STAG3 promotes exit from pluripotency through post-transcriptional mRNA regulation in the cytoplasm

Reviewer #1 (Public Review):

The paper titled "STAG3 promotes exit from pluripotency through post-transcriptional mRNA regulation in the cytoplasm" suggests a new and unexpected role for STAG3, a protein traditionally associated with the cohesin complex during meiosis, in regulating the exit from pluripotency in mouse embryonic stem cells (mESCs). While STAG3 is traditionally studied for its role in meiosis, this paper reveals that STAG3 is expressed in mouse embryonic stem cells (mESCs) and primordial germ cell-like cells (PGCLCs) and may be necessary for PGCLC-like specification and exit from pluripotency. In ESCs, the study reports that STAG3 is found in the cytoplasm, where it interacts with various RNA-binding proteins (RBPs) and localizes to centrosomes. Knockdown of STAG3 disrupts centrosome stability and RNA-induced silencing complex (RISC) components, leading to the misregulation of mRNAs such as DPPA3, Nanog, and TNRC6C. In summary, this study expands the known functions of STAG3 beyond cohesin, highlighting a potential role in cytoplasmic post-transcriptional regulation.

The authors perform a comprehensive characterization of RNA and protein changes in ESCs and differentiated cells upon loss of STAG3, providing preliminary and intriguing insights. However, there are several aspects that require further exploration:

(1) A rescue experiment for the STAG3 RNAi is missing, making it unclear whether the observed effects are indeed due to the knockdown of STAG3.

(2) While the paper identifies several interactions and effects of STAG3, it lacks detailed mechanistic insights into how STAG3 regulates specific mRNAs and proteins. Specifically, it is unclear which proteins directly interact with STAG3 or recruit STAG3 to RNP complexes. AlphaFold may help in this analysis.

(3) It is unclear whether this is an alternative STAG3 isoform or if STAG3 is modified. What dictates its interaction with cohesin versus RNPs?

(5) Are there unique features or sequence barcodes present on the misregulated RNAs?

(6) Does STAG3 associate with a single type of RNP or is it present in all types?

Reviewer #2 (Public Review):

Summary:

This manuscript addresses the intriguing topic of the potential roles of germline-specific proteins in early development. While this issue is quite interesting and generally under-explored, the work falls short of making truly tangible inroads.

Strengths:

The strength of the study is in new proteomic datasets.

Weaknesses:

The manuscript makes some strong statements, beginning with the title "STAG3 (1) promotes exit from pluripotency (2) through post-transcriptional mRNA regulation in the cytoplasm".

Upon reviewing the data it appears that neither (1) or (2) here have strong foundations based on experiments presented. While intriguing, the experimental evidence is still rather inconclusive.

The potential involvement of STAG3 in PGC specification is the most intriguing aspect of this study. Unfortunately, it is not going far enough to derive a fully meaningful biological conclusion. In fact, DPPPA3-GFP, PRDM-GFP, and PGC marker expression results are contradictory and do not form a coherent picture of the biological effect of STAG3 depletion. No effect of the knock-down in PGC specification when PRDM is scored (line 167) is particularly worrisome. As for finding a cytoplasmic role of STAG3, the data also remain inconclusive.