Lin28-mediated control of let-7 microRNA expression by alternative TUTases Zcchc11 (TUT4) and Zcchc6 (TUT7)

Abstract

The pluripotency factor Lin28 recruits a 3' terminal uridylyl transferase (TUTase) to selectively block let-7 microRNA biogenesis in undifferentiated cells. Zcchc11 (TUTase4/TUT4) was previously identified as an enzyme responsible for Lin28-mediated pre-let-7 uridylation and control of let-7 expression. Here we investigate the protein and RNA determinants for this interaction. Biochemical dissection and reconstitution assays reveal the TUTase domains necessary and sufficient for Lin28-enhanced pre-let-7 uridylation. A single C2H2-type zinc finger domain of Zcchc11 was found to be responsible for the functional interaction with Lin28. We identify Zcchc6 (TUTase7) as an alternative TUTase that functions with Lin28 in vitro, and accordingly, we find Zcchc11 and Zcchc6 redundantly control let-7 biogenesis in embryonic stem cells. Our study indicates that Lin28 uses two different TUTases to control let-7 expression and has important implications for stem cell biology as well as cancer.

FIGURE 1.

Domains of Zcchc11 required for Lin28-mediated pre-let-7 uridylation. (A) Schematic representation of Zcchc11 and truncations used for in vitro uridylation assays. (B) Uridylation assays with synthetic pre-let-7g carried out using Flag immunopurified (IP) Zcchc11 variants and IP Lin28. α-Flag Western blots show similar amounts of IP Zcchc11 within experiments. (C) Summary of Zcchc11 domain requirements from in vitro uridylation assays. (D) Reconstitution of Lin28-enhanced pre-let-7 uridylation with recombinant proteins. Zcchc11 truncation ΔPneumoG/C purified from either HEK293T (IP) or E. coli (Recombinant) was incubated with either Flag-Lin28 (IP) or 6x-His Lin28 (r.Lin28) in a uridylation assay with synthetic pre-let-7g. (Left) Schematic representation of the domains present in ΔPneumoG/C.

FIGURE 2.

The preE of let-7 is sufficient to direct both Lin28 binding and uridylation of pre-let-7. (A) Diagram of synthetic RNAs used for in vitro uridylation assays. (Pre-let-7g) Endogenous precursor let-7g miRNA sequence; (Pre-miR-21) endogenous precursor miR-21 sequence; (Pre-7S21L) synthetic RNA consisting of the miR-21 preE and let-7g stem; (Pre-21S7L) synthetic RNA consisting of the let-7g preE and miR-21 stem sequences. (B, left) Uridylation assay as in Figure 1 using WT Flag IP-mZcchc11, with or without Flag IP-mLin28, and the indicated precursor miRNAs. (Right) 5′ end-labeled RNAs showing equal amounts. (C) (Top) Diagram of synthetic RNAs used for in vitro uridylation assays. (Bottom left) Uridylation assays and (bottom right) 5′ end-labeled RNAs showing equal amounts as in B.

FIGURE 3.

Zcchc11 and Zcchc6 have a highly similar domain organization and can both mediate Lin28-dependent pre-let-7 uridylation in vitro. (A) Schematic showing the domain similarities between hZcchc11 and hZcchc6 with the N-terminal C2H2 zinc finger highlighted and critical zinc finger residues in bold. (B) Uridylation assay with Flag-IP WT mZcchc11 and a mutant harboring point mutations in two conserved asparates required for catalysis, with or without Flag-IP Lin28. (C) Alignment of the nucleotidyl transferase (Ntr) domains of hZcchc11 and hZcchc6. Aspartic acid residues critical for catalysis are boxed. (D) α-Flag WB showing relative amounts of Flag-hZcchc11 and Flag-hZcchc6 (left) or Flag-hLin28A and Flag-hLin28B (right). (E) EMSA showing similar amounts of functional Flag-hLin28A and Flag-hLin28B used in uridylation assays. (F) Uridylation assays using Flag-hZcchc11 or Flag-hZcchc6 with either Flag-hLin28A or Flag-hLin28B. (G) Uridylation assay with Flag-hZcchc6 and r.Lin28A.

FIGURE 4.

Zcchc11 and Zcchc6 function redundantly to suppress let-7 expression in embryonic cells. (A) qRT–PCR analysis of mature let-7g and mature miR-21 levels in P19 embryonal carcinoma cells after transfection with the indicated siRNAs (left). mRNA levels of the indicated genes in P19 cells after transfection with the indicated siRNAs (right). (B) qRT–PCR as in A in V6.5 mouse embryonic stem cells. For all experiments, miRNA levels were normalized to sno-142 and mRNA levels were normalized to β-actin. Error bars represent SD of experiments in triplicate.