UV Cross-linking Assays Research Articles

MiR-204 suppresses porcine reproductive and respiratory syndrome virus (PRRSV) replication via inhibiting LC3B-mediated autophagy

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) has been regarded as a persistent challenge for the swine farms worldwide. microRNAs (miRNAs) play key roles in regulating almost every important biological process, including virus-host interaction. In this study, we found that miR-204 was highly expressed in cells that were not permissive to PRRSV infection compared with cells susceptible to PRRSV infection. Subsequently, we demonstrated that overexpression of miR-204 significantly inhibited PRRSV replication in porcine alveolar macrophages (PAMs). Through bioinformatic analysis, we found that there existed a potential binding site of miR-204 on the 3′UTR of microtubule associated protein 1 light chain 3B (MAP1LC3B, LC3B), a hallmark of autophagy. Applying experiments including luciferase reporter assay and UV cross-linking and immunoprecipitation (CLIP) assay, we demonstrated that miR-204 directly targeted LC3B, thereby downregulating autophagy. Meanwhile, we investigated the interplay between autophagy and PRRSV replication in PAMs, confirming that PRRSV infection induces autophagy, which in turn facilitates viral replication. Overall, we verify that miR-204 suppresses PRRSV replication via inhibiting LC3B-mediated autophagy in PAMs. These findings will provide a novel potential approach for us to develop antiviral therapeutic agents and controlling measures for future PRRSV outbreaks.

The Non-Canonical Iron-Responsive Element of IRE-tvcp12 Hairpin Structure at the 3'-UTR of Trichomonas vaginalis TvCP12 mRNA That Binds TvHSP70 and TvACTN-3 Can Regulate mRNA Stability and Amount of Protein.

Trichomonas vaginalis is one of the most common sexually transmitted parasites in humans. This protozoan has high iron requirements for growth, metabolism, and virulence. However, iron concentrations also differentially modulate T. vaginalis gene expression as in the genes encoding cysteine proteinases TvCP4 and TvCP12. Our goal was to identify the regulatory mechanism mediating the upregulation of tvcp12 under iron-restricted (IR) conditions. Here, we showed by RT-PCR, Western blot, and immunocytochemistry assays that IR conditions increase mRNA stability and amount of TvCP12. RNA electrophoretic mobility shift assay (REMSA), UV cross-linking, and competition assays demonstrated that a non-canonical iron-responsive element (IRE)-like structure at the 3'-untranslated region of the tvcp12 transcript (IRE-tvcp12) specifically binds to human iron regulatory proteins (IRPs) and to atypical RNA-binding cytoplasmic proteins from IR trichomonads, such as HSP70 and α-Actinin 3. These data were confirmed by REMSA supershift and Northwestern blot assays. Thus, our findings show that a positive gene expression regulation under IR conditions occurs at the posttranscriptional level possibly through RNA-protein interactions between atypical RNA-binding proteins and non-canonical IRE-like structures at the 3'-UTR of the transcript by a parallel mechanism to the mammalian IRE/IRP system that can be applied to other iron-regulated genes of T. vaginalis.

MiR-142-3p suppresses porcine reproductive and respiratory syndrome virus (PRRSV) infection by directly targeting Rac1

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) has been recognized as one of the severest epidemics in pigs worldwide. microRNAs (miRNAs) play important roles in a variety of biological processes, including cell differentiation, proliferation and death, as well as viral infections and antiviral immune responses. In this study, we found that miR-142–3p was expressed lower in cells susceptible to PRRSV infection than in cells less or no permissive to PRRSV infection. Subsequently, we showed that overexpression of miR-142–3p remarkably inhibited PRRSV infection in PAMs, while blockage of endogenous miR-142–3p significantly enhanced PRRSV replication. Then, we demonstrated that miR-142–3p directly targeted Ras-related C3 botulinum toxin substrate 1 (Rac1), a member of Rho GTPases family, by using luciferase reporter assay and UV cross-linking and immunoprecipitation (CLIP) assay. Importantly, we verified that miR-142–3p inhibited PRRSV entry into PAMs and accordingly suppressed PRRSV infection by downregulating Rac1 expression. These findings reveal an important role of miR-142–3p in modulating PRRSV infection and provide us with some ideas for developing novel antiviral therapy against PRRSV infection.

Synergistic roles for human U1 snRNA stem-loops in pre-mRNA splicing

ABSTRACT During spliceosome assembly, interactions that bring the 5′ and 3′ ends of an intron in proximity are critical for the production of mature mRNA. Here, we report synergistic roles for the stem-loops 3 (SL3) and 4 (SL4) of the human U1 small nuclear RNA (snRNA) in maintaining the optimal U1 snRNP function, and formation of cross-intron contact with the U2 snRNP. We find that SL3 and SL4 bind distinct spliceosomal proteins and combining a U1 snRNA activity assay with siRNA-mediated knockdown, we demonstrate that SL3 and SL4 act through the RNA helicase UAP56 and the U2 protein SF3A1, respectively. In vitro analysis using UV crosslinking and splicing assays indicated that SL3 likely promotes the SL4-SF3A1 interaction leading to enhancement of A complex formation and pre-mRNA splicing. Overall, these results highlight the vital role of the distinct contacts of SL3 and SL4 in bridging the pre-mRNA bound U1 and U2 snRNPs during the early steps of human spliceosome assembly.

ALYREF Drives Cancer Cell Proliferation Through an ALYREF-MYC Positive Feedback Loop in Glioblastoma.

BackgroundWhile RNA-binding proteins (RBPs) are known to affect RNA homeostasis during cancer cell initiation and development, their characteristics and biological function in glioblastoma (GBM) remain unclear.MethodsDifferences in RBP expression were explored by differential analysis of The Cancer Genome Atlas-GBM and Genotype-Tissue Expression (GTEx) datasets. Real-time PCR was conducted to verify the expressional levels of Aly/REF export factor (ALYREF) in normal brain and GBM tissues. Proliferative assays were performed to investigate molecular functions of ALYREF in GBM cells in vitro and in vivo. Real-time PCR and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to analyze the ALYREF downstream signaling pathways. A chromatin immunoprecipitation (ChIP) assay was performed to identify key transcriptional factors that regulate ALYREF expression at RNA level. UV crosslinking, immunoprecipitation (CLIP) and RNA stability assays were conducted to reveal the bound RNAs and their stability regulated by ALYREF.ResultsThe results showed that ALYREF is frequently increased in GBM tissues, and its mRNA expression is regulated by the MYC proto-oncogene, bHLH transcription factor (MYC). Inhibition of ALYREF expression decreased GBM cell proliferative ability in vitro and tumor formation in vivo. KEGG analysis revealed that high ALYREF expression in GBM tissues was enriched in the upregulation of oncogenic pathways such as the Wnt/β-catenin signaling pathway. The CLIP assay showed that ALYREF drives GBM carcinogenesis by binding to and stabilizing MYC mRNAs. Overexpression of MYC restored the oncogenic property of ALYREF-deficient GBM cells.ConclusionOur data showed that ALYREF is regulated by MYC at the transcriptional level. ALYREF drives GBM cell proliferation by activating the Wnt/β-catenin signaling pathway and stabilizing MYC mRNA, suggesting that an ALYREF-MYC positive feedback loop might be a potential therapeutic target for treating GBM patients.

PTBP3 Induced Inhibition of Differentiation of Gastric Cancer Cells Through Alternative Splicing of Id1.

Overexpression of PTBP3, a factor involved in alternative splicing, may inhibit the differentiation of leukemia cells. However, its role in gastric cancer differentiation and the specific pathways involved are unclear. In this study, we found that PTBP3 was upregulated in the poorly differentiated gastric cancer tissues. Patients with high levels of PTBP3 expression had significantly shorter survival than those with low PTBP3 expression. In gastric cancer cells, the regulatory effect of PTBP3 on alternative splicing of the Id1 gene was investigated. Following sodium butyrate-induced differentiation of MKN45 cells, the expression of Id1a decreased, but the expression of Id1b increased. RNA interference and overexpression experiments showed that PTBP3 upregulated Id1a expression and downregulated Id1b expression. RNA immunoprecipitation (RIP) assays indicated PTBP3 could interact with Id1. UV cross-linking assays indicated that PTBP3 interacted with the CU rich region of the Id1 gene. Two-hybrid experiments and a gel mobility shift assays found that Id1b had a more potent affinity for Hes1 than Id1a. Chromatin immunoprecipitation (ChIP) assays verified the association of Hes1 and the promoter of PTBP3 gene. Luciferase assays revealed that Hes1 bound the N-box sequence in the PTBP3 promoter. After silencing or overexpression of Hes1, PTBP3 protein expression remained unchanged. Thus, the loss of feedback regulation among PTBP3, Id1, and Hes1 in gastric cancer cells may be one of the causes of inhibited differentiation and malignant proliferation of these cells.

Approaches for Investigating Translational Regulation Controlled by PARP1: Biotin-Based UV Cross-Linking and Luciferase Reporter Assay.

The RNA-binding proteins (RBPs) play a pivotal role in controlling gene expression through posttranscriptional processes. As the trans-acting factors, RBPs interact with the cis-regulatory elements located within mRNAs to regulate mRNA translational efficiency. Adding a new-layer regulation, recent studies suggest that poly(ADP-ribosyl)ation of the RNA-binding proteins often inhibit the RNA-binding ability of RBPs, thus regulating RBP-dependent mRNA metabolism including translational control. Here, we describe a biotin-based UV cross-linking method to determine if excessive accumulation of pADPr in the cell disrupts the interaction between RBPs and their target mRNAs. In addition, we illustrate the protocol of using the luciferase reporter assay to determine the effect of poly(ADP-ribosyl)ation on mRNA translation.

The eIF3 complex of Trypanosoma brucei: composition conservation does not imply the conservation of structural assembly and subunits function.

The multisubunit eukaryotic initiation factor 3 (eIF3) plays multiple roles in translation but is poorly understood in trypanosomes. The putative subunits eIF3a and eIF3f of Trypanosoma brucei (TbIF3a and TbIF3f) were overexpressed and purified, and 11 subunits were identified, TbIF3a through l minus j, which form a tight complex. Both TbIF3a and TbIF3f are essential for the viability of T. brucei. RNAi knockdown of either of them severely reduced total translation and the ratio of the polysome/80S peak area. TbIF3f and TbIF3a RNAi cell lines were modified to express tagged-TbIF3a and -TbIF3f, respectively. RNAi in combination with affinity purification assays indicated that both subunits are variably required for TbIF3 stability and integrity. The relative abundance of other subunits in the TbIF3f-tag complex changed little upon TbIF3a depletion; while only subunits TbIF3b, i, and e copurified comparably with TbIF3a-tag upon TbIF3f depletion. A genome-wide UV-crosslinking assay showed that several TbIF3 subunits have direct RNA-binding activity, with TbIF3c showing the strongest signal. In addition, CrPV IRES, but neither EMCV IRES nor HCV IRES, was found to mediate translation in T. brucei. These results together imply that the structure of TbIF3 and the subunits function have trypanosome-specific features, although the composition is evolutionarily conserved.

The Chromatin Remodeler ISW1 Is a Quality Control Factor that Surveys Nuclear mRNP Biogenesis

Chromatin dynamics play an essential role in regulating DNA transaction processes, but it is unclear whether transcription-associated chromatin modifications control the mRNA ribonucleoparticles (mRNPs) pipeline from synthesis to nuclear exit. Here, we identify the yeast ISW1 chromatin remodeling complex as an unanticipated mRNP nuclear export surveillance factor that retains export-incompetent transcripts near their transcription site. This tethering activity of ISW1 requires chromatin binding and is independent of nucleosome sliding activity or changes in RNA polymerase II processivity. Combination of invivo UV-crosslinking and genome-wide RNA immunoprecipitation assays show that Isw1 and its cofactors interact directly with premature mRNPs. Our results highlight that the concerted action of Isw1 and the nuclear exosome ensures accurate surveillance mechanism that proofreads the efficiency of mRNA biogenesis.

DHX29 reduces leaky scanning through an upstream AUG codon regardless of its nucleotide context.

During eukaryotic translation initiation, the 43S preinitiation complex (43S PIC), consisting of the 40S ribosomal subunit, eukaryotic initiation factors (eIFs) and initiator tRNA scans mRNA to find an appropriate start codon. Key roles in the accuracy of initiation codon selection belong to eIF1 and eIF1A, whereas the mammalian-specific DHX29 helicase substantially contributes to ribosomal scanning of structured mRNAs. Here, we show that DHX29 stimulates the recognition of the AUG codon but not the near-cognate CUG codon regardless of its nucleotide context during ribosomal scanning. The stimulatory effect depends on the contact between DHX29 and eIF1A. The unique DHX29 N-terminal domain binds to the ribosomal site near the mRNA entrance, where it contacts the eIF1A OB domain. UV crosslinking assays revealed that DHX29 may rearrange eIF1A and eIF2α in key nucleotide context positions of ribosomal complexes. Interestingly, DHX29 impedes the 48S initiation complex formation in the absence of eIF1A perhaps due to forming a physical barrier that prevents the 43S PIC from loading onto mRNA. Mutational analysis allowed us to split the mRNA unwinding and codon selection activities of DHX29. Thus, DHX29 is another example of an initiation factor contributing to start codon selection.

Post-Transcriptional Regulation of Endothelial Nitric Oxide Synthase Expression by Polypyrimidine Tract-Binding Protein 1.

Endothelial nitric oxide synthase (eNOS) is an important regulator of vascular function and its expression is regulated at post-transcriptional levels through a yet unknown mechanism. The purpose of this study is to elucidate the post-transcriptional factors regulating eNOS expression and function in endothelium. To elucidate the molecular basis of tumor necrosis factor (TNF)-α-mediated eNOS mRNA instability, biotinylated eNOS 3'-untranslational region (UTR) was used to purify its associated proteins by RNA affinity chromatography from cytosolic fractions of TNF-α-stimulated human umbilical vein endothelial cells (HUVECs). We identified 2 cytosolic proteins, with molecular weight of 52 and 57 kDa, which specifically bind to eNOS 3'-UTR in response to TNF-α stimulation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis identified the 57-kDa protein as polypyrimidine tract-binding protein 1 (PTB1). RNA gel mobility shift and UV cross-linking assays demonstrated that PTB1 binds to a UCUU-rich sequence in eNOS 3'-UTR, and the C-terminal half of PTB1 is critical to this interaction. Importantly, PTB1 overexpression leads to decreased activity of luciferase gene fused with eNOS 3'-UTR as well as reduced eNOS expression and activity in human ECs. In HUVECs, we show that TNF-α markedly increased PTB1 expression, whereas adenovirus-mediated PTB1 overexpression decreased eNOS mRNA stability and reduced protein expression and endothelium-dependent relaxation. Furthermore, knockdown of PTB1 substantially attenuated TNF-α-induced destabilization of eNOS transcript and downregulation of eNOS expression. These results indicate that PTB1 is essential for regulating eNOS expression at post-transcriptional levels and suggest a novel therapeutic target for treatment of vascular diseases associated with inflammatory endothelial dysfunction.

Cellular Proteins Act as Bridge Between 5' and 3' Ends of the Coxsackievirus B3 Mediating Genome Circularization During RNA Translation.

The positive single-stranded RNA genome of the Coxsackievirus B3 (CVB3) contains a 5' untranslated region (UTR) which hosts the internal ribosome entry site (IRES) element that governs cap-independent translation initiation and a polyadenylated 3' UTR which is required for stimulating the IRES activity. Viral RNA genomes could circularize to regulate initiation of translation and RNA synthesis at 5' and 3' ends. Interactions could either take place by direct RNA-RNA contacts, through cellular protein bridges mediating RNA circularization or both. Accordingly, we aimed to assess the nature of molecular interactions between these two regions and to evaluate cellular factors required for mRNA 3' end-mediated stimulation of CVB3 IRES-driven translation. By gel shift assays, we have showed that combining, in vitro, 5' and 3' UTR fragments had no discernible effect on the structures of RNAs, arguing against the presence of specific canonical RNA-RNA cyclization sequences between these two regions. Competitive UV crosslinking assays using BHK-21 cell extract showed common cellular proteins eIF3b, PTB, and La binding to both 5'- and 3' end RNAs. PCBP 1-2 and PABP were shown to bind, respectively, to 5' and 3' UTR probes. Taking together, these data suggest that CVB3 5'-3' end bridging occurs through 5' UTR-protein-protein-3' UTR interactions and not through RNA-RNA direct contact. The dual involvement of the 3' and 5' UTRs in controlling viral translation and RNA synthesis highlights the relevance of these regions in the infectious virus life cycle, making them suitable candidates for targeted CVB3 antiviral therapy.

Molecular characterization of Plasmodium falciparum Bruno/CELF RNA binding proteins

The human malaria parasite Plasmodium falciparum employs intricate post-transcriptional regulatory mechanisms in different stages of its life cycle. Despite the importance of post-transcriptional regulation, key elements of these processes, namely RNA binding proteins (RBPs), are poorly characterized. In this study, the RNA binding properties of P. falciparum proteins were characterized including two putative members of the Bruno/CELF family of RBPs (PfCELF1 and PfCELF2), dihydrofolate reductase-thymidylate synthase (PfDHFR-TS), and adenosine deaminase (PfAda). RNA binding activity was tested using UV-crosslinking and electrophoretic mobility shift assays. PfCELF1 and PfDHFR-TS demonstrated RNA binding activity, whereas PfAda and PfCELF2 were RBP-negative. Intracellular protein localization of RBPs was studied using GFP-tagged transgenic parasite lines. PfCELF1 protein may shuttle between nucleus and cytoplasm, as shown by a predominantly nuclear PfCELF1 cell population and another predominantly cytoplasmic. In contrast, PfDHFR-TS protein is predominantly cytoplasmic. PfCELF1 may thus have several roles, including pre-mRNA processing. The mRNA targets of these P. falciparum proteins were investigated by ribonomics using DNA microarrays. A sequence motif similar to that recognized by CELF proteins in other species is common in the introns of target mRNAs identified for PfCELF1, suggesting that nuclear-localized PfCELF1 may regulate pre-mRNA splicing in P. falciparum, as has been found for CELF proteins in other species. In contrast, none or very few mRNA targets were found for the other proteins, suggesting that they do not have biologically relevant roles as RBPs in the asexual stages of P. falciparum.

Characterization of RNA binding protein RBP-P reveals a possible role in rice glutelin gene expression and RNA localization

RNA binding proteins (RBPs) play an important role in mRNA metabolism including synthesis, maturation, transport, localization, and stability. In developing rice seeds, RNAs that code for the major storage proteins are transported to specific domains of the cortical endoplasmic reticulum (ER) by a regulated mechanism requiring RNA cis-localization elements, or zipcodes. Putative trans-acting RBPs that recognize prolamine RNA zipcodes required for restricted localization to protein body-ER have previously been identified. Here, we describe the identification of RBP-P using a Northwestern blot approach as an RBP that recognizes and binds to glutelin zipcode RNA, which is required for proper RNA localization to cisternal-ER. RBP-P protein expression coincides with that of glutelin during seed maturation and is localized to both the nucleus and cytosol. RNA-immunoprecipitation and subsequent RT-PCR analysis further demonstrated that RBP-P interacts with glutelin RNAs. In vitro RNA-protein UV-crosslinking assays showed that recombinant RBP-P binds strongly to glutelin mRNA, and in particular, 3' UTR and zipcode RNA. RBP-P also exhibited strong binding activity to a glutelin intron sequence, suggesting that RBP-P might participate in mRNA splicing. Overall, these results support a multifunctional role for RBP-P in glutelin mRNA metabolism, perhaps in nuclear pre-mRNA splicing and cytosolic localization to the cisternal-ER.

α-Actinin TvACTN3 ofTrichomonas vaginalisIs an RNA-Binding Protein That Could Participate in Its Posttranscriptional Iron Regulatory Mechanism

Trichomonas vaginalis is a sexually transmitted flagellated protist parasite responsible for trichomoniasis. This parasite is dependent on high levels of iron, favoring its growth and multiplication. Iron also differentially regulates some trichomonad virulence properties by unknown mechanisms. However, there is evidence to support the existence of gene regulatory mechanisms at the transcriptional and posttranscriptional levels that are mediated by iron concentration in T. vaginalis. Thus, the goal of this study was to identify an RNA-binding protein in T. vaginalis that interacts with the tvcp4 RNA stem-loop structure, which may participate in a posttranscriptional iron regulatory mechanism mediated by RNA-protein interactions. We performed RNA electrophoretic mobility shift assay (REMSA) and supershift, UV cross-linking, Northwestern blot, and western blot (WB) assays using cytoplasmic protein extracts from T. vaginalis with the tvcp4 RNA hairpin structure as a probe. We identified a 135-kDa protein isolated by the UV cross-linking assays as α-actinin 3 (TvACTN3) by MALDI-TOF-MS that was confirmed by LS-MS/MS and de novo sequencing. TvACTN3 is a cytoplasmic protein that specifically binds to hairpin RNA structures from trichomonads and humans when the parasites are grown under iron-depleted conditions. Thus, TvACTN3 could participate in the regulation of gene expression by iron in T. vaginalis through a parallel posttranscriptional mechanism similar to that of the IRE/IRP system.

A Human Proteome Microarray Identifies that the Heterogeneous Nuclear Ribonucleoprotein K (hnRNP K) Recognizes the 5′ Terminal Sequence of the Hepatitis C Virus RNA

Stem-loop I (SL1) located in the 5' untranslated region of the hepatitis C virus (HCV) genome initiates binding to miR-122, a microRNA required for hepatitis HCV replication. However, proteins that bind SL1 remain elusive. In this study, we employed a human proteome microarray, comprised of ∼17,000 individually purified human proteins in full-length, and identified 313 proteins that recognize HCV SL1. Eighty-three of the identified proteins were annotated as liver-expressing proteins, and twelve of which were known to be associated with hepatitis virus. siRNA-induced silencing of eight out of 12 candidate genes led to at least 25% decrease in HCV replication efficiency. In particular, knockdown of heterogeneous nuclear ribonucleoprotein K (hnRNP K) reduced HCV replication in a concentration-dependent manner. Ultra-violet-crosslinking assay also showed that hnRNP K, which functions in pre-mRNA processing and transport, showed the strongest binding to the HCV SL1. We observed that hnRNP K, a nuclear protein, is relocated in the cytoplasm in HCV-expressing cells. Immunoprecipitation of the hnRNP K from Huh7.5 cells stably expressing HCV replicon resulted in the co-immunoprecipitation of SL1. This work identifies a cellular protein that could have an important role in the regulation of HCV RNA gene expression and metabolism.

Zinc‐induced modulation of SRSF6 activity alters Bim splicing to promote generation of the most potent apoptotic isoform BimS

Bim is a member of the pro-apoptotic BH3-only Bcl-2 family of proteins. Bim gene undergoes alternative splicing to produce three predominant splicing variants (BimEL, BimL and BimS). The smallest variant BimS is the most potent inducer of apoptosis. Zinc (Zn(2+)) has been reported to stimulate apoptosis in various cell types. In this study, we examined whether Zn(2+) affects the expression of Bim in human neuroblastoma SH-SY5Y cells. Zn(2+) triggered alterations in Bim splicing and induced preferential generation of BimS, but not BimEL and BimL, in a dose- and time-dependent manner. Other metals (cadmium, cobalt and copper) and stresses (oxidative, endoplasmic reticulum and genotoxic stresses) had little or no effect on the expression of BimS. To address the mechanism of Zn(2+)-induced preferential generation of BimS, which lacks exon 4, we developed a Bim mini-gene construct. Deletion analysis using the Bim mini-gene revealed that predicted binding sites of the SR protein SRSF6, also known as SRp55, are located in the intronic region adjacent to exon 4. We also found that mutations in the predicted SRSF6-binding sites abolished generation of BimS mRNA from the mutated Bim mini-gene. In addition, a UV cross-linking assay followed by Western blotting showed that SRSF6 directly bound to the predicted binding site and Zn(2+) suppressed this binding. Moreover, Zn(2+) stimulated SRSF6 hyper-phosphorylation. TG003, a cdc2-like kinase inhibitor, partially prevented Zn(2+)-induced generation of BimS and SRSF6 hyper-phosphorylation. Taken together, our findings suggest that Zn(2+) inhibits the activity of SRSF6 and promotes elimination of exon 4, leading to preferential generation of BimS.

RNA helicase is involved in the expression and replication of classical swine fever virus and interacts with untranslated region

To investigate whether cytoplasmic RNA helicase A (RHA) influences the expression and replication of classical swine fever virus (CSFV), an siRNA molecule targeted to RHA was transfected into PK-15 cells. The siRNA was found to reduce cytoplasmic RHA. In CSFV subgenomic replicon transfected cells, incubation with the siRNAs negatively impacted viral NS3 and RNA production. In the CSFV infected cells, treatment with the siRNA resulted in a significant reduction of viral replication by 65–70%. Furthermore, affinity chromatography and UV-crosslinking assays revealed that RHA can bind the 5′ and 3′ terminal region of CSFV 3′-untranslated region (UTR), the 5′ terminal region and domain III of CSFV 5′ UTR. All these regions are important for viral replication and translation. These data showed that RHA is involved in the expression and replication of CSFV and might participate in modulation of RNA synthesis, replication and translation of CSFV by binding these regions.

Alternative splicing of the neurofibromatosis type 1 pre-mRNA is regulated by the muscleblind-like proteins and the CUG-BP and ELAV-like factors

BackgroundAlternative splicing is often subjected to complex regulatory control that involves many protein factors and cis-acting RNA sequence elements. One major challenge is to identify all of the protein players and define how they control alternative expression of a particular exon in a combinatorial manner. The Muscleblind-like (MBNL) and CUG-BP and ELAV-Like family (CELF) proteins are splicing regulatory proteins, which function as antagonists in the regulation of several alternative exons. Currently only a limited number of common targets of MBNL and CELF are known that are antagonistically regulated by these two groups of proteins.ResultsRecently, we identified neurofibromatosis type 1 (NF1) exon 23a as a novel target of negative regulation by CELF proteins. Here we report that MBNL family members are positive regulators of this exon. Overexpression of MBNL proteins promote exon 23a inclusion in a low MBNL-expressing cell line, and simultaneous siRNA-mediated knockdown of MBNL1 and MBNL2 family members in a high MBNL-expressing cell line promotes exon 23a skipping. Importantly, these two groups of proteins antagonize each other in regulating inclusion of exon 23a. Furthermore, we analyzed the binding sites of these proteins in the intronic sequences upstream of exon 23a by UV cross-linking assays. We show that in vitro, in addition to the previously identified preferred binding sequence UGCUGU, the MBNL proteins need the neighboring sequences for optimal binding.ConclusionThis study along with our previous work that demonstrated roles for Hu, CELF, and TIA-1 and TIAR proteins in the regulation of NF1 exon 23a establish that this exon is under tight, complex control.

5-Aminoimidazole-4-carboxamide ribonucleoside stabilizes low density lipoprotein receptor mRNA in hepatocytes via ERK-dependent HuR binding to an AU-rich element

Objective5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) has pleiotropic and beneficial effects on metabolic disorders. However, the effects of AICAR on low density lipoprotein (LDL) metabolism are poorly understood. Methods and resultsAICAR induces increased LDLR mRNA levels and increased LDLR protein production in hepatocytes. The AICAR-dependent LDLR mRNA increase was partially mediated by mRNA stabilization in an extracellular signal-regulated kinase1/2 (ERK1/2)-dependent manner, but not by the AMP-activated protein kinase (AMPK) activation. Reporter assays using a variety of constructs harboring the 3′-untranslated region (UTR) of human LDLR mRNA revealed that the most upstream AU-rich element (ARE) was critical for these AICAR effects. Using UV cross-linking assays, we found increased binding of three cytoplasmic proteins to this ARE region in response to AICAR and identified a 35-kDa protein as Human antigen R (HuR). Blocking ERK signaling pathway activation resulted in attenuated HuR binding. Silencing HuR expression by RNA interference hindered AICAR-induced LDLR mRNA stability, whereas its overexpression stabilized this mRNA. ConclusionsAICAR-dependent LDLR mRNA stabilization is mediated, at least in part, by HuR binding to the ARE1 region. Given that AICAR enhanced LDL uptake in hepatocytes, our findings warrant further studies using animal models to develop a novel LDL-cholesterol lowering agent as a possible strategy to treat atherosclerosis-related cardiovascular diseases.