Post-transcriptional Regulatory Protein Research Articles

The Post-Transcriptional Regulatory Protein CsrA Amplifies Its Targetome through Direct Interactions with Stress-Response Regulatory Hubs: The EvgA and AcnA Cases.

Global rewiring of bacterial gene expressions in response to environmental cues is mediated by regulatory proteins such as the CsrA global regulator from E. coli. Several direct mRNA and sRNA targets of this protein have been identified; however, high-throughput studies suggest an expanded RNA targetome for this protein. In this work, we demonstrate that CsrA can extend its network by directly binding and regulating the evgA and acnA transcripts, encoding for regulatory proteins. CsrA represses EvgA and AcnA expression and disrupting the CsrA binding sites of evgA and acnA, results in broader gene expression changes to stress response networks. Specifically, altering CsrA-evgA binding impacts the genes related to acidic stress adaptation, and disrupting the CsrA-acnA interaction affects the genes involved in metal-induced oxidative stress responses. We show that these interactions are biologically relevant, as evidenced by the improved tolerance of evgA and acnA genomic mutants depleted of CsrA binding sites when challenged with acid and metal ions, respectively. We conclude that EvgA and AcnA are intermediate regulatory hubs through which CsrA can expand its regulatory role. The indirect CsrA regulation of gene networks coordinated by EvgA and AcnA likely contributes to optimizing cellular resources to promote exponential growth in the absence of stress.

Contribution of Hfq to gene regulation and virulence in Histophilus somni.

Histophilus somni is one of the predominant bacterial pathogens responsible for bovine respiratory and systemic diseases in cattle. Despite the identification of numerous H. somni virulence factors, little is known about the regulation of such factors. The post-transcriptional regulatory protein Hfq may play a crucial role in regulation of components that affect bacterial virulence. The contribution of Hfq to H. somni phenotype and virulence was investigated following creation of an hfq deletion mutant of H. somni strain 2336 (designated H. somni 2336Δhfq). A comparative analysis of the mutant to the wild-type strain was carried out by examining protein and carbohydrate phenotype, RNA sequence, intracellular survival in bovine monocytes, serum susceptibility, and virulence studies in mouse and calf models. H. somni 2336Δhfq exhibited a truncated lipooligosaccharide (LOS) structure, with loss of sialylation. The mutant demonstrated increased susceptibility to intracellular and serum-mediated killing compared to the wild-type strain. Transcriptomic analysis displayed significant differential expression of 832 upregulated genes and 809 downregulated genes in H. somni 2336Δhfq compared to H. somni strain 2336, including significant downregulation of lsgB and licA, which contribute to LOS oligosaccharide synthesis and sialylation. A substantial number of differentially expressed genes were associated with polysaccharide synthesis and other proteins that could influence virulence. The H. somni 2336Δhfq mutant strain was attenuated in a mouse septicemia model and somewhat attenuated in a calf intrabronchial challenge model. H. somni was recovered less frequently from nasopharyngeal swabs, endotracheal aspirates, and lung tissues of calves challenged with H. somni 2336Δhfq compared to the wild-type strain, and the percentage of abnormal lung tissue in calves challenged with H. somni 2336Δhfq was lower than in calves challenged with the wild-type strain. In conclusion, our results support that Hfq accounts for the regulation of H. somni virulence factors.

Identification of a 6-RBP gene signature for a comprehensive analysis of glioma and ischemic stroke: Cognitive impairment and aging-related hypoxic stress.

There is mounting evidence that ischemic cerebral infarction contributes to vascular cognitive impairment and dementia in elderly. Ischemic stroke and glioma are two majorly fatal diseases worldwide, which promote each other's development based on some common underlying mechanisms. As a post-transcriptional regulatory protein, RNA-binding protein is important in the development of a tumor and ischemic stroke (IS). The purpose of this study was to search for a group of RNA-binding protein (RBP) gene markers related to the prognosis of glioma and the occurrence of IS, and elucidate their underlying mechanisms in glioma and IS. First, a 6-RBP (POLR2F, DYNC1H1, SMAD9, TRIM21, BRCA1, and ERI1) gene signature (RBPS) showing an independent overall survival prognostic prediction was identified using the transcriptome data from TCGA-glioma cohort (n = 677); following which, it was independently verified in the CGGA-glioma cohort (n = 970). A nomogram, including RBPS, 1p19q codeletion, radiotherapy, chemotherapy, grade, and age, was established to predict the overall survival of patients with glioma, convenient for further clinical transformation. In addition, an automatic machine learning classification model based on radiomics features from MRI was developed to stratify according to the RBPS risk. The RBPS was associated with immunosuppression, energy metabolism, and tumor growth of gliomas. Subsequently, the six RBP genes from blood samples showed good classification performance for IS diagnosis (AUC = 0.95, 95% CI: 0.902–0.997). The RBPS was associated with hypoxic responses, angiogenesis, and increased coagulation in IS. Upregulation of SMAD9 was associated with dementia, while downregulation of POLR2F was associated with aging-related hypoxic stress. Irf5/Trim21 in microglia and Taf7/Trim21 in pericytes from the mouse cerebral cortex were identified as RBPS-related molecules in each cell type under hypoxic conditions. The RBPS is expected to serve as a novel biomarker for studying the common mechanisms underlying glioma and IS.

Low CPEB1 levels may predict the benefit of 5-fluorouracil treatment in patients with colon or stomach adenocarcinoma.

BackgroundFor patients with colon or stomach adenocarcinoma, 5-fluorouracil (5-FU) is an essential component of systemic chemotherapy in the palliative and adjuvant settings. The post-transcriptional regulatory factor cytoplasmic polyadenylation element-binding protein 1 (CPEB1) has been reported to be linked to tumor metastasis. This study aimed to investigate the relationship between CPEB1 expression and 5-FU treatment response in patients with colon and stomach adenocarcinomas.MethodsThe expression of CPEB1 in stomach adenocarcinoma and colorectal cancer (CRC) tissues and in cell lines was determined by quantitative real-time PCR (qRT-PCR) and immunohistochemistry analyses. Transwell assays were employed to analyze the effects of CPEB1 on the migration and invasion abilities of gastric cancer (GC) and CRC cells.ResultsThe expression levels of CPEB1 were increased in colon and stomach adenocarcinoma and were negatively correlated with malignancy and poor patient survival. Data suggested that patients with CRC or GC who had strong CPEB1 expression responded poorly to 5-FU treatment. Furthermore, knockdown of CPEB1 inhibited the migration and invasion of CRC and GC cells via a mechanism involving decreased expression of matrix metalloprotein (MMP)2, 7, and 9. Finally, our methylated RNA immunoprecipitation PCR (meRIP qPCR) data suggested that the increased CPEB1 expression in colon and stomach adenocarcinomas might be mediated by FTO (FTO alpha-ketoglutarate dependent dioxygenase)-dependent m6A demethylation of CPEB1 mRNA.ConclusionsOur results indicate that the level of CPEB1 expression may be valuable for predicting the benefit of 5-FU treatment for patients with colon and stomach adenocarcinomas. We therefore propose that low CPEB1 expression may represent a novel biomarker for personalized 5-FU therapy.

Recombinant Transcription Factors (TFs) Fused to ZEBRA Minimal Transduction Domain (MD) for Modulation of mRNA Transcripts.

Transcription factors (TFs) are key players in the control of gene expression and consequently all major cellular process, ranging from cell fate determination to cell cycle control and response to the environment.In particular cases, their ectopic expression has shown great promise in cell reprogramming for regenerative medicine, ontogenesis studies, and cell modeling. The current reprogramming methods mainly rely on gene transfer, therefore require technological improvements to limit genetic imprinting and improve safety. Direct protein delivery could represent an attractive alternative. Cell-penetrating peptides (CPPs) fused to recombinant TFs or other proteins involved in the epigenetic definition of cells have great potential in this context. We have thus developed the direct vectorization of Oct4, Sox2, or Nanog TFs and the posttranscriptional regulatory RNA-binding protein Lin28a by using the minimal transduction domain (MD11) of Epstein-Barr virus ZEBRA protein.This section describes the molecular cloning and production of different TFs fused to ZEBRA MD11 domain in the E. coli expression system. We also include the optimized purification conditions for each recombinant protein. The treatment of primary fibroblasts as well as cord blood-derived hematopoietic stem cells is also described. Finally, the transcriptional activation of the target genes following the transfer of TFs analyzed by quantitative PCR is presented.Our work primarily finds applications for advanced medicinal products, an area that requires novel therapy designs and delivery systems devoid of genetic material transfer to improve safety.

Identification of the Functions and Prognostic Values of RNA Binding Proteins in Bladder Cancer.

Post-transcriptional regulation plays a leading role in gene regulation and RNA binding proteins (RBPs) are the most important posttranscriptional regulatory protein. RBPs had been found to be abnormally expressed in a variety of tumors and is closely related to its occurrence and progression. However, the exact mechanism of RBPs in bladder cancer (BC) is unknown. We downloaded transcriptomic data of BC from the Cancer Genome Atlas (TCGA) database and used bioinformatics techniques for subsequent analysis. A total of 116 differentially expressed RBPs were selected, among which 61 were up-regulated and 55 were down-regulated. We then identified 12 prognostic RBPs including CTIF, CTU1, DARS2, ENOX1, IGF2BP2, LIN28A, MTG1, NOVA1, PPARGC1B, RBMS3, TDRD1, and ZNF106, and constructed a prognostic risk score model. Based on this model we found that patients in the high-risk group had poorer overall survival (P < 0.001), and the area under the receiver operator characteristic curve for this model was 0.677 for 1 year, 0.697 for 3 years, and 0.709 for 5 years. Next, we drew a nomogram based on the risk score and other clinical variables, which showed better predictive performance. Our findings contribute to a better understanding of the pathogenesis, progression and metastasis of BC. The model of these 12 genes has good predictive value and may have good prospects for improving clinical treatment regimens and patient prognosis.

CPEB1 deletion is not a common explanation for premature ovarian insufficiency in a Chinese cohort

PurposePremature ovarian insufficiency (POI), which is characterized by early menopause before the age of 40 years, affects approximately 1–5% of women. Cytoplasmic polyadenylation element binding protein 1 (CPEB1) is a post-transcriptional regulatory protein that is highly expressed in germ cells and promotes oocytes maturation, and several studies have found microdeletions of chromosome 15q25.2, which contains the CPEB1 gene, in POI patients. However, the deleted region also includes other plausible genes, and thus the contribution of CPEB1 to POI is uncertain. The present study aimed to determine the relationship between CPEB1 deletion and POI in a Chinese cohort.Material and methodsQuantitative real-time polymerase chain reaction (qPCR) with primers for exon 4 and exon 11 of CPEB1 was performed to detect the CPEB1 deletion in 323 patients with POI and in 300 healthy controls. Subsequent qPCR with primers for each exon of CPEB1 was performed to precisely localize the deletion locus.ResultsOne patient with primary amenorrhea was found to carry a heterozygous deletion of exons 8–12 of the CPEB1 gene.ConclusionOur study is the first to search for CPEB1 deletions in POI patients using a simple qPCR method, and we show that CPEB1 deletion is not a common cause for POI in a Chinese cohort.

Hfq modulates global protein pattern and stress response in Bordetella pertussis

B. pertussis is the etiological agent of whooping cough, a highly contagious respiratory disease which remains uncontrolled worldwide. Understanding how this pathogen responds to the environmental changes and adapts to different niches found inside the host might contribute to gain insight into bacterial pathogenesis. Comparative analyses of previous transcriptomic and proteomic data suggested that post-transcriptional regulatory mechanisms modulate B. pertussis virulence in response to iron availability. Iron scarcity represents one of the major stresses faced by bacterial pathogens inside the host. In this study, we used gel-free nanoLC-MS/MS-based proteomics to investigate whether Hfq, a highly conserved post-transcriptional regulatory protein, is involved in B. pertussis adaptation to low iron environment. To this end, we compared the protein profiles of wild type B. pertussis and its isogenic hfq deletion mutant strain under iron-replete and iron-depleted conditions. Almost of 33% of the proteins identified under iron starvation was found to be Hfq-dependent. Among them, proteins involved in oxidative stress tolerance and virulence factors that play a key role in the early steps of host colonization and bacterial persistence inside the host cells. Altogether these results suggest that Hfq shapes the infective phenotype of B. pertussis. SignificanceIn the last years, it became evident that post-transcriptional regulation of gene expression in ba cteria plays a central role in host-pathogen interactions. Hfq is a bacterial protein that regulates gene expression at post-transcriptional level found pivotal in the establishment of successful infections. In this study, we investigated the role of Hfq in Bordetella pertussis response to iron starvation, one of the main stresses imposed by the host. The data demonstrate that Hfq regulates the abundance of a significant number of B. pertussis proteins in response to iron starvation. Among them, virulence factors and proteins involved in oxidative stress tolerance, key players in host colonization and intracellular bacterial survival. Altogether, our results suggest a relevant role of Hfq in B. pertussis adaptation to the different niches found inside the host eventually granting bacterial pathogenesis.

The RNA-binding protein Celf1 post-transcriptionally regulates p27Kip1 and Dnase2b to control fiber cell nuclear degradation in lens development.

Opacification of the ocular lens, termed cataract, is a common cause of blindness. To become transparent, lens fiber cells undergo degradation of their organelles, including their nuclei, presenting a fundamental question: does signaling/transcription sufficiently explain differentiation of cells progressing toward compromised transcriptional potential? We report that a conserved RNA-binding protein Celf1 post-transcriptionally controls key genes to regulate lens fiber cell differentiation. Celf1-targeted knockout mice and celf1-knockdown zebrafish and Xenopus morphants have severe eye defects/cataract. Celf1 spatiotemporally down-regulates the cyclin-dependent kinase (Cdk) inhibitor p27Kip1 by interacting with its 5’ UTR and mediating translation inhibition. Celf1 deficiency causes ectopic up-regulation of p21Cip1. Further, Celf1 directly binds to the mRNA of the nuclease Dnase2b to maintain its high levels. Together these events are necessary for Cdk1-mediated lamin A/C phosphorylation to initiate nuclear envelope breakdown and DNA degradation in fiber cells. Moreover, Celf1 controls alternative splicing of the membrane-organization factor beta-spectrin and regulates F-actin-crosslinking factor Actn2 mRNA levels, thereby controlling fiber cell morphology. Thus, we illustrate new Celf1-regulated molecular mechanisms in lens development, suggesting that post-transcriptional regulatory RNA-binding proteins have evolved conserved functions to control vertebrate oculogenesis.

Global role of the bacterial post-transcriptional regulator CsrA revealed by integrated transcriptomics

CsrA is a post-transcriptional regulatory protein that is widely distributed among bacteria. This protein influences bacterial lifestyle decisions by binding to the 5′ untranslated and/or early coding regions of mRNA targets, causing changes in translation initiation, RNA stability, and/or transcription elongation. Here, we assess the contribution of CsrA to gene expression in Escherichia coli on a global scale. UV crosslinking immunoprecipitation and sequencing (CLIP-seq) identify RNAs that interact directly with CsrA in vivo, while ribosome profiling and RNA-seq uncover the impact of CsrA on translation, RNA abundance, and RNA stability. This combination of approaches reveals unprecedented detail about the regulatory role of CsrA, including novel binding targets and physiological roles, such as in envelope function and iron homeostasis. Our findings highlight the integration of CsrA throughout the E. coli regulatory network, where it orchestrates vast effects on gene expression.

Identification of a homogenous structural basis for oligomerization by retroviral Rev-like proteins

BackgroundRev-like proteins are post-transcriptional regulatory proteins found in several retrovirus genera, including lentiviruses, betaretroviruses, and deltaretroviruses. These essential proteins mediate the nuclear export of incompletely spliced viral RNA, and act by tethering viral pre-mRNA to the host CRM1 nuclear export machinery. Although all Rev-like proteins are functionally homologous, they share less than 30% sequence identity. In the present study, we computationally assessed the extent of structural homology among retroviral Rev-like proteins within a phylogenetic framework.ResultsWe undertook a comprehensive analysis of overall protein domain architecture and predicted secondary structural features for representative members of the Rev-like family of proteins. Similar patterns of α-helical domains were identified for Rev-like proteins within each genus, with the exception of deltaretroviruses, which were devoid of α-helices. Coiled-coil oligomerization motifs were also identified for most Rev-like proteins, with the notable exceptions of HIV-1, the deltaretroviruses, and some small ruminant lentiviruses. In Rev proteins of primate lentiviruses, the presence of predicted coiled-coil motifs segregated within specific primate lineages: HIV-1 descended from SIVs that lacked predicted coiled-coils in Rev whereas HIV-2 descended from SIVs that contained predicted coiled-coils in Rev. Phylogenetic ancestral reconstruction of coiled-coils for all Rev-like proteins predicted a single origin for the coiled-coil motif, followed by three losses of the predicted signal. The absence of a coiled-coil signal in HIV-1 was associated with replacement of canonical polar residues with non-canonical hydrophobic residues. However, hydrophobic residues were retained in the key ‘a’ and ‘d’ positions, and the α-helical region of HIV-1 Rev oligomerization domain could be modeled as a helical wheel with two predicted interaction interfaces. Moreover, the predicted interfaces mapped to the dimerization and oligomerization interfaces in HIV-1 Rev crystal structures. Helical wheel projections of other retroviral Rev-like proteins, including endogenous sequences, revealed similar interaction interfaces that could mediate oligomerization.ConclusionsSequence-based computational analyses of Rev-like proteins, together with helical wheel projections of oligomerization domains, reveal a conserved homogeneous structural basis for oligomerization by retroviral Rev-like proteins.

Modulation of the cytoplasmic functions of mammalian post-transcriptional regulatory proteins by methylation and acetylation: a key layer of regulation waiting to be uncovered?

Post-transcriptional control of gene expression is critical for normal cellular function and viability and many of the proteins that mediate post-transcriptional control are themselves subject to regulation by post-translational modification (PTM), e.g. phosphorylation. However, proteome-wide studies are revealing new complexities in the PTM status of mammalian proteins, in particular large numbers of novel methylated and acetylated residues are being identified. Here we review studied examples of methylation/acetylation-dependent regulation of post-transcriptional regulatory protein (PTRP) function and present collated PTM data that points to the huge potential for regulation of mRNA fate by these PTMs.

Abstract 3542: Inhibition of HuR effectively suppresses ovarian tumor growth in mice

Abstract Ovarian cancer is the most deadly gynecologic cancer. To address the clinical need for a more effective therapy than currently exists, this investigation focused on targeting a post-transcriptional (RNA to protein) regulatory RNA-binding protein, HuR (Human antigen R, aka ELAVL1). HuR regulates the expression of multiple genes known to function in tumor cell survival and in drug resistance. It is abundant and hyper-functional in ovarian cancer cells as compared to normal cells, thus providing a therapeutic window. We investigated the effect of inhibiting HuR expression in ovarian tumor cells in culture and in ovarian tumors in mice. We generated A2780 and OVCAR5 cells that constitutively express either shHuR or a control shRNA, and assayed cell proliferation and the ability of these cells to produce colonies in a soft agar. Cell proliferation in both shRNA-expressing cell lines was significantly reduced compared to control shRNA-expressing cells (19% and 11% in A2780 and OVCAR5, respectively; p&amp;lt;0.005). A2780-shRNA cells produced 65% fewer colonies in soft agar a compared to parental A2780 cells and control shRNA-expressing cells. To evaluate the effect of HuR suppression on tumor growth, C57BL/6 female mice were injected intraperitoneally with ID8-luc murine ovarian cancer cells; mice developed tumors throughout the peritoneal cavity. Approximately 4 weeks following cell injection, tumor-bearing mice were optically imaged to establish baseline biolumiscence, an indicator of ID8-luc-derived tumor load. A novel CpG-free DNA dendrimer vector, 3DNA, was derivatized with folate, aiming for tumor-targeted siHuR delivery upon systemic administration to mice. Mice were treated twice a week for four weeks (retro-orbital injection) with one of three 3DNA formulations or with 0.9% saline (n = 5-6/group). The 3DNA formulations were: 1) Folate-derivatized 3DNA/siHuR; 2) 3DNA/siHuR; and 3) Folate-derivatized 3DNA/control siRNA. Tumor response to treatment was assessed once a week by optical imaging. Using baseline tumor load as the comparator, tumor growth was suppressed ∼3-fold in mice treated for 4 weeks with folate-derivatized 3DNA/siHuR, the highest suppression of all treatment groups, including non-folate derivatized 3DNA/siHuR (p = 0.01), folate-derivatized 3DNA/control siRNA (p = 0.05), and 0.9% saline (p = 0.04). No overt toxicity was observed. A biodistribution study in which CY3-labeled folate-derivatized 3DNA was systemically administered resulted in very high fluorescence in ovarian tumors with little to no fluorescence in normal tissues, thus supporting the tumor-targeting potential of folate and the enhanced therapeutic effect observed in folate-derivatized 3DNA/siHuR treated mice. These results support further pursuit of HuR-targeted therapy as a complementary approach to current, active therapies for ovarian cancer for overcoming drug resistance and inhibiting tumor growth. Citation Format: Janet A. Sawicki, Yu-Hung Huang, Jonathan R. Brody, Robert C. Getts, Kelly Rhodes, Jackie Gerhart. Inhibition of HuR effectively suppresses ovarian tumor growth in mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3542. doi:10.1158/1538-7445.AM2015-3542

Changes in proteome of the Δhfq strain derived from Francisella tularensis LVS correspond with its attenuated phenotype.

The posttranscriptional regulatory protein Hfq was shown to be an important determinant of the stress resistance and full virulence in the dangerous human pathogen Francisella tularensis. Transcriptomics brought rather limited clues to the precise contribution of Hfq in virulence. To reveal the molecular basis of the attenuation caused by hfq inactivation, we employed iTRAQ in the present study and compared proteomes of the parent and isogenic Δhfq strains. We show that Hfq modulates the level of 76 proteins. Most of them show decreased abundance in the ∆hfq mutant, thereby indicating that Hfq widely acts rather as a positive regulator of Francisella gene expression. Several key Francisella virulence factors including those encoded within the Francisella pathogenicity island were found among the downregulated proteins, which is in a good agreement with the attenuated phenotype of the Δhfq strain. To further validate the iTRAQ exploratory findings, we subsequently performed targeted LC-SRM analysis of selected proteins. This accurate quantification method corroborated the trends found in the iTRAQ data.

Unperturbed posttranscriptional regulatory Rev protein function and HIV-1 replication in astrocytes.

Astrocytes protect neurons, but also evoke proinflammatory responses to injury and viral infections, including HIV. There is a prevailing notion that HIV-1 Rev protein function in astrocytes is perturbed, leading to restricted viral replication. In earlier studies, our finding of restricted viral entry into astrocytes led us to investigate whether there are any intracellular restrictions, including crippled Rev function, in astrocytes. Despite barely detectable levels of DDX3 (Rev-supporting RNA helicase) and TRBP (anti-PKR) in primary astrocytes compared to astrocytic cells, Rev function was unperturbed in wild-type, but not DDX3-ablated astrocytes. As in permissive cells, after HIV-1 entry bypass in astrocytes, viral-encoded Tat and Rev proteins had robust regulatory activities, leading to efficient viral replication. Productive HIV-1 infection in astrocytes persisted for several weeks. Our findings on HIV-1 entry bypass in astrocytes demonstrated that the intracellular environment is conducive to viral replication and that Tat and Rev functions are unperturbed.

Cell-Based Screening Assay for Antiviral Compounds Targeting the Ability of Herpesvirus Posttranscriptional Regulatory Proteins To Stabilize Viral mRNAs

Each human herpesvirus expresses a multifunctional regulatory protein that is essential for lytic viral replication. A cell-based assay targeting the function of these proteins was developed based on the finding that Epstein-Barr virus (EBV) SM and Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 stabilize specific target mRNAs. Both proteins facilitate the accumulation of lytic transcripts by incompletely characterized posttranscriptional mechanisms. SM and ORF57 exhibit target gene specificity and enhance the accumulation of certain EBV and KSHV mRNAs that are poorly expressed in their absence. Conversely, SM- and ORF57-independent viral and cellular transcripts accumulate efficiently, and their expression does not respond to SM or ORF57. Fusion of an ORF57-responsive transcript to ORF57-independent transcripts demonstrated that ORF57 dependence is cis-dominant. EBV SM also enhanced the accumulation of such fused mRNA transcripts. These data suggest that the coding regions of specific viral transcripts confer instability even when fused to heterologous genes. The findings were used to develop a reporter assay that measures EBV SM function in rescuing the expression of poorly expressed transcripts by posttranscriptional mechanisms. The assay represents a method for the screening of small interfering RNAs (siRNAs) and compounds to investigate the mechanism of action of SM and its homologs and potentially to aid in the discovery of novel antiviral agents.

RNA mimicry, a decoy for regulatory proteins

Small non-coding RNA molecules (sRNA) are key regulators participating in complex networks, which adapt metabolism in response to environmental changes. In this issue of Molecular Microbiology, and in a related paper in Proc. Natl. Acad. Sci. USA, Moreno et al. (2011) and Sonnleitner et al. (2009) report on novel sRNAs, which act as decoys to inhibit the activity of the master post-transcriptional regulatory protein Crc. Crc is a key protein involved in carbon catabolite repression that optimizes metabolism improving the adaptation of the bacteria to their diverse habitats. Crc is a novel RNA-binding protein that regulates translation of multiple target mRNAs. Two regulatory sRNAs in Pseudomonas putida mimic the natural mRNA targets of Crc and counteract the action of Crc by sequestrating the protein when catabolite repression is absent. Crc trapping by a sRNA is a mechanism reminiscent to the regulation of the repressor of secondary metabolites (RsmA) in Pseudomonas, and highlights the suitability of RNA-dependent regulation to rapidly adjust cell growth in response to environmental changes.

The Sm-like RNA chaperone Hfq mediates transcription antitermination at Rho-dependent terminators

In Escherichia coli, the essential motor protein Rho promotes transcription termination in a tightly controlled manner that is not fully understood. Here, we show that the general post-transcriptional regulatory protein Hfq associates with Rho to regulate Rho function. The Hfq:Rho complex can be further stabilized by RNA bridging both factors in a configuration that inhibits the ATP hydrolysis and duplex unwinding activities of Rho and that mediates transcription antitermination at Rho-dependent terminators in vitro and in vivo. Antitermination at a prototypical terminator (λtR1) requires Hfq binding to an A/U-rich transcript region directly upstream from the terminator. Antitermination is modulated by trans-acting factors (NusG or nucleic acid competitors) that affect Hfq association with Rho or RNA. These data unveil a new Hfq function and a novel transcription regulatory mechanism with potentially important implications for bacterial RNA metabolism, gene silencing, and pathogenicity.

ICP27 protein of Marek’s disease virus interacts with SR proteins and inhibits the splicing of cellular telomerase chTERT and viral vIL8 transcripts

All herpesviruses have a post-transcriptional regulatory protein that prevents precursor mRNA splicing and leads to the shutting off of host protein synthesis. The ICP27 protein of herpes simplex virus 1 (HSV-1) is the prototype of these proteins. Marek's disease virus (MDV-1), an alphaherpesvirus that induces lymphoma in birds, also has an ICP27 protein that is produced in lytic MDV-1-infected cells. We characterized this protein. We demonstrated ICP27 production in latently infected MSB-1 cells, but only on MDV-1 reactivation. ICP27 was found predominantly in specific structures within the nucleus. The ICP27 of MDV-1 colocalized and interacted with SR proteins. We demonstrated inhibitory effects of MDV-1 ICP27 on the splicing of both the viral vIL8 and cellular chTERT (telomerase reverse transcriptase) genes. Thus, the ICP27 of MDV-1 plays a similar role to the ICP27 of HSV-1 and may be involved in MDV-1 replication and the development of Marek's disease.

General and Target-Specific RNA Binding Properties of Epstein-Barr Virus SM Posttranscriptional Regulatory Protein

Epstein-Barr virus (EBV) SM protein is an essential nuclear shuttling protein expressed by EBV early during the lytic phase of replication. SM acts to increase EBV lytic gene expression by binding EBV mRNAs and enhancing accumulation of the majority of EBV lytic cycle mRNAs. SM increases target mRNA stability and nuclear export, in addition to modulating RNA splicing. SM and its homologs in other herpesvirus have been hypothesized to function in part by binding viral RNAs and recruiting cellular export factors. Although activation of gene expression by SM is gene specific, it is unknown whether SM binds to mRNA in a specific manner or whether its RNA binding is target independent. SM-mRNA complexes were isolated from EBV-infected B-lymphocyte cell lines induced to permit lytic EBV replication, and a quantitative measurement of mRNAs corresponding to all known EBV open reading frames was performed by real-time quantitative reverse transcription-PCR. The results showed that although SM has broad RNA binding properties, there is a clear hierarchy of affinities among EBV mRNAs with respect to SM complex formation. In vitro binding assays with two of the most highly SM-associated transcripts suggested that SM binds preferentially to specific sequences or structures present in noncoding regions of some EBV mRNAs. Furthermore, the presence of these sequences conferred responsiveness to SM. These data are consistent with a mechanism of action similar to that of hnRNPs, which exert sequence-specific effects on gene expression despite having multiple degenerate consensus binding sites common to a large number of RNAs.