Characterization of METTL16 as a cytoplasmic RNA binding protein


Autoři: Daniel J. Nance aff001;  Emily R. Satterwhite aff001;  Brinda Bhaskar aff001;  Sway Misra aff001;  Kristen R. Carraway aff001;  Kyle D. Mansfield aff001
Působiště autorů: Department of Biochemistry and Molecular Biology, Brody School of Medicine, East Carolina University, Greenville, North Carolina, United States of America aff001
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: 10.1371/journal.pone.0227647

Souhrn

mRNA modification by N6-methyladenosine (m6A) is involved in many post-transcriptional regulation processes including mRNA stability, splicing and promotion of translation. Accordingly, the recently identified mRNA methylation complex containing METTL3, METTL14, and WTAP has been the subject of intense study. However, METTL16 (METT10D) has also been identified as an RNA m6A methyltransferase that can methylate both coding and noncoding RNAs, but its biological role remains unclear. While global studies have identified many potential RNA targets of METTL16, only a handful, including the long noncoding RNA MALAT1, the snRNA U6, as well as the mRNA MAT2A have been verified and/or studied to any great extent. In this study we identified/verified METTL16 targets by immunoprecipitation of both endogenous as well as exogenous FLAG-tagged protein. Interestingly, exogenously overexpressed METTL16 differed from the endogenous protein in its relative affinity for RNA targets which prompted us to investigate METTL16's localization within the cell. Surprisingly, biochemical fractionation revealed that a majority of METTL16 protein resides in the cytoplasm of a number of cells. Furthermore, siRNA knockdown of METTL16 resulted in expression changes of a few mRNA targets suggesting that METTL16 may play a role in regulating gene expression. Thus, while METTL16 has been reported to be a nuclear protein, our findings suggest that METTL16 is also a 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.

Klíčová slova:

Cytoplasm – Immunohistochemistry techniques – Immunoprecipitation – Messenger RNA – Methylation – Non-coding RNA sequences – Small interfering RNAs – Small nuclear RNA


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