Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 23
Filter
Add more filters










Publication year range
1.
Nat Commun ; 13(1): 7904, 2022 12 23.
Article in English | MEDLINE | ID: mdl-36550132

ABSTRACT

N6-methyladenosine (m6A), the most abundant modification of mRNA, is essential for normal development and dysregulation promotes cancer. m6A is highly enriched in the 3' untranslated region (UTR) of a large subset of mRNAs to influence mRNA stability and/or translation. However, the mechanism responsible for the observed m6A distribution remains enigmatic. Here we find the exon junction complex shapes the m6A landscape by blocking METTL3-mediated m6A modification close to exon junctions within coding sequence (CDS). Depletion of EIF4A3, a core component of the EJC, causes increased METTL3 binding and m6A modification of short internal exons, and sites close to exon-exon junctions within mRNA. Reporter gene experiments further support the role of splicing and EIF4A3 deposition in controlling m6A modification via the local steric blockade of METTL3. Our results explain how characteristic patterns of m6A mRNA modification are established and uncover a role of the EJC in shaping the m6A epitranscriptome.


Subject(s)
Cell Nucleus , RNA Splicing , RNA Splicing/genetics , Cell Nucleus/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Exons/genetics , RNA Stability/genetics
2.
Mol Ther Nucleic Acids ; 12: 591-599, 2018 Sep 07.
Article in English | MEDLINE | ID: mdl-30195795

ABSTRACT

RNAi-based gene therapy using miRNA-adapted short hairpin RNAs (shRNAmiR) is a powerful approach to modulate gene expression. However, we have observed low viral titers with shRNAmiR-containing recombinant vectors and hypothesized that this could be due to cleavage of viral genomic RNA by the endogenous microprocessor complex during virus assembly. To test this hypothesis, we targeted DROSHA, the core component of the microprocessor complex, and successfully generated monoallelic and biallelic DROSHA knockout (KO) HEK293T cells for vector production. DROSHA KO was verified by polymerase chain reaction (PCR) and western blot analysis. We produced lentiviral vectors containing Venus with or without shRNA hairpins and generated virus supernatants using DROSHA KO packaging cells. We observed an increase in the fluorescence intensity of hairpin-containing Venus transcripts in DROSHA KO producer cells consistent with reduced microprocessor cleavage of encoded mRNA transcripts, and recovery in the viral titer of hairpin-containing vectors compared with non-hairpin-containing constructs. We confirmed the absence of significant shRNAmiR processing by northern blot analysis and showed that this correlated with an increase in the amount of full-length vector genomic RNA. These findings may have important implications in future production of viral shRNAmiR-containing vectors for RNAi-based therapy.

3.
Hum Mol Genet ; 27(9): 1654-1663, 2018 05 01.
Article in English | MEDLINE | ID: mdl-29509898

ABSTRACT

Tuberous sclerosis complex (TSC) is an autosomal dominant disease caused by germline inactivating mutations of TSC1 or TSC2. In TSC-associated tumors of the brain, heart, skin, kidney and lung, inactivation of both alleles of TSC1 or TSC2 leads to hyperactivation of the mTORC1 pathway. The TSC/mTORC1 pathway is a key regulator of cellular processes related to growth, proliferation and autophagy. We and others have previously found that mTORC1 regulates microRNA biogenesis, but the mechanisms are not fully understood. Microprocessor, a multi-protein complex including the nuclease Drosha, processes the primary miR transcript. Using a dual-luciferase reporter, we found that inhibition of mTORC1 or downregulation of Raptor decreased Microprocessor activity, while loss of TSC2 led to a striking increase (∼5-fold) in Microprocessor activity. To determine the global impact of TSC2 on microRNAs we quantitatively analyzed 752 microRNAs in Tsc2-expressing and Tsc2-deficient cells. Out of 259 microRNAs expressed in both cell lines, 137 were significantly upregulated and 24 were significantly downregulated in Tsc2-deficient cells, consistent with the increased Microprocessor activity. Microprocessor activity is known to be regulated in part by GSK3ß. We found that total GSK3ß levels were higher in Tsc2-deficient cells, and the increase in Microprocessor activity associated with Tsc2 loss was reversed by three different GSK3ß inhibitors. Furthermore, mTOR inhibition increased the levels of phospho-GSK3ß (S9), which negatively affects Microprocessor activity. Taken together these data reveal that TSC2 regulates microRNA biogenesis and Microprocessor activity via GSK3ß.


Subject(s)
Glycogen Synthase Kinase 3 beta/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , MicroRNAs/genetics , Animals , Cell Proliferation/genetics , Cell Proliferation/physiology , Glycogen Synthase Kinase 3 beta/genetics , HeLa Cells , Humans , Immunoblotting , Mechanistic Target of Rapamycin Complex 1/genetics , Mice , RNA, Small Interfering/genetics , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Tuberous Sclerosis Complex 1 Protein/genetics , Tuberous Sclerosis Complex 1 Protein/metabolism , Tuberous Sclerosis Complex 2 Protein/genetics , Tuberous Sclerosis Complex 2 Protein/metabolism
4.
Mol Ther ; 25(9): 1999-2013, 2017 09 06.
Article in English | MEDLINE | ID: mdl-28619647

ABSTRACT

Patient-derived induced pluripotent stem cells (iPSCs) hold great promise for autologous cell replacement. However, for many inherited diseases, treatment will likely require genetic repair pre-transplantation. Genome editing technologies are useful for this application. The purpose of this study was to develop CRISPR-Cas9-mediated genome editing strategies to target and correct the three most common types of disease-causing variants in patient-derived iPSCs: (1) exonic, (2) deep intronic, and (3) dominant gain of function. We developed a homology-directed repair strategy targeting a homozygous Alu insertion in exon 9 of male germ cell-associated kinase (MAK) and demonstrated restoration of the retinal transcript and protein in patient cells. We generated a CRISPR-Cas9-mediated non-homologous end joining (NHEJ) approach to excise a major contributor to Leber congenital amaurosis, the IVS26 cryptic-splice mutation in CEP290, and demonstrated correction of the transcript and protein in patient iPSCs. Lastly, we designed allele-specific CRISPR guides that selectively target the mutant Pro23His rhodopsin (RHO) allele, which, following delivery to both patient iPSCs in vitro and pig retina in vivo, created a frameshift and premature stop that would prevent transcription of the disease-causing variant. The strategies developed in this study will prove useful for correcting a wide range of genetic variants in genes that cause inherited retinal degeneration.


Subject(s)
CRISPR-Cas Systems , Gene Editing , Gene Targeting , Induced Pluripotent Stem Cells/metabolism , Retinal Degeneration/genetics , Stem Cell Transplantation , Alleles , Animals , Cell Line , Gene Order , Genetic Loci , Genetic Therapy , Genetic Vectors/genetics , Homologous Recombination , Humans , Induced Pluripotent Stem Cells/cytology , Introns , Mutation , Protein Serine-Threonine Kinases/genetics , RNA, Guide, Kinetoplastida , Retinal Degeneration/therapy , Stem Cell Transplantation/methods , Transplantation, Autologous
5.
BMC Biol ; 15(1): 45, 2017 05 19.
Article in English | MEDLINE | ID: mdl-28526034

ABSTRACT

BACKGROUND: Intravascular leukocyte recruitment in most vertebrate tissues is restricted to postcapillary and collecting venules, whereas capillaries and arterioles usually support little or no leukocyte adhesion. This segmental restriction is thought to be mediated by endothelial, rather than hemodynamic, differences. The underlying mechanisms are largely unknown, in part because effective tools to distinguish, isolate, and analyze venular endothelial cells (V-ECs) and non-venular endothelial cells (NV-ECs) have been unavailable. We hypothesized that the atypical chemokine receptor DARC (Duffy Antigen Receptor for Chemokines, a.k.a. ACKR1 or CD234) may distinguish V-ECs versus NV-ECs in mice. METHODS: We generated a rat-anti-mouse monoclonal antibody (MAb) that specifically recognizes the erythroid and endothelial forms of native, surface-expressed DARC. Using this reagent, we characterized DARC expression and distribution in the microvasculature of murine tissues. RESULTS: DARC was exquisitely restricted to post-capillary and small collecting venules and completely absent from arteries, arterioles, capillaries, veins, and most lymphatics in every tissue analyzed. Accordingly, intravital microscopy showed that adhesive leukocyte-endothelial interactions were restricted to DARC+ venules. DARC was detectable over the entire circumference of V-ECs, but was more concentrated at cell-cell junctions. Analysis of single-cell suspensions suggested that the frequency of V-ECs among the total microvascular EC pool varies considerably between different tissues. CONCLUSIONS: Immunostaining of endothelial DARC allows the identification and isolation of intact V-ECs from multiple murine tissues. This strategy may be useful to dissect the mechanisms underlying segmental microvascular specialization in healthy and diseased tissues and to characterize the role of EC subsets in tissue-homeostasis, immune surveillance, infection, inflammation, and malignancies.


Subject(s)
Duffy Blood-Group System , Endothelial Cells , Endothelium, Vascular , Gene Expression Regulation , Mice , Receptors, Cell Surface , Animals , Mice/genetics , Mice/metabolism , Duffy Blood-Group System/genetics , Duffy Blood-Group System/metabolism , Endothelial Cells/cytology , Endothelial Cells/metabolism , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Mice, Inbred BALB C , Mice, Inbred C57BL , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Veins/metabolism
6.
Nat Cell Biol ; 19(1): 60-67, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27992407

ABSTRACT

Signalling and post-transcriptional gene control are both critical for the regulation of pluripotency, yet how they are integrated to influence cell identity remains poorly understood. LIN28 (also known as LIN28A), a highly conserved RNA-binding protein, has emerged as a central post-transcriptional regulator of cell fate through blockade of let-7 microRNA biogenesis and direct modulation of mRNA translation. Here we show that LIN28 is phosphorylated by MAPK/ERK in pluripotent stem cells, which increases its levels via post-translational stabilization. LIN28 phosphorylation had little impact on let-7 but enhanced the effect of LIN28 on its direct mRNA targets, revealing a mechanism that uncouples LIN28's let-7-dependent and -independent activities. We have linked this mechanism to the induction of pluripotency by somatic cell reprogramming and the transition from naive to primed pluripotency. Collectively, our findings indicate that MAPK/ERK directly impacts LIN28, defining an axis that connects signalling, post-transcriptional gene control, and cell fate regulation.


Subject(s)
MAP Kinase Signaling System , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , RNA-Binding Proteins/metabolism , Transcription, Genetic , Amino Acid Sequence , Animals , Blotting, Western , HeLa Cells , Humans , Immunoprecipitation , Mass Spectrometry , Mice , Mice, Transgenic , MicroRNAs/metabolism , Mouse Embryonic Stem Cells/metabolism , Phosphorylation , Protein Domains , Protein Stability
7.
Mol Cell ; 62(3): 335-345, 2016 05 05.
Article in English | MEDLINE | ID: mdl-27117702

ABSTRACT

METTL3 is an RNA methyltransferase implicated in mRNA biogenesis, decay, and translation control through N(6)-methyladenosine (m(6)A) modification. Here we find that METTL3 promotes translation of certain mRNAs including epidermal growth factor receptor (EGFR) and the Hippo pathway effector TAZ in human cancer cells. In contrast to current models that invoke m(6)A reader proteins downstream of nuclear METTL3, we find METTL3 associates with ribosomes and promotes translation in the cytoplasm. METTL3 depletion inhibits translation, and both wild-type and catalytically inactive METTL3 promote translation when tethered to a reporter mRNA. Mechanistically, METTL3 enhances mRNA translation through an interaction with the translation initiation machinery. METTL3 expression is elevated in lung adenocarcinoma and using both loss- and gain-of-function studies, we find that METTL3 promotes growth, survival, and invasion of human lung cancer cells. Our results uncover an important role of METTL3 in promoting translation of oncogenes in human lung cancer.


Subject(s)
Adenocarcinoma/enzymology , Lung Neoplasms/enzymology , Methyltransferases/metabolism , Peptide Chain Initiation, Translational , RNA, Messenger/metabolism , A549 Cells , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Adenocarcinoma of Lung , Cell Movement , Cell Proliferation , Cell Survival , ErbB Receptors/biosynthesis , ErbB Receptors/genetics , Eukaryotic Initiation Factor-3/metabolism , Gene Expression Regulation, Neoplastic , Humans , Intracellular Signaling Peptides and Proteins/biosynthesis , Intracellular Signaling Peptides and Proteins/genetics , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Methyltransferases/genetics , Neoplasm Invasiveness , RNA Interference , RNA, Messenger/genetics , Ribosomes/enzymology , Signal Transduction , Trans-Activators , Transcription Factors , Transcriptional Coactivator with PDZ-Binding Motif Proteins , Transfection , Up-Regulation
8.
Cell Rep ; 13(2): 260-6, 2015 Oct 13.
Article in English | MEDLINE | ID: mdl-26440890

ABSTRACT

Let-7 microRNAs (miRNAs) are critical regulators of animal development, stem cell differentiation, glucose metabolism, and tumorigenesis. Mammalian genomes contain 12 let-7 isoforms that suppress expression of a common set of target mRNAs. LIN28 proteins selectively block let-7 biogenesis in undifferentiated cells and in cancer. The current model for coordinate let-7 repression involves the LIN28 cold-shock domain (CSD) binding the terminal loop and the two CCHC-type zinc fingers recognizing a GGAG sequence motif in precursor let-7 (pre-let-7) RNAs. Here, we perform a systematic analysis of all let-7 miRNAs and find that a single let-7 family member, human let-7a-3 (and its murine ortholog let-7c-2), escapes LIN28-mediated regulation. Mechanistically, we find that the pre-let-7c-2 loop precludes LIN28A binding and regulation. These findings refine the current model of let-7 regulation by LIN28 proteins and have important implications for understanding the LIN28/let-7 axis in development and disease.


Subject(s)
MicroRNAs/genetics , RNA-Binding Proteins/metabolism , Animals , Base Sequence , HEK293 Cells , HeLa Cells , Hep G2 Cells , Humans , Mice , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , RNA-Binding Proteins/chemistry
9.
Cell ; 156(5): 893-906, 2014 Feb 27.
Article in English | MEDLINE | ID: mdl-24581491

ABSTRACT

Global downregulation of microRNAs (miRNAs) is commonly observed in human cancers and can have a causative role in tumorigenesis. The mechanisms responsible for this phenomenon remain poorly understood. Here, we show that YAP, the downstream target of the tumor-suppressive Hippo-signaling pathway regulates miRNA biogenesis in a cell-density-dependent manner. At low cell density, nuclear YAP binds and sequesters p72 (DDX17), a regulatory component of the miRNA-processing machinery. At high cell density, Hippo-mediated cytoplasmic retention of YAP facilitates p72 association with Microprocessor and binding to a specific sequence motif in pri-miRNAs. Inactivation of the Hippo pathway or expression of constitutively active YAP causes widespread miRNA suppression in cells and tumors and a corresponding posttranscriptional induction of MYC expression. Thus, the Hippo pathway links contact-inhibition regulation to miRNA biogenesis and may be responsible for the widespread miRNA repression observed in cancer.


Subject(s)
MicroRNAs/metabolism , Neoplasms/genetics , Cell Count , Cell Cycle Proteins , Cell Line , DEAD-box RNA Helicases/metabolism , Hippo Signaling Pathway , Humans , MicroRNAs/genetics , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Signal Transduction , Transcription Factors/metabolism , Transcriptome
10.
Nature ; 497(7448): 244-8, 2013 May 09.
Article in English | MEDLINE | ID: mdl-23594738

ABSTRACT

The pluripotency factor Lin28 blocks the expression of let-7 microRNAs in undifferentiated cells during development, and functions as an oncogene in a subset of cancers. Lin28 binds to let-7 precursor (pre-let-7) RNAs and recruits 3' terminal uridylyl transferases to selectively inhibit let-7 biogenesis. Uridylated pre-let-7 is refractory to processing by Dicer, and is rapidly degraded by an unknown RNase. Here we identify Dis3l2 as the 3'-5' exonuclease responsible for the decay of uridylated pre-let-7 in mouse embryonic stem cells. Biochemical reconstitution assays show that 3' oligouridylation stimulates Dis3l2 activity in vitro, and knockdown of Dis3l2 in mouse embryonic stem cells leads to the stabilization of pre-let-7. Our study establishes 3' oligouridylation as an RNA decay signal for Dis3l2, and identifies the first physiological RNA substrate of this new exonuclease, which is mutated in the Perlman syndrome of fetal overgrowth and causes a predisposition to Wilms' tumour development.


Subject(s)
Exonucleases/metabolism , Exoribonucleases/metabolism , Fetal Macrosomia/enzymology , Fetal Macrosomia/genetics , MicroRNAs/metabolism , RNA Stability , RNA-Binding Proteins/metabolism , Ribonucleases/metabolism , Wilms Tumor/enzymology , Wilms Tumor/genetics , Animals , Cells, Cultured , Embryonic Stem Cells/metabolism , Fetal Macrosomia/metabolism , HEK293 Cells , Humans , Mice , MicroRNAs/genetics , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Processing, Post-Transcriptional , Substrate Specificity , Uridine Monophosphate/analogs & derivatives , Uridine Monophosphate/metabolism , Wilms Tumor/etiology , Wilms Tumor/metabolism
11.
Cell ; 147(1): 81-94, 2011 Sep 30.
Article in English | MEDLINE | ID: mdl-21962509

ABSTRACT

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.


Subject(s)
Glucose/metabolism , MicroRNAs/metabolism , Animals , Diabetes Mellitus, Type 2/metabolism , Gene Expression Regulation , Genome-Wide Association Study , Humans , Insulin Resistance , Mice , Mice, Knockout , Mice, Transgenic , MicroRNAs/genetics , Obesity/genetics , Obesity/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism
12.
Adv Exp Med Biol ; 700: 56-66, 2011.
Article in English | MEDLINE | ID: mdl-21755473

ABSTRACT

The Microprocessor, comprising the ribonuclease Drosha and its essential cofactor, the double-stranded RNA-binding protein, DGCR8, is essential for the first step of the miRNA biogenesis pathway. It specifically cleaves double-stranded RNA within stem-loop structures of primary miRNA transcripts (pri-miRNAs) to generate precursor (pre-miRNA) intermediates. Pre-miRNAs are subsequently processed by Dicer to their mature ∼22 nt form. Thus, Microprocessor is essential for miRNA maturation, and pri-miRNA cleavage by this complex defines one end of the mature miRNA. Moreover, it is emerging that dysregulation of the Microprocessor is associated with various human diseases. It is therefore important to understand the mechanisms by which the expression of the subunits of the Microprocessor is regulated. Recent findings have uncovered a post-transcriptional mechanism that maintains the integrity of the Microprocessor. These studies revealed that the Microprocessor is involved in the processing of the messenger RNA (mRNA) that encodes DGCR8. This regulatory feedback loop, along with the reported role played by DGCR8 in the stabilization of Drosha protein, is part of a newly identified regulatory mechanism controlling Microprocessor activity.


Subject(s)
RNA Processing, Post-Transcriptional , RNA-Binding Proteins , Humans , MicroRNAs/genetics , Microcomputers , RNA, Double-Stranded , RNA-Binding Proteins/metabolism
13.
Nat Struct Mol Biol ; 18(3): 323-7, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21297638

ABSTRACT

MicroRNAs (miRNAs) are a class of small, noncoding RNAs that function by regulating gene expression post-transcriptionally. Alterations in miRNA expression can strongly influence cellular physiology. Here we demonstrated cross-regulation between two components of the RNA interference (RNAi) machinery in human cells. Inhibition of exportin-5, the karyopherin responsible for pre-miRNA export, downregulated expression of Dicer, the RNase III required for pre-miRNA maturation. This effect was post-transcriptional and resulted from an increased nuclear localization of Dicer mRNA. In vitro assays and cellular RNA immunoprecipitation experiments showed that exportin-5 interacted directly with Dicer mRNA. Titration of exportin-5 by overexpression of either pre-miRNA or the adenoviral VA1 RNA resulted in loss of Dicer mRNA-exportin-5 interaction and reduction of Dicer level. This saturation also occurred during adenoviral infection and enhanced viral replication. Our study reveals an important cross-regulatory mechanism between pre-miRNA or viral small RNAs and Dicer through exportin-5.


Subject(s)
DEAD-box RNA Helicases/genetics , Gene Expression Regulation , Karyopherins/metabolism , MicroRNAs/metabolism , RNA, Messenger/metabolism , RNA, Viral/metabolism , Ribonuclease III/genetics , Adenoviridae/genetics , Adenoviridae/physiology , Adenoviridae Infections/virology , DEAD-box RNA Helicases/metabolism , HeLa Cells , Humans , Karyopherins/genetics , MicroRNAs/genetics , Protein Binding , RNA Interference , RNA, Messenger/genetics , RNA, Viral/genetics , Ribonuclease III/metabolism , Virus Replication
14.
Nat Cell Biol ; 12(10): 928-9, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20885420

ABSTRACT

Pumilio proteins PUM1 and PUM2 are shown to regulate microRNA-dependent gene silencing by induction of a conformational switch in the 3' untranslated region of p27 mRNA. This conformational change is required for efficient microRNA-mediated repression of this cell-cycle regulator in rapidly proliferating cells.


Subject(s)
Cyclin-Dependent Kinase Inhibitor p27/genetics , MicroRNAs/metabolism , RNA-Binding Proteins/metabolism , 3' Untranslated Regions/genetics , Animals , Cell Cycle , Cell Proliferation , Gene Silencing , Humans , Nucleic Acid Conformation
15.
Adv Exp Med Biol ; 700: 56-66, 2010.
Article in English | MEDLINE | ID: mdl-21627030

ABSTRACT

The Microprocessor, comprising the ribonuclease Drosha and its essential cofactor, the double-stranded RNA-binding protein, DGCR8, is essential for the first step of the miRNA biogenesis pathway. It specifically cleaves double-stranded RNA within stem-loop structures of primary miRNA transcripts (pri-miRNAs) to generate precursor (pre-miRNA) intermediates. Pre-miRNAs are subsequently processed by Dicer to their mature 22 nt form. Thus, Microprocessor is essential for miRNA maturation, and pri-miRNA cleavage by this complex defines one end of the mature miRNA. Moreover, it is emerging that dysregulation of the Microprocessor is associated with various human diseases. It is therefore important to understand the mechanisms by which the expression of the subunits of the Microprocessor is regulated. Recent findings have uncovered a post-transcriptional mechanism that maintains the integrity of the Microprocessor. These studies revealed that the Microprocessor is involved in the processing of the messenger RNA (mRNA) that encodes DGCR8. This regulatory feedback loop, along with the reported role played by DGCR8 in the stabilization of Drosha protein, is part ofa newly identified regulatory mechanism controlling Microprocessor activity.


Subject(s)
Gene Expression Regulation , MicroRNAs/physiology , Proteins/genetics , Ribonuclease III/genetics , Animals , Homeostasis , Humans , Proteins/physiology , RNA-Binding Proteins , Ribonuclease III/physiology
16.
RNA ; 15(6): 1005-11, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19383765

ABSTRACT

The Microprocessor, comprising the RNase III Drosha and the double-stranded RNA binding protein DGCR8, is essential for microRNA (miRNA) biogenesis. In the miRNA processing pathway certain hairpin structures within primary miRNA (pri-miRNA) transcripts are specifically cleaved by the Microprocessor to release approximately 60-70-nucleotide precursor miRNA (pre-miRNA) intermediates. Although both Drosha and DGCR8 are required for Microprocessor activity, the mechanisms regulating the expression of these proteins are unknown. Here we report that the Microprocessor negatively regulates DGCR8 expression. Using in vitro reconstitution and in vivo studies, we demonstrate that a hairpin, localized in the 5' untranslated region (5'UTR) of DGCR8 mRNA, is cleaved by the Microprocessor. Accordingly, knockdown of Drosha leads to an increase in DGCR8 mRNA and protein levels in cells. Furthermore, we found that the DGCR8 5'UTR confers Microprocessor-dependent repression of a luciferase reporter gene in vivo. Our results uncover a novel feedback loop that regulates DGCR8 levels.


Subject(s)
MicroRNAs/metabolism , Proteins/genetics , RNA, Messenger/metabolism , Ribonuclease III/metabolism , 5' Untranslated Regions , Cells, Cultured , HeLa Cells , Humans , Proteins/metabolism , RNA Processing, Post-Transcriptional , RNA-Binding Proteins , Ribonuclease III/genetics , Transfection
17.
Retrovirology ; 6: 26, 2009 Mar 09.
Article in English | MEDLINE | ID: mdl-19272132

ABSTRACT

The rate of HIV-1 gene expression is a key step that determines the kinetics of virus spread and AIDS progression. Viral entry and gene expression were described to be the key determinants for cell permissiveness to HIV. Recent reports highlighted the involvement of miRNA in regulating HIV-1 replication post-transcriptionally. In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression. Here we show that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterize some of the proteins required for miRNA-mediated silencing (miRNA effectors). RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes. Interestingly, knockdown of RCK/p54 or DGCR8 resulted in virus reactivation in PBMCs isolated from HIV infected patients treated with suppressive HAART.


Subject(s)
Gene Expression Regulation, Viral , HIV-1/physiology , MicroRNAs/metabolism , RNA, Viral/metabolism , Virus Replication/physiology , Argonaute Proteins , Cell Line , Cells, Cultured , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Eukaryotic Initiation Factor-2/metabolism , Gene Knockdown Techniques , HeLa Cells , Humans , Polyribosomes/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , RNA Interference/physiology , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Virus Latency/physiology
19.
Nat Cell Biol ; 9(3): 331-8, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17293853

ABSTRACT

The p300-CBP-associated factor (PCAF) is a histone acetyltransferase (HAT) involved in the reversible acetylation of various transcriptional regulators, including the tumour suppressor p53. It is implicated in many cellular processes, such as transcription, differentiation, proliferation and apoptosis. We observed that knockdown of PCAF expression in HeLa or U2OS cell lines induces stabilization of the oncoprotein Hdm2, a RING finger E3 ligase primarily known for its role in controlling p53 stability. To investigate the molecular basis of this effect, we examined whether PCAF is involved in Hdm2 ubiquitination. Here, we show that PCAF, in addition to its acetyltransferase activity, possesses an intrinsic ubiquitination activity that is critical for controlling Hdm2 expression levels, and thus p53 functions. Our data highlight a regulatory crosstalk between PCAF and Hdm2 activities, which is likely to have a central role in the subtle control of p53 activity after DNA damage.


Subject(s)
Cell Cycle Proteins/metabolism , Histone Acetyltransferases/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolism , Apoptosis/drug effects , Apoptosis/genetics , Apoptosis/radiation effects , Binding Sites/genetics , Catalytic Domain/genetics , Cell Cycle Proteins/genetics , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/genetics , DNA Damage/drug effects , DNA Damage/radiation effects , Gene Expression/drug effects , Gene Expression/radiation effects , HeLa Cells , Histone Acetyltransferases/genetics , Humans , Mutation , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-mdm2/genetics , RNA, Antisense/genetics , RNA, Small Interfering/genetics , Transcription Factors/genetics , Transfection , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics , Ultraviolet Rays , Zinostatin/pharmacology , p300-CBP Transcription Factors
20.
Science ; 315(5818): 1579-82, 2007 Mar 16.
Article in English | MEDLINE | ID: mdl-17322031

ABSTRACT

MicroRNAs (miRNAs) are single-stranded noncoding RNAs of 19 to 25 nucleotides that function as gene regulators and as a host cell defense against both RNA and DNA viruses. We provide evidence for a physiological role of the miRNA-silencing machinery in controlling HIV-1 replication. Type III RNAses Dicer and Drosha, responsible for miRNA processing, inhibited virus replication both in peripheral blood mononuclear cells from HIV-1-infected donors and in latently infected cells. In turn, HIV-1 actively suppressed the expression of the polycistronic miRNA cluster miR-17/92. This suppression was found to be required for efficient viral replication and was dependent on the histone acetyltransferase Tat cofactor PCAF. Our results highlight the involvement of the miRNA-silencing pathway in HIV-1 replication and latency.


Subject(s)
HIV-1/physiology , Leukocytes, Mononuclear/virology , MicroRNAs/genetics , RNA Interference , Virus Replication , 3' Untranslated Regions , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line , Gene Expression Regulation , Gene Products, tat/metabolism , HIV-1/genetics , HeLa Cells , Histone Acetyltransferases/genetics , Histone Acetyltransferases/metabolism , Humans , Jurkat Cells , Leukocytes, Mononuclear/enzymology , Oligonucleotide Array Sequence Analysis , RNA, Small Interfering/genetics , Ribonuclease III/genetics , Ribonuclease III/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transfection , Virus Latency , p300-CBP Transcription Factors , tat Gene Products, Human Immunodeficiency Virus
SELECTION OF CITATIONS
SEARCH DETAIL
...