Poster abstracts
Poster number 157 submitted by Megan Westwood
Investigation of the structure of pre-miR-122 and the features that impact Dicer processing efficiency
Megan N. Westwood (Biophysics Program, University of Michigan), Sicong Ma (Biophysics Program, University of Michigan), Sarah C. Keane (Biophysics Program, Department of Chemistry, University of Michigan )
Abstract:
Mature microRNAs (miRs or miRNAs) play a critical role in post-transcriptional regulation of gene expression, regulating more than half of human messenger (m) RNAs. MiRNAs are transcribed from DNA into primary miRNAs. Primary (pri) miRNAs are processed in the nucleus by the microprocessor complex, which cleaves at the base of the pri-miRNA stem to make precursor (pre) miRNAs. The pre-miRNA is then exported to the cytoplasm by exportin 5 where it is subsequently processed by the Dicer:transactivation response element RNA-binding protein (TRBP) complex, which removes the apical loop to generate the mature miRNA duplex. The mature miRNA interacts with Argonaute proteins and makes sequence-specific interactions with mRNAs to induce degradation or repress translation. MiR-122 is a tissue-specific miRNA highly expressed in the liver. Aberrant expression of miR-122 is a hallmark of various tumors and cancers including hepatocellular carcinoma. Unlike many pre-miRNAs, there are no known protein binding partners for pre-miR-122 that regulate its biogenesis post-transcriptionally. Therefore, I hypothesize that pre-miR-122 has intrinsic structural properties that regulate its processing. Thermal denaturation studies revealed stabilization of the RNA structure at low pH and RNA secondary structure predictions suggested several alternative structures were possible in the apical loop. I am using nuclear magnetic resonance (NMR) spectroscopy to determine the structure of pre-miR-122 and to examine potential alternative structures that may exist in the pre-miR-122 apical loop. Future work will involve in vitro Dicer-TRBP processing assays on wildtype pre-miR-122 and mutants that are designed to stabilize specific alternative structures to assess which structural features regulate its biogenesis.
References:
Zhang, X.; Zeng, Y. The Terminal Loop Region Controls MicroRNA Processing by Drosha and Dicer. Nucleic Acids Res. 2010, 38 (21), 7689–7697.
Fareh, M.; Yeom, K.-H.; Haagsma, A. C.; Chauhan, S.; Heo, I.; Joo, C. TRBP Ensures Efficient Dicer Processing of Precursor MicroRNA in RNA-Crowded Environments. Nat. Commun. 2016, 7 (1), 13694.
Ma, S.; Kotar, A.; Hall, I.; Grote, S.; Rouskin, S.; Keane, S. C. Structure of Pre-miR-31 Reveals an Active Role in Dicer–TRBP Complex Processing. Proc. Natl. Acad. Sci. 2023, 120 (39), e2300527120.
Keywords: miRNA, biogenesis, NMR