[Show abstract][Hide abstract] ABSTRACT: Skeletal muscle contains long multinucleated and contractile structures known as muscle fibers, which arise from the fusion
of myoblasts into multinucleated myotubes during myogenesis. The myogenic regulatory factor (MRF) MYF5 is the earliest to
be expressed during myogenesis and functions as a transcription factor in muscle progenitor cells (satellite cells) and myocytes.
In mouse C2C12 myocytes, MYF5 is implicated in the initial steps of myoblast differentiation into myotubes. Here, using ribonucleoprotein
immunoprecipitation (RIP) analysis, we discovered a novel function for MYF5 as an RNA-binding protein which associated with
a subset of myoblast mRNAs. One prominent MYF5 target was Ccnd1 mRNA, which encodes the key cell cycle regulator CCND1 (Cyclin D1). Biotin-RNA pulldown, UV-crosslinking and gel shift experiments
indicated that MYF5 was capable of binding the 3′ untranslated region (UTR) and the coding region (CR) of Ccnd1 mRNA. Silencing MYF5 expression in proliferating myoblasts revealed that MYF5 promoted CCND1 translation and modestly increased
transcription of Ccnd1 mRNA. Accordingly, overexpressing MYF5 in C2C12 cells upregulated CCND1 expression while silencing MYF5 reduced myoblast
proliferation as well as differentiation of myoblasts into myotubes. Moreover, MYF5 silencing reduced myogenesis, while ectopically
restoring CCND1 abundance partially rescued the decrease in myogenesis seen after MYF5 silencing. We propose that MYF5 enhances
early myogenesis in part by coordinately elevating Ccnd1 transcription and Ccnd1 mRNA translation.
Preview · Article · Jan 2016 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: The function of the vast majority of mammalian long noncoding (lnc) RNAs remains unknown. Here, analysis of a highly abundant
mammalian lncRNA, OIP5-AS1, known as cyrano in zebrafish, revealed that OIP5-AS1 reduces cell proliferation. In human cervical carcinoma HeLa cells, the RNA-binding protein HuR, which enhances cell proliferation,
associated with OIP5-AS1 and stabilized it. Tagging OIP5-AS1 with MS2 hairpins to identify associated microRNAs revealed that miR-424 interacted with OIP5-AS1 and competed with HuR for binding to OIP5-AS1. We further identified a ‘sponge’ function for OIP5-AS1, as high levels of OIP5-AS1 increased HuR-OIP5-AS1 complexes and prevented HuR interaction with target mRNAs, including those that encoded proliferative proteins, while conversely,
lowering OIP5-AS1 increased the abundance of HuR complexes with target mRNAs. We propose that OIP5-AS1 serves as a sponge or a competing endogenous (ce)RNA for HuR, restricting its availability to HuR target mRNAs and thereby
repressing HuR-elicited proliferative phenotypes.
Full-text · Article · Jan 2016 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: The tRNA methytransferase NSun2 promotes cell proliferation, but the molecular mechanism has not been elucidated. Here, we report that NSun2 regulates CDK1 expression in a cell cycle-dependent manner. Knockdown of NSun2 decreased CDK1 protein level, while overexpression of NSun2 elevated it without altering
mRNA levels. Further studies revealed that NSun2 methylated
and in cells and that methylation by NSun2 enhanced CDK1 translation. Importantly, NSun2-mediated regulation of CDK1 expression had an impact on the cell division cycle. These results provide new insight for the regulation of CDK1 during the cell division cycle.
No preview · Article · Sep 2015 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: Vascular endothelial growth factor (VEGF) A is a master regulator of neovascularization and angiogenesis. VEGFA is potently induced by hypoxia and by pathological conditions including diabetic retinopathy and tumorigenesis. Fine-tuning of VEGFA expression by different stimuli is important for maintaining tissue vascularization and organ homeostasis. Here, we tested the effect of the hypoxia mimetic cobalt chloride (CoCl2) on VEGFA expression in human cervical carcinoma HeLa cells. We found that CoCl2 increased the levels of VEGFA mRNA and VEGFA protein without affecting VEGFA mRNA stability. Biotin pulldown analysis to capture the RNA-binding proteins (RBPs) bound to VEGFA mRNA followed by mass spectrometry analysis revealed that the RBP HuR [human antigen R, a member of the embryonic lethal abnormal vision (ELAV) family of proteins], interacts with VEGFA mRNA. VEGFA mRNA-tagging experiments showed that exposure to CoCl2 increases the interaction of HuR with VEGFA mRNA and promoted the colocalization of HuR and the distal part of the VEGFA 3'-untranslated region (UTR) in the cytoplasm. We propose that under hypoxia-like conditions, HuR enhances VEGFA mRNA translation.
[Show abstract][Hide abstract] ABSTRACT: Post-transcriptional gene regulation is robustly regulated by RNA-binding proteins (RBPs). Here we describe the collection of RNAs regulated by AUF1 (AU-binding factor 1), an RBP linked to cancer, inflammation and aging. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis reveals that AUF1 primarily recognizes U-/GU-rich sequences in mRNAs and noncoding RNAs and influences target transcript fate in three main directions. First, AUF1 lowers the steady-state levels of numerous target RNAs, including long noncoding RNA NEAT1, in turn affecting the organization of nuclear paraspeckles. Second, AUF1 does not change the abundance of many target RNAs, but ribosome profiling reveals that AUF1 promotes the translation of numerous mRNAs in this group. Third, AUF1 unexpectedly enhances the steady-state levels of several target mRNAs encoding DNA-maintenance proteins. Through its actions on target RNAs, AUF1 preserves genomic integrity, in agreement with the AUF1-elicited prevention of premature cellular senescence.
Full-text · Article · Nov 2014 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: Noncoding RNAs (ncRNAs) and RNA-binding proteins are potent post-transcriptional regulators of gene expression. The ncRNA
7SL is upregulated in cancer cells, but its impact upon the phenotype of cancer cells is unknown. Here, we present evidence that
7SL forms a partial hybrid with the 3′-untranslated region (UTR) of TP53 mRNA, which encodes the tumor suppressor p53. The interaction of 7SL with TP53 mRNA reduced p53 translation, as determined by analyzing p53 expression levels, nascent p53 translation and TP53 mRNA association with polysomes. Silencing 7SL led to increased binding of HuR to TP53 mRNA, an interaction that led to the promotion of p53 translation and increased p53 abundance. We propose that the competition
between 7SL and HuR for binding to TP53 3′UTR contributes to determining the magnitude of p53 translation, in turn affecting p53 levels and the growth-suppressive
function of p53. Our findings suggest that targeting 7SL may be effective in the treatment of cancers with reduced p53 levels.
Full-text · Article · Aug 2014 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: The mammalian RNA-binding protein AUF1 (AU-binding factor 1, also known as heterogeneous nuclear ribonucleoprotein D [hnRNP
D]) binds to numerous mRNAs and influences their posttranscriptional fate. Given that many AUF1 target mRNAs encode muscle-specific
factors, we investigated the function of AUF1 in skeletal muscle differentiation. In mouse C2C12 myocytes, where AUF1 levels
rise at the onset of myogenesis and remain elevated throughout myocyte differentiation into myotubes, RNP immunoprecipitation
(RIP) analysis indicated that AUF1 binds prominently to Mef2c (myocyte enhancer factor 2c) mRNA, which encodes the key myogenic transcription factor MEF2C. By performing mRNA half-life
measurements and polysome distribution analysis, we found that AUF1 associated with the 3′ untranslated region (UTR) of Mef2c mRNA and promoted MEF2C translation without affecting Mef2c mRNA stability. In addition, AUF1 promoted Mef2c gene transcription via a lesser-known role of AUF1 in transcriptional regulation. Importantly, lowering AUF1 delayed myogenesis,
while ectopically restoring MEF2C expression levels partially rescued the impairment of myogenesis seen after reducing AUF1
levels. We propose that MEF2C is a key effector of the myogenesis program promoted by AUF1.
Full-text · Article · Jun 2014 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: The primarily neuronal RNA-binding protein HuD is implicated in learning and memory. Here, we report the identification of several HuD target transcripts linked to Alzheimer's disease (AD) pathogenesis. HuD interacted with the 3' UTRs of APP mRNA (encoding amyloid precursor protein) and BACE1 mRNA (encoding β-site APP-cleaving enzyme 1) and increased the half-lives of these mRNAs. HuD also associated with and stabilized the long noncoding (lnc)RNA BACE1AS, which partly complements BACE1 mRNA and enhances BACE1 expression. Consistent with HuD promoting production of APP and APP-cleaving enzyme, the levels of APP, BACE1, BACE1AS, and Aβ were higher in the brain of HuD-overexpressing mice. Importantly, cortex (superior temporal gyrus) from patients with AD displayed significantly higher levels of HuD and, accordingly, elevated APP, BACE1, BACE1AS, and Aβ than did cortical tissue from healthy age-matched individuals. We propose that HuD jointly promotes the production of APP and the cleavage of its amyloidogenic fragment, Aβ.
[Show abstract][Hide abstract] ABSTRACT: Although mammalian long non-coding (lnc)RNAs are best known for modulating transcription, their post-transcriptional influence on mRNA splicing, stability and translation is emerging. Here we report a post-translational function for the lncRNA HOTAIR as an inducer of ubiquitin-mediated proteolysis. HOTAIR associates with E3 ubiquitin ligases bearing RNA-binding domains, Dzip3 and Mex3b, as well as with their respective ubiquitination substrates, Ataxin-1 and Snurportin-1. In this manner, HOTAIR facilitates the ubiquitination of Ataxin-1 by Dzip3 and Snurportin-1 by Mex3b in cells and in vitro, and accelerates their degradation. HOTAIR levels are highly upregulated in senescent cells, causing rapid decay of targets Ataxin-1 and Snurportin-1, and preventing premature senescence. These results uncover a role for a lncRNA, HOTAIR, as a platform for protein ubiquitination.
Full-text · Article · Dec 2013 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: In response to stress conditions, many mammalian mRNAs accumulate in stress granules (SGs) together with numerous RNA-binding
proteins that control mRNA turnover and translation. However, the signaling cascades that modulate the presence of ribonucleoprotein
(RNP) complexes in SGs are poorly understood. Here, we investigated the localization of human antigen R (HuR), an mRNA-stabilizing
RNA-binding protein, in SGs following exposure to the stress agent arsenite. Unexpectedly, the mobilization of HuR to SGs
was prevented through the activation of Janus kinase 3 (JAK3) by the vitamin K3 analog menadione. JAK3 phosphorylated HuR
at tyrosine 200, in turn inhibiting HuR localization in SGs, reducing HuR interaction with targets SIRT1 and VHL mRNAs, and accelerating target mRNA decay. Our findings indicate that HuR is tyrosine-phosphorylated by JAK3, and link this
modification to HuR subcytoplasmic localization and to the fate of HuR target mRNAs.
Full-text · Article · Oct 2013 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: A hallmark trait of cellular senescence is the acquisition of a senescence-associated secretory phenotype (SASP). SASP factors include cytokines and their receptors (IL-6, IL-8, osteoprotegerin, GM-CSF), chemokines and their ligands (MCP-1, HCC4), and oncogenes (Gro1 and Gro2), many of them encoded by mRNAs whose stability and translation are tightly regulated. Using two models of human fibroblast senescence (WI-38 and IDH4 cells), we report the identification of RNA-binding protein NF90 as a post-transcriptional repressor of several SASP factors. In 'young', proliferating fibroblasts, NF90 was highly abundant, associated with numerous SASP mRNAs, and inhibited their expression. By contrast, senescent cells expressed low levels of NF90, thus allowing SASP factor expression to increase. NF90 elicited these effects mainly by repressing the translation of target SASP mRNAs, since silencing NF90 did not increase the steady-state levels of SASP mRNAs but elevated key SASP factors including MCP-1, GROa, IL-6, and IL-8. Our findings indicate that NF90 contributes to maintaining low levels of SASP factors in non-senescent cells, while NF90 reduction in senescent cells allows SASP factor expression to rise.
[Show abstract][Hide abstract] ABSTRACT: Mammalian long intergenic noncoding RNAs (lincRNAs) are best known for modulating transcription. Here we report a posttranscriptional function for lincRNA-p21 as a modulator of translation. Association of the RNA-binding protein HuR with lincRNA-p21 favored the recruitment of let-7/Ago2 to lincRNA-p21, leading to lower lincRNA-p21 stability. Under reduced HuR levels, lincRNA-p21 accumulated in human cervical carcinoma HeLa cells, increasing its association with JUNB and CTNNB1 mRNAs and selectively lowering their translation. With elevated HuR, lincRNA-p21 levels declined, which in turn derepressed JunB and β-catenin translation and increased the levels of these proteins. We propose that HuR controls translation of a subset of target mRNAs by influencing lincRNA-p21 levels. Our findings uncover a role for lincRNA as a posttranscriptional inhibitor of translation.
[Show abstract][Hide abstract] ABSTRACT: The microRNA miR-519 robustly inhibits cell proliferation, in turn triggering senescence and decreasing tumor growth. However,
the molecular mediators of miR-519-elicited growth inhibition are unknown. Here, we systematically investigated the influence
of miR-519 on gene expression profiles leading to growth cessation in HeLa human cervical carcinoma cells. By analyzing miR-519-triggered
changes in protein and mRNA expression patterns and by identifying mRNAs associated with biotinylated miR-519, we uncovered
two prominent subsets of miR-519-regulated mRNAs. One subset of miR-519 target mRNAs encoded DNA maintenance proteins (including
DUT1, EXO1, RPA2, and POLE4); miR-519 repressed their expression and increased DNA damage, in turn raising the levels of the
cyclin-dependent kinase (cdk) inhibitor p21. The other subset of miR-519 target mRNAs encoded proteins that control intracellular
calcium levels (notably, ATP2C1 and ORAI1); their downregulation by miR-519 aberrantly elevated levels of cytosolic [Ca2+] storage in HeLa cells, similarly increasing p21 levels in a manner dependent on the Ca2+-activated kinases CaMKII and GSK3β. The rises in levels of DNA damage, the Ca2+ concentration, and p21 levels stimulated an autophagic phenotype in HeLa and other human carcinoma cell lines. As a consequence,
ATP levels increased, and the level of activity of the AMP-activated protein kinase (AMPK) declined, further contributing
to the elevation in the abundance of p21. Our results indicate that miR-519 promotes DNA damage, alters Ca2+ homeostasis, and enhances energy production; together, these processes elevate the expression level of p21, promoting growth
inhibition and cell survival.
[Show abstract][Hide abstract] ABSTRACT: Although expression of the mammalian RNA-binding protein HuD was considered to be restricted to neurons, we report that HuD is present in pancreatic β cells, where its levels are controlled by the insulin receptor pathway. We found that HuD associated with a 22-nucleotide segment of the 5' untranslated region (UTR) of preproinsulin (Ins2) mRNA. Modulating HuD abundance did not alter Ins2 mRNA levels, but HuD overexpression decreased Ins2 mRNA translation and insulin production, and conversely, HuD silencing enhanced Ins2 mRNA translation and insulin production. Following treatment with glucose, HuD rapidly dissociated from Ins2 mRNA and enabled insulin biosynthesis. Importantly, HuD-knockout mice displayed higher insulin levels in pancreatic islets, while HuD-overexpressing mice exhibited lower insulin levels in islets and in plasma. In sum, our results identify HuD as a pivotal regulator of insulin translation in pancreatic β cells.
[Show abstract][Hide abstract] ABSTRACT: The cellular abundance of topoisomerase IIα (TOP2A) critically maintains DNA topology after replication and determines the
efficacy of TOP2 inhibitors in chemotherapy. Here, we report that the RNA-binding protein HuR, commonly overexpressed in cancers,
binds to the TOP2A 3′-untranslated region (3′UTR) and increases TOP2A translation. Reducing HuR levels triggered the recruitment of TOP2A transcripts to RNA-induced silencing complex (RISC) components and to cytoplasmic processing bodies. Using a novel MS2-tagged
RNA precipitation method, we identified microRNA miR-548c-3p as a mediator of these effects and further uncovered that the
interaction of miR-548c-3p with the TOP2A 3′UTR repressed TOP2A translation by antagonizing the action of HuR. Lowering TOP2A by silencing HuR or by overexpressing
miR-548c-3p selectively decreased DNA damage after treatment with the chemotherapeutic agent doxorubicin. In sum, HuR enhances
TOP2A translation by competing with miR-548c-3p; their combined actions control TOP2A expression levels and determine the
effectiveness of doxorubicin.
[Show abstract][Hide abstract] ABSTRACT: RNA-binding proteins (RBPs) regulate gene expression at many post-transcriptional levels, including mRNA stability and translation.
The RBP nucleolin, with four RNA-recognition motifs, has been implicated in cell proliferation, carcinogenesis and viral infection.
However, the subset of nucleolin target mRNAs and the influence of nucleolin on their expression had not been studied at a
transcriptome-wide level. Here, we globally identified nucleolin target transcripts, many of which encoded cell growth- and
cancer-related proteins, and used them to find a signature motif on nucleolin target mRNAs. Surprisingly, this motif was very
rich in G residues and was not only found in the 3′-untranslated region (UTR), but also in the coding region (CR) and 5′-UTR.
Nucleolin enhanced the translation of mRNAs bearing the G-rich motif, since silencing nucleolin did not change target mRNA
stability, but decreased the size of polysomes forming on target transcripts and lowered the abundance of the encoded proteins.
In summary, nucleolin binds G-rich sequences in the CR and UTRs of target mRNAs, many of which encode cancer proteins, and
enhances their translation.
Full-text · Article · Jul 2011 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Ionizing radiation (IR) triggers adaptive changes in gene expression. Here, we show that survival after IR strongly depends on the checkpoint kinase Chk2 acting upon its substrate HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. Microarray analysis showed that in human HCT116 colorectal carcinoma cells (WT), IR-activated Chk2 triggered the dissociation of virtually all of HuR-bound mRNAs, since IR did not dissociate HuR target mRNAs in Chk2-null (CHK2-/-) HCT116 cells. Accordingly, several HuR-interacting mRNAs encoding apoptosis- and proliferation-related proteins (TJP1, Mdm2, TP53BP2, Bax, K-Ras) dissociated from HuR in WT cells, but remained bound and showed altered post-transcriptional regulation in CHK2-/- cells. Use of HuR mutants that were not phosphorylatable by Chk2 (HuR(3A)) and HuR mutants mimicking constitutive phosphorylation by Chk2 (HuR(3D)) revealed that dissociation of HuR target transcripts enhanced cell survival. We propose that the release of HuR-bound mRNAs via an IR-Chk2-HuR regulatory axis improves cell outcome following IR.