LncRNA-Snhg3 Aggravates Hepatic Steatosis by Regulating PPARγ via SND1/H3K27me3

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors investigate the contributions of the long noncoding RNA snhg3 in liver metabolism and MAFLD. The authors conclude that liver-specific loss or overexpression of Snhg3 impacts hepatic lipid content and obesity through epigenetic mechanisms. More specifically, the authors invoke that the nuclear activity of Snhg3 aggravates hepatic steatosis by altering the balance of activating and repressive chromatin marks at the Pparg gene locus. This regulatory circuit is dependent on a transcriptional regulator SNG1.

Strengths:

The authors developed a tissue-specific lncRNA knockout and KI models. This effort is certainly appreciated as few lncRNA knockouts have been generated in the context of metabolism. Furthermore, lncRNA effects can be compensated in a whole organism or show subtle effects in acute versus chronic perturbation, rendering the focus on in vivo function important and highly relevant. In addition, Snhg3 was identified through a screening strategy and as a general rule the authors the authors attempt to follow unbiased approaches to decipher the mechanisms of Snhg3.

Weaknesses:

Despite efforts at generating a liver-specific knockout, the phenotypic characterization is not focused on the key readouts. Notably missing are rigorous lipid flux studies and targeted gene expression/protein measurement that would underpin why the loss of Snhg3 protects from lipid accumulation. Along those lines, claims linking the Snhg3 to MAFLD would be better supported with careful interrogation of markers of fibrosis and advanced liver disease. In other areas, significance is limited since the presented data is either not clear or rigorous enough. Finally, there is an important conceptual limitation to the work since PPARG is not established to play a major role in the liver.

Reviewer #2 (Public Review):

Through RNA analysis, Xie et al found LncRNA Snhg3 was one of the most down-regulated Snhgs by a high-fat diet (HFD) in mouse liver. Consequently, the authors sought to examine the mechanism through which Snhg3 is involved in the progression of metabolic dysfunction-associated fatty liver diseases (MASLD) in HFD-induced obese (DIO) mice. Interestingly, liver-specific Sngh3 knockout was reduced, while Sngh3 over-expression potentiated fatty liver in mice on an HFD. Using the RNA pull-down approach, the authors identified SND1 as a potential Sngh3 interacting protein. SND1 is a component of the RNA-induced silencing complex (RISC). The authors found that Sngh3 increased SND1 ubiquitination to enhance SND1 protein stability, which then reduced the level of repressive chromatin H3K27me3 on PPARg promoter. The upregulation of PPARg, a lipogenic transcription factor, thus contributed to hepatic fat accumulation.

The authors propose a signaling cascade that explains how LncRNA sngh3 may promote hepatic steatosis. Multiple molecular approaches have been employed to identify molecular targets of the proposed mechanism, which is a strength of the study. There are, however, several potential issues to consider before jumping to a conclusion.

(1) First of all, it's important to ensure the robustness and rigor of each study. The manuscript was not carefully put together. The image qualities for several figures were poor, making it difficult for the readers to evaluate the results with confidence. The biological replicates and numbers of experimental repeats for cell-based assays were not described. When possible, the entire immunoblot imaging used for quantification should be presented (rather than showing n=1 representative). There were multiple mislabels in figure panels or figure legends (e.g., Figure 2I, Figure 2K, and Figure 3K). The b-actin immunoblot image was reused in Figure 4J, Figure 5G, and Figure 7B with different exposure times. These might be from the same cohort of mice. If the immunoblots were run at different times, the loading control should be included on the same blot as well.

(2) The authors can do a better job in explaining the logic for how they came up with the potential function of each component of the signaling cascade. Sngh3 is down-regulated by HFD. However, the evidence presented indicates its involvement in promoting steatosis. In Figure 1C, one would expect PPARg expression to be up-regulated (when Sngh3 was down-regulated). If so, the physiological observation conflicts with the proposed mechanism. In addition, SND1 is known to regulate RNA/miRNA processing. How do the authors rule out this potential mechanism? How about the hosting snoRNA, Snord17? Does it involve the progression of NASLD?

(3) The role of PPARg in fatty liver diseases might be a rodent-specific phenomenon. PPARg agonist treatment in humans may actually reduce ectopic fat deposition by increasing fat storage in adipose tissues. The relevance of the findings to human diseases should be discussed.