Human Lin28 forms a high-affinity 1:1 complex with the 106~363 cluster miRNA miR-363

TY - JOUR

T1 - Human Lin28 forms a high-affinity 1:1 complex with the 106~363 cluster miRNA miR-363

AU - Peters, Daniel T

AU - Fung, Herman Kh

AU - Levdikov, Vladimir M

AU - Irmscher, Tobias

AU - Warrander, Fiona C

AU - Greive, Sandra J

AU - Kovalevskiy, Oleg

AU - Isaacs, Harry V

AU - Coles, Mark Christopher

AU - Antson, Alfred A

N1 - © 2016 American Chemical Society.

PY - 2016/8/25

Y1 - 2016/8/25

N2 - Lin28A is a post-transcriptional regulator of gene expression that interacts with and negatively regulates the biogenesis of let-7 family miRNAs. Recent data suggested that Lin28A also binds the putative tumour suppressor miR-363, a member of the 106~363 cluster of miRNAs. Affinity toward this miRNA and the stoichiometry of the protein-RNA complex are unknown. Characterisation of human Lin28's interaction with RNA has been complicated by difficulties in producing stable RNA-free protein. We have engineered a maltose binding protein fusion with Lin28, which binds let-7 miRNA with a Kd of 54.1 ± 4.2 nM, in agreement with previous data on a murine homologue. We show that human Lin28A binds miR-363 with 1:1 stoichiometry and with similar, if not higher, affinity (Kd = 16.6 ± 1.9 nM). Further analysis suggests that the interaction of the N-terminal cold shock domain of Lin28A with RNA is salt-dependent, supporting a model where the cold shock domain allows the protein to sample RNA substrates through transient electrostatic interactions.

AB - Lin28A is a post-transcriptional regulator of gene expression that interacts with and negatively regulates the biogenesis of let-7 family miRNAs. Recent data suggested that Lin28A also binds the putative tumour suppressor miR-363, a member of the 106~363 cluster of miRNAs. Affinity toward this miRNA and the stoichiometry of the protein-RNA complex are unknown. Characterisation of human Lin28's interaction with RNA has been complicated by difficulties in producing stable RNA-free protein. We have engineered a maltose binding protein fusion with Lin28, which binds let-7 miRNA with a Kd of 54.1 ± 4.2 nM, in agreement with previous data on a murine homologue. We show that human Lin28A binds miR-363 with 1:1 stoichiometry and with similar, if not higher, affinity (Kd = 16.6 ± 1.9 nM). Further analysis suggests that the interaction of the N-terminal cold shock domain of Lin28A with RNA is salt-dependent, supporting a model where the cold shock domain allows the protein to sample RNA substrates through transient electrostatic interactions.

U2 - 10.1021/acs.biochem.6b00682

DO - 10.1021/acs.biochem.6b00682

M3 - Article

C2 - 27559824

SN - 0006-2960

VL - 55

SP - 5021−5027

JO - Biochemistry

JF - Biochemistry

ER -