Author response:
The following is the authors’ response to the previous reviews.
eLife assessment
This study explores the role of one the most abundant circRNAs, circHIPK3, in bladder cancer cells, providing convincing data that circHIPK3 depletion affects thousands of genes and that those downregulated (including STAT3) share an 11-mer motif with circHIPK3, corresponding to a binding site for IGF2BP2. The experiments demonstrate that circHIPK3 can compete with the downregulated mRNAs targets for IGF2BP2 binding and that IGF2BP2 depletion antagonizes the effect of circHIPK3 depletion by upregulating the genes containing the 11mer motif. These valuable findings contribute to the growing recognition of the complexity of cancer signaling regulation and highlight the intricate interplay between circRNAs and protein-coding genes in tumorigenesis.
Public Reviews:
Reviewer #1 (Public Review):
In this work the authors propose a new regulatory role for one the most abundant circRNAs, circHIPK3. They demonstrate that circHIPK3 interacts with an RNA binding protein (IGF2BP2), sequestering it away from its target mRNAs. This interaction is shown to regulates the expression of hundreds of genes that share a specific sequence motif (11-mer motif) in their untranslated regions (3'-UTR), identical to one present in circHIPK3 where IGF2BP2 binds. The study further focuses on the specific case of STAT3 gene, whose mRNA product is found to be downregulated upon circHIPK3 depletion. This suggests that circHIPK3 sequesters IGF2BP2, preventing it from binding to and destabilizing STAT3 mRNA. The study presents evidence supporting this mechanism and discusses its potential role in tumor cell progression. These findings contribute to the growing complexity of understanding cancer regulation and highlight the intricate interplay between circRNAs and protein-coding genes in tumorigenesis.
Strengths:
The authors show mechanistic insight into a proposed novel "sponging" function of
circHIPK3 which is not mediated by sequestering miRNAs but rather a specific RNA binding protein (IGF2BP2). They address the stoichiometry of the molecules involved in the interaction, which is a critical aspect that is frequently overlooked in this type of studies. They provide both genome-wide analysis and a specific case (STAT3) which is relevant for cancer progression. Overall, the authors have significantly improved their manuscript in their revised version.
Weaknesses:
While the authors have performed northern blots to measure circRNA levels, an estimation of the circRNA overexpression efficiency, namely the circular-to-linear expression ratio, would be desired. The seemingly contradictory effects of circHIPK3 and STAT3 depletion in cancer progression, are now addressed by the authors in their revised manuscript, incorporating potential reasons that might explain such complexity.
We have now included a full version of the northern blot, where no discernible linear precursor can be detected, supporting efficient circHIPK3 WT and circHIPK3 MUT production (please see the detailed description in the specific comments below). We agree that the observations about STAT3 homeostasis and cancer progression, is not a straightforward extrapolation as discussed.
Reviewer #2 (Public Review):
Summary:
The authors have diligently addressed most of the points raised during the review process (except the important point of "additional in vitro experiments [...] needed to investigate the implication of circHIPK3 in bladder cancer cell phenotype" for which no additional experiments were performed), resulting in an improvement in the study. The data are now described with clarity and conciseness, enhancing the overall quality of the manuscript.
Strengths:
New, well-defined molecular mechanism of circRNAs involvement in bladder cancer.
Weaknesses:
Lack of solid translational significance data.
The focus of this study has been to disclose molecular mechanisms of action by circHIPK3, with implications for cancer. We agree that further studies are needed to fully understand the impact of circHIPK3 in bladder cancer.
Reviewer #3 (Public Review):
In Okholm et al., the authors evaluate the functional impact of circHIPK3 in bladder cancer cells. By knocking down circHIPK3 and performing an RNA-seq analysis, the authors found thousands of deregulated genes which look unaffected by miRNAs sponging function and that are, instead, enriched for a 11-mer motif. Further investigations showed that the 11mer motif is shared with the circHIPK3 and able to bind the IGF2BP2 protein. The authors validated the binding of IGF2BP2 and demonstrated that IGF2BP2 KD antagonizes the effect of circHIPK3 KD and leads to the upregulation of genes containing the 11-mer. Among the genes affected by circHIPK3 KD and IGF2BP2 KD, resulting in downregulation and upregulation respectively, the authors found the STAT3 gene, which also consistently has concomitant upregulation of one of its targets TP53. The authors propose a mechanism of competition between circHIPK3 and IGF2BP2 triggered by IGF2BP2 nucleation, potentially via phase separation.
Strengths:
Although the number of circRNAs continues to grow, this field lacks many instances of detailed molecular investigations. The presented work critically addresses some of the major piaalls in the field of circRNAs, and there has been a careful analysis of aspects frequently poorly investigated. Experiments involving use of time-point knockdown followed by RNAseq, investigation of miRNA-sponge function of circHIPK3, identification of 11-mer motif, identification and validation of IGF2BP2, and the analysis of copy number ratio between circHIPK3 and IGF2BP2 in assessing the potential ceRNA mode of action are thorough and convincing.
Weaknesses:
It is unclear why the authors used certain bladder cancer cells versus non-bladder cells in some experiments. The efficacy of certain experiments (specifically rescue experiments) and some control conditions is still questionable. Overall, the presented study adds some further knowledge in describing circHIPK3 function, its capability to regulate some downstream genes, and its interaction and competition for IGF2BP2.
We have provided a discussion and argumentation of how certain bladder cancer cells (and non-bladder cancer cells) have been used in this study in our previous rebuttal letter and also clarified this further in the materials and methods section in the first revision. Regarding control conditions for experiments, we believe we have included all necessary controls and explanations for these in the revised version (please see the detailed description in the specific comments below).
Recommendations for the authors:
Reviewer #1 (Recommendations For The Authors):
Major points about revised manuscript
(1) In Supplementary Figure S5H, the membrane may have been trimmed too closely to the circRNA band, potentially resulting in the absence of the linear RNA band. Could the authors provide a full image of the membrane that includes the loading points? Having access to the complete image would allow for a more comprehensive evaluation of the results, including the presence or absence of expected linear and circular RNA bands.
I have taken the liberty to move this “major point” from the public review section, as I believe it would be too detailed for this section. We have included the full section of the northern blot, according to the reviewers recommendations.
As described in the previous rebuttal letter our northern blots suffered from heavy background signal arising from the rRNA bands, which was the reason for cuttng the northern blot in the previous version of Supplementary figure S5H. We have now shown the entire blot as suggested by the reviewer, so that the reader can more clearly inspect any potential linear precursor band. We previously stated that we could not assess the circular-to-linear ratio due to background signal, since a potential linear HIPK3 precursor RNA could be masked by the rRNA signal. However, the theoretical size of a linear precursor is ~2.9 kb – a region where we do not detect any distinct bands (just above the 18S band), making a rather efficient circularization very likely. In support of this claim, we are using the Laccase2 vector described in Kramer et al, 2015 (Genes dev), which is proven to produce high levels of circHIPK3 compared to negligable amounts of linear precursor (although in a different cell line). We have also included a 5.8S rRNA probe to control for loading and RNase R activity (can also be ascertained by the disappearence of 18S/28S bands). Since we do not have the option to use another probe (limited by the BSJ-specific probe) and it is not practical to deplete for rRNA from 20 µg samples of total RNA, prior to running the northern blot, we find that this data sufficiently proves that our vector constructs produce a decent amount of RNase R-resistant circHIPK3, with no visible/discernible linear precursor.
Minor points about revised manuscript
(1) In Supplementary Figure S3B, the authors offer no explanation as to why genes that become upregulated upon circHIPK3 knockdown generally contain more circHIPK3-RBP binding sites other than for IGF2BP2. A clarification would be of help.
Again, this issue has been addressed in the previous rebuttal letter. Our response is repeated below:
We do not have any evidence to explain this observation. One possibility is that other RBPs elicit mRNA-stabilizing effects on average, whereas abundant IGF2BP2 (~ 120.000200.000 copies per cell) now able to bind more target mRNAs and elicit destabilization. This remains highly speculative though.
(2) In Supplementary Figure S3D, the authors' claim that the 11-mer motif is found more bound to IGF2BP2 than for other circHIPK3-RBPs should referred to the corresponding dataset/reference.
Again, this issue has been addressed in the previous rebuttal letter. Our response is repeated below:
This information is stated in the figure legend (K562) and we have now included it in the main text as well: “We evaluated how often binding sites of circHIPK3-RBPs overlap the 11-mer motif and found that this is more often the case for IGF2BP2 binding sites than binding sites of the other circHIPK3-RBPs when scrutinizing K562 datasets (Supplementary Figure S3D)”.
(3) In the rescue experiment where both circHIPK3 and IGF2BP2 are downregulated, using the term "normalization" to mean reestablishing normal levels of gene expression can lead to confusion with the concept of normalization as it is commonly understood in the context of data processing (i.e. the mathematical process of adjusting data to account for various factors that might affect measurements). I would recommend the authors to use a term that more specifically describes the biological process they are referring to, such as "restoration of normal expression levels" or simply "return to normal levels".
We agree that this term could be misunderstood. This has now been changed as recommended.
(4) The figure legend of Supplementary Figure 5F is wrongly labeled. The legend for panel F actually corresponds to panel G and vice versa.
This has now been corrected.
Reviewer #2 (Recommendations For The Authors):
The authors have diligently addressed most of the points raised during the review process (except the important point of "additional in vitro experiments [...] needed to investigate the implication of circHIPK3 in bladder cancer cell phenotype" for which no additional experiments were performed), resulting in an improvement in the study. The data are now described with clarity and conciseness, enhancing the overall quality of the manuscript. Therefore, I support the publication of this work.
We thank the reviewer for the positive comments.
Reviewer #3 (Recommendations For The Authors):
Please ensure that when the changes are made (especially for major points) by addressing the reviewer's comments, these are all appropriately incorporated in the text (for example the use of Act B as a low affinity positive control (now in Fig 4A), is not explained in the text neither the legends/methods)
This has now been included.
Please ensure that all the legends correspond to the right figures (eg: Supplementary Figure with rescue experiment is 5F, but the corresponding legend in the manuscript is the S5G).
This has now been corrected.
Please for future reviewing processes ensure the new parts are properly highlighted or coloured differently in the manuscript
This has now been done more thoroughly.
