Negative regulation of miRNAs sorting in EVs: the RNA-binding protein PCBP2 impairs SYNCRIP-mediated miRNAs EVs loading

Reviewer #1 (Public review):

In this study, Marocco and colleagues perform a deep characterization of the complex molecular mechanism guiding the recognition of a particular CELLmotif previously identified in hepatocytes in another publication. Having miR-155-3p with or without this CELLmotif as the initial focus, the authors identify 21 proteins differentially binding to these two miRNA versions. From there, they decided to focus on PCBP2. They elegantly demonstrate PCBP2 binding to the miR-155-3p WT version but not to the CELLmotif-mutated version. miR-155-3p contains a hEXOmotif identified in a different report, whose recognition is largely mediated by another RNA-binding protein called SYNCRIP. Interestingly, mutation of the hEXOmotif contained in miR-155-3p did not only blunt SYNCRIP binding but also PCBP2 binding despite the maintenance of the CELLmotif. This indicates that somehow SYNCRIP binding is a pre-requisite for PCBP2 binding. EMSA assay confirms that SYNCRIP is necessary for PCBP2 binding to miR-155-3p, while PCBP2 is not needed for SYNCRIP binding. The authors aim to extend these findings to other miRNAs containing both motifs. For that, they perform a small-RNA-Seq of EVs released from cells knockdown for PCBP2 versus control cells, identifying a subset of miRNAs whose expression either increases or decreases. The assumption is that those miRNAs containing PCBP2-binding CELLmotif should now be less retained in the cell and go more to extracellular vesicles, thus reflecting a higher EV expression. The specific subset of miRNAs having both the CELLmotif and hEXOmotif (9 miRNAs) whose expressions increase in EVs due to PCBP2 reduction is also affected by knocking-down SYNCRIP in the sense that reduction of SYNCRIP leads to lower EV sorting. Further experiments confirm that PCBP2 and SYNCRIP bind to these 9 miRNAs and that knocking down SYNCRIP impairs their EV sorting.

While the process studied in this work is novel and interesting, there are several aspects of this manuscript that should be improved:

(1) First of all, the nature of the CELLmotif and the hEXOmotif they are studying is extremely confusing. For the CELLmotif, the authors seem to focus on the Core CELLmotif AUU A/G in some experiments and the extended 7-nucleotide version in others. The fact that these CELLmotif and hEXOmotif are not shown anywhere in the figures (I mean with the full nucleotide variability described in the original publications) but only referred to in the text further complicates the identification of the motifs and the understanding of the experiments. Moreover, I am not convinced that the sequences they highlight in grey correspond to the original CELLmotif in all cases. For instance, in the miR-155-3p sequence, GCAUU is highlighted in grey. However, the original CELLmotif is basically 7-nucleotide long: C, A/U, G/A/C, U, U/A, C/G/A, A/U/C or CAGUUCA in its more abundant version. I can only see clearly the presence of the Core CELLmotif AUUA in miR-155-3p; however, the last A is not highlighted in grey. It is true that there is some nucleotide variability in each position in the originally reported CELLmotif by the authors in ref. 5 and the hEXOmotifs in ref. 7; however, not all nucleotides are equally likely to be found in each position. This fact seems to be not to be taken into account by the authors as they took basically any sequence with any length and almost sequence combination as valid CELLmotif. This means that I cannot identify the CELLmotif in many cases among the ones they highlight in grey. Instead, they should really focus on the most predominant CELLmotif sequence or, instead, take a reduced subset of "more abundant" CELLmotif versions from the ones that could fit in the originally described CELLmotif. Altogether, the authors need to explain much better what they have considered as the CELLmotif, what is the Core CELLmotif and what is hEXOmotif in each case and restrict to the most likely versions of the CELLmotif and hEXOmotif.

(2) Validation of EV isolation method: first, a large part of Supplementary Figure 2 is not readable. EV markers seem to be enriched in EV isolates; however, more EV and cell markers should be assayed to fulfill MISEV guidelines.

(3) A key variable is missing in Supplementary Figure 2, which is whether PCBP2 or SYNCRIP knockdowns impair EV secretion rates. A quantification of the nr vesicles released per cell upon knocking down each of these factors would be essential to rule out that any of the effects seen throughout the paper are not due to reduced or enhanced EV production rather than miRNA sorting/retention.

(4) The EMSA experiment is important to support their claims. Given the weak bands that are shown, the authors need to show all their replicates to convince the readers that it is reproducible.

(5) Although the bindings of SYNCRIP and PCBP2 to miR-155-3p and other miRNAs having both hEXOmotif and CELLmotif seem clear, the need for SYNCRIP binding to allow for PCBP2-mediated cellular retention is counterintuitive. What happens to those miRNAs that only contain a CELLmotif in terms of cellular retention and SYNCRIP dependence for cellular retention? In this regard, a representative miRNA (miR-31-3p) is analyzed in several experiments, showing that PCBP2 does not bind to it unless a hEXOmotif is introduced (Figure 3). However, this type of experiment should definitely be extended to other miRNAs containing only CELLmotif without hEXOmotif.

(6) Along the same line, I am missing another important experiment: the artificial incorporation of CELLmotif. For example, miR-365-2-5p lacks a CELLmotif but has a hEXOmotif. Does PCBP2 bind to this miRNA upon incorporation of CELLmotif? Does this lead now to enhanced cellular retention of this miRNA?

(7) What would be the net effect of knocking down both SYNCRIP and PCBP2 at the same time? Would this neutralize each other's effect or would the lack of one impose on the other? That could help in understanding the complex interplay between these two factors for mediating cellular retention and EV sorting.

(8) The authors have here a great opportunity to shed some light on an unclear aspect of miRNA EV sorting and cellular retention: whether the RBPs go together with the miRNA to the EVs or not. While the original paper describing hEXOmotif found SYNCRIP in EVs, another publication (Jeppesen et al, Cell 2019; PMID: 30951670) later found this RBP being very scarce in small EVs compared to cellular bodies or large EVs (Supplementary Tables 3 and 4 in that publication). Can the authors find SYNCRIP and PCBP2 in the EVs? Another important question would be the colocalization of these RBPs in the place where the miRNA selection is supposed to take place: in multivesicular bodies (MVB). Is there a colocalization of these RBPs with MVBs in the cell?

(9) In Figure 4C, the authors state in the text that CELLmotif and hEXOmotif are present in extra-seed region; however, for miR-181d-5p and miR-122-3p this is not true as their CELLmotifs fall within the seed sequence.

(10) The authors need to describe how they calculate the EV/cell ratio in gene expression in some experiments (for instance, Figures 1H, 4D, etc). Did they use any housekeeping gene for EV RNA content, the same RNA load, or some other alternative method to normalize EV vs cell RNA content?

(11) I would suggest that the authors speculate a bit in the discussion section on how the interaction between PCBP2 and SYNCRIP takes place. Do they contain any potential interacting domain? The binding of one to the miRNA would impose a topological interference on the binding of the other?

Reviewer #2 (Public review):

Summary:

The author of this manuscript aimed to uncover the mechanisms behind miRNA retention within cells. They identified PCBP2 as a crucial factor in this process, revealing a novel role for RNA-binding proteins. Additionally, the study discovered that SYNCRIP is essential for PCBP2's function, demonstrating the cooperative interaction between these two proteins. This research not only sheds light on the intricate dynamics of miRNA retention but also emphasizes the importance of protein interactions in regulating miRNA behavior within cells.

Strengths:

This paper makes important progress in understanding how miRNAs are kept inside cells. It identifies PCBP2 as a key player in this process, showing a new role for proteins that bind RNA. The study also finds that SYNCRIP is needed for PCBP2 to work, highlighting how these proteins work together. These discoveries not only improve our knowledge of miRNA behavior but also suggest new ways to develop treatments by controlling miRNA locations to influence cell communication in diseases. The use of liver cell models and thorough experiments ensures the results are reliable and show their potential for RNA-based therapies

Weaknesses:

Despite its strengths, the manuscript has several notable limitations. The study's exclusive focus on hepatocytes limits the applicability of the findings to other cell types and physiological contexts. While the interaction between PCBP2 and SYNCRIP is well-characterized, the manuscript lacks detailed insights into the structural basis of this interaction and the dynamic regulation of their binding. The generalization of the findings to a broader spectrum of miRNAs and RNA-binding proteins (RBPs) remains underexplored, leaving gaps in understanding the full scope of miRNA compartmentalization.

Furthermore, the therapeutic implications of these findings, though promising, are not directly connected to specific disease models or clinical scenarios, reducing their immediate translational impact. The manuscript would also benefit from a deeper discussion of potential upstream regulators of PCBP2 and SYNCRIP and the influence of cellular or environmental factors on their activity. Additionally, it is important to note that SYNCRIP has already been recognized as a major regulator of miRNA loading in extracellular vesicles (EVs). However, the purity of EVs is a concern, as the author only performed crude extraction methods without further purification using an iodixanol density gradient. The study also lacks in vivo evidence of PCBP2's role in exosomal miRNA export.