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

Thornton, James E.; Chang, Hung Chi; Piskounova, Elena; Gregory, Richard I.

doi:10.1261/rna.034538.112

Cited by 187 publications

(215 citation statements)

References 51 publications

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“…1A). 25 We therefore subcloned the cDNA encoding this minimal region of Zcchc11 with a FLAG tag epitope sequence at the 3 0 end into the prokaryotic expression vector pETDuet-1. After expression was induced with isopropyl b-D-1-thiogalactopyranoside (IPTG) at 20 C overnight, the recombinant Zcchc11 (rZcchc11) protein was isolated by anti-FLAG affinity purification.…”

Section: Purification and Characterization Of Recombinant Zcchc11mentioning

confidence: 99%

“…25 Immunoprecipitation and recombinant protein purification Immunoprecipitation and recombinant protein purification were performed as previously described. 25 Briefly, Flag-Lin28 expression plasmids were transfected into HEK293T cells with Lipofectamine 2000 (Invitrogen). 48 hours later, whole cell lysate was collected for the purification of Flag-Lin28 with antiFlag M2 beads (Sigma).…”

Section: Plasmidsmentioning

confidence: 99%

“…25 0.01 mM unlabeled synthetic pre-let-7a1 or mature let-7i guide RNA (Dharmacon) were incubated with purified proteins and buffer containing [a-32 P] UTP at 37 C for 1h. A limited amount of (125 nM) [a-32 P] UTP was added in the uridylation system, therefore the pre-let-7a1 or mature let-7i are predominantly monouridylated in these reaction conditions.…”

Section: In Vitro Uridylation Assaymentioning

confidence: 99%

“…[23][24][25] These enzymes are recruited by Lin28 to let-7 precursors (pre-let-7) where they catalyze the addition of an oligo(U)-tail. Uridylated pre-let-7 is resistant to Dicer processing and degraded by the 3 0 -5 0 exonuclease Dis3l2.…”

Section: Introductionmentioning

confidence: 99%

“…Uridylated pre-let-7 is resistant to Dicer processing and degraded by the 3 0 -5 0 exonuclease Dis3l2. [23][24][25][26][27] TUTase activity is required for the Lin28-mediated inhibition of let-7 biogenesis, and RNAi-mediated knockdown of Zcchc11 has been shown to inhibit tumorigenesis of human breast, ovarian, melanoma, prostate, and liver cancer cells in mouse xenograft assays. 28,29 Moreover, Zcchc11 knockdown inhibited the lung metastasis of liver cancer cells.…”

Section: Introductionmentioning

confidence: 99%

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Identification of small molecule inhibitors of Zcchc11 TUTase activity

Lin

Gregory

2015

RNA Biology

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The RNA-binding protein Lin28 regulates the expression of the let-7 family of microRNAs (miRNAs) during early embryonic development. Lin28 recruits the 3 0 terminal uridylyl transferase (TUTase) Zcchc11 (TUT4) and/or Zcchc6 (TUT7) to precursor let-7 RNA (pre-let-7) to selectively block let-7 biogenesis. Uridylated pre-let-7 is targeted for decay by the downstream exonuclease Dis3l2 thereby preventing processing to mature let-7. Activation of this oncogenic pathway via up-regulation of Lin28 expression promotes cellular transformation, drives tumorigenesis in mouse models, and is frequently observed in a wide variety of cancer. Recent proof-of-principle experiments showed that Zcchc11 knockdown inhibits the tumorigenicity of Lin28-expressing human cancer cells and established this enzyme as a possible new therapeutic target for human malignancies. However, there are currently no known pharmacological agents capable of targeting this novel enzyme. In this study we developed and applied a sensitive biochemical assay that monitors Zcchc11 activity. Using this assay we performed an automated high-throughput screen of »15,000 chemicals to identify putative TUTase inhibitors. Several of these small molecules were validated as specific inhibitors of Zcchc11 activity. Our results demonstrate the feasibility of screening for TUTase inhibitors and present a relatively simple platform that can be exploited for future drug discovery efforts aimed at restoring let-7 expression in cancer.

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Section: Purification and Characterization Of Recombinant Zcchc11mentioning

confidence: 99%

Section: Plasmidsmentioning

confidence: 99%

Section: In Vitro Uridylation Assaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Identification of small molecule inhibitors of Zcchc11 TUTase activity

Lin

Gregory

2015

RNA Biology

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The role of Lin28A and Lin28B in cancer beyond Let‐7

Cotino‐Nájera,

García‐Villa,

Cruz‐Rosales

et al. 2024

FEBS Letters

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Lin28A and Lin28B are paralogous RNA‐binding proteins that play fundamental roles in development and cancer by regulating the microRNA family of tumor suppressor Let‐7. Although Lin28A and Lin28B share some functional similarities with Let‐7 inhibitors, they also have distinct expression patterns and biological functions. Increasing evidence indicates that Lin28A and Lin28B differentially impact cancer stem cell properties, epithelial‐mesenchymal transition, metabolic reprogramming, and other hallmarks of cancer. Therefore, it is important to understand the overexpression of Lin28A and Lin28B paralogs in specific cancer contexts. In this review, we summarize the main similarities and differences between Lin28A and Lin28B, their implications in different cellular processes, and their role in different types of cancer. In addition, we provide evidence of other specific targets of each lin28 paralog, as well as the lncRNAs and miRNAs that promote or inhibit its expression, and how this impacts cancer development and progression.

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SUMOylation modulates the LIN28A‐let‐7 signaling pathway in response to cellular stresses in cancer cells

et al. 2020

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LIN28A is a conserved RNA‐binding protein that inhibits the biogenesis of let‐7 microRNAs, thus promoting cancer progression. However, mechanisms underlying the activation of the LIN28A‐let‐7 signaling pathway remain poorly understood. Here, we show that LIN28A is SUMOylated in vivo and in vitro at K15, which is increased by hypoxia but reduced by chemotherapy drugs such as Cisplatin and Paclitaxel. SUMOylation of LIN28A aggravates its inhibition of let‐7 maturation, resulting in a stark reduction in let‐7, which promotes cancer cell proliferation, migration, invasion, and tumor growth in vivo. Mechanistically, SUMOylation of LIN28A increases its binding affinity with the precursor let‐7 (pre‐let‐7), which subsequently enhances LIN28A‐mediated recruitment of terminal uridylyltransferase TUT4 and simultaneously blocks DICER processing of pre‐let‐7, thereby reducing mature let‐7 production. These effects are abolished in SUMOylation‐deficient mutant LIN28A‐K15R. In summary, these findings shed light on a novel mechanism by which SUMOylation could regulate the LIN28A‐let‐7 pathway in response to cellular stress in cancer cells.

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

Cited by 187 publications

References 51 publications

Identification of small molecule inhibitors of Zcchc11 TUTase activity

Identification of small molecule inhibitors of Zcchc11 TUTase activity

The role of Lin28A and Lin28B in cancer beyond Let‐7

SUMOylation modulates the LIN28A‐let‐7 signaling pathway in response to cellular stresses in cancer cells

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