Investigating the Impact of Mettl16 Cellular Localization on RNA Binding Preferences
Nance, Daniel Joel
Recently, mRNA modification by N6-methyladenosine (m6A) has been shown to be involved in post-transcriptional regulation processes including mRNA stability, splicing and promotion of translation. Accordingly, the mRNA methylation complex of Mettl3/14/WTAP has been the subject of intense study. However, we and others have also identified Mettl16 as an RNA m6A methyltransferase that can methylate both coding and noncoding RNAs, but its biological role remains unclear. Mettl16’s RNA targets have been identified and include the long noncoding RNA MALAT1, the snRNA U6, as well as many mRNAs including MAT2A, HIF1, and SEMA4F. To investigate the functional role of Mettl16 we knocked out Mettl16 via CRISPR but were unable to obtain viable clones suggesting that it may be essential for cell growth; a finding supported by the literature. Transient knockdown via siRNA was successful, but no effect on RNA target expression or translation was observed. We are currently developing a tetracycline inducible METTL16 knockdown system to examine the effects of more long-term depletion. We have been successful at overexpressing METTL16 and identifying additional RNA targets by immunoprecipitation. Interestingly, when overexpressing exogenous Mettl16 we have observed differences from the endogenous protein in both the RNA targets as well as the relative affinity for targets. We hypothesize that this difference may be related to cellular localization as the endogenous protein appears to be mainly cytoplasmic while a significant fraction of the overexpressed protein is nuclear. Thus, while METTL16 has been asserted to be a nuclear protein, our findings depict METTL16 as a primarily cytoplasmic methyltransferase that may alter its RNA binding preferences depending on its cellular localization. Future studies will seek to confirm differences between cytoplasmic and nuclear RNA targets in addition to exploring the physiological role of Mettl16 through long-term knockdown.
Nance, Daniel Joel. (May 2019). Investigating the Impact of Mettl16 Cellular Localization on RNA Binding Preferences (Honors Thesis, East Carolina University). Retrieved from the Scholarship. (http://hdl.handle.net/10342/7338.)
Nance, Daniel Joel. Investigating the Impact of Mettl16 Cellular Localization on RNA Binding Preferences. Honors Thesis. East Carolina University, May 2019. The Scholarship. http://hdl.handle.net/10342/7338. February 21, 2020.
Nance, Daniel Joel, “Investigating the Impact of Mettl16 Cellular Localization on RNA Binding Preferences” (Honors Thesis., East Carolina University, May 2019).
Nance, Daniel Joel. Investigating the Impact of Mettl16 Cellular Localization on RNA Binding Preferences [Honors Thesis]. Greenville, NC: East Carolina University; May 2019.
East Carolina University