Dataset Information

Human Lin28 Forms a High-Affinity 1:1 Complex with the 106~363 Cluster miRNA miR-363.

ABSTRACT: Lin28A is a post-transcriptional regulator of gene expression that interacts with and negatively regulates the biogenesis of let-7 family miRNAs. Recent data suggested that Lin28A also binds the putative tumor suppressor miR-363, a member of the 106~363 cluster of miRNAs. Affinity for this miRNA and the stoichiometry of the protein-RNA complex are unknown. Characterization of human Lin28's interaction with RNA has been complicated by difficulties in producing stable RNA-free protein. We have engineered a maltose binding protein fusion with Lin28, which binds let-7 miRNA with a Kd of 54.1 ± 4.2 nM, in agreement with previous data on a murine homologue. We show that human Lin28A binds miR-363 with a 1:1 stoichiometry and with a similar, if not higher, affinity (Kd = 16.6 ± 1.9 nM). Further analysis suggests that the interaction of the N-terminal cold shock domain of Lin28A with RNA is salt-dependent, supporting a model in which the cold shock domain allows the protein to sample RNA substrates through transient electrostatic interactions.

SUBMITTER: Peters DT

PROVIDER: S-EPMC5193468 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Human Lin28 Forms a High-Affinity 1:1 Complex with the 106~363 Cluster miRNA miR-363.

Peters Daniel T DT Fung Herman K H HK Levdikov Vladimir M VM Irmscher Tobias T Warrander Fiona C FC Greive Sandra J SJ Kovalevskiy Oleg O Isaacs Harry V HV Coles Mark M Antson Alfred A AA

Biochemistry 20160901 36

Lin28A is a post-transcriptional regulator of gene expression that interacts with and negatively regulates the biogenesis of let-7 family miRNAs. Recent data suggested that Lin28A also binds the putative tumor suppressor miR-363, a member of the 106~363 cluster of miRNAs. Affinity for this miRNA and the stoichiometry of the protein-RNA complex are unknown. Characterization of human Lin28's interaction with RNA has been complicated by difficulties in producing stable RNA-free protein. We have eng ...[more]

PMID: 27559824

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Project description:MicroRNAs (miRs) have been identified as potent regulators of both normal development and the hallmarks of cancer. Targeting of microRNAs has been shown to have preclinical promise, and select miR-based therapies are now in clinical trials. Ewing Sarcoma is a biologically aggressive pediatric cancer with little change in clinical outcomes despite improved chemotherapeutic regimens. There is a substantial need for new therapies to improve Ewing Sarcoma outcomes and to prevent chemotherapy-related secondary sequelae. Most Ewing Sarcoma tumors are driven by the EWS/Fli-1 fusion oncoprotein, acting as a gain-of-function transcription factor causing dysregulation of a variety of targets, including microRNAs. Our previous studies, and those of others, have identified upregulation of miRs belonging to the related miR-17~92a, miR-106b~25, and miR-106a~363 clusters in Ewing Sarcoma. However, the functional consequences of this have not been characterized, nor has miR blockade been explored as an anti-cancer strategy in Ewing Sarcoma. To simulate a potential therapeutic approach, we examined the effects of blockade of these clusters, and their component miRs. Using colony formation as a read-out, we find that blockade of selected individual cluster component miRs, using specific inhibitors, has little or no effect. Combinatorial inhibition using miR "sponge" methodology, on the other hand, is inhibitory to colony formation, with blockade of whole clusters generally more effective than blockade of miR families. We show that a miR-blocking sponge directed against the poorly characterized miR-106a~363 cluster is a particularly potent inhibitor of clonogenic growth in a subset of Ewing Sarcoma cell lines. We further identify upregulation of miR-15a as a downstream mechanism contributing to the miR-106a~363 sponge growth-inhibitory effect. Taken together, our studies provide support for a pro-oncogenic role of the miR-106a~363 cluster in Ewing Sarcoma, and identify miR-106a~363 blockade, as well as miR-15a replacement, as possible strategies for inhibition of Ewing Sarcoma growth.

Project description:BackgroundExcessive abdominal fat deposition in commercial broilers presents an obstacle to profitable meat quality, feed utilization, and reproduction. Abdominal fat deposition depends on the proliferation of preadipocytes and their maturation into adipocytes, which involves a cascade of regulatory molecules. Accumulating evidence has shown that microRNAs (miRNAs) serve as post-transcriptional regulators of adipogenic differentiation in mammals. However, the miRNA-mediated molecular mechanisms underlying abdominal fat deposition in chickens are still poorly understood. This study aimed to investigate the biological functions and regulatory mechanism of miRNAs in chicken abdominal adipogenesis.ResultsWe established a chicken model of abdominal adipocyte differentiation and analyzed miRNA and mRNA expression in abdominal adipocytes at different stages of differentiation (0, 12, 48, 72, and 120 h). A total of 217 differentially expressed miRNAs (DE-miRNAs) and 3520 differentially expressed genes were identified. Target prediction of DE-miRNAs and functional enrichment analysis revealed that the differentially expressed targets were significantly enriched in lipid metabolism-related signaling pathways, including the PPAR signaling and MAPK signaling pathways. A candidate miRNA, gga-miR-106-5p, exhibited decreased expression during the proliferation and differentiation of abdominal preadipocytes and was downregulated in the abdominal adipose tissues of fat chickens compared to that of lean chickens. gga-miR-106-5p was found to inhibit the proliferation and adipogenic differentiation of chicken abdominal preadipocytes. A dual-luciferase reporter assay suggested that the KLF15 gene, which encodes a transcriptional factor, is a direct target of gga-miR-106-5p. gga-miR-106-5p suppressed the post-transcriptional activity of KLF15, which is an activator of abdominal preadipocyte proliferation and differentiation, as determined with gain- and loss-of-function experiments.Conclusionsgga-miR-106-5p functions as an inhibitor of abdominal adipogenesis by targeting the KLF15 gene in chickens. These findings not only improve our understanding of the specific functions of miRNAs in avian adipogenesis but also provide potential targets for the genetic improvement of excessive abdominal fat deposition in poultry.

Dataset Information

Human Lin28 Forms a High-Affinity 1:1 Complex with the 106~363 Cluster miRNA miR-363.

Publications

Human Lin28 Forms a High-Affinity 1:1 Complex with the 106~363 Cluster miRNA miR-363.

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