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1.
Int J Mol Sci ; 25(11)2024 May 23.
Article in English | MEDLINE | ID: mdl-38891874

ABSTRACT

Formin Homology Proteins (Formins) are a highly conserved family of cytoskeletal regulatory proteins that participate in a diverse range of cellular processes. FMNL2 is a member of the Diaphanous-Related Formin sub-group, and previous reports suggest FMNL2's role in filopodia assembly, force generation at lamellipodia, subcellular trafficking, cell-cell junction assembly, and focal adhesion formation. How FMNL2 is recruited to these sites of action is not well understood. To shed light on how FMNL2 activity is partitioned between subcellular locations, we used biotin proximity labeling and proteomic analysis to identify an FMNL2 interactome. The interactome identified known and new FMNL2 interacting proteins with functions related to previously described FMNL2 activities. In addition, our interactome predicts a novel connection between FMNL2 and extracellular vesicle assembly. We show directly that FMNL2 protein is present in exosomes.


Subject(s)
Formins , Formins/metabolism , Humans , Proteomics/methods , Exosomes/metabolism , Mass Spectrometry/methods , Protein Binding , HEK293 Cells , Protein Interaction Maps
2.
Cancers (Basel) ; 15(17)2023 Aug 30.
Article in English | MEDLINE | ID: mdl-37686616

ABSTRACT

Advances in sequencing technologies and the bioinformatic analysis of big data facilitate the study of jumping genes' activity in the human genome in cancer from a broad perspective. Retrotransposons, which move from one genomic site to another by a copy-and-paste mechanism, are regulated by various molecular pathways that may be disrupted during tumorigenesis. Active retrotransposons can stimulate type I IFN responses. Although accumulated evidence suggests that retrotransposons can induce inflammation, the research investigating the exact mechanism of triggering these responses is ongoing. Understanding these mechanisms could improve the therapeutic management of cancer through the use of retrotransposon-induced inflammation as a tool to instigate immune responses to tumors.

3.
J Extracell Vesicles ; 11(10): e12220, 2022 10.
Article in English | MEDLINE | ID: mdl-36214496

ABSTRACT

One of the functions of small extracellular vesicles (sEVs) which has received the most attention is their capacity to deliver RNA into the cytoplasm of target cells. These studies have often been performed by transfecting RNAs into sEV-producing cells, to later purify and study sEV delivery of RNA. Transfection complexes and other delivery vehicles accumulate in late endosomes where sEV are formed and over 50% of transfection complexes or delivery vehicles administered to cells are released again to the extracellular space by exocytosis. This raises the possibility that transfection complexes could alter sEVs and contaminate sEV preparations. We found that widely used transfection reagents including RNAiMax and INTERFERin accumulated in late endosomes. These transfection complexes had a size similar to sEV and were purified by ultracentrifugation like sEV. Focusing on the lipid-based transfection reagent RNAiMax, we found that preparations of sEV from transfected cells contained lipids from transfection complexes and transfected siRNA was predominantly in particles with the density of transfection complexes, rather than sEV. This suggests that transfection complexes, such as lipid-based RNAiMax, may frequently contaminate sEV preparations and could account for some reports of sEV-mediated delivery of nucleic acids. Transfection of cells also impaired the capacity of sEVs to deliver stably-expressed siRNAs, suggesting that transfection of cells may alter sEVs and prevent the study of their endogenous capacity to deliver RNA to target cells.


Subject(s)
Extracellular Vesicles , Lipids , RNA, Small Interfering , Transfection , Ultracentrifugation
4.
Nat Commun ; 13(1): 1898, 2022 04 07.
Article in English | MEDLINE | ID: mdl-35393414

ABSTRACT

Recent advances in cancer therapeutics clearly demonstrate the need for innovative multiplex therapies that attack the tumour on multiple fronts. Oncolytic or "cancer-killing" viruses (OVs) represent up-and-coming multi-mechanistic immunotherapeutic drugs for the treatment of cancer. In this study, we perform an in-vitro screen based on virus-encoded artificial microRNAs (amiRNAs) and find that a unique amiRNA, herein termed amiR-4, confers a replicative advantage to the VSVΔ51 OV platform. Target validation of amiR-4 reveals ARID1A, a protein involved in chromatin remodelling, as an important player in resistance to OV replication. Virus-directed targeting of ARID1A coupled with small-molecule inhibition of the methyltransferase EZH2 leads to the synthetic lethal killing of both infected and uninfected tumour cells. The bystander killing of uninfected cells is mediated by intercellular transfer of extracellular vesicles carrying amiR-4 cargo. Altogether, our findings establish that OVs can serve as replicating vehicles for amiRNA therapeutics with the potential for combination with small molecule and immune checkpoint inhibitor therapy.


Subject(s)
Extracellular Vesicles , MicroRNAs , Neoplasms , Oncolytic Virotherapy , Oncolytic Viruses , Humans , MicroRNAs/genetics , Neoplasms/therapy , Oncolytic Viruses/genetics
5.
NAR Cancer ; 3(1): zcaa040, 2021 Mar.
Article in English | MEDLINE | ID: mdl-33447827

ABSTRACT

It is challenging to identify the causes and consequences of retrotransposon expression in human disease due to the hundreds of active genomic copies and their poor conservation across species. We profiled genomic insertions of retrotransposons in ovarian cancer. In addition, in ovarian and breast cancer we analyzed RNAs exhibiting Bayesian correlation with retrotransposon RNA to identify causes and consequences of retrotransposon expression. This strategy finds divergent inflammatory responses associated with retrotransposon expression in ovarian and breast cancer and identifies new factors inducing expression of endogenous retrotransposons including anti-viral responses and the common tumor suppressor BRCA1. In cell lines, mouse ovarian epithelial cells and patient-derived tumor spheroids, BRCA1 promotes accumulation of retrotransposon RNA. BRCA1 promotes transcription of active families of retrotransposons and their insertion into the genome. Intriguingly, elevated retrotransposon expression predicts survival in ovarian cancer patients. Retrotransposons are part of a complex regulatory network in ovarian cancer including BRCA1 that contributes to patient survival. The described strategy can be used to identify the regulators and impacts of retrotransposons in various contexts of biology and disease in humans.

6.
J Neurochem ; 156(4): 524-538, 2021 02.
Article in English | MEDLINE | ID: mdl-32683701

ABSTRACT

Many of the genes whose mutation causes Amyotrophic Lateral Sclerosis (ALS) are RNA-binding proteins which localize to stress granules, while others impact the assembly, stability, and elimination of stress granules. This has led to the hypothesis that alterations in the dynamics of stress granules and RNA biology cause ALS. Genetic mutations in Superoxide Dismutase 1 (SOD1) also cause ALS. Evidence demonstrates that SOD1 harboring ALS-linked mutations is recruited to stress granules, induces changes in alternative splicing, and could be an RNA-binding protein. Whether SOD1 inclusions contain RNA in disease models and whether SOD1 directly binds RNA remains uncertain. We applied methods including cross-linking immunoprecipitation and in vitro gel shift assays to detect binding of SOD1 to RNA in vitro, in cells with and without stress granules, and in mice expressing human SOD1 G93A. We find that SOD1 localizes to RNA-rich structures including stress granules, and SOD1 inclusions in mice contain mRNA. However, we find no evidence that SOD1 directly binds RNA. This suggests that SOD1 may impact stress granules, alternative splicing and RNA biology without binding directly to RNA.


Subject(s)
Cytoplasmic Granules/metabolism , Mutation/physiology , RNA/metabolism , Superoxide Dismutase/metabolism , Animals , Cytoplasmic Granules/chemistry , Cytoplasmic Granules/genetics , HeLa Cells , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Protein Binding/physiology , Protein Structure, Tertiary , RNA/analysis , RNA/genetics , Superoxide Dismutase/analysis , Superoxide Dismutase/genetics
7.
Nat Biomed Eng ; 4(1): 52-68, 2020 01.
Article in English | MEDLINE | ID: mdl-31937944

ABSTRACT

A small percentage of the short interfering RNA (siRNA) delivered via passive lipid nanoparticles and other delivery vehicles reaches the cytoplasm of cells. The high doses of siRNA and delivery vehicle that are thus required to achieve therapeutic outcomes can lead to toxicity. Here, we show that the integration of siRNA sequences into a Dicer-independent RNA stem-loop based on pre-miR-451 microRNA-which is highly enriched in small extracellular vesicles secreted by many cell types-reduces the expression of the genes targeted by the siRNA in the liver, intestine and kidney glomeruli of mice at siRNA doses that are at least tenfold lower than the siRNA doses typically delivered via lipid nanoparticles. Small extracellular vesicles that efficiently package siRNA can significantly reduce its therapeutic dose.


Subject(s)
Extracellular Vesicles/metabolism , MicroRNAs/metabolism , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/metabolism , Animals , Cell Line, Tumor , Drug Delivery Systems/methods , Gene Expression/drug effects , Humans , Mice , MicroRNAs/chemistry , Motor Neurons/drug effects , Nanoparticles/administration & dosage , RNA Interference , RNA, Small Interfering/chemistry
8.
Cell Rep ; 26(8): 2150-2165.e5, 2019 02 19.
Article in English | MEDLINE | ID: mdl-30784596

ABSTRACT

The autophagy pathway is an essential facet of the innate immune response, capable of rapidly targeting intracellular bacteria. However, the initial signaling regulating autophagy induction in response to pathogens remains largely unclear. Here, we report that AMPK, an upstream activator of the autophagy pathway, is stimulated upon detection of pathogenic bacteria, before bacterial invasion. Bacterial recognition occurs through the detection of outer membrane vesicles. We found that AMPK signaling relieves mTORC1-mediated repression of the autophagy pathway in response to infection, positioning the cell for a rapid induction of autophagy. Moreover, activation of AMPK and inhibition of mTORC1 in response to bacteria is not accompanied by an induction of bulk autophagy. However, AMPK signaling is required for the selective targeting of bacteria-containing vesicles by the autophagy pathway through the activation of pro-autophagic kinase complexes. These results demonstrate a key role for AMPK signaling in coordinating the rapid autophagic response to bacteria.


Subject(s)
Autophagy-Related Protein-1 Homolog/metabolism , Bacterial Outer Membrane/metabolism , Class III Phosphatidylinositol 3-Kinases/metabolism , Macroautophagy , Protein Kinases/metabolism , AMP-Activated Protein Kinase Kinases , Animals , Cells, Cultured , HCT116 Cells , HEK293 Cells , Host-Pathogen Interactions , Humans , MCF-7 Cells , Macrophages/metabolism , Macrophages/microbiology , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice , Mice, Inbred NOD , Salmonella/pathogenicity
9.
Mol Cell Oncol ; 5(3): e1445941, 2018.
Article in English | MEDLINE | ID: mdl-30250898

ABSTRACT

Autophagy-related-5 (Atg5) and Autophagy-related-16-Like-1 (Atg16L1) canonically participate in autophagy. Recent research demonstrates that apart from this, they also control production of extracellular vesicles called exosomes by regulating acidification of late endosomes. Atg5-mediated exosome production increased migration and metastasis of breast cancer cells suggesting exosomes may perform some functions ascribed to autophagy.

10.
Nat Commun ; 9(1): 2794, 2018 07 18.
Article in English | MEDLINE | ID: mdl-30022074

ABSTRACT

Mutations in proteins like FUS which cause Amyotrophic Lateral Sclerosis (ALS) result in the aberrant formation of stress granules while ALS-linked mutations in other proteins impede elimination of stress granules. Repeat expansions in C9ORF72, the major cause of ALS, reduce C9ORF72 levels but how this impacts stress granules is uncertain. Here, we demonstrate that C9ORF72 associates with the autophagy receptor p62 and controls elimination of stress granules by autophagy. This requires p62 to associate via the Tudor protein SMN with proteins, including FUS, that are symmetrically methylated on arginines. Mice lacking p62 accumulate arginine-methylated proteins and alterations in FUS-dependent splicing. Patients with C9ORF72 repeat expansions accumulate symmetric arginine dimethylated proteins which co-localize with p62. This suggests that C9ORF72 initiates a cascade of ALS-linked proteins (C9ORF72, p62, SMN, FUS) to recognize stress granules for degradation by autophagy and hallmarks of a defect in this process are observable in ALS patients.


Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Autophagy/genetics , C9orf72 Protein/genetics , RNA-Binding Protein FUS/genetics , Sequestosome-1 Protein/genetics , Survival of Motor Neuron 1 Protein/genetics , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Animals , Arginine/metabolism , C9orf72 Protein/metabolism , Cell Line, Tumor , Cytoplasmic Granules/metabolism , Cytoplasmic Granules/pathology , Embryo, Mammalian , HeLa Cells , Humans , Methylation , Mice , Mice, Knockout , Motor Neurons/cytology , Motor Neurons/metabolism , Primary Cell Culture , Protein-Arginine N-Methyltransferases/genetics , Protein-Arginine N-Methyltransferases/metabolism , RNA-Binding Protein FUS/metabolism , Sequestosome-1 Protein/metabolism , Stress, Physiological , Survival of Motor Neuron 1 Protein/metabolism
11.
Dev Cell ; 43(6): 716-730.e7, 2017 12 18.
Article in English | MEDLINE | ID: mdl-29257951

ABSTRACT

Autophagy and autophagy-related genes (Atg) have been attributed prominent roles in tumorigenesis, tumor growth, and metastasis. Extracellular vesicles called exosomes are also implicated in cancer metastasis. Here, we demonstrate that exosome production is strongly reduced in cells lacking Atg5 and Atg16L1, but this is independent of Atg7 and canonical autophagy. Atg5 specifically decreases acidification of late endosomes where exosomes are produced, disrupting the acidifying V1V0-ATPase by removing a regulatory component, ATP6V1E1, into exosomes. The effect of Atg5 on exosome production promotes the migration and in vivo metastasis of orthotopic breast cancer cells. These findings uncover mechanisms controlling exosome release and identify means by which autophagy-related genes can contribute to metastasis in autophagy-independent pathways.


Subject(s)
Autophagy-Related Protein 5/metabolism , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Vacuolar Proton-Translocating ATPases/metabolism , Animals , Autophagy/physiology , Autophagy-Related Protein 5/genetics , Autophagy-Related Protein 7/genetics , Autophagy-Related Protein 7/metabolism , Cell Line, Tumor/metabolism , Endosomes/metabolism , Exosomes/metabolism , Female , Humans , Lysosomes/metabolism , Mice , Mice, Inbred BALB C , Neoplasm Metastasis , Vacuolar Proton-Translocating ATPases/genetics
12.
EMBO Rep ; 16(10): 1334-57, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26265008

ABSTRACT

In embryonic stem cells (ESCs), gene regulatory networks (GRNs) coordinate gene expression to maintain ESC identity; however, the complete repertoire of factors regulating the ESC state is not fully understood. Our previous temporal microarray analysis of ESC commitment identified the E3 ubiquitin ligase protein Makorin-1 (MKRN1) as a potential novel component of the ESC GRN. Here, using multilayered systems-level analyses, we compiled a MKRN1-centered interactome in undifferentiated ESCs at the proteomic and ribonomic level. Proteomic analyses in undifferentiated ESCs revealed that MKRN1 associates with RNA-binding proteins, and ensuing RIP-chip analysis determined that MKRN1 associates with mRNAs encoding functionally related proteins including proteins that function during cellular stress. Subsequent biological validation identified MKRN1 as a novel stress granule-resident protein, although MKRN1 is not required for stress granule formation, or survival of unstressed ESCs. Thus, our unbiased systems-level analyses support a role for the E3 ligase MKRN1 as a ribonucleoprotein within the ESC GRN.


Subject(s)
Embryonic Stem Cells/physiology , Gene Regulatory Networks/genetics , Nerve Tissue Proteins/genetics , Ribonucleoproteins/genetics , Animals , Cytoplasm/metabolism , Genomics , Mice , Nerve Tissue Proteins/chemistry , Proteomics , RNA/metabolism , RNA-Binding Proteins/metabolism , Ribonucleoproteins/chemistry , Ubiquitin-Protein Ligases/metabolism
14.
Nat Commun ; 5: 5276, 2014 Nov 04.
Article in English | MEDLINE | ID: mdl-25366815

ABSTRACT

Many cytoplasmic substrates degraded by autophagy have been identified; however, the impact of RNA degradation by autophagy remains uncertain. Retrotransposons comprise 40% of the human genome and are a major source of genetic variation among species, individuals and cells. Retrotransposons replicate via a copy-paste mechanism involving a cytoplasmic RNA intermediate. Here we report that autophagy degrades retrotransposon RNA from both long and short interspersed elements, preventing new retrotransposon insertions into the genome. Retrotransposon RNA localizes to RNA granules, whose selective degradation is facilitated by the autophagy receptors NDP52 and p62. Accordingly, NDP52 and p62 control retrotransposon insertion in the genome. Mice lacking a copy of Atg6/Beclin1, a gene critical for autophagy, also accumulate both retrotransposon RNA and genomic insertions. Thus, autophagy physiologically buffers genetic variegation by degrading retrotransposon RNA. This may contribute to the increased tumorigenesis occuring when autophagy is inhibited and suggest a role for autophagy in tempering evolutionary change.


Subject(s)
Alu Elements , Autophagy , Long Interspersed Nucleotide Elements , Nuclear Proteins/metabolism , RNA-Binding Proteins/metabolism , Animals , Apoptosis Regulatory Proteins/metabolism , Beclin-1 , Genomic Instability , HEK293 Cells , HeLa Cells , Humans , Membrane Proteins/metabolism , Mice , Phagosomes/metabolism , RNA/metabolism
15.
RNA ; 20(2): 143-9, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24355758

ABSTRACT

Pharmacologic target gene modulation is the primary objective for RNA antagonist strategies and gene therapy. Here we show that mRNAs encoding tissue-specific gene transcripts can be detected in biological fluids and that RNAi-mediated target gene silencing in the liver and brain results in quantitative reductions in serum and cerebrospinal fluid mRNA levels, respectively. Further, administration of an anti-miRNA oligonucleotide resulted in decreased levels of the miRNA in circulation. Moreover, ectopic expression of an adenoviral transgene in the liver was quantified based on measurement of serum mRNA levels. This noninvasive method for monitoring tissue-specific RNA modulation could greatly advance the clinical development of RNA-based therapeutics.


Subject(s)
Gene Knockdown Techniques , RNA Interference , RNA, Messenger/blood , Aged , Animals , Biomarkers, Tumor/blood , Biomarkers, Tumor/genetics , Gene Expression , Glypicans/genetics , Humans , Liver/metabolism , Macaca fascicularis , Male , Organ Specificity , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Rats , Rats, Sprague-Dawley , Species Specificity , alpha-Fetoproteins/genetics
16.
Autophagy ; 9(5): 781-3, 2013 May.
Article in English | MEDLINE | ID: mdl-23422216

ABSTRACT

MicroRNAs (miRNAs) form a class of ~21 nucleotide (nt) RNAs that post-transcriptionally repress partially complementary messenger RNAs. miRNA-mediated silencing is critical for control of many key biological processes such as tumorigenesis, neuronal synaptic plasticity and defense against bacteria and viruses. Thus, unsurprisingly, miRNA biogenesis, abundance and action are under refined feedback control that is only beginning to be experimentally uncovered. We recently discovered that DICER1 and EIF2C/AGO are targeted for degradation by autophagy as miRNA-free entities by the selective autophagy receptor CALCOCO2/NDP52 (calcium binding and coiled-coil domain 2/nuclear dot protein, 52 kDa). Strikingly, autophagy establishes a checkpoint for continued loading of miRNA, and this checkpoint is required for maintenance of miRNA abundance and proper miRNA activity. This newfound role for autophagy in miRNA biology suggests that human diseases exhibiting misregulated autophagy may be interdependent with defects in miRNA-mediated regulation of gene networks.


Subject(s)
Autophagy/genetics , Homeostasis/genetics , MicroRNAs/metabolism , Eukaryotic Initiation Factors/metabolism , Humans , MicroRNAs/genetics , Models, Biological , RNA Interference , Ribonuclease III/metabolism , Ubiquitination
17.
Nat Cell Biol ; 14(12): 1314-21, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23143396

ABSTRACT

MicroRNAs (miRNAs) form a class of short RNAs (∼ 21 nucleotides) that post-transcriptionally regulate partially complementary messenger RNAs. Each miRNA may target tens to hundreds of transcripts to control key biological processes. Although the biochemical reactions underpinning miRNA biogenesis and activity are relatively well defined and the importance of their homeostasis is increasingly evident, the processes underlying regulation of the miRNA pathway in vivo are still largely elusive. Autophagy, a degradative process in which cytoplasmic material is targeted into double-membrane vacuoles, is recognized to critically contribute to cellular homeostasis. Here, we show that the miRNA-processing enzyme, DICER (also known as DICER1), and the main miRNA effector, AGO2 (also known as eukaryotic translation initiation factor 2C, 2 (EIF2C2)), are targeted for degradation as miRNA-free entities by the selective autophagy receptor NDP52 (also known as calcium binding and coiled-coil domain 2 (CALCOCO2)). Autophagy establishes a checkpoint required for continued loading of miRNA into AGO2; accordingly, NDP52 and autophagy are required for homeostasis and activity of the tested miRNAs. Autophagy also engages post-transcriptional regulation of the DICER mRNA, underscoring the importance of fine-tuned regulation of the miRNA pathway. These findings have implications for human diseases linked to misregulated autophagy, DICER- and miRNA-levels, including cancer.


Subject(s)
Argonaute Proteins/metabolism , Autophagy/physiology , MicroRNAs/metabolism , Ribonuclease III/metabolism , Argonaute Proteins/genetics , Autophagy/genetics , Cell Line , Cell Line, Tumor , HeLa Cells , Humans , Immunoprecipitation , Microscopy, Confocal , Microscopy, Electron , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Ribonuclease III/genetics
18.
Nat Struct Mol Biol ; 19(5): 517-24, S1, 2012 Apr 08.
Article in English | MEDLINE | ID: mdl-22484317

ABSTRACT

Despite intense research in the context of neurodegenerative diseases associated with its misfolding, the endogenous human prion protein PrP(C) (or PRNP) has poorly understood physiological functions. Whereas most PrP(C) is exposed to the extracellular environment, conserved domains result in transmembrane forms of PrP(C) that traffic in the endolysosomal system and are linked to inherited and infectious neuropathologies. One transmembrane PrP(C) variant orients the N-terminal 'octarepeat' domain into the cytoplasm. Here we demonstrate that the octarepeat domain of human PrP(C) contains GW/WG motifs that bind Argonaute (AGO) proteins, the essential components of microRNA (miRNA)-induced silencing complexes (miRISCs). Transmembrane PrP(C) preferentially binds AGO, and PrP(C) promotes formation or stability of miRISC effector complexes containing the trinucleotide repeat-containing gene 6 proteins (TNRC6) and miRNA-repressed mRNA. Accordingly, effective repression of several miRNA targets requires PrP(C). We propose that dynamic interactions between PrP(C)-enriched endosomes and subcellular foci of AGO underpin these effects.


Subject(s)
Argonaute Proteins/metabolism , MicroRNAs/metabolism , PrPC Proteins/metabolism , Amino Acid Sequence , Animals , Argonaute Proteins/analysis , Autoantigens/metabolism , Cell Line , Humans , Mice , Molecular Sequence Data , PrPC Proteins/analysis , PrPC Proteins/chemistry , Protein Binding , Protein Structure, Tertiary , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Ribonuclease III/metabolism
19.
Methods Mol Biol ; 725: 63-76, 2011.
Article in English | MEDLINE | ID: mdl-21528447

ABSTRACT

Most complexes involved in RNA silencing were thought to be concentrated in cytoplasmic sites called P-bodies in the absence of stress. Accumulating evidence suggests that distinct cellular organelles or sites may be involved in the maturation of RNA-induced silencing complexes (RISC), decapping and deadenylation of miRNA-repressed mRNA, transport of translationally repressed mRNA, and disassembly of RISC complexes. Significant fractions of proteins essential for RNA silencing associate with membranes in general (GW182, AGO, and DICER), or more specifically with endoplasmic reticulum and Golgi (AGO), or endosomes and multivesicular bodies (AGO, GW182). In contrast, mRNA decapping and decay occur mainly in the cytoplasm. Continuous density gradients capable of partitioning these cellular compartments are valuable tools in efforts to decipher the complexes, trafficking and regulation of RISC throughout its biogenesis, action and turnover.


Subject(s)
Autoantigens/isolation & purification , Autoantigens/metabolism , Biochemistry/methods , Eukaryotic Initiation Factors/isolation & purification , Eukaryotic Initiation Factors/metabolism , RNA-Binding Proteins/isolation & purification , RNA-Binding Proteins/metabolism , Transport Vesicles/metabolism , Biological Transport/physiology , Cell Line , Centrifugation, Density Gradient , Fractional Precipitation , HeLa Cells , Humans , RNA/isolation & purification , RNA/metabolism
20.
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
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