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Gracjan Michlewski


Nila Roy Choudhury, Jakub Stanislaw Nowak, Santosh Kumar
Michlewski lab website

Regulation of MicroRNA Processing and Function

MicroRNAs (miRNAs) are conserved non-coding RNAs that regulate gene expression by targeting partially complementary sequences in the mRNAs. Each miRNA potentially regulates hundreds of mRNA targets, thus controlling a variety of biological  processes, including mammalian cellular differentiation and development. In spite of widespread efforts to understand the roles of individual miRNAs little progress has been made towards unravelling the regulation of their biogenesis.

RNA-binding proteins control gene expression in all living cells by regulating all aspects of RNA biology. Because RNA binding motives are short and abundant, a typical RNA-binding protein has thousands of RNA targets. Strikingly, many RNA-binding proteins are multifunctional and, depending on their substrates, can have positive, negative or passive effects on the related molecular process. Although considerable progress has been made in cataloguing RNA-Protein  (RNP) complexes, it is very hard to predict their molecular and physiological function only based on their binary interaction.

My group has focused on elucidating the cis and trans- acting factors of tissue-specific miRNA biogenesis in mammalian cells. We have  identified proteins that regulate the production of brain-enriched and brain- specific miRNAs, as well as novel  RNA-binding protein, responsible for the selective uridylation and degradation of miRNA precursors in embryonic cells.
We have  demonstrated that the expression profile  of brain-enriched miRNA-7, which is processed from a ubiquitous  pre-mRNA transcript coding for hnRNP K protein, is achieved by inhibition of its biogenesis in non- brain  cells (Choudhury et al., 2013).  By identifying MSI2 and HuR proteins as inhibitors of miRNA-7 maturation in non-brain cells we provided the first insight into the regulation of brain-enriched miRNA processing by defined tissue-specific factors. Furthermore, we showed that brain-specific miRNA-9 is regulated transcriptionally and post-transcriptionally during neuronal differentiation (Nowak et al., 2014). We revealed that Lin28a, an RNA- binding  protein progressively switched off during differentiation, inhibits  the processing of brain-specific miRNA-9 by inducing the degradation of its precursor transcript during early stages of neuronal differentiation. Finally, we have  identified the E3 ubiquitin ligaseTrim25 as a RNA-dependent co-factor for Lin28a/TuT4- mediated uridylation of let-7 precursors (Choudhury et al., 2014). This demonstrated for the first time that a protein-modifying enzyme, recently shown to bind RNA, can guide the function of RNA-protein complexes in cis. Our findings have  far reaching consequences for our understanding of how RNA-binding proteins commit to a specific molecular function and how, through targeting miRNA biogenesis pathway, they contribute to control of gene expression mammalian cells.

Selected publications:

Choudhury, N.R., Nowak, J.S., Zou, J., Rappsilber, J., Spoel, S.H., and Michlewski, G. (2014). Trim25 Is an RNA- Specific Activator  of Lin28a/TuT4-Mediated Uridylation. Cell reports 9,

Nowak, J.S., Choudhury, N.R., de Lima Alves, F., Rappsilber, J., and Michlewski, G. (2014). Lin28a regulates neuronal differentiation and controls miR-9 production. Nature communications 5, 3687.

Choudhury, N.R., de Lima Alves, F., de Andres-Aguayo, L., Graf, T., Caceres, J.F., Rappsilber, J., and Michlewski, G. (2013). Tissue-specific control of brain- enriched miR-7 biogenesis. Genes & development 27, 24-38.

Figure 1. Structural and sequence context of the GGAG motif determine Lin28a binding and functionality. (A) Schematic of the secondary structure of wild-type and conserved terminal loop (CTL) mutants of pri-let-7a-1. The mutated nucleotides are in green and the GGAG motif is in red. (B) Western blot analysis  of Lin28a and DHX9 proteins in RNA pull-downs from day 0 (d0) P19 teratocarcinoma embryonic cell extract using wild-type pre-let-7 or its CTL mutants. (C) In vitro processing uridylation assays performed with internally radiolabeled pre-let-7a transcripts in the presence of d0 P19 cell extract. (−) represents an untreated control. Reactions were supplemented with 0.25 mM UTP. The products were analyzed on an 8% denaturing polyacrylamide gel.