We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 4

1.
Figure 2

Figure 2. Zcchc11 TUTase activity is Lin28-dependent. From: Lin28 recruits the TUTase Zcchc11 to inhibit let-7 maturation in embryonic stem cells.

(a) Uridylation assays performed with pre-let-7g in the presence of unlabelled UTP, ATP, CTP or GTP. (b) TUTase assay performed with pre-let-7a with a catalytically inactive Zcchc11 protein. A mutant Zcchc11 protein with two mutated aspartate residues (D1008A, D1010A in the NTr) was used in the uridylation assay. c, Uridylation assay with synthetic pre-miR-21 as a substrate in the uridylation assay as in Fig 1(c).

John P. Hagan, et al. Nat Struct Mol Biol. ;16(10):1021-1025.
2.
Figure 3

Figure 3. Inhibition of Zcchc11 selectively elevates mature let-7 levels in embryonic cells. From: Lin28 recruits the TUTase Zcchc11 to inhibit let-7 maturation in embryonic stem cells.

(a) Real-time PCR analysis of mRNA and miRNA levels in response to the indicated siRNA transfection of mouse P19 cells. Data is plotted as −ΔΔCt. Relative fold change equals 2(−ΔΔCt). Positive and negative −ΔΔCt values reflect upregulation and loss, respectively. (b) Knockdown of Zcchc11 in mouse ES cells. Depicted errors bars are ±s.d. (c) Mature microRNA levels upon HA-Zcchc11 and HA-mutant Zcchc11 overexpression in P19 cells. Presented p-values are based on comparisons between control siRNA versus siRNA set for a given gene knockdown. Depicted errors bars are ±s.d.

John P. Hagan, et al. Nat Struct Mol Biol. ;16(10):1021-1025.
3.
Figure 1

Figure 1. Identification of Zcchc11 as a candidate pre-let-7 TUTase. From: Lin28 recruits the TUTase Zcchc11 to inhibit let-7 maturation in embryonic stem cells.

(a) Diagrammatic representation of mouse Zcchc11 protein, Protein accession number (NCBI): AAI50792.1. Poly(A) Polymerase (PAP) domains are depicted in light gray, Zinc finger CCHC domains are in black, Nucleotidyltransferase (NTr) domain is in dark gray. aa, amino acids. (b) EMSA performed using radiolabeled pre-let-7g. Both recombinant Lin28 (5 μM) and Flag-Lin28 immunoprecipitated from ES cells were analyzed for ribonucleocomplex formation with pre-let-7g. (c) In vitro pre-let-7 uridylation assay. Synthetic pre-let-7a-1 was incubated with Flag-Lin28, HA-Zcchc11, r.Lin28, or a combination of Lin28 and Zcchc11 proteins. rPAP protein (8 units) was used as a positive control.

John P. Hagan, et al. Nat Struct Mol Biol. ;16(10):1021-1025.
4.
Figure 4

Figure 4. Zcchc11 inhibition exclusively affects let-7 biogenesis and leads to repression of let-7 target gene expression. From: Lin28 recruits the TUTase Zcchc11 to inhibit let-7 maturation in embryonic stem cells.

(a) The TaqMan Rodent MicroRNA A Array v2.0 was used to examine global miRNA levels. Independent siRNA sets were used as indicated in the axis legends and data plotted as −ΔΔCt. Left and right panels depict the results for Lin28 and Zcchc11, respectively. For each panel, the upper right quadrant contains those miRNAs that are reproducibly upregulated by both siRNA knockdown sets. Depicted errors bars are ±s.e.m. (b) Luciferase reporter assays to evaluate the in vivo relevance of elevated mature let-7 levels. Renilla luciferase lacked (termed “Absent”), contained an imperfect, or had a perfect let-7 binding site in its 3′UTR. Depicted errors bars are ±s.d.

John P. Hagan, et al. Nat Struct Mol Biol. ;16(10):1021-1025.

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk