous small RNA absorption is still unclear, especially the mechanism of promoting small RNA absorption at low pH is still unknown.

On November 6, 2023, a research paper entitled "Cryo-EM structures of human SID-1 transmembrane family proteins and implications for their low-pH-dependent RNA transport activity" was published on《Cell Research》by SUN Fei's group at the Institute of Biophysics, JI Xiaoyun's group and ZHANG Chenyu's group at the School of Life Sciences, Nanjing University. In this study, cryo-EM single-particle technology was used to simultaneously resolve the homodimer three-dimensional structures of human SIDT1 and SIDT2 proteins for the first time and which is equally important with that the team found SIDT1 and SIDT2 can bind miRNAs in a pH-dependent manner and induce further oligomerization of SIDT1 and SIDT2. This study revealed the molecular mechanism of the acidic environment to promote the absorption of small RNAs at the molecular level, and displayed the underlying molecular mechanism of SIDT1 and SIDT2 mediating nucleic acid transport, which has potential significance for in-depth understanding of the functional regulation of SIDT1 and SIDT2 in the acidic microenvironment and the development of RNA delivery systems.

Several difficulties that the expression and purification of membrane protein samples and the serious orientation advantage in the preparation of frozen samples were all overcome by the research team for the first time. Both SIDT1 and SIDT2 contain an extracellular domain (ECD), a transmembrane domain (TMD) consisting of 11 transmembrane helices, and a flexible intracellular domain (ICD). Through structural analysis, the researchers found that the structures of SIDT1 and SIDT2 were highly similar, suggesting that they may have similar biological functions. The researchers also identified evolutionarily conserved sites, such as disulfide bonds and glycosylation sites, which play a crucial role in the stability and structural integrity of SIDT1 and SIDT2 under acidic conditions. Additionly, the polymerization status of SIDT1 and SIDT2 in vitro in living cells were examined which showed both proteins exist as dimers or oligomers and TMD is essential for maintaining dimers. These findings provide important information for understanding the function of SIDT1 and SIDT2 in vivo.

Fig.1 Cryo-EM structure of SIDT1 and SIDT2 homodimers

In order to uncover the molecular mechanism by which SIDT1 and SIDT2 mediate the transport of small RNAs, the researchers further explored the binding of their ECDs to small RNAs. The results showed that the ECD of SIDT1 and SIDT2 could effectively bind to small RNAs in a pH-dependent manner. Under acidic conditions, the affinity of the ECD for small RNAs increases, and this binding further triggers the oligomerization of the ECD. This finding overturns the previously reported view that SIDT1^ECDand SIDT2^ECDcan only bind longer double-stranded RNAs (> 100 bp). At the same time, the researchers also found that the binding of ECD proteins to nucleic acids under acidic conditions was not affected by the specificity of nucleic acid species and single and double strands, and this binding was pH-dependent. Finally, through analytical ultracentrifugation and gel filtration chromatography, the research team also found that SIDT1 and SIDT2 bind to nucleic acids under acidic conditions to induce the oligomerization of proteins, indicating that they have the potential to form nucleic acid pores. This study revealed the key role of low pH environment in promoting the uptake of exogenous small RNAs by SIDT1 and SIDT2, and strengthened the theoretical basis of the molecular mechanism that promotes the transmembrane uptake of small RNAs at low pH.

In summary, the cryo-EM structures of the transmembrane family proteins SIDT1 and SIDT2 in mammalian cells were analyzed, and the pH-dependent binding and oligomerization of SIDT1 and SIDT2 to RNA were proposed for the first time. Therefore, this study provides a molecular basis for the nucleic acid transport of SID-1 transmembrane family proteins, and also provides a direct theoretical molecular basis for the phenomenon of "gene cross-border regulation". ZHENG Le and YANG Tingting, Ph.D. students from the School of Life Sciences, Nanjing University, GUO Hangtian, a postdoctoral fellow from the School of Life Sciences, Nanjing University, QI Chen, a Ph.D. student from the Institute of Biophysics, Chinese Academy of Sciences, and LU Yuchi, a Ph.D. student from the Institute of Immunochemistry, ShanghaiTech University, are the co-first authors of the paper. Prof. JI Xiaoyun and Prof. ZHANG Chenyu from the School of Life Sciences of Nanjing University, Prof. SUN Fei and Prof. ZHU Yun from the Institute of Biophysics of the Chinese Academy of Sciences, and Dr. GUO Hangtian from the School of Life Sciences, Nanjing University are the co-corresponding authors of the paper. This research work was supported by the National Key R&D Program of China and the National Natural Science Foundation of China. Some of the sample preparation, data collection and analysis were strongly supported and helped by the relevant staff of the Bioimaging Center of the Protein Science Research Platform of the Institute of Biophysics.

Article link：https://www.nature.com/articles/s41422-023-00893-1

Related Link：https://life.nju.edu.cn/cc/9e/c12937a642206/page.htm

Contact: ZHU Yun

Institute of Biophysics, Chinese Academy of Sciences

Beijing 100101, China

Email: zhunyun@ibp.ac.cn

(Reported by Prof. SUN Fei’s group)