Title
Restoration of miRNA-149 expression by TmPyP4 induced unfolding of quadruplex within its precursor
Author
Arpita Ghosh, Mary Krishnan Ekka, Arpita Tawani, Amit Kumar, Debojyoti Chakraborty, Souvik Maiti
Year
2018
Journal
Biochemistry
Abstract
Non-coding RNAs are functional RNA molecules that get transcribed from DNA but are not translated into proteins; yet, they can regulate gene expression at transcriptional and post-transcriptional levels. Secondary structures present within these RNAs play a major role in determining their nature of function. In case of miRNAs, the precursor miRNA have hairpin stem loop structure which is required for Dicer recognition and further maturation. Shift in the hairpin to G-quadruplex structure depends upon the nucleotide sequence, the cellular microenvironment and might affect the miRNA maturation and other downstream activity. One such structure, G-quadruplex, can modulate the miRNA regulatory role. Formation of G-quadruplex within precursor miRNA-149 has been shown to inhibit Dicer processing activity followed by suppression of miRNA-149 maturation and thereby rendering it pathogenic for cancer. As suppressed expression of miRNA-149 markedly increases cell proliferation, here, in this report, we show that suppression of cell proliferation by the upregulated miRNA-149 could be rescued by unfolding the quadruplex present in pre-miRNA-149 by TmPyP4 (Porphyrin) treatment. Using UV-visible spectroscopy, circular dichroism and isothermal titration calorimetry, we observed that TmPyP4 binds strongly to quadruplex and unfolds it, which was further verified by NMR Spectroscopy. In-cellulo, qRT-PCR measurements of miRNA-149 in MCF-7 breast cancer cells showed concentration dependent enhancement of mature miRNA-149 upon treatment of TmPyP4. As a consequence of enhanced miRNA-149 levels, we could even observe the reduction in miRNA-149 target protein ZBTB2 that eventually lead to reduced cell proliferation.
Instrument
J-815
Keywords
Circular dichroism, DNA structure, Chemical stability, Ligand binding, Biochemistry