A small RNA makes a Bic difference

Highly specific gene knockouts in mice for the microRNA miR155 show multiple defects in adaptive immunity

Differential Regulation of the Melanoma Proteome by eIF4A1 and eIF4E.

Small molecules and antisense oligonucleotides that inhibit the translation initiation factors eIF4A1 and eIF4E have been explored as broad-based therapeutic agents for cancer treatment, based on the frequent upregulation of these two subunits of the eIF4F cap-binding complex in many cancer cells. Here, we provide support for these therapeutic approaches with mechanistic studies of eIF4F-driven tumor progression in a preclinical model of melanoma. Silencing eIF4A1 or eIF4E decreases melanoma proliferation and invasion. There were common effects on the level of cell-cycle proteins that could explain the antiproliferative effects in vitro Using clinical specimens, we correlate the common cell-cycle targets of eIF4A1 and eIF4E with patient survival. Finally, comparative proteomic and transcriptomic analyses reveal extensive mechanistic divergence in response to eIF4A1 or eIF4E silencing. Current models indicate that eIF4A1 and eIF4E function together through the 5'UTR to increase translation of oncogenes. In contrast, our data demonstrate that the common effects of eIF4A1 and eIF4E on translation are mediated by the coding region and 3'UTR. Moreover, their divergent effects occur through the 5'UTR. Overall, our work shows that it will be important to evaluate subunit-specific inhibitors of eIF4F in different disease contexts to fully understand their anticancer actions. Cancer Res; 77(3); 613-22. ©2016 AACR

RATA: A method for high-throughput identification of RNA bound transcription factors.

Long non-coding RNAs (lncRNAs) regulate critical cellular processes and their dysregulation contributes to multiple diseases. Although only a few lncRNAs have defined mechanisms, many of these characterized lncRNAs interact with transcription factors to regulate gene expression, suggesting a common mechanism of action. Identifying RNA-bound transcription factors is especially challenging due to inefficient RNA immunoprecipitation and low abundance of many transcription factors. Here we describe a highly sensitive, user-friendly, and inexpensive technique called RATA (RNA-associated transcription factor array), which utilizes a MS2-aptamer pulldown strategy coupled with transcription factor activation arrays for identification of transcription factors associated with a nuclear RNA of interest. RATA requires only ~5 million cells and standard molecular biology reagents for multiplexed identification of up to 96 transcription factors in 2-3 d. Thus, RATA offers significant advantages over other technologies for analysis of RNA-transcription factor interactions

The TAL1 complex targets the FBXW7 tumor suppressor by activating miR-223 in human T cell acute lymphoblastic leukemia

The oncogenic transcription factor TAL1/SCL is aberrantly expressed in 60% of cases of human T cell acute lymphoblastic leukemia (T-ALL) and initiates T-ALL in mouse models. By performing global microRNA (miRNA) expression profiling after depletion of TAL1, together with genome-wide analysis of TAL1 occupancy by chromatin immunoprecipitation coupled to massively parallel DNA sequencing, we identified the miRNA genes directly controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. The most dynamically regulated miRNA was miR-223, which is bound at its promoter and up-regulated by the TAL1 complex. miR-223 expression mirrors TAL1 levels during thymic development, with high expression in early thymocytes and marked down-regulation after the double-negative-2 stage of maturation. We demonstrate that aberrant miR-223 up-regulation by TAL1 is important for optimal growth of TAL1-positive T-ALL cells and that sustained expression of miR-223 partially rescues T-ALL cells after TAL1 knockdown. Overexpression of miR-223 also leads to marked down-regulation of FBXW7 protein expression, whereas knockdown of TAL1 leads to up-regulation of FBXW7 protein levels, with a marked reduction of its substrates MYC, MYB, NOTCH1, and CYCLIN E. We conclude that TAL1-mediated up-regulation of miR-223 promotes the malignant phenotype in T-ALL through repression of the FBXW7 tumor suppressor

Repression of tumor suppressor miR-451 is essential for NOTCH1-induced oncogenesis in T-ALL

miR-451 represses expression of Myc and acts as a tumor suppressor in murine and human T cell acute lymphoblastic leukemia

The TAL1 complex targets the FBXW7 tumor suppressor by activating miR-223 in human T cell acute lymphoblastic leukemia

The oncogenic transcription factor TAL1/SCL is aberrantly expressed in 60% of cases of human T cell acute lymphoblastic leukemia (T-ALL) and initiates T-ALL in mouse models. By performing global microRNA (miRNA) expression profiling after depletion of TAL1, together with genome-wide analysis of TAL1 occupancy by chromatin immunoprecipitation coupled to massively parallel DNA sequencing, we identified the miRNA genes directly controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. The most dynamically regulated miRNA was miR-223, which is bound at its promoter and up-regulated by the TAL1 complex. miR-223 expression mirrors TAL1 levels during thymic development, with high expression in early thymocytes and marked down-regulation after the double-negative-2 stage of maturation. We demonstrate that aberrant miR-223 up-regulation by TAL1 is important for optimal growth of TAL1-positive T-ALL cells and that sustained expression of miR-223 partially rescues T-ALL cells after TAL1 knockdown. Overexpression of miR-223 also leads to marked down-regulation of FBXW7 protein expression, whereas knockdown of TAL1 leads to up-regulation of FBXW7 protein levels, with a marked reduction of its substrates MYC, MYB, NOTCH1, and CYCLIN E. We conclude that TAL1-mediated up-regulation of miR-223 promotes the malignant phenotype in T-ALL through repression of the FBXW7 tumor suppressor.National Cancer Institute (U.S.) (5P01CA109901)National Cancer Institute (U.S.) (5P01CA68484)National Cancer Institute (U.S.) (1K99CA157951)National Institutes of Health (U.S.). Intramural Research ProgramCenter for Cancer Research (National Cancer Institute (U.S.)

Feed-Forward Microprocessing and Splicing Activities at a MicroRNA–Containing Intron

The majority of mammalian microRNA (miRNA) genes reside within introns of protein-encoding and non-coding genes, yet the mechanisms coordinating primary transcript processing into both mature miRNA and spliced mRNA are poorly understood. Analysis of melanoma invasion suppressor miR-211 expressed from intron 6 of melastatin revealed that microprocessing of miR-211 promotes splicing of the exon 6–exon 7 junction of melastatin by a mechanism requiring the RNase III activity of Drosha. Additionally, mutations in the 5′ splice site (5′SS), but not in the 3′SS, branch point, or polypyrimidine tract of intron 6 reduced miR-211 biogenesis and Drosha recruitment to intron 6, indicating that 5′SS recognition by the spliceosome promotes microprocessing of miR-211. Globally, knockdown of U1 splicing factors reduced intronic miRNA expression. Our data demonstrate novel mutually-cooperative microprocessing and splicing activities at an intronic miRNA locus and suggest that the initiation of spliceosome assembly may promote microprocessing of intronic miRNAs

MicroRNA Expression Profiling Identifies Activated B Cell Status in Chronic Lymphocytic Leukemia Cells

Chronic lymphocytic leukemia (CLL) is thought to be a disease of resting lymphocytes. However, recent data suggest that CLL cells may more closely resemble activated B cells. Using microRNA (miRNA) expression profiling of highly-enriched CLL cells from 38 patients and 9 untransformed B cells from normal donors before acute CpG activation and 5 matched B cells after acute CpG activation, we demonstrate an activated B cell status for CLL. Gene set enrichment analysis (GSEA) identified statistically-significant similarities in miRNA expression between activated B cells and CLL cells including upregulation of miR-34a, miR-155, and miR-342-3p and downregulation of miR-103, miR-181a and miR-181b. Additionally, decreased levels of two CLL signature miRNAs miR-29c and miR-223 are associated with ZAP70+ and IgVH unmutated status and with shorter time to first therapy. These data indicate an activated B cell status for CLL cells and suggest that the direction of change of individual miRNAs may predict clinical course in CLL

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery