Selective microRNA uridylation by Zcchc6 (TUT7) and Zcchc11 (TUT4)

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.

Identification of small molecule inhibitors of Zcchc11 TUTase activity

“…Since TUTase activity is required for the Lin28 mediated repression of let-7 biogenesis, and is likely to be highly "druggable," targeting TUTase activity might represent a promising strategy to inhibit the Lin28 oncogenic pathway in cancers. In addition, the TUTases were recently reported to uridylate miRNA and mRNA independent of Lin28, 30,46,47 suggesting that TUTases are significant players in regulating posttranscriptional gene expression through uridylation. Therefore, potent TUTase inhibitors might not only be used for drug development against the Lin28 oncogenic pathway, but could also be used to study uridylation-mediated RNA decay.…”

“…30 We furthermore experimentally defined a sequence motif present in a small subset of mature miRNAs that confers this preferential uridylation activity. 30 Consistent with these findings, rZcchc11 was capable of uridylating mature let-7 RNA in vitro (Fig. 1E,F).…”

Literature review of baseline information to support the risk assessment of RNAi‐based GM plants

“…Data from zebrafish also contributed to understanding other miRNA regulations. Two related terminal uridyl transferases (TUTases), Zcchc6 (TUT7) and Zcchc11 (TUT4), selectively 3' monouridylate a subset of miRNAs (Thornton et al, 2014). TUTase inhibition in zebrafish embryos causes developmental defects and aberrant Hox gene expression The present document has been produced and adopted by the bodies identified above as authors.…”

“…( Thornton et al, 2014). Another miRNA regulator discovered in the zebrafish is dead end 1 (DND1), which is negatively regulating miRNA targeting.…”

Nucleotide specificity of the human terminal nucleotidyltransferase Gld2 (TUT2)

“…These untemplated nucleotide additions are an efficient means to control the levels of active miRNAs in the cell. The seemingly innocuous addition of a single nucleotide can initiate miRNA maturation, stabilization, or convert an active miRNA to an inactive form (Thornton et al 2014). While multiple adenine residues are added to mRNA for stabilization, extending the transcript life span (Norbury 2013), multiple uridine residues mark both miRNA and mRNA for degradation (Mullen and Marzluff 2008;Rissland and Norbury 2009;Lim et al 2014).…”