The Non-Canonical Aspects of MicroRNAs: Many Roads to Gene Regulation

MicroRNAs (miRNAs) are critical regulators of gene expression. As miRNAs are frequently deregulated in many human diseases, including cancer and immunological disorders, it is important to understand their biological functions. Typically, miRNA-encoding genes are transcribed by RNA Polymerase II and generate primary transcripts that are processed by RNase III-endonucleases DROSHA and DICER into small RNAs of approximately 21 nucleotides. All miRNAs are loaded into Argonaute proteins in the RNA-induced silencing complex (RISC) and act as post-transcriptional regulators by binding to the 3′- untranslated region (UTR) of mRNAs. This seed-dependent miRNA binding inhibits the translation and/or promotes the degradation of mRNA targets. Surprisingly, recent data presents evidence for a target-mediated decay mechanism that controls the level of specific miRNAs. In addition, several non-canonical miRNA-containing genes have been recently described and unexpected functions of miRNAs have been identified. For instance, several miRNAs are located in the nucleus, where they are involved in the transcriptional activation or silencing of target genes. These epigenetic modifiers are recruited by RISC and guided by miRNAs to specific loci in the genome. Here, we will review non-canonical aspects of miRNA biology, including novel regulators of miRNA expression and functions of miRNAs in the nucleus.

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Analysis of miRNAs in the Heads of Different Castes of the Bumblebee Bombus lantschouensis (Hymenoptera: Apidae)

Insects ◽

10.3390/insects10100349 ◽

2019 ◽

Vol 10 (10) ◽

pp. 349 ◽

Cited By ~ 2

Author(s):

Liu ◽

Huang ◽

Zhang ◽

Liu ◽

Keyword(s):

Small Rna ◽

Small Rnas ◽

Target Genes ◽

Solexa Sequencing ◽

Biological Functions ◽

Putative Target ◽

Differentially Expressed Mirnas ◽

Rna Deep Sequencing ◽

Small Rna Deep Sequencing ◽

Go Terms

Bumblebees are important insect pollinators for many wildflowers and crops. MicroRNAs (miRNAs) are endogenous non-coding small RNAs that regulate different biological functions in insects. In this study, the miRNAs in the heads of the three castes of the bumblebee Bombus lantschouensis were identified and characterized by small RNA deep sequencing. The significant differences in the expression of miRNAs and their target genes were analyzed. The results showed that the length of the small RNA reads from males, queens, and workers was distributed between 18 and 30 nt, with a peak at 22 nt. A total of 364 known and 89 novel miRNAs were identified from the heads of the three castes. The eight miRNAs with the highest expressed levels in males, queens, and workers were identical, although the order of these miRNAs based on expression differed. The male vs. queen, male vs. worker, and worker vs. queen comparisons identified nine, fourteen, and four miRNAs with significant differences in expression, respectively. The different castes were clustered based on the differentially expressed miRNAs (DE miRNAs), and the expression levels of the DE miRNAs obtained by RT-qPCR were consistent with the read counts obtained through Solexa sequencing. The putative target genes of these DE miRNAs were enriched in 29 Gene Ontology (GO) terms, and catalytic activity was the most enriched GO term, as demonstrated by its association with 2837 target genes in the male vs. queen comparison, 3535 target genes in the male vs. worker comparison, and 2185 target genes in the worker vs. queen comparison. This study highlights the characteristics of the miRNAs in the three B. lantschouensis castes and will aid further studies on the functions of miRNAs in bumblebees.

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Regulation of Thermotolerance by Stress-Induced Transcription Factors in Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.00029-08 ◽

2008 ◽

Vol 7 (5) ◽

pp. 783-790 ◽

Cited By ~ 32

Author(s):

Noritaka Yamamoto ◽

Yuka Maeda ◽

Aya Ikeda ◽

Hiroshi Sakurai

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Factors ◽

High Temperature ◽

Heat Shock ◽

Cell Growth ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Target Genes ◽

Recovery Period ◽

Before And After

ABSTRACT The heat shock transcription factor Hsf1 and the general stress transcription factors Msn2 and Msn4 (Msn2/4) are major regulators of the heat shock response in Saccharomyces cerevisiae. Here, we show that transcriptional activation of their target genes, including HSP104, an antistress chaperone gene, is obligatory for thermotolerance. Although Hsf1 activity might be necessary before the exposure of cells to high temperature, severe heat shock induced the binding of hyperphosphorylated Hsf1 to its target promoters. However, promoter-bound, phosphorylated Hsf1 was inactive for transcription because RNA polymerase II was inactive at high temperatures. Rather, our results suggest that Hsf1 activates the transcription of most of its target genes during the recovery period following severe heat shock. This delayed upregulation by Hsf1, which would be induced by misfolded proteins that accumulate in severely heat-shocked cells, is required for the resumption of normal cell growth. In contrast, the factors Msn2/4 were not involved in the delayed upregulation of genes and were dispensable for cell growth during the recovery period, suggesting that they play a role before the exposure to high temperature. These results show that Hsf1 and Msn2/4 act differentially before and after exposure to extreme temperatures to ensure cell survival and growth.

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NACK and INTEGRATOR act coordinately to activate Notch-mediated transcription in tumorigenesis

Cell Communication and Signaling ◽

10.1186/s12964-021-00776-1 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Elena Shersher ◽

Mohini Lahiry ◽

Annamil Alvarez-Trotta ◽

Giulia Diluvio ◽

David J. Robbins ◽

...

Keyword(s):

Liquid Chromatography ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Target Genes ◽

Quantitative Polymerase Chain Reaction ◽

Size Exclusion ◽

M Cell ◽

Exclusion Chromatography ◽

Liquid Chromatography Tandem Mass ◽

Eac Cells

Abstract Background Notch signaling drives many aspects of neoplastic phenotype. Here, we report that the Integrator complex (INT) is a new component of the Notch transcriptional supercomplex. Together with Notch Activation Complex Kinase (NACK), INT activates Notch1 target genes by driving RNA polymerase II (RNAPII)-dependent transcription, leading to tumorigenesis. Methods Size exclusion chromatography and CBF-1/RBPJ/Suppressor of Hairless/Lag-1 (CSL)-DNA affinity fast protein liquid chromatography (FPLC) was used to purify Notch/CSL-dependent complexes for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Chromatin immunoprecipitation (ChIP) and quantitative polymerase chain reaction (qPCR) were performed to investigate transcriptional regulation of Notch target genes. Transfection of Notch Ternary Complex components into HEK293T cells was used as a recapitulation assay to study Notch-mediated transcriptional mechanisms. Gene knockdown was achieved via RNA interference and the effects of protein depletion on esophageal adenocarcinoma (EAC) proliferation were determined via a colony formation assay and murine xenografts. Western blotting was used to examine expression of INT subunits in EAC cells and evaluate apoptotic proteins upon INT subunit 11 knockdown (INTS11 KD). Gene KD effects were further explored via flow cytometry. Results We identified the INT complex as part of the Notch transcriptional supercomplex. INT, together with NACK, activates Notch-mediated transcription. While NACK is required for the recruitment of RNAPII to a Notch-dependent promoter, the INT complex is essential for RNAPII phosphorylated at serine 5 (RNAPII-S5P), leading to transcriptional activation. Furthermore, INT subunits are overexpressed in EAC cells and INTS11 KD results in G2/M cell cycle arrest, apoptosis, and cell growth arrest in EAC. Conclusions This study identifies the INT complex as a novel co-factor in Notch-mediated transcription that together with NACK activates Notch target genes and leads to cancer cell proliferation.

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Activation Functions 1 and 2 of Nuclear Receptors: Molecular Strategies for Transcriptional Activation

Molecular Endocrinology ◽

10.1210/me.2002-0384 ◽

2003 ◽

Vol 17 (10) ◽

pp. 1901-1909 ◽

Cited By ~ 155

Author(s):

Anette Wärnmark ◽

Eckardt Treuter ◽

Anthony P. H. Wright ◽

Jan-Åke Gustafsson

Keyword(s):

Transcription Factors ◽

Rna Polymerase Ii ◽

Nuclear Receptors ◽

Transcriptional Activation ◽

Molecular Mechanisms ◽

Target Genes ◽

Current Knowledge ◽

Preinitiation Complex ◽

Activation Domains ◽

Terminal Domain

Abstract Nuclear receptors (NRs) comprise a family of ligand inducible transcription factors. To achieve transcriptional activation of target genes, DNA-bound NRs directly recruit general transcription factors (GTFs) to the preinitiation complex or bind intermediary factors, so-called coactivators. These coactivators often constitute subunits of larger multiprotein complexes that act at several functional levels, such as chromatin remodeling, enzymatic modification of histone tails, or modulation of the preinitiation complex via interactions with RNA polymerase II and GTFs. The binding of NR to coactivators is often mediated through one of its activation domains. Many NRs have at least two activation domains, the ligand-independent activation function (AF)-1, which resides in the N-terminal domain, and the ligand-dependent AF-2, which is localized in the C-terminal domain. In this review, we summarize and discuss current knowledge regarding the molecular mechanisms of AF-1- and AF-2-mediated gene activation, focusing on AF-1 and AF-2 conformation and coactivator binding.

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Activator Gcn4p and Cyc8p/Tup1p Are Interdependent for Promoter Occupancy at ARG1 In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.25.24.11171-11183.2005 ◽

2005 ◽

Vol 25 (24) ◽

pp. 11171-11183 ◽

Cited By ~ 19

Author(s):

Soon-ja Kim ◽

Mark J. Swanson ◽

Hongfang Qiu ◽

Chhabi K. Govind ◽

Alan G. Hinnebusch

Keyword(s):

Amino Acid ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Target Genes ◽

Direct Role ◽

Valine Biosynthesis ◽

Promoter Occupancy ◽

Yeast Deletion Library ◽

Deletion Library

ABSTRACT The Cyc8p/Tup1p complex mediates repression of diverse genes in Saccharomyces cerevisiae and is recruited by DNA binding proteins specific for the different sets of repressed genes. By screening the yeast deletion library, we identified Cyc8p as a coactivator for Gcn4p, a transcriptional activator of amino acid biosynthetic genes. Deletion of CYC8 confers sensitivity to an inhibitor of isoleucine/valine biosynthesis and impairs activation of Gcn4p-dependent reporters and authentic amino acid biosynthetic target genes. Deletion of TUP1 produces similar but less severe activation defects in vivo. Although expression of Gcn4p is unaffected by deletion of CYC8, chromatin immunoprecipitation assays reveal a strong defect in binding of Gcn4p at the target genes ARG1 and ARG4 in cyc8Δ cells and to a lesser extent in tup1Δ cells. The defects in Gcn4p binding and transcriptional activation in cyc8Δ cells cannot be overcome by Gcn4p overexpression but are partially suppressed in tup1Δ cells. The impairment of Gcn4p binding in cyc8Δ and tup1Δ cells is severe enough to reduce recruitment of SAGA, Srb mediator, TATA binding protein, and RNA polymerase II to the ARG1 and ARG4 promoters, accounting for impaired transcriptional activation of these genes in both mutants. Cyc8p and Tup1p are recruited to the ARG1 and ARG4 promoters, consistent with a direct role for this complex in stimulating Gcn4p occupancy of the upstream activation sequence (UAS). Interestingly, Gcn4p also stimulates binding of Cyc8p/Tup1p at the 3′ ends of these genes, raising the possibility that Cyc8p/Tup1p influences transcription elongation. Our findings reveal a novel coactivator function for Cyc8p/Tup1p at the level of activator binding and suggest that Gcn4p may enhance its own binding to the UAS by recruiting Cyc8p/Tup1p.

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Hypoxic Regulation of Gene Transcription and Chromatin: Cause and Effect

International Journal of Molecular Sciences ◽

10.3390/ijms21218320 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8320

Author(s):

Jessica D. Kindrick ◽

David R. Mole

Keyword(s):

Transcriptional Activation ◽

Cellular Response ◽

Target Genes ◽

Hypoxia Inducible Factor ◽

Transcriptional Response ◽

Epigenetic Landscape ◽

Low Oxygen ◽

Epigenetic Modifiers ◽

Oxygen Sensitive ◽

Cell Type Specific

Cellular responses to low oxygen (hypoxia) are fundamental to normal physiology and to the pathology of many common diseases. Hypoxia-inducible factor (HIF) is central to this by enhancing the transcriptional activity of many hundreds of genes. The cellular response to HIF is cell-type-specific and is largely governed by the pre-existing epigenetic landscape. Prior to activation, HIF-binding sites and the promoters of HIF-target genes are already accessible, in contact with each other through chromatin looping and display markers of activity. However, hypoxia also modulates the epigenetic environment, both in parallel to and as a consequence of HIF activation. This occurs through a combination of oxygen-sensitive changes in enzyme activity, transcriptional activation of epigenetic modifiers, and localized recruitment to chromatin by HIF and activated RNApol2. These hypoxic changes in the chromatin environment may both contribute to and occur as a consequence of transcriptional regulation. Nevertheless, they have the capacity to both modulate and extend the transcriptional response to hypoxia.

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A common intermediary factor (p52/54) recognizing "acidic blob"-type domains is required for transcriptional activation by the Jun proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5508 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5508-5515 ◽

Cited By ~ 15

Author(s):

T Oehler ◽

P Angel

Keyword(s):

Transcription Factors ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Target Genes ◽

Serum Response Factor ◽

Limiting Factor ◽

Regulation Of Transcription ◽

Physical Interaction ◽

Main Component

The ability of the c-Jun protein, the main component of the transcription factor AP1, to interact directly or indirectly with the RNA polymerase II-initiation complex to activate transcription was investigated by in vivo transcription interference ("squelching") experiments. Coexpression of a Jun mutant lacking its DNA binding domain strongly represses the activity of wild-type c-Jun. Repression depends on the presence of the transactivation domains (TADs), suggesting that a limiting factor interacting with the TADs is essential to link Jun and the components of the transcriptional machinery. The activity of this intermediary factor(s) is restricted to TADs characterized by an abundance of negatively charged amino acids, as demonstrated by the abilities of the TADs of JunB, GAL4, and VP16 to repress c-Jun activity. Depending on the presence of the TADs of Jun, we found physical interaction between Jun and a cluster of three proteins with molecular masses of 52, 53, and 54 kDa (p52/54). Association between Jun and p52/54 is strongly reduced in the presence of VP16, suggesting that the two proteins compete for binding to p52/54. Transcription factors containing a different type of TAD (e.g., GHF1, estrogen receptor, or serum response factor) fail to inhibit Jun activity, suggesting that these proteins act through a different mechanism. We consider the requirement of Jun to interact with p52/54 utilized by other transcription factors a new mechanism in the regulation of transcription of Jun-dependent target genes.

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TEsmall identifies small RNAs associated with targeted inhibitor resistance in melanoma

10.1101/359471 ◽

2018 ◽

Author(s):

Kathryn O’Neill ◽

Wen-Wei Liao ◽

Ami Patel ◽

Molly Gale Hammell

Keyword(s):

Small Rna ◽

Small Rnas ◽

Target Genes ◽

Mapk Pathway ◽

Single Agent ◽

Resistance Mechanisms ◽

Melanoma Cells ◽

Cancer Therapies ◽

Mrna Targets ◽

Inhibitor Resistance

MicroRNAs (miRNAs) are small 21-22nt RNAs that act to regulate the expression of mRNA target genes through direct binding to mRNA targets. While miRNAs typically dominate small RNA transcriptomes, many other classes are present including tRNAs, snoRNAs, snRNAs, Y-RNAs, piRNAs, and siRNAs. Interactions between processing machinery and targeting networks of these various small RNA classes remains unclear, largely because these small RNAs are typically analyzed separately. Here we present TEsmall, a tool that allows for the simultaneous processing and analysis of small RNAs from each annotated class in a single integrated workflow. The pipeline begins with raw fastq reads and proceeds all the way to producing count tables formatted for differential expression. Several interactive charts are also produced to look at overall distributions in length and annotation classes. We next applied the TEsmall pipeline to small RNA libraries generated from melanoma cells responding to targeted inhibitors of the MAPK pathway. Targeted oncogene inhibitors have emerged as way to tailor cancer therapies to the particular mutations present in a given tumor. While these targeted strategies are typically effective for short intervals, the emergence of resistance is extremely common, limiting the effectiveness of single-agent therapeutics and driving the need for a better understanding of resistance mechanisms. Using TEsmall, we identified several microRNAs and other small RNA classes that are enriched in inhibitor resistant melanoma cells in multiple melanoma cell lines and may be able to serve as markers of resistant populations more generally.

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The Mediator Subunit MED16 Transduces NRF2-Activating Signals into Antioxidant Gene Expression

Molecular and Cellular Biology ◽

10.1128/mcb.00785-15 ◽

2015 ◽

Vol 36 (3) ◽

pp. 407-420 ◽

Cited By ~ 28

Author(s):

Hiroki Sekine ◽

Keito Okazaki ◽

Nao Ota ◽

Hiroki Shima ◽

Yasutake Katoh ◽

...

Keyword(s):

Gene Expression ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Molecular Mechanisms ◽

Target Genes ◽

Antioxidant Response ◽

Wild Type ◽

Antioxidant Genes ◽

Antioxidant Gene Expression

The KEAP1-NRF2 system plays a central role in cytoprotection. NRF2 is stabilized in response to electrophiles and activates transcription of antioxidant genes. Although robust induction of NRF2 target genes confers resistance to oxidative insults, how NRF2 triggers transcriptional activation after binding to DNA has not been elucidated. To decipher the molecular mechanisms underlying NRF2-dependent transcriptional activation, we purified the NRF2 nuclear protein complex and identified the Mediator subunits as NRF2 cofactors. Among them, MED16 directly associated with NRF2. Disruption ofMed16significantly attenuated the electrophile-induced expression of NRF2 target genes but did not affect hypoxia-induced gene expression, suggesting a specific requirement for MED16 in NRF2-dependent transcription. Importantly, we found that 75% of NRF2-activated genes exhibited blunted inductions by electrophiles inMed16-deficient cells compared to wild-type cells, which strongly argues that MED16 is a major contributor supporting NRF2-dependent transcriptional activation. NRF2-dependent phosphorylation of the RNA polymerase II C-terminal domain was absent inMed16-deficient cells, suggesting that MED16 serves as a conduit to transmit NRF2-activating signals to RNA polymerase II. MED16 indeed turned out to be essential for cytoprotection against oxidative insults. Thus, the KEAP1-NRF2-MED16 axis has emerged as a new regulatory pathway mediating the antioxidant response through the robust activation of NRF2 target genes.

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