RNA Destabilization Research Articles

Hepatic tristetraprolin promotes insulin resistance through RNA destabilization of FGF21.

The role of posttranscriptional metabolic gene regulatory programs in diabetes is not well understood. Here, we show that the RNA-binding protein tristetraprolin (TTP) is reduced in the livers of diabetic mice and humans and is transcriptionally induced in response to insulin treatment in murine livers in vitro and in vivo. Liver-specific Ttp-KO (lsTtp-KO) mice challenged with high-fat diet (HFD) have improved glucose tolerance and peripheral insulin sensitivity compared with littermate controls. Analysis of secreted hepatic factors demonstrated that fibroblast growth factor 21 (FGF21) is posttranscriptionally repressed by TTP. Consistent with increased FGF21, lsTtp-KO mice fed HFD have increased brown fat activation, peripheral tissue glucose uptake, and adiponectin production compared with littermate controls. Downregulation of hepatic Fgf21 via an adeno-associated virus-driven shRNA in mice fed HFD reverses the insulin-sensitizing effects of hepatic Ttp deletion. Thus, hepatic TTP posttranscriptionally regulates systemic insulin sensitivity in diabetes through liver-derived FGF21.

Silencing messages in a unique way.

A global analysis of translation efficiency and RNA accumulation revealed that microRNAs in the unicellular green alga Chlamydomonas regulate target gene expression via RNA destabilization and translational repression like land plants and metazoans, yet in a different way.

RNA Binding Protein Human Antigen R Is Translocated in Astrocytes following Spinal Cord Injury and Promotes the Inflammatory Response.

Inflammation plays a prominent role in the events following traumatic injury to the central nervous system (CNS). The initial inflammatory response is driven by mediators such as tumor necrosis factor α and interleukin 1β, which are produced by activated astrocytes and microglia at the site of injury. These factors are regulated post-transcriptionally by RNA binding proteins (RBP) that interact with adenylate and uridylate-rich elements (ARE) in the 3'-untranslated region of the messenger RNA (mRNA). Human antigen R (HuR) is one of these RBPs and generally functions as a positive regulator of ARE-containing mRNAs. Here, we hypothesized that HuR plays an important role in the induction of cytokine and chemokines in astrocytes following traumatic injury. Using a mouse model of spinal cord injury, we found HuR to be extensively translocated to the cytoplasm in astrocytes at the level of injury, consistent with its activation. In an in vitro stretch injury model of CNS trauma, we observed a similar cytoplasmic shift of HuR in astrocytes and an attenuation of cytokine induction with HuR knockdown. RNA kinetics and luciferase assays suggested that the effect was more related to transcription than RNA destabilization. A small molecule inhibitor of HuR suppressed cytokine induction of injured astrocytes and reduced chemoattraction for neutrophils and microglia. In summary, HuR is activated in astrocytes in the early stages of CNS trauma and positively regulates the molecular response of key inflammatory mediators in astrocytes. Our findings suggest that HuR may be a therapeutic target in acute CNS trauma for blunting secondary tissue injury triggered by the inflammatory response.

Proteomic Analysis Implicates Dominant Alterations of RNA Metabolism and the Proteasome Pathway in the Cellular Response to Carbon-Ion Irradiation.

Radiotherapy with heavy ions is considered advantageous compared to irradiation with photons due to the characteristics of the Braggs peak and the high linear energy transfer (LET) value. To understand the mechanisms of cellular responses to different LET values and dosages of heavy ion radiation, we analyzed the proteomic profiles of mouse embryo fibroblast MEF cells exposed to two doses from different LET values of heavy ion 12C. Total proteins were extracted from these cells and examined by Q Exactive with Liquid Chromatography (LC)—Electrospray Ionization (ESI) Tandem MS (MS/MS). Using bioinformatics approaches, differentially expressed proteins with 1.5 or 2.0-fold changes between different dosages of exposure were compared. With the higher the dosage and/or LET of ion irradiation, the worse response the cells were in terms of protein expression. For instance, compared to the control (0 Gy), 771 (20.2%) proteins in cells irradiated at 0.2 Gy of carbon-ion radiation with 12.6 keV/μm, 313 proteins (8.2%) in cells irradiated at 2 Gy of carbon-ion radiation with 12.6 keV/μm, and 243 proteins (6.4%) in cells irradiated at 2 Gy of carbon-ion radiation with 31.5 keV/μm exhibited changes of 1.5-fold or greater. Gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, Munich Information Center for Protein Sequences (MIPS) analysis, and BioCarta analysis all indicated that RNA metabolic processes (RNA splicing, destabilization and deadenylation) and proteasome pathways may play key roles in the cellular response to heavy-ion irradiation. Proteasome pathways ranked highest among all biological processes associated with heavy carbon-ion irradiation. In addition, network analysis revealed that cellular pathways involving proteins such as Col1a1 and Fn1 continued to respond to high dosages of heavy-ion irradiation, suggesting that these pathways still protect cells against damage. However, pathways such as those involving Ikbkg1 responded better at lower dosages than at higher dosages, implying that cell damage would occur when the networks involving these proteins stop responding. Our investigation provides valuable proteomic information for elucidating the mechanism of biological effects induced by carbon ions in general.

Cotranslational microRNA mediated messenger RNA destabilization.

MicroRNAs are small (22 nucleotide) regulatory molecules that play important roles in a wide variety of biological processes. These RNAs, which bind to targeted mRNAs via limited base pairing interactions, act to reduce protein production from those mRNAs. Considerable evidence indicates that miRNAs destabilize targeted mRNAs by recruiting enzymes that function in normal mRNA decay and mRNA degradation is widely thought to occur when mRNAs are in a ribosome free state. Nevertheless, when examined, miRNA targeted mRNAs are invariably found to be polysome associated; observations that appear to be at face value incompatible with a simple decay model. Here, we provide evidence that turnover of miRNA-targeted mRNAs occurs while they are being translated. Cotranslational mRNA degradation is initiated by decapping and proceeds 5' to 3' behind the last translating ribosome. These results provide an explanation for a long standing mystery in the miRNA field.

Transcriptome-Wide Cleavage Site Mapping on Cellular mRNAs Reveals Features Underlying Sequence-Specific Cleavage by the Viral Ribonuclease SOX.

Many viruses express factors that reduce host gene expression through widespread degradation of cellular mRNA. An example of this class of proteins is the mRNA-targeting endoribonuclease SOX from the gamma-herpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV). Previous studies indicated that cleavage of messenger RNAs (mRNA) by SOX occurs at specific locations defined by the sequence of the target RNA, which is at odds with the down-regulation of a large portion of cellular transcripts. In this study, we address this paradox by using high-throughput sequencing of cleavage intermediates combined with a custom bioinformatics-based analysis pipeline to identify SOX cleavage sites across the mRNA transcriptome. These data, coupled with targeted mutagenesis, reveal that while cleavage sites are specific and reproducible, they are defined by a degenerate sequence motif containing a small number of conserved residues rather than a strong consensus sequence. This degenerate element is well represented in both human and KSHV mRNA, and its presence correlates with RNA destabilization by SOX. This represents a new endonuclease targeting strategy, in which use of a degenerate targeting element enables RNA cleavage at specific locations without restricting the range of targets. Furthermore, it shows that strong target selectivity can be achieved without a high degree of sequence specificity.

When core competence is not enough: functional interplay of the DEAD-box helicase core with ancillary domains and auxiliary factors in RNA binding and unwinding.

DEAD-box helicases catalyze RNA duplex unwinding in an ATP-dependent reaction. Members of the DEAD-box helicase family consist of a common helicase core formed by two RecA-like domains. According to the current mechanistic model for DEAD-box mediated RNA unwinding, binding of RNA and ATP triggers a conformational change of the helicase core, and leads to formation of a compact, closed state. In the closed conformation, the two parts of the active site for ATP hydrolysis and of the RNA binding site, residing on the two RecA domains, become aligned. Closing of the helicase core is coupled to a deformation of the RNA backbone and destabilization of the RNA duplex, allowing for dissociation of one of the strands. The second strand remains bound to the helicase core until ATP hydrolysis and product release lead to re-opening of the core. The concomitant disruption of the RNA binding site causes dissociation of the second strand. The activity of the helicase core can be modulated by interaction partners, and by flanking N- and C-terminal domains. A number of C-terminal flanking regions have been implicated in RNA binding: RNA recognition motifs (RRM) typically mediate sequence-specific RNA binding, whereas positively charged, unstructured regions provide binding sites for structured RNA, without sequence-specificity. Interaction partners modulate RNA binding to the core, or bind to RNA regions emanating from the core. The functional interplay of the helicase core and ancillary domains or interaction partners in RNA binding and unwinding is not entirely understood. This review summarizes our current knowledge on RNA binding to the DEAD-box helicase core and the roles of ancillary domains and interaction partners in RNA binding and unwinding by DEAD-box proteins.

Metastasis-suppressor transcript destabilization through TARBP2 binding of mRNA hairpins.

Aberrant regulation of RNA stability has an important role in many disease states. Deregulated post-transcriptional modulation, such as that governed by microRNAs targeting linear sequence elements in messenger RNAs, has been implicated in the progression of many cancer types. A defining feature of RNA is its ability to fold into structures. However, the roles of structural mRNA elements in cancer progression remain unexplored. Here we performed an unbiased search for post-transcriptional modulators of mRNA stability in breast cancer by conducting whole-genome transcript stability measurements in poorly and highly metastatic isogenic human breast cancer lines. Using a computational framework that searches RNA sequence and structure space, we discovered a family of GC-rich structural cis-regulatory RNA elements, termed sRSEs for structural RNA stability elements, which are significantly overrepresented in transcripts displaying reduced stability in highly metastatic cells. By integrating computational and biochemical approaches, we identified TARBP2, a double-stranded RNA-binding protein implicated in microRNA processing, as the trans factor that binds the sRSE family and similar structural elements--collectively termed TARBP2-binding structural elements (TBSEs)--in transcripts. TARBP2 is overexpressed in metastatic cells and metastatic human breast tumours and destabilizes transcripts containing TBSEs. Endogenous TARBP2 promotes metastatic cell invasion and colonization by destabilizing amyloid precursor protein (APP) and ZNF395 transcripts, two genes previously associated with Alzheimer's and Huntington's disease, respectively. We reveal these genes to be novel metastasis suppressor genes in breast cancer. The cleavage product of APP, extracellular amyloid-α peptide, directly suppresses invasion while ZNF395 transcriptionally represses a pro-metastatic gene expression program. The expression levels of TARBP2, APP and ZNF395 in human breast carcinomas support their experimentally uncovered roles in metastasis. Our findings establish a non-canonical and direct role for TARBP2 in mammalian gene expression regulation and reveal that regulated RNA destabilization through protein-mediated binding of mRNA structural elements can govern cancer progression.

Association between circulating microRNAs, cardiovascular risk factors and outcome in patients with acute myocardial infarction

MicroRNAs (miRNAs) are highly conserved short noncoding RNAs that regulate gene expression through repression of target messenger RNA translation or destabilization. MicroRNAs regulate multiple biological functions in the healthy and diseased heart, thereby contributing to disease development. The biomarker value of circulating miRNAs has been extensively tested, especially for acute myocardial infarction (AMI). Initial small scale studies reported robust diagnostic value for AMI for cardiac-enriched miRNAs [1].

Comparative Functional Analysis of ZFP36 Genes during Xenopus Development

ZFP36 constitutes a small family of RNA binding proteins (formerly known as the TIS11 family) that target mRNA and promote their degradation. In mammals, ZFP36 proteins are encoded by four genes and, although they show similar activities in a cellular RNA destabilization assay, there is still a limited knowledge of their mRNA targets and it is not known whether or not they have redundant functions. In the present work, we have used the Xenopus embryo, a model system allowing gain- and loss-of-function studies, to investigate, whether individual ZFP36 proteins had distinct or redundant functions. We show that overexpression of individual amphibian zfp36 proteins leads to embryos having the same defects, with alteration in somites segmentation and pronephros formation. In these embryos, members of the Notch signalling pathway such as hairy2a or esr5 mRNA are down-regulated, suggesting common targets for the different proteins. We also show that mouse Zfp36 protein overexpression gives the same phenotype, indicating an evolutionary conserved property among ZFP36 vertebrate proteins. Morpholino oligonucleotide-induced loss-of-function leads to defects in pronephros formation, reduction in tubule size and duct coiling alterations for both zfp36 and zfp36l1, indicating no functional redundancy between these two genes. Given the conservation in gene structure and function between the amphibian and mammalian proteins and the conserved mechanisms for pronephros development, our study highlights a potential and hitherto unreported role of ZFP36 gene in kidney morphogenesis.

From microRNAs to targets: pathway discovery in cell fate transitions

MicroRNAs (miRNAs) are 22 nt non-coding RNAs that regulate expression of downstream targets by messenger RNA (mRNA) destabilization and translational inhibition. A large number of eukaryotic mRNAs are targeted by miRNAs, with many individual mRNAs being targeted by multiple miRNAs. Further, a single miRNA can target hundreds of mRNAs, making these small RNAs powerful regulators of cell fate decisions. Such regulation by miRNAs has been observed in the maintenance of the embryonic stem cell (ESC) cell cycle and during ESC differentiation. MiRNAs can also promote the dedifferentiation of somatic cells to induced pluripotent stem cells. During this process they target multiple downstream genes, which represent important nodes of key cellular processes. Here, we review these findings and discuss how miRNAs may be used as tools to discover novel pathways that are involved in cell fate transitions using dedifferentiation of somatic cells to induced pluripotent stem cells as a case study.

The ribosomal protein RACK1 is required for microRNA function in both C. elegans and humans.

Despite the importance of microRNAs (miRNAs) in gene regulation, it is unclear how the miRNA-Argonaute complex--or miRNA-induced silencing complex (miRISC)--can regulate the translation of their targets in such diverse ways. We demonstrate here a direct interaction between the miRISC and the ribosome by showing that a constituent of the eukaryotic 40S subunit, receptor for activated C-kinase (RACK1), is important for miRNA-mediated gene regulation in animals. In vivo studies demonstrate that RACK1 interacts with components of the miRISC in nematodes and mammals. In both systems, the alteration of RACK1 expression alters miRNA function and impairs the association of the miRNA complex with the translating ribosomes. Our data indicate that RACK1 can contribute to the recruitment of miRISC to the site of translation, and support a post-initiation mode of miRNA-mediated gene repression.

The Oligoadenylate Synthetase Family: An Ancient Protein Family with Multiple Antiviral Activities

The 2'-5' oligoadenylate synthetases (OAS) are interferon-induced antiviral enzymes that recognize virally produced dsRNA and initiate RNA destabilization through activation of RNase L within infected cells. However, recent evidence points toward several RNase L-independent pathways, through which members of the OAS family can exert antiviral activity. The crystal structure of OAS led to a novel insight into the catalytic mechanism, and revealed a remarkable similarity between OAS, Polyadenosine polymerase, and the class I CCA-adding enzyme from Archeoglobus fulgidus. This, combined with a variety of bioinformatic data, leads to the definition of a superfamily of template independent polymerases and proved that the OAS family are ancient proteins, which probably arose as early as the beginning of metazoan evolution.

Abstract 4070: Mutant tristetraprolin enhances growth factor downregulation and inhibition of cell growth in malignant glioma

Abstract Background: Tristetraprolin (TTP) negatively regulates short-lived mRNAs by binding to AU-rich elements in the 3′ untranslated region (3′UTR). A number of mRNA targets, including vascular endothelial growth factor (VEGF), and interleukin (IL)-8, play an important role in tumor progression. We have shown previously that TTP downregulates IL-8 and VEGF in malignant glioma (MG) cells via RNA destabilization. In primary glioblastoma tumors, TTP is heavily phosphorylated which may render the protein less active. Hypothesis: We postulate that the destabilizing effect of TTP on target growth factor mRNAs is held in check by hyperphosphorylation which leads to altered subcellular localization and protein stability. Methods: Wild-type TTP and two mutant forms in which select serines were exchanged with alanines were cloned with a Flag epitope into pTRE2 and pEGFP vectors. U251 Tet On MG cells were transfected with pTRE2–TTP plasmids to produce stable clones. TTP expression was induced using doxycycline. Cellular location was assessed by fluorescence microscopy and Western blotting. Protein stability was measured by assessment of TTP levels following cycloheximide treatment. VEGF and IL-8 mRNA and protein levels were measured by qRT-PCR and ELISA. For assessment of RNA half-lives, cells were treated with actinomycin D. Cell growth, proliferation and cell viability were assessed using biochemical assays, soft agar, and Trypan Blue method. Results: Both IL-8 and VEGF mRNA levels were significantly lowered in the mutant TTP clones compared to wild-type. Half-lives of these mRNAs were also shortened, and ELISA analysis showed a correspondingly greater decrease in protein expression. There was a marked impact on cellular phenotype with greater inhibition of proliferation and loss of cell viability. A Soft agar colony formation was decreased by five-fold in mutant clones versus wild-type and a greater than ten-fold decrease versus the parent cell line. With TNF-α, there was a significant shift of mutant TTP to the nuclear compartment compared to WT TTP. Treatment with cycloheximide revealed that the WT TTP was less stable compared to dephosphorylated mutant. Conclusion: Removal of key serine residues from TTP enhances its function as an RNA destabilizer of VEGF and IL-8 mRNs and as a negative regulator of tumor cell growth in MG. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4070.

Recognition of Adenosine Residues by the Active Site of Poly(A)-specific Ribonuclease

Poly(A)-specific ribonuclease (PARN) is a mammalian 3'-exoribonuclease that degrades poly(A) with high specificity. To reveal mechanisms by which poly(A) is recognized by the active site of PARN, we have performed a kinetic analysis using a large repertoire of trinucleotide substrates. Our analysis demonstrated that PARN harbors specificity for adenosine recognition in its active site and that the nucleotides surrounding the scissile bond are critical for adenosine recognition. We propose that two binding pockets, which interact with the nucleotides surrounding the scissile bond, play a pivotal role in providing specificity for the recognition of adenosine residues by the active site of PARN. In addition, we show that PARN, besides poly(A), also quite efficiently degrades poly(U), approximately 10-fold less efficiently than poly(A). The poly(U)-degrading property of PARN could be of biological significance as oligo(U) tails recently have been proposed to play a role in RNA stabilization and destabilization.

A single-base resolution map of an archaeal transcriptome

Organisms of the third domain of life, the Archaea, share molecular characteristics both with Bacteria and Eukarya. These organisms attract scientific attention as research models for regulation and evolution of processes such as transcription, translation, and RNA processing. We have reconstructed the primary transcriptome of Sulfolobus solfataricus P2, one of the most widely studied model archaeal organisms. Analysis of 625 million bases of sequenced cDNAs yielded a single-base-pair resolution map of transcription start sites and operon structures for more than 1000 transcriptional units. The analysis led to the discovery of 310 expressed noncoding RNAs, with an extensive expression of overlapping cis-antisense transcripts to a level unprecedented in any bacteria or archaea but resembling that of eukaryotes. As opposed to bacterial transcripts, most Sulfolobus transcripts completely lack 5'-UTR sequences, suggesting that mRNA/ncRNA interactions differ between Bacteria and Archaea. The data also reveal internal hotspots for transcript cleavage linked to RNA degradation and predict sequence motifs that promote RNA destabilization. This study highlights transcriptome sequencing as a key tool for understanding the mechanisms and extent of RNA-based regulation in Bacteria and Archaea.

Piwi-interacting RNAs (piRNAs) in animals: The story so far

Piwi-interacting RNAs (piRNAs) are small RNA molecules of between 24 to 31 nucleotides (nts) sequence and interact with Piwi subfamily proteins. These piRNAs play important regulatory roles in germline stem cell maintenance, epigenetic regulation, and transposition. Although the biogenesis pathways of piRNAs are not well understood but recent scientific advances have revealed some important aspects of their synthesis. Studies on regulatory mechanism of piRNAs are in their early stages. Yet, the current findings suggest that piRNA based regulation has direct implications on germline development and transposon silencing through heterochromatin formation or RNA destabilization. This review summarizes the knowledge about all aspects of piRNAs since its discovery.

Posttranscriptional Regulation of Chicken ccn2 Gene Expression by Nucleophosmin/B23 during Chondrocyte Differentiation

CCN2/CTGF is a multifunctional factor that plays a crucial role in the growth and differentiation of chondrocytes. The chicken ccn2 gene is regulated not only at the transcriptional level but also by the interaction between a posttranscriptional element in the 3' untranslated region (3'-UTR) and a cofactor. In the present study, we identified a nucleophosmin (NPM) (also called B23) as this cofactor. Binding of NPM to the element was confirmed, and subsequent analysis revealed a significant correlation between the decrease in cytosolic NPM and the increased stability of the ccn2 mRNA during chondrocyte differentiation in vivo. Furthermore, recombinant chicken NPM enhanced the degradation of chimeric RNAs containing the posttranscriptional cis elements in a chicken embryonic fibroblast extract in vitro. It is noteworthy that the RNA destabilization effect by NPM was far more prominent in the cytosolic extract of chondrocytes than in that of fibroblasts, representing a chondrocyte-specific action of NPM. Stimulation by growth factors to promote differentiation changed the subcellular distribution of NPM in chondrocytes, which followed the expected patterns from the resultant change in the ccn2 mRNA stability. Therefore, the present study reveals a novel aspect of NPM as a key player in the posttranscriptional regulation of ccn2 mRNA during the differentiation of chondrocytes.

Tristetraprolin Down-regulates Interleukin-8 and Vascular Endothelial Growth Factor in Malignant Glioma Cells

Malignant gliomas are highly aggressive tumors of the central nervous system that rely on production of growth factors for tumor progression. Vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and tumor necrosis factor-alpha, for example, are up-regulated in these tumors to promote angiogenesis and proliferation. RNA stability, mediated through adenine and uridine-rich elements (ARE) in the 3' untranslated region, is a critical control point for regulating these growth factors. RNA half-life is predominantly governed by a balance between stabilizing and destabilizing factors that bind to ARE. We have previously shown that the stabilizing factor HuR is overexpressed in malignant gliomas and linked to RNA stabilization of angiogenic growth factors. Here, we report that the destabilizing factor tristetraprolin (TTP) is also ubiquitously expressed in primary malignant glioma tissues and cell lines. In contrast to benign astrogliotic tissues, however, the protein was hyperphosphorylated, with evidence implicating the p38/mitogen-activated protein kinase (MAPK) pathway. Conditional overexpression of TTP as a transgene in malignant glioma cells led to RNA destabilization of IL-8 and VEGF and down-regulation of protein production. Analysis of in vivo RNA binding indicated a shift of mRNA toward ectopic TTP and away from endogenous HuR. This biochemical phenotype was associated with a decrease in cell proliferation, loss of cell viability, and apoptosis. We postulate that hyperphosphorylation of TTP via p38/MAPK promotes progression of malignant gliomas by negatively regulating its RNA destabilizing function.

MicroRNA Inhibition of Translation Initiation in Vitro by Targeting the Cap-Binding Complex eIF4F

MicroRNAs (miRNAs) play an important role in gene regulatory networks in animals. Yet, the mechanistic details of their function in translation inhibition or messenger RNA (mRNA) destabilization remain controversial. To directly examine the earliest events in this process, we have developed an in vitro translation system using mouse Krebs-2 ascites cell-free extract that exhibits an authentic miRNA response. We show here that translation initiation, specifically the 5' cap recognition process, is repressed by endogenous let-7 miRNAs within the first 15 minutes of mRNA exposure to the extract when no destabilization of the transcript is observed. Our results indicate that inhibition of translation initiation is the earliest molecular event effected by miRNAs. Other mechanisms, such as mRNA degradation, may subsequently consolidate mRNA silencing.