Structural basis for RNA recognition by the N-terminal tandem RRM domains of human RBM45.

RBM45 is an RNA-binding protein involved in neural development, whose aggregation is associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). However, the mechanisms of RNA-binding and aggregation of RBM45 remain unelucidated. Here, we report the crystal structure of the N-terminal tandem RRM domains of human RBM45 in complex with single-stranded DNA (ssDNA). Our structural and biochemical results revealed that both the RRM1 and RRM2 of RBM45 recognized the GAC sequence of RNA/ssDNA. Two aromatic residues and an arginine residue in each RRM were critical for RNA-binding, and the interdomain linker was also involved in RNA-binding. Two RRMs formed a pair of antiparallel RNA-binding sites, indicating that the N-terminal tandem RRM domains of RBM45 bound separate GAC motifs in one RNA strand or GAC motifs in different RNA strands. Our findings will be helpful in the identification of physiologic targets of RBM45 and provide evidence for understanding the physiologic and pathologic functions of RBM45.

Akkermansia muciniphila and its outer membrane protein Amuc_1100 prophylactically attenuate 5-fluorouracil-induced intestinal mucositis.

5-Fluorouracil (5-FU) is a chemotherapy drug used to treat tumors. Previous studies have shown that Akkermansia muciniphila (A. muciniphila) and its outer membrane protein, Amuc_1100, alleviate dextran sodium sulfate (DSS)-induced colitis in mice. We investigated the effects of both A. muciniphila and Amuc_1100 on 5-FU-induced intestinal mucosal damage in mice. C57BL/6 mice were gavaged with A. muciniphila or Amuc_1100 daily before, during, and after 5-FU injection for a total of 14 days. By evaluating diarrheal toxicity scores, body weight changes, colonic anatomy images, and histopathology of intestinal injury in these mice, we found that A. muciniphila and Amuc_1100 alleviated 5-FU-induced intestinal mucositis. Quantitative polymerase chain reaction assays of intestinal cytokine mRNA levels demonstrated that both A. muciniphila and Amuc_1100 attenuated the upregulation of intestinal Tumor Necrosis Factor-α (TNF-α) and interleukin-6 (IL-6) induced by 5-FU treatment. In addition, they both reduced 5-FU-induced the NLR family pyrin domain containing 3 (NLRP3) inflammatory vesicle activation. Furthermore, by monitoring the mRNA expression of tight junction proteins in the intestine, we found that A. muciniphila and Amuc_1100 were capable of restoring the damaged intestinal barrier. Notably, A. muciniphila and Amuc_1100 also played a role in altering the structure of the intestinal microbial community. The present study revealed the protective role of both A. muciniphila and Amuc_1100 in the intestinal mucositis caused by 5-FU, providing new insights into treatment options.

Functional and structural characterization of a GH3 ß-N-acetylhexosaminidase from Akkermansia muciniphila involved in mucin degradation.

Akkermansia muciniphila is a probiotic that colonizes the outer layer of intestinal mucus and is negatively associated with metabolic disorders. Amuc_2109 protein, a ß-N-acetylhexosaminidase from A. muciniphila, may be involved in the degradation of mucins and is associated with intestinal health. Here, we reported the crystal structure of Amuc_2109, which belongs to the GH family 3 enzymes and fell into the canonical (α/ß)8 TIM barrel structure with GlcNAc bound to the active center. Biochemical assay characterization of Amuc_2109 revealed that Amuc_2109 is a GlcNAc-specific glycosidase active over a wide temperature and pH range, reflecting the survival advantage of Amuc_2109 in the intestinal environment. Our structural and biochemical results will contribute to the understanding of the catalytic mechanism of the GH3 ß-N-acetylhexosaminidase and help to gain insight into the molecular mechanism of complex carbohydrate utilization and restoration of the intestinal barrier in A. muciniphila.

Structural basis for mRNA recognition by human RBM38.

RNA-binding protein RBM38 was reported to bind the mRNA of several p53-related genes through its RRM domain and to up-regulate or down-regulate protein translation by increasing mRNA stability or recruitment of other effector proteins. The recognition mechanism, however, for RNA-binding of RBM38 remains unclear. Here, we report the crystal structure of the RRM domain of human RBM38 in complex with a single-stranded RNA. Our structural and biological results revealed that RBM38 recognizes G(U/C/A)GUG sequence single-stranded RNA in a sequence-specific and structure-specific manner. Two phenylalanine stacked with bases of RNA were crucial for RNA binding, and a series of hydrogen bonds between the base atoms of RNA and main-chain or side-chain atoms of RBM38 determine the sequence-specific recognition. Our results revealed the RNA-recognition mechanism of human RBM38 and provided structural information for understanding the RNA-binding property of RBM38.

A ß-N-acetylhexosaminidase Amuc_2109 from Akkermansia muciniphila protects against dextran sulfate sodium-induced colitis in mice by enhancing intestinal barrier and modulating gut microbiota.

Inflammatory bowel disease (IBD) is associated with the microbial composition of the gut and its metabolites. Akkermansia muciniphila is a probiotic that exerts a significant alleviative or therapeutic effect on host enteritis. This study was designed to determine the protective effect and potential mechanism underlying the secretion of ß-acetylaminohexosidase (Amuc_2109) by A. muciniphila against dextran sulfate sodium (DSS)-induced colitis in mice. C57BL/6 mice were gavaged with Amuc_2109 for 21 days, and during the last seven days of treatment, they drank DSS dissolved in their drinking water to induce colitis. Our results showed that supplementation with Amuc_2109 improved DSS-induced colitis as evidenced by lowered disease activity index (DAI) scores, reduced weight loss, increased colon length, and inhibited oxidative stress. In addition, Amuc_2109 inhibited the overexpression of inflammatory cytokines (TNF-α, IL-1ß, IL-6) and the NLR family pyrin domain containing 3 (NLRP3) inflammasome in DSS-induced colitis. Furthermore, supplementation with Amuc_2109 also restored the mRNA expression of tight junction proteins (ZO-1, occludin, claudin-1). Further analysis of fecal microbial 16S rRNA sequences showed that Amuc_2109 reshaped the intestinal microbiota. While the anti-inflammatory effects of Amuc_2109 were only manifested with the wild-type protein, the anti-inflammatory effects were completely lost after the mutation of its key catalytic amino acids rendered Amuc_2109 inactive. In summary, these findings demonstrate the potential of Amuc_2109, as a therapeutic agent for ulcerative colitis. We posit that it will provide additional assistance in the prevention and treatment of mucus layer-related diseases such as ulcerative colitis.