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Gracjan Michlewski


Nila Roy Choudhury, Jakub Nowak
Michlewski lab website

Regulation of MicroRNA Processing and Function

MicroRNAs (miRs) constitute a large family of short (21-23nt), conserved non-coding RNAs. They regulate gene expression by targeting partially complementary sequences in the mRNAs. Each microRNA has the capacity to regulate several, possibly hundreds, of mRNA targets, thus controlling a variety of biological processes, including cell cycle, developmental timing, differentiation, metabolism, neuronal patterning and ageing. In spite of the great effort to understand the various biological roles of individual microRNAs there is a huge void of knowledge about the regulation of their own biogenesis. It is known that several tissue-specific microRNAs are under transcriptional control; however, it is unknown whether post-transcriptional processes contribute to establishing their levels. Our group is focused on elucidating the cis and trans-acting factors that play significant roles in microRNA processing and function.

Recently, we showed that brain-specific miR-9 is regulated transcriptionally and post-transcriptionally during the neuronal differentiation. We demonstrated that miR-9 is more efficiently processed in differentiated than undifferentiated cells. We revealed that Lin28a inhibits the processing of miR-9 by inducing the degradation of its precursor through a uridylation-independent mechanism. Furthermore, we showed that constitutive expression of untagged but not GFP-tagged Lin28a causes a severe neuronal differentiation phenotype, which coincides with reduced miR-9 levels. Using an inducible Tet-On 3G system we demonstrated that Lin28a can also reduce miR-9 levels in differentiated cells. Altogether, we shed more light on the role of Lin28a in the neuronal differentiation as well as showed novel, substrate-dependent mechanism of Lin28a action. Finally, our results provide new evidence that transcriptionally controlled microRNAs can undergo extensive and complementary post-transcriptional regulation.

By combining a wide spectrum of molecular biology and biochemistry techniques, including functional assays in mammalian cultured cells, RNA deep sequencing, RNA chromatography combined with SILAC Mass Spectrometry, RNA structure probing and in vitro processing assays, we are dissecting the fine details of microRNA biogenesis pathways. Currently, we are investigating the mechanisms controlling the abundance of the brain-specific microRNAs, as well as the contribution of microRNAs to induced Pluripotent Stem (iPS) cell formation. If successful, our projects will have far reaching consequences for our understanding of how microRNAs regulate gene expression, providing novel cutting-edge avenues for future research and potential targets for novel therapies.

Selected publications:

Selected Publications: Michlewski, G., Guil, S., Semple, C.A., and Cáceres, J.F. (2008) Posttranscriptional regulation of miRNAs harboring conserved terminal loops. Mol Cell 32, 383-393
Michlewski, G., and Cáceres, J.F. (2010) Antagonistic role of hnRNP A1 and KSRP in the regulation of let-7a biogenesis. Nat Struct Mol Biol 17, 1011-1018.
Choudhury, N.R., de Lima Alves, F., de Andrés-Aguayo, L., Graf, T., Cáceres, J.F., Rappsilber, J., and Michlewski, G. (2013) Tissue-specific control of brain-enriched miR-7 biogenesis. Genes Dev 27, 24-38.

Figure 1. SILAC combined with RNA pull-down and Mass Spectrometry reveals putative regulators of brain-specific microRNA biogenesis. A. Schematic of the method. P19 cells are grown in “light” medium containing or “heavy” medium. Cells grown in “light medium” are subjected to retinoic acid-induced neuronal differentiation until day 9 (d9). Next, RNA pull-down are performed with agarose beads covalently linked to microRNA CTLs (Conserved Terminal Loops) or pre-microRNAs and incubated with premixed extracts from “light” d9 or “heavy” day 0 (d0) P19 cells. After RNase treatment, the supernatants are subjected to quantitative mass spectrometry, which identifies putative microRNA biogenesis factors. B. The graph represents the fold enrichment of proteins that bind to the miR-9-1 CTL in experiments with “heavy” d0 P19 cell extracts compared with “light” d9 P19 cell extracts. The values are presented on a log10 scale. The Lin28a protein is indicated in red.

Figure 2. Lin28a-mediated inhibition of miR-9. A. In undifferentiated cells and during early differentiation Lin28a together with yet unknown effector protein, inhibit production of miR-9 by inducing the degradation of its precursor.
B. In differentiated neuronal cells lack of Lin28a allows for efficient processing by Dicer and miR-9 maturation.