Characterization of METTL16 as a cytoplasmic RNA binding protein

Daniel J Nance,Sway Misra,Brinda Bhaskar,Kristen R Carraway,Kyle D Mansfield,Emily R Satterwhite,Alexander F Palazzo

doi:10.1371/journal.pone.0227647

Daniel J Nance, Sway Misra + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0227647

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Journal: PLOS ONE	Publication Date: Jan 15, 2020
Citations: 44	License type: CC BY 4.0

Affiliation: East Carolina University

Abstract

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.

Highlights

The amount of the RNA target in the GFP or METTL16 IP was compared to the input levels from 50% of the sample to generate a relative enrichment (Fig 1B, left panels)
Fold enrichment of the RNA target in the METTL16 IP compared to the GFP IP was calculated (Fig 1B, right panels) by dividing the METTL16 relative enrichment by the GFP relative enrichment value
By comparing enrichment to both the input as well as a negative IP we can get a better sense of the nature of the binding and have greater confidence in identifying METTL16 binding targets

Summary

Introduction

METTL16 has been shown to bind and methylate mRNAs, including MAT2A, which can regulate its alternative splicing in response to cellular SAM levels [29, 31, 32]. Low intracellular levels of SAM do not allow for efficient methylation by METTL16, increasing METTL16 occupancy on the mRNA which results in increased splicing of the retained intron This increases stabilization and translation of the MAT2A mRNA and production of SAM synthetase which increases SAM levels in the cell. Our results suggest that the majority of METTL16 protein resides in the cytoplasm of a number of cell types, and that knockdown of METTL16 protein can affect the expression of a few of its mRNA binding targets These results suggest that METTL16 may have additional roles in the cell that could contribute to its essentiality

Results

Discussion

Materials and methods