The distinct RNA-interaction modes of a small ZnF domain underlay TUT4(7) diverse action in miRNA regulation

ABSTRACT

TUT4 and the closely related TUT7 are non-templated poly(U) polymerases required at different stages of development, and their mis-regulation or mutation has been linked to important cancer pathologies. While TUT4(7) interaction with its pre-miRNA targets has been characterized in detail, the molecular bases of the broader target recognition process are unclear. Here, we examine RNA binding by the ZnF domains of the protein. We show that TUT4(7) ZnF2 contains two distinct RNA binding surfaces that are used in the interaction with different RNA nucleobases in different targets, i.e that this small domain encodes diversity in TUT4(7) selectivity and molecular function. Interestingly and unlike other well-characterized CCHC ZnFs, ZnF2 is not physically coupled to the flanking ZnF3 and acts independently in miRNA recognition, while the remaining CCHC ZnF of TUT4(7), ZnF1, has lost its intrinsic RNA binding capability. Together, our data suggest that the ZnFs of TUT4(7) are independent units for RNA and, possibly, protein-protein interactions that underlay the protein’s functional flexibility and are likely to play an important role in building its interaction network.

KEYWORDS:

• TUT4/zcchc11
• pre-let7i miRNA
• ssRNA recognition
• nuclear magnetic resonance
• Biolayer Interferometry

Acknowledgments

NMR spectra were recorded at the MRC Biomedical NMR and UCL NMR facilities and we thank Geoff Kelly, Alain Oregioni and Angelo Pinto De Figueiredo for assistance.

Disclosure statement

The authors declare no competing financial interests.

Author Contributions

Cloning of the TUT4 ZnFs constructs was performed by KMC and EC. The protein samples were prepared by KMC and BCA. NMR experiments were recorded and analysed by KMC, BCA and GN. BLI experiments were recorded by BCA and analysed by BCA and SRM. The project was designed by A.R. and the manuscript was written by A.R. and KMC with contributions from all authors.

Data Sharing Statement

The plasmids expressing the constructs discussed in this study will be made available by the authors upon request. The resonances assignment of hTUT4 ZnF2 protein will be available the BMRB database (https://bmrb.io/) upon publication of the manuscript.

Supplementary material

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by the UK Medical Research Council (MC_PC_13051, U117574558, MR/S000305/1) to A.R. It was also supported by University College London and by the Francis Crick Institute which receives its core funding from Cancer Research UK (FC001178) the UK Medical Research Council (FC001178) and the Wellcome Trust (FC001178). The work was supported by the Francis Crick Institute also through provision of access to the MRC Biomedical NMR Centre (Francis Crick Institute core funding by Cancer Research UK (FC001029), the UK Medical Research Council (FC001029), and the Wellcome Trust (FC001029).)