Abstract
Recent small RNA sequencing data has uncovered 3′ end modification of mature microRNAs (miRNAs). This non-templated nucleotide addition can impact miRNA gene regulatory networks through the control of miRNA stability or by interfering with the repression of target mRNAs. The miRNA modifying enzymes responsible for this regulation remain largely uncharacterized. Here we describe the ability for two related terminal uridyl transferases (TUTases), Zcchc6 (TUT7) and Zcchc11 (TUT4), to 3′ mono-uridylate a specific subset of miRNAs involved in cell differentiation and Homeobox (Hox) gene control. Zcchc6/11 selectively uridylates these miRNAs in vitro, and we biochemically define a bipartite sequence motif that is necessary and sufficient to confer Zcchc6/11 catalyzed uridylation. Depletion of these TUTases in cultured cells causes the selective loss of 3′ mono-uridylation of many of the same miRNAs. Upon TUTase-dependent loss of uridylation, we observe a concomitant increase in non-templated 3′ mono-adenylation. Furthermore, TUTase inhibition in Zebrafish embryos causes developmental defects and aberrant Hox expression. Our results uncover the molecular basis for selective miRNA mono-uridylation by Zcchc6/11, highlight the precise control of different 3′ miRNA modifications in cells and have implications for miRNA and Hox gene regulation during development.
Highlights
MicroRNAs are small non-coding RNAs that negatively regulate gene expression by binding to complementary sites in the 3 untranslated regions (3 UTRs) of messenger RNAs and induce gene silencing via the RNA-induced silencing complex (RISC) [1]
To further understand the role of Zcchc11 and Zcchc6 mature miRNA uridylation, we investigated whether these two terminal uridyl transferases (TUTases) have an inherent preference toward various miRNA substrates and found that a simple sequence motif exists that is necessary and sufficient for targeting by Zcchc11 and Zcchc6 in vitro
Consistent with the preferential uridylation activity we observed, we found that purified Zcchc11 preferentially associates with let-7 guide RNA in an electromobility shift assay (EMSA) (Figure 4g)
Summary
MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression by binding to complementary sites in the 3 untranslated regions (3 UTRs) of messenger RNAs (mRNAs) and induce gene silencing via the RNA-induced silencing complex (RISC) [1]. While the canonical pathway of miRNA biogenesis is largely understood, there are numerous modifications made to nascent miRNA species that impact their expression and function [2,3]. Many of these modifications are appreciated in their ability to enhance or disrupt miRNA processing and repressive ability, while others are only recently being described and characterized [3,4,5]. Given that much of this activity is restricted to certain cellular contexts, the selective mechanisms of mature miRNA modification may play important roles in development and tissue specification
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