Stepwise assembly of multiple Lin28 proteins on the terminal loop of let-7 miRNA precursors

Lin28 inhibits the biogenesis of let-7 miRNAs through direct interactions with let-7 precursors. Previous studies have described seemingly inconsistent Lin28 binding sites on pre-let-7 RNAs. Here, we reconcile these data by examining the binding mechanism of Lin28 to the terminal loop of pre-let-7g (TL-let-7g) using biochemical and biophysical methods. First, we investigate Lin28 binding to TL-let-7g variants and short RNA fragments and identify three independent binding sites for Lin28 on TL-let-7g. We then determine that Lin28 assembles in a stepwise manner on TL-let-7g to form a stable 1:3 complex. We show that the cold-shock domain (CSD) of Lin28 is responsible for remodelling the terminal loop of TL-let-7g, whereas the NCp7-like domain facilitates the initial binding of Lin28 to TL-let-7g. This stable binding of multiple Lin28 molecules to the terminal loop of pre-let-7g extends to other precursors of the let-7 family, but not to other pre-miRNAs tested. We propose a model for stepwise assembly of the 1:1, 1:2 and 1:3 pre-let-7g/Lin28 complexes. Stepwise multimerization of Lin28 on pre-let-7 is required for maximum inhibition of Dicer cleavage for a least one member of the let-7 family and may be important for orchestrating the activity of the several factors that regulate let-7 biogenesis.     

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

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.     

Short loop-targeting oligoribonucleotides antagonize Lin28 and enable pre-let-7 processing and suppression of cell growth in let-7-deficient cancer cells

MicroRNAs (miRNAs) originate from stem-loop-containing precursors (pre-miRNAs, pri-miRNAs) and mature by means of the Drosha and Dicer endonucleases and their associated factors. The let-7 miRNAs have prominent roles in developmental differentiation and in regulating cell proliferation. In cancer, the tumor suppressor function of let-7 is abrogated by overexpression of Lin28, one of several RNA-binding proteins that regulate let-7 biogenesis by interacting with conserved motifs in let-7 precursors close to the Dicer cleavage site. Using in vitro assays, we have identified a binding site for short modified oligoribonucleotides (‘looptomirs’) overlapping that of Lin28 in pre-let-7a-2. These looptomirs selectively antagonize the docking of Lin28, but still permit processing of pre-let-7a-2 by Dicer. Looptomirs restored synthesis of mature let-7 and inhibited growth and clonogenic potential in Lin28 overexpressing hepatocarcinoma cells, thereby demonstrating a promising new means to rescue defective miRNA biogenesis in Lin28-dependent cancers.     

Importance of the NCp7-like domain in the recognition of pre-let-7g by the pluripotency factor Lin28

The pluripotency factor Lin28 is a highly conserved protein comprising a unique combination of RNA-binding motifs, an N-terminal cold-shock domain and a C-terminal region containing two retroviral-type CCHC zinc-binding domains. An important function of Lin28 is to inhibit the biogenesis of the let-7 family of microRNAs through a direct interaction with let-7 precursors. Here, we systematically characterize the determinants of the interaction between Lin28 and pre-let-7 g by investigating the effect of protein and RNA mutations on in vitro binding. We determine that Lin28 binds with high affinity to the extended loop of pre-let-7 g and that its C-terminal domain contributes predominantly to the affinity of this interaction. We uncover remarkable similarities between this C-terminal domain and the NCp7 protein of HIV-1, not only in terms of primary structure but also in their modes of RNA binding. This NCp7-like domain of Lin28 recognizes a G-rich bulge within pre-let-7 g, which is adjacent to one of the Dicer cleavage sites. We hypothesize that the NCp7-like domain initiates RNA binding and partially unfolds the RNA. This partial unfolding would then enable multiple copies of Lin28 to bind the extended loop of pre-let-7 g and protect the RNA from cleavage by the pre-microRNA processing enzyme Dicer.     

An RNA-binding Protein,Lin28, Recognizes and Remodels G-quartets in the MicroRNAs (miRNAs) and mRNAs It Regulates

Lin28 is an evolutionarily conserved RNA-binding protein that inhibits processing of pre-let-7 microRNAs (miRNAs) and regulates translation of mRNAs that control developmental timing, pluripotency, metabolism, and tumorigenesis. The RNA features that mediate Lin28 binding to the terminal loops of let-7 pre-miRNAs and to Lin28-responsive elements (LREs) in mRNAs are not well defined. Here we show that Lin28 target datasets are enriched for RNA sequences predicted to contain stable planar structures of 4 guanines known as G-quartets (G4s). The imino NMR spectra of pre-let-7 loops and LREs contain resonances characteristic of G4 hydrogen bonds. These sequences bind to a G4-binding fluorescent dye, N-methyl-mesoporphyrin IX (NMM). Mutations and truncations in the RNA sequence that prevent G4 formation also prevent Lin28 binding. The addition of Lin28 to a pre-let-7 loop or an LRE reduces G4 resonance intensity and NMM binding, suggesting that Lin28 may function to remodel G4s. Further, we show that NMM inhibits Lin28 binding. Incubation of a human embryonal carcinoma cell line with NMM reduces its stem cell traits. In particular it increases mature let-7 levels, decreases OCT4, HMGA1, CCNB1, CDK4, and Lin28A protein, decreases sphere formation, and inhibits colony formation. Our results suggest a previously unknown structural feature of Lin28 targets and a new strategy for manipulating Lin28 function.     

The Lin28 cold-shock domain remodels pre-let-7 microRNA

The RNA-binding protein Lin28 regulates the processing of a developmentally important group of microRNAs, the let-7 family. Lin28 blocks the biogenesis of let-7 in embryonic stem cells and thereby prevents differentiation. It was shown that both RNA-binding domains (RBDs) of this protein, the cold-shock domain (CSD) and the zinc-knuckle domain (ZKD) are indispensable for pri- or pre-let-7 binding and blocking its maturation. Here, we systematically examined the nucleic acid-binding preferences of the Lin28 RBDs and determined the crystal structure of the Lin28 CSD in the absence and presence of nucleic acids. Both RNA-binding domains bind to single-stranded nucleic acids with the ZKD mediating specific binding to a conserved GGAG motif and the CSD showing only limited sequence specificity. However, only the isolated Lin28 CSD, but not the ZKD, can bind with a reasonable affinity to pre-let-7 and thus is able to remodel the terminal loop of pre-let-7 including the Dicer cleavage site. Further mutagenesis studies reveal that the Lin28 CSD induces a conformational change in the terminal loop of pre-let-7 and thereby facilitates a subsequent specific binding of the Lin28 ZKD to the conserved GGAG motif.     

Role of ribosomal protein RPS2 in controlling let-7a expression in human prostate cancer

We discovered that an inverse relationship exists in the expression of ras/c-myc and ribosomal protein RPS2 with pre-let-7a-1/let-7a/let-7f miRNA and prostate tumor cell malignancy. Nonmalignant IBC-10a cells expressed low levels of ras/RPS2 and elevated pre-let-7a-1/let-7a/let-7f miRNA, whereas the reverse occurred in malignant PCa-20a and PC-3ML cells. Stable transfection of IBC-10a cells with pBABE.ras and pBABE.RPS2 induced ras, c-myc, and RPS2 expression, whereas the levels of let-7a/let-7f miRNA dropped to near zero. Conversely, in pBABE.pre-let-7a-1 transfected PCa-20a and PC-3ML clones, let-7a/let-7f increased whereas ras, RPS2, and c-myc dropped greater than 5-fold. Electrophoretic mobility shift assays, antibody "supershift" assays and immunoprecipitation assays revealed that RPS2 specifically binds pre-let-7a-1 to block RNA processing. Immunoflourescent studies and Northern blots confirmed that RPS2 complexes with pre-let-7a-1 (i.e., in episomal structures) to block processing to let-7a/let-7f, indicating RPS2 may prevent let-7a miRNA expression to indirectly promote oncogene expression. Functional studies further showed that the colony-forming ability (CFA) and invasive activities of IBC-10a cells were significantly enhanced in pBABE-ras.IBC-10a and pBABE-RPS2-IBC-10a clones. Conversely, with the "knockdown" of ras and RPS2 in malignant PC-3ML cells (i.e., in pLKO.TRC.shRNA.ras.PC3-ML, pLKO.TRC.shRNA.RPS2.PC-3ML transfected cells), there was both a loss of these functions and a loss of tumorigenesis in SCID mice. Likewise, with the overexpression of let-7a/let-7f in pBABE.pre-let-7a-1.PC-3ML clones (and PCa-20a clones), CFAs, invasive activities in vitro, and tumorigenesis in vivo were significantly reduced. These results show for the first time that RPS2 blocks pre-let-7a-1 processing to enable ras and c-myc expression and the transformation of primary tumor cells.     

Lin28 Induces Epithelial-to-Mesenchymal Transition and Stemness via Downregulation of Let-7a in Breast Cancer Cells

The RNA-binding protein Lin28 is known to promote malignancy by inhibiting the biogenesis of let-7, which functions as a tumor suppressor. However, the role of the Lin28/let-7 axis in the epithelial-to-mesenchymal transition (EMT) and stemness in breast cancer has not been clearly expatiated. In our previous study, we demonstrated that let-7 regulates self-renewal and tumorigenicity of breast cancer stem cells. In the present study, we demonstrated that Lin28 was highly expressed in mesenchymal (M) type cells (MDA-MB-231 and SK-3rd), but it was barely detectable in epithelial (E) type cells (MCF-7 and BT-474). Lin28 remarkably induced the EMT, increased a higher mammosphere formation rate and ALDH activity and subsequently promoted colony formation, as well as adhesion and migration in breast cancer cells. Furthermore, we demonstrated that Lin28 induced EMT in breast cancer cells via downregulation of let-7a. Strikingly, Lin28 overexpression was found in breast cancers that had undergone metastasis and was strongly predictive of poor prognoses in breast cancers. Given that Lin28 induced the EMT via let-7a and promoted breast cancer metastasis, Lin28 may be a therapeutic target for the eradication of breast cancer metastasis.     

Post-transcriptional fine-tuning of COP9 signalosome subunit biosynthesis is regulated by the c-Myc/Lin28B/let-7 pathway

The COP9 signalosome (CSN) complex controls protein degradation via the ubiquitin (Ub) proteasome system (UPS) in eukaryotes. In mammalian cells, the multimeric CSN is composed of eight subunits (CSN1 - CSN8). It regulates cullin-RING Ub ligases (CRLs), which target essential regulatory proteins for ubiquitination and subsequent degradation. Thereby, the CSN cooperates with the UPS in a variety of essential cellular functions, including DNA repair, cell cycle and differentiation. Although functions of the CSN have been elucidated, mechanisms and regulatory principles of its de novo formation are completely unknown. Here, we show that there is a fundamental mechanism that allows a coordinated expression of all CSN subunits, a prerequisite for CSN assembly. CSN subunit mRNAs are targets of miRNAs of the let-7 family suppressing CSN subunit expression in human cells. Factors that reduce or block let-7 miRNAs induce the coordinated expression of CSN subunits. For instance, over-expression of CSN1 specifically traps let-7a-1 miRNA and elevates CSN subunit levels by two- to fourfold in a coordinated manner. CSN subunit expression is also increased by specific miRNA inhibitors or by interferon (IFN)-mediated induction of STAT1 and c-Myc reducing levels of let-7 miRNAs. Activation of STAT1 by IFNα or IFNγ induces c-Myc, which increases CSN subunit expression via the Lin28B/let-7 regulatory pathway. By contrast, a let-7a-1 mimic reduces CSN subunit expression. Our data show that let-7 miRNAs control the fine-tuning and coordinated expression of subunits for CSN de novo formation, presumably a general regulatory principle for other Zomes complexes as well.     

过表达Lin28a/Lin28b对let-7家族活性的影响

研究在HeLaS3细胞中过表达Lin28a/Lin28b对let-7家族miRNA表达水平和活性的影响。首先,构建Lin28a和Lin28b的表达载体pAAV2neo-Lin28a和pAAV2neo-Lin28b,分别转染HeLaS3细胞并筛选获得Lin28a和Lin28b的稳定表达细胞株HeLaS3/pAAV2neo-Lin28a和HeLaS3/pAAV2neo-Lin28b。然后,以pAAV2neo-Gluc-(Fluc)为基本骨架,构建并获得检测let-7家族miRNA活性的8种质粒型载体,并包装为相应的重组腺相关病毒(Recombinant adeno-associated virus,rAAV),作为检测miRNA靶序列介导的转录后抑制活性的传感器,命名为Asensor。在此基础上,以HeLaS3细胞为对照,用Western blot检测HeLaS3/pAAV2neo-Lin28a和HeLaS3/pAAV2neo-Lin28b细胞中Lin28a和Lin28b表达水平,用QRT-PCR测定let-7家族各成员表达水平,用Asensor检测let-7家族各成员活性。Western blot结果显示,HeLaS3/pAAV2neo-Lin28a和HeLaS3/pAAV2neo-Lin28b均能有效地表达Lin28a和Lin28b蛋白;QRT-PCR检测结果显示,相比于HeLaS3细胞,HeLaS3/pAAV2neo-Lin28a细胞中let-7家族各成员表达水平都下降(除let-7e外),但不同成员下降幅度存在差异;Asensor检测结果显示,let-7家族所有成员活性水平都下降,但不同成员下降幅度也存在差异,且同一成员活性水平与表达水平及其下降趋势也不一致。相比于HeLaS3细胞,HeLaS3/pAAV2neo-Lin28b细胞中let-7家族成员的表达和活性水平均明显下降,但表达水平的下降幅度比HeLaS3/pAAV2neo-Lin28a细胞大,而活性的下降幅度却与之相近。本研究建立了一种检测和比较miRNA靶序列介导的转录后抑制活性的方法,首次研究了过表达Lin28a和Lin28b对细胞中的let-7家族miRNA活性影响,并发现Lin28a和Lin28b对let-7家族miRNA表达水平的影响和对其相应活性的影响不一致性,说明在检测miRNA表达水平的同时检测miRNA活性能更全面揭示miRNA的调节功能,为进一步研究let-7家族的调控机制奠定了基础。     

A LIN28-dependent structural change in pre-let-7g directly inhibits dicer processing

Several recent studies have provided evidence that LIN28, a cytoplasmic RNA-binding protein, inhibits the biogenesis of members of the let-7 microRNA family at the Dicer step in both mammals and Caenorhabditis elegans. However, the precise mechanism of inhibition is still poorly understood. Here we report on an in vitro study, which combined RNase footprinting, gel shift binding assays, and processing assays, to investigate the molecular basis and function of the interaction between the native let-7g precursor (pre-let-7g) and LIN28. We have mapped the structure of pre-let-7g and identified some regions of the terminal loop of pre-let-7g that physically interact with LIN28. We have also identified a conformational change upon LIN28 binding that results in the unwinding of an otherwise double-stranded region at the Dicer processing site of pre-let-7g. Furthermore, we showed that a mutant pre-let-7g that displays an open upper stem inhibited pre-let-7g Dicer processing to the same extent as LIN28. The data support a mechanism by which LIN28 can directly inhibit let-7g biogenesis at the Dicer processing step.     

The Lin28/let-7 axis regulates glucose metabolism

The let-7 tumor suppressor microRNAs are known for their regulation of oncogenes, while the RNA-binding proteins Lin28a/b promote malignancy by inhibiting let-7 biogenesis. We have uncovered unexpected roles for the Lin28/let-7 pathway in regulating metabolism. When overexpressed in mice, both Lin28a and LIN28B promote an insulin-sensitized state that resists high-fat-diet induced diabetes. Conversely, muscle-specific loss of Lin28a or overexpression of let-7 results in insulin resistance and impaired glucose tolerance. These phenomena occur, in part, through the let-7-mediated repression of multiple components of the insulin-PI3K-mTOR pathway, including IGF1R, INSR, and IRS2. In addition, the mTOR inhibitor, rapamycin, abrogates Lin28a-mediated insulin sensitivity and enhanced glucose uptake. Moreover, let-7 targets are enriched for genes containing SNPs associated with type 2 diabetes and control of fasting glucose in human genome-wide association studies. These data establish the Lin28/let-7 pathway as a central regulator of mammalian glucose metabolism.     

Ectopic over-expression of tristetraprolin in human cancer cells promotes biogenesis of let-7 by down-regulation of Lin28

Tristetraprolin (TTP) is a AU-rich element (ARE) binding protein and exhibits suppressive effects on cell growth through down-regulation of ARE-containing oncogenes. The let-7 microRNA has emerged as a significant factor in tumor suppression. Both TTP and let-7 are often repressed in human cancers, thereby promoting oncogenesis by derepressing their target genes. In this work, an unexpected link between TTP and let-7 has been found in human cancer cells. TTP promotes an increase in expression of mature let-7, which leads to the inhibition of let-7 target gene CDC34 expression and suppresses cell growth. This event is associated with TTP-mediated inhibition of Lin28, which has emerged as a negative modulator of let-7. Lin28 mRNA contains ARE within its 3'-UTR and TTP enhances the decay of Lin28 mRNA through binding to its 3'-UTR. This suggests that the TTP-mediated down-regulation of Lin28 plays a key role in let-7 miRNA biogenesis in cancer cells.