circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA

Reviewer #1 (Public Review):

In this work the authors propose a new regulatory role for one the most abundant circRNAs, circHIPK3, by showing that it interacts with an RNA binding protein (IGF2BP2) and, by sequestering it, it regulates the expression of hundreds of genes containing a sequence (11-mer motif) in their untranslated regions (3'-UTR). This sequence is also present in circHIPK3, precisely where IGF2BP2 binds. The study further focuses on one specific case, the STAT3 gene, whose mRNA product is downregulated upon circHIPK3 depletion apparently through sequestering IGF2BP2, which otherwise binds to and stabilizes STAT3 mRNA. The study presents mechanistic insight into the interactions, sequence motifs, and stoichiometries of the molecules involved in this new mode of regulation. Altogether, this new mechanism seems to underlie the effects of circHIPK3 in cancer progression.

Strengths:
The authors show mechanistic insight into a proposed novel "sponging" function of circHIPK3 which is not mediated by sequestering miRNAs but rather by 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 study. They provide both genome-wide analysis and a specific case (STAT3) that is relevant for cancer progression.

Weaknesses:
One of the central conclusions of the manuscript, namely that circHIPK3 sequesters IGF2BP2 and thereby regulates target mRNAs, lacks more direct experimental evidence such as rescue experiments where both species are simultaneously knocked down. CircRNA overexpression lacks a demonstration of circularization efficiencies. There seem to be contradictory effects of circHIPK3 and STAT3 depletion in cancer progression, namely that while circHIPK3 is frequently downregulated in cancer, circHIPK3 downregulation in this study leads to downregulation of STAT3. This does not seem to fit the fact that STAT3 is normally activated in a wide diversity of cancers and is positively associated with cell proliferation. The result is neither consistent with the fact that circHIPK3 expression positively correlates with good clinical outcomes. Overall, the authors have achieved some of their aims but additional controls would be advisable to fully support their conclusions.

Reviewer #2 (Public Review):

The manuscript by Okholm and colleagues identified an interesting new instance of ceRNA involving a circular RNA. The data are clearly presented and support the conclusions. Quantification of the copy number of circRNA and quantification of the protein were performed, and this is important to support the ceRNA mechanism.

Reviewer #3 (Public Review):

In Okholm et al., the authors evaluate the functional impact of circHIPK3 in bladder cancer cells. By knocking it down and performing an RNA-seq analysis, the authors found thousands of deregulated genes that look unaffected by miRNAs sponging function and that are, instead, enriched for an 11-mer motif. Further investigations showed that the 11-mer 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. This was accompanied by consistent 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:
The number of circRNAs continues to drastically grow; however, the field lacks detailed molecular investigations. The presented work critically addresses some of the major pitfalls in the field of circRNAs and there has been a careful analysis of aspects frequently poorly investigated. The time-point KD followed by RNA-seq, investigation of the miRNAs-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 have been extensively explored and, comprehensively are convincing.

Weaknesses:
In some parts, the manuscript lacks appropriate internal controls (eg: comparison with normal bladder cells, linear transcript measurements upon the KD, RIP internal controls/ WB analysis, etc), statistical analysis and significance (in some qPCRs), exhaustive description in the methods of microscopy and image analysis, western blot, and a separate section of cell lines used. The use of certain cell lines bladder cancer cells vs non-bladder cells in some experiments for the purpose of the study is also unclear.

Overall, the presented study adds new knowledge in describing circHIPK3 function, its capability to regulate some downstream genes and its interaction and competition for IGF2BP2. However, whereas the experimental part appears technically logical, it remains unclear the overall goal of this study and the final conclusions. The mechanism of condensation proposed, although interesting and encouraging, would need further experimental support and information, especially in the context of cancer.

In summary, this study is a promising step forward in the comprehension of the functional role of circHIPK3. These data could possibly help to better understand the circHIPK3 role in cancer.