Recruitment of the m 6 A/Am demethylase FTO to target RNAs by the telomeric zinc finger protein ZBTB48

  1. Syed Nabeel-Shah
  2. Shuye Pu
  3. Giovanni L. Burke
  4. Nujhat Ahmed
  5. Ulrich Braunschweig
  6. Shaghayegh Farhangmehr
  7. Hyunmin Lee
  8. Mingkun Wu
  9. Zuyao Ni
  10. Hua Tang
  11. Guoqing Zhong
  12. Edyta Marcon
  13. Zhaolei Zhang
  14. Benjamin J. Blencowe
  15. Jack F. Greenblatt

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

N6-methyladenosine (m6A), the most abundant internal modification on eukaryotic mRNA, and N6, 2′-O-dimethyladenosine (m6Am), are epitranscriptomic marks that function in multiple aspects of posttranscriptional regulation. Fat mass and obesity-associated protein (FTO) can remove both m 6 A and m6Am; however, little is known about how FTO achieves its substrate selectivity. Here, we demonstrate that ZBTB48, a C2H2-zinc finger protein that functions in telomere maintenance, associates with FTO and binds both mRNA and the telomere-associated regulatory RNA TERRA to regulate the functional interactions of FTO with target transcripts. Specifically, depletion of ZBTB48 affects targeting of FTO to sites of m6A/m6Am modification, changes cellular m6A/m6Am levels and, consequently, alters decay rates of target RNAs. ZBTB48 ablation also accelerates growth of HCT-116 colorectal cancer cells and modulates FTO- dependent regulation of Metastasis-associated protein 1 (MTA1) transcripts by controlling the binding to MTA1 mRNA of the m6A reader IGF2BP2. Our findings thus uncover a previously unknown mechanism of posttranscriptional regulation in which ZBTB48 co-ordinates RNA- binding of the m6A/m6Am demethylase FTO to control expression of its target RNAs.

Article activity feed

  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    The authors do not wish to provide a response at this time

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    In the manuscript by Nabeel-Shah et al. the authors identify ZBTB48 as a novel interactor of FTO. They show that ZBTB48 helps recruiting FTO to mRNA as well as the noncoding RNA Terra. Their results further suggest that this recruitment is required for FTO to demethylated m6A and m6Am RNA modification in target RNAs. This affects cellular rates of RNA turnover. Furthermore, the mechanism is involved in repressing colorectal cancer cell growth.

    Overall, the authors present a new role of ZBTB48 in m6A and m6Am mediated RNA metabolism. They also suggest a nice model of ZBTB48 action, via the recruitment of the demethylase FTO. These findings will be of interest for the general RNA community and might be also relevant for cancer treatment. However, I have some concerns about the quality and analysis of the obtained data, especially the iCLIP and miCLIP experiments. The concerns are detailed below and should be addressed before publication.

    Major concerns:

    1. Figure 1B) I think that the results shown in the autoradiograph are not very convincing and suggest that the purification of ZBTB48 is not very clean. The radioactive signal covers the lane from 50 kDa to 200 kDa. The ZBTB48 alone is running around 80 kDa. If the purification is specific most of the signal should be above 80 kDa. Maybe it helps to also use higher RNAse concentrations: Having very short RNA pieces will allow to evaluate specificity of the purification, since protein-RNA complexes will just a bit above the size of the protein. This also applies to Figures S1E,F.

    This concern could also apply to the generated iCLIP libraries and indicates that at least part of the obtained reads does not originate from ZBTB48 crosslinked RNA. The validation of protein-RNA interactions with RIP shown in Figure S2E supports the quality of the iCLIP data. Here some control RNAs should be analyzed to show that the RIP is not unspecifically enriching any RNA.

    1. Regarding the co-occurrence of m6A sites as well as FTO and ZBTB48 binding sites shown in Figures 2B, C, F and G. The CLIP signal is a lot affected by crosslinking bias and read mappability. Therefore, to make these results more convincing it would be important to include additional iCLIP datasets (published other RBPs) for comparison.
    2. Regarding Figure 2D and related analyses: I very much like this experiment and the results obtained here! I just wondered where the remaining reads go in the ZBTB48 knockdown. The introns? Maybe this becomes clearer in the meta profile representation used in Figure 1F.

    For me the changes in the 5' UTR look most dramatic. Maybe this means that ZBTB48 is most important for recruitment of FTO to m6Am sites. Therefore, I think it would be good to differentiate the analyses in the remainder of the manuscript for m6A and m6Am sites. I first step would be to treat sites in the 5'UTR separate from the CDS and 3'UTR sites in the following analysis.

    1. I have some concerns about the analysis of the miCLIP data. In Figure 3A antibody crosslinking in all conditions appears similar. Yet in panel B there seems less signal for FTO and ZBTB48 overexpression in all areas. Have there been more reads generated for the GFP control? Where is the rest of the reads going? I think it would be required here to identify peaks that significantly change between the conditions. Then ask do those peaks coincide with DRACH and were are they located. Also, in the Genome browser pictures the signal is going down at all locations. Why is that? Usually, miCLIP generates a lot of background peaks. These should be unchanged.
    2. I am a bit confused by the author's interpretation of the results shown in Figure 5E. For me this plot shows that target transcripts are less downregulated and less upregulated than non-targets? Basically they are less regulated overall. In this context I also think that the representation in of the mean in figure 5E and F is misleading. Data distribution should be visible as violin or boxplot.

    Minor points:

    Figure 1D) I think the legend in the panel is confusing and does not add information. Especially since its in a different order than the categories on the y-Axis.

    Figure S2D) I wondered if the authors controlled for mappability of reads when picking random sites. How do the authors account for that in iCLIP there will be less reads in introns compared to exons?

    5E,F boxplots would be more suitable than barplots.xx

    Figure 2I: Please do not use "+ve"

    Figures with Microscopy pictures of cells have no scale bars or way too small.

    The model that is shown in figure 7 is somehow misleading as it shows FTO binding only to ZBTB48 and not to the RNA.

    Significance

    Overall, the authors present a new role of ZBTB48 in m6A and m6Am mediated RNA metabolism. They also suggest a nice model of ZBTB48 action, via the recruitment of the demethylase FTO. These findings will be of interest for the general RNA community and might be also relevant for cancer treatment. However, I have some concerns about the quality and analysis of the obtained data, especially the iCLIP and miCLIP experiments. The concerns are detailed below and should be addressed before publication.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, Nabeel-Shah et al. identified that the telomeric zinc finger protein ZBTB48 help recruit FTO onto target RNA, including mRNAs and the telomere-associated regulatory RNA TERRA, to achieve cellular RNA m6A/m6Am demethylation, thereby regulating cell biology such as tumour growth studied in this study. The biochemistry and molecular biology experiments were done in HEK293 cells, and the cell models for tumour growth was done in HCT116 cells. However, I have identified both major and minor concerns that, when addressed, could further strengthen the findings and enhance the impact on the field.

    Major concerns

    1. Regarding the ZBTB48/FTO targets and m6A/m6Am level that are regulated by ZBTB48/FTO axis. Wei et al Molecular Cell 2018 (PMID: 30197295) (Figure 1B) quantified the m6A/m6Am levels upon knockdown of FTO in HeLa, HEK293, and 3T3-L1 cells, and found that the m6A/m6Am levels are generally mildly (10-20%) yet significantly upregulated on poly(A)+ RNAs. Here shown in the Figure 4A in this manuscript, the authors show that (1) siFTO and siZBTB48 led to ~2-3 fold upregulation of m6A and m6Am levels using total RNAs (dominant by rRNA in the population), and (2) that the m6A and m6Am levels are similar between siFTO and siZBTB48. Regarding (1), can the authors explain the discrepancy? This point is also relevant to the m6AIP-qRT-PCR results in this manuscript. Regarding (2), does this result suggest that ZBTB48 helps FTO to demethylate nearly all its targets, rather than a subset?
    2. Considering the significance of how FTO achieves target specificity, can the author anticipate the extent of applicability of the proposed model? Does the interaction between ZBTB48 and FTO also exist in various human and mouse cell lines? If confirmed, this discovery would hold substantial value for the field. This interaction at least needs to be confirmed in HCT116 cells used for tumour growth model in this study.

    Minor concerns.

    1. The authors realised that knockdown of ZBTB48 does not change FTO levels, whereas overexpression of ZBTB48 leads to elevated FTO. It is unclear about the rationale behind overexpression studies?
    2. Considering the multifunctional nature of ZBTB48, it's important to disentangle transcriptional and post-transcriptional roles of ZBTB48 to draw conclusions. I appreciate the analysis conducted in this manuscript. Is it possible to overexpress an DNA-binding mutant of ZBTB48 or RNA-binding mutant of ZBTB48 proteins?
    3. In Fig 2B, the innet panel does not match the metagene profile regarding the difference.
    4. In Fig 5H, there is a lack of experiments for comparison, i.e. siFTO and o/e FTO.
    5. Page 3, "Genome-wide studies" should be "Transcriptome-wide studies".

    Significance

    Strengths: The biochemistry and molecular biology experiments are comprehensive and well designed. The analysis is robust, and the conclusions generally align with the presented data.

    Limitations: Cell line-specific and/or species-specific interaction?

    Advance: filling a gap in our knowledge of how FTO achieves target specificity.

    Audience: Basica research in the field of RNA modifications and RNA Biology.

    My expertise: Bioinformatics, gene regulation by RNA m6A modification

  4. Version published to 10.1101/2024.01.15.575768v2 on bioRxiv
  5. Version published to 10.1101/2024.01.15.575768v1 on bioRxiv