Pat1 proteins: regulating mRNAs from birth to death?

2012 ◽  
Vol 3 (4) ◽  
pp. 295-306
Author(s):  
Nancy Standart ◽  
Aline Marnef
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Mrna Decay ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Viral Gene Expression ◽  
Model Systems ◽  
Viral Gene ◽  
Nuclear Speckles ◽  
Mrna Targets

AbstractThe Pat1 protein family has been the subject of several recent extensive investigations of diverse model systems ranging from yeast, flies and worms to man, using a variety of experimental approaches. Although some contradictions remain, the emerging consensus view is that these RNA-binding proteins act in mRNA decay by physically linking deadenylation with decapping and by regulating gene expression as translational repressors. These multiple functions are present in the single invertebrate Pat1 proteins, whereas, in vertebrates, one Pat1 variant represses translation in early development, while a somatic version synthesised in embrogenesis and in adults acts in mRNA decay. At steady state, Pat1 proteins are found enriched in cytoplasmic P(rocessing)-bodies, and related mRNP complexes and granules. Evidence recently obtained from mammalian tissue culture cells shows that Pat1 shuttles in and out of the nucleus, where it localises to nuclear speckles, PML bodies and nucleolar caps, which suggests RNA-related nuclear functions. Less well understood, Pat1 proteins may play additional roles in miRNA silencing and/or biogenesis, as well in the regulation of viral gene expression. Due to the relatively low level of sequence conservation between Pat1 proteins from different species and lacking any discernable motifs, determining their functional domains has proved difficult, as is obtaining a simple unified view of the location of the binding sites of their interacting proteins in all examined species. Questions that remain to be addressed include the following: 1) What are their roles in the nucleus? 2) What is the link, if one exists, between their cytoplasmic and nuclear roles? 3) Do they have specific mRNA targets? 4) Which signalling pathways regulate their P-body localisation in mammalian cells, which may affect quiescent cell survival?

scholarly journals Dysregulation of M segment gene expression contributes to influenza A virus host restriction

2019 ◽  
Author(s):  
Brenda M. Calderon ◽  
Shamika Danzy ◽  
Gabrielle K. Delima ◽  
Nathan T. Jacobs ◽  
Ketaki Ganti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Influenza A Virus ◽  
Mammalian Cells ◽  
Influenza A ◽  
Viral Gene Expression ◽  
Host Adaptation ◽  
Model Systems ◽  
Viral Gene ◽  
M2 Protein ◽  
Viral Growth

AbstractThe M segment of the 2009 pandemic influenza A virus (IAV) has been implicated in its emergence into human populations. To elucidate the genetic contributions of the M segment to host adaptation, and the underlying mechanisms, we examined a panel of isogenic viruses that carry avian- or human-derived M segments. Avian, but not human, M segments restricted viral growth and transmission in mammalian model systems, and the restricted growth correlated with increased expression of M2 relative to M1. M2 overexpression was associated with intracellular accumulation of autophagosomes, which was alleviated by interference of the viral proton channel activity by amantadine treatment. As M1 and M2 are expressed from the M mRNA through alternative splicing, we separated synonymous and non-synonymous changes that differentiate human and avian M segments and found that dysregulation of gene expression leading to M2 overexpression diminished replication, irrespective of amino acid composition of M1 or M2. Moreover, in spite of efficient replication, virus possessing a human M segment that expressed avian M2 protein at low level did not transmit efficiently. We conclude that (i) determinants of transmission reside in the IAV M2 protein, and that (ii) control of M segment gene expression is a critical aspect of IAV host adaptation needed to prevent M2-mediated dysregulation of vesicular homeostasis.Author summaryInfluenza A virus (IAV) pandemics arise when a virus adapted to a non-human host overcomes species barriers to successfully infect humans and sustain human-to-human transmission. To gauge the adaptive potential and therefore pandemic risk posed by a particular IAV, it is critical to understand the mechanisms underlying viral adaptation to human hosts. Here, we focused on the role of one of IAV’s eight gene segments, the M segment, in host adaptation. Comparing the growth of IAVs with avian- and human-derived M segments in avian and mammalian systems revealed that the avian M segment restricts viral growth specifically in mammalian cells. We show that the mechanism underlying this host range phenotype is a dysregulation of viral gene expression when the avian IAV M segment is transcribed in mammalian cells. In particular, excess production of the M2 protein results in viral interference with cellular functions on which the virus relies. Our results therefore reveal that the use of cellular machinery to control viral gene expression leaves the virus vulnerable to over- or under-production of critical viral gene products in a new host species.

scholarly journals Hepatitis B virus Core protein nuclear interactome identifies SRSF10 as a host RNA-binding protein restricting HBV RNA production

2020 ◽  
Author(s):  
Hélène Chabrolles ◽  
Héloïse Auclair ◽  
Serena Vegna ◽  
Thomas Lahlali ◽  
Caroline Pons ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Core Protein ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
B Virus ◽  
Regulatory Functions

AbstractDespite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.Author SummaryChronic infection with Hepatitis B virus (HBV) affects more than 250 millions of people world-wide and is a major global cause of liver cancer. Current treatments lead to a significant reduction of viremia in patients. However, viral clearance is rarely obtained and the persistence of the HBV genome in the hepatocyte’s nucleus generates a stable source of viral RNAs and subsequently proteins which play important roles in immune escape mechanisms and liver disease progression. Therapies aiming at efficiently and durably eliminating viral gene expression are still required. In this study, we identified the nuclear partners of the HBV Core protein (HBc) to understand how this structural protein, responsible for capsid assembly in the cytoplasm, could also regulate viral gene expression. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs). One of these RBPs, SRSF10, was demonstrated to restrict HBV RNA levels and a drug, able to alter its phosphorylation, behaved as an antiviral compound capable of reducing viral gene expression. Altogether, this study sheds new light novel regulatory functions of HBc and provides information relevant for the development of antiviral strategies aiming at preventing viral gene expression.

Protein-RNA interactome analysis reveals wide association of KSHV ORF57 with host non-coding RNAs and polysomes

2021 ◽  
Author(s):  
Beatriz Alvarado-Hernandez ◽  
Yanping Ma ◽  
Nishi R. Sharma ◽  
Vladimir Majerciak ◽  
Alexei Lobanov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Splicing ◽  
Rna Binding ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
28S Rrna ◽  
Protein Coding ◽  
Infected Cells ◽  
Non Coding Rnas ◽  
Rna Interaction

Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF57 is an RNA-binding post-transcriptional regulator. We recently applied an affinity-purified anti-ORF57 antibody to conduct ORF57-CLIP (Cross-linking Immunoprecipitation) in combination with RNA-sequencing (CLIP-seq) and analyzed the genome-wide host RNA transcripts in association with ORF57 in BCBL-1 cells with lytic KSHV infection. Mapping of the CLIPed RNA reads to the human genome (GRCh37) revealed that most of the ORF57-associated RNA reads were from rRNAs. The remaining RNA reads mapped to several classes of host non-coding and protein-coding mRNAs. We found ORF57 binds and regulates expression of a subset of host lncRNAs, including LINC00324, LINC00355, and LINC00839 which are involved in cell growth. ORF57 binds snoRNAs responsible for 18S and 28S rRNA modifications, but does not interact with fibrillarin and NOP58. We validated ORF57 interactions with 67 snoRNAs by ORF57-RNA immunoprecipitation (RIP)-snoRNA-array assays. Most of the identified ORF57 rRNA binding sites (BS) overlap with the sites binding snoRNAs. We confirmed ORF57-snoRA71B RNA interaction in BCBL-1 cells by ORF57-RIP and Northern blot analyses using a 32 P-labeled oligo probe from the 18S rRNA region complementary to snoRA71B. Using RNA oligos from the rRNA regions that ORF57 binds for oligo pulldown-Western blot assays, we selectively verified ORF57 interactions with 5.8S and 18S rRNAs. Polysome profiling revealed that ORF57 associates with both monosomes and polysomes and its association with polysomes increases PABPC1 binding to, but prevent Ago2 from polysomes. Our data indicate a functional correlation with ORF57 binding and suppression of Ago2 activities for ORF57 promotion of gene expression. Significance As an RNA-binding protein, KSHV ORF57 regulates RNA splicing, stability, and translation and inhibits host innate immunity by blocking the formation of RNA granules in virus infected cells. In this report, ORF57 was found to interact many host non-coding RNAs, including lncRNAs, snoRNAs and ribosomal RNAs to carry out additional unknown functions. ORF57 binds a group of lncRNAs via the identified RNA motifs by ORF57 CLIP-seq to regulate their expression. ORF57 associates with snoRNAs independently of fibrillarin and NOP58 proteins, and with ribosomal RNA in the regions that commonly bind snoRNAs. Knockdown of fibrillarin expression decreases the expression of snoRNAs and CDK4, but not affect viral gene expression. More importantly, we found that ORF57 binds translationally active polysomes and enhances PABPC-1 but prevents Ago2 association with polysomes. Data provide a compelling evidence on how ORF57 in KSHV infected cells might regulate protein synthesis by blocking Ago2’s hostile activities on translation.

scholarly journals CELF RNA binding proteins promote axon regeneration in C. elegans and mammals through alternative splicing of Syntaxins

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Lizhen Chen ◽  
Zhijie Liu ◽  
Bing Zhou ◽  
Chaoliang Wei ◽  
Yu Zhou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Alternative Splicing ◽  
Binding Proteins ◽  
Rna Binding ◽  
Axon Regeneration ◽  
Rna Binding Proteins ◽  
Mrna Isoforms ◽  
C Elegans ◽  
Mrna Targets

Axon injury triggers dramatic changes in gene expression. While transcriptional regulation of injury-induced gene expression is widely studied, less is known about the roles of RNA binding proteins (RBPs) in post-transcriptional regulation during axon regeneration. In C. elegans the CELF (CUGBP and Etr-3 Like Factor) family RBP UNC-75 is required for axon regeneration. Using crosslinking immunoprecipitation coupled with deep sequencing (CLIP-seq) we identify a set of genes involved in synaptic transmission as mRNA targets of UNC-75. In particular, we show that UNC-75 regulates alternative splicing of two mRNA isoforms of the SNARE Syntaxin/unc-64. In C. elegans mutants lacking unc-75 or its targets, regenerating axons form growth cones, yet are deficient in extension. Extending these findings to mammalian axon regeneration, we show that mouse Celf2 expression is upregulated after peripheral nerve injury and that Celf2 mutant mice are defective in axon regeneration. Further, mRNAs for several Syntaxins show CELF2 dependent regulation. Our data delineate a post-transcriptional regulatory pathway with a conserved role in regenerative axon extension.

scholarly journals Posttranscriptional gene regulation by RNA-binding proteins during oxidative stress: implications for cellular senescence

2008 ◽  
Vol 389 (3) ◽  
pp. 243-255 ◽  
Author(s):  
Kotb Abdelmohsen ◽  
Yuki Kuwano ◽  
Hyeon Ho Kim ◽  
Myriam Gorospe
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Cellular Senescence ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Oxidant Damage ◽  
Regulated Gene Expression

AbstractTo respond adequately to oxidative stress, mammalian cells elicit rapid and tightly controlled changes in gene expression patterns. Besides alterations in the subsets of transcribed genes, two posttranscriptional processes prominently influence the oxidant-triggered gene expression programs: mRNA turnover and translation. Here, we review recent progress in our knowledge of theturnover andtranslationregulatory (TTR) mRNA-bindingproteins (RBPs) that influence gene expression in response to oxidative damage. Specifically, we identify oxidant damage-regulated mRNAs that are targets of TTR-RBPs, we review the oxidant-triggered signaling pathways that govern TTR-RBP function, and we examine emerging evidence that TTR-RBP activity is altered with senescence and aging. Given the potent influence of TTR-RBPs upon oxidant-regulated gene expression profiles, we propose that the senescence-associated changes in TTR-RBPs directly contribute to the impaired responses to oxidant damage that characterize cellular senescence and advancing age.

scholarly journals Genetic Modification of Oncolytic Newcastle Disease Virus for Cancer Therapy

2016 ◽  
Vol 90 (11) ◽  
pp. 5343-5352 ◽  
Author(s):  
Xing Cheng ◽  
Weijia Wang ◽  
Qi Xu ◽  
James Harper ◽  
Danielle Carroll ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Culture ◽  
Cancer Therapy ◽  
Newcastle Disease Virus ◽  
Newcastle Disease ◽  
Mammalian Cells ◽  
Oncolytic Virus ◽  
Viral Gene Expression ◽  
Clinical Development ◽  
Viral Gene

ABSTRACTClinical development of a mesogenic strain of Newcastle disease virus (NDV) as an oncolytic agent for cancer therapy has been hampered by its select agent status due to its pathogenicity in avian species. Using reverse genetics, we have generated a lead candidate oncolytic NDV based on the mesogenic NDV-73T strain that is no longer classified as a select agent for clinical development. This recombinant NDV has a modification at the fusion protein (F) cleavage site to reduce the efficiency of F protein cleavage and an insertion of a 198-nucleotide sequence into the HN-L intergenic region, resulting in reduced viral gene expression and replication in avian cells but not in mammalian cells. In mammalian cells, except for viral polymerase (L) gene expression, viral gene expression is not negatively impacted or increased by the HN-L intergenic insertion. Furthermore, the virus can be engineered to express a foreign gene while still retaining the ability to grow to high titers in cell culture. The recombinant NDV selectively replicates in and kills tumor cells and is able to drive potent tumor growth inhibition following intratumoral or intravenous administration in a mouse tumor model. The candidate is well positioned for clinical development as an oncolytic virus.IMPORTANCEAvian paramyxovirus type 1, NDV, has been an attractive oncolytic agent for cancer virotherapy. However, this virus can cause epidemic disease in poultry, and concerns about the potential environmental and economic impact of an NDV outbreak have precluded its clinical development. Here we describe generation and characterization of a highly potent oncolytic NDV variant that is unlikely to cause Newcastle disease in its avian host, representing an essential step toward moving NDV forward as an oncolytic agent. Several attenuation mechanisms have been genetically engineered into the recombinant NDV that reduce chicken pathogenicity to a level that is acceptable worldwide without impacting viral production in cell culture. The selective tumor replication of this recombinant NDV, bothin vitroandin vivo, along with efficient tumor cell killing makes it an attractive oncolytic virus candidate that may provide clinical benefit to patients.

scholarly journals Structural features within the NORAD long noncoding RNA underlie efficient repression of Pumilio activity

2021 ◽  
Author(s):  
Omer Ziv ◽  
Svetlana Farberov ◽  
Jian You Lau ◽  
Eric A Miska ◽  
Grzegorz Kudla ◽  
...  
Keyword(s):  
Rna Structure ◽  
Mammalian Cells ◽  
Noncoding Rna ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Structural Features ◽  
Structural Domain ◽  
Mrna Targets ◽  
Structural Principles ◽  
Pumilio Proteins

It is increasingly appreciated that long non-coding RNAs (lncRNAs) carry out important functions in mammalian cells, but how these are encoded in their sequences and manifested in their structures remains largely unknown. Some lncRNAs bind to and modulate the availability of RNA binding proteins, but the structural principles that underlie this mode of regulation are underexplored. Here, we focused on the NORAD lncRNA, which binds Pumilio proteins and modulates their ability to repress hundreds of mRNA targets. We probed the RNA structure and long-range RNA-RNA interactions formed by NORAD inside cells, under different stressful conditions. We discovered that NORAD structure is highly modular, and consists of well-defined domains that contribute independently to NORAD function. We discovered that NORAD structure spatially clusters the Pumilio binding sites along NORAD in a manner that contributes to the de-repression of Pumilio target proteins. Following arsenite stress, the majority of NORAD structure undergoes relaxation and forms inter-molecular interactions with RNAs that are targeted to stress granules. NORAD sequence thus dictates elaborated structural domain organization that facilitates its function on multiple levels, and which helps explain the extensive evolutionary sequence conservation of NORAD regions that are not predicted to directly bind Pumilio proteins.

scholarly journals Multiple RSV strains infecting HEp-2 and A549 cells reveal cell line-dependent differences in resistance to RSV infection.

2021 ◽  
Author(s):  
Anubama Rajan ◽  
Felipe-Andres Piedra ◽  
Letisha Aideyan ◽  
Trevor McBride ◽  
Matthew J Robertson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Cell Lines ◽  
A549 Cells ◽  
Viral Gene Expression ◽  
Model Systems ◽  
Viral Gene ◽  
Transformed Cell ◽  
Syncytial Virus ◽  
Transformed Cell Lines

Respiratory syncytial virus (RSV) is a leading cause of pediatric acute respiratory infection worldwide. There are currently no approved vaccines or antivirals to combat RSV disease. A few transformed cell lines and two historic strains have been extensively used to study RSV. Here we report a thorough molecular and cell biological characterization of HEp-2 and A549 cells infected with four strains of RSV representing both major subgroups as well as historic and more contemporaneous genotypes -- [RSV/A/Tracy (GA1), RSV/A/Ontario (ON), RSV/B/18537 (GB1), RSV/B/Buenos Aires (BA)] -- via measurements of viral replication kinetics and viral gene expression, immunofluorescence-based imaging of gross cellular morphology and cell-associated RSV, and measurements of host response including transcriptional changes and levels of secreted cytokines and growth factors. Our findings strongly suggest 1) the existence of a conserved difference in gene expression between RSV subgroups A and B; 2) the A549 cell line is a more stringent and natural host of replicating RSV than the HEp-2 cell line; and 3) consistent with previous studies, determining the full effects of viral genetic variation in RSV pathogenesis requires model systems as tractable as transformed cell lines but better representative of the human host.

scholarly journals Influenza Virus Usurps an Interferon-Induced Translational Program to Promote Viral Replication

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 134
Author(s):  
Mitchell P. Ledwith ◽  
Vy Tran ◽  
Thiprampai Thamamongood ◽  
Christina A. Higgins ◽  
Shashank Tripathi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Influenza Virus ◽  
Viral Replication ◽  
Rna Binding ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Translational Efficiency ◽  
A Genome ◽  
Cellular Mrnas ◽  
Stimulation Of

Hosts mount prudently tuned responses to viral infection in an attempt to block nearly every step of the replication cycle. Viruses must adapt to replicate in this hostile antiviral cellular state. Interferon stimulation or pathogen challenge robustly induces expression of IFIT (interferon-induced proteins with tetratricopeptide repeats) proteins. IFITs are a family of proteins that bind RNA and play antiviral roles during infection. Thus, we were surprised to identify the IFIT family as top candidate proviral host factors for influenza A virus (IAV) in a genome-wide CRISPR–Cas9 knockout screen. We validated the proviral activity of IFIT2 by showing that IFIT2-deficient cells support lower levels of IAV replication and exhibit defects in viral gene expression. The molecular functions of IFIT2, let alone how they are used by influenza virus, are unknown. Using CLIP-seq, we showed that IFIT2 binds directly to viral and cellular mRNAs in AU-rich regions largely in the 3’UTR, with a preference for a subset of interferon-stimulated mRNAs. IFIT2 also associates with actively translating ribosomes in infected cells to facilitate the translation of viral messages. IFIT2-responsive elements from an IAV mRNA were sufficient to confer translational enhancement to exogenous transcripts in cis. Conversely, mutation of these elements or the use of an IFIT2 RNA-binding mutant ablated stimulation of viral gene expression. Together, these data link the RNA-binding capability of IFIT2 to changes in translational efficiency of target viral mRNAs and the stimulation of viral replication. They establish a model for the normal function of IFIT2 as an antiviral protein affecting the post-transcriptional fate of cellular mRNAs and explain how influenza virus repurposes IFIT2 to support viral replication. Our work highlights a new node for the regulation of translation during interferon responses and highlights how canonical antiviral responses may be repurposed to support viral replication.

Real-time analysis of the transcriptional regulation of HIV and hCMV promoters in single mammalian cells

1995 ◽  
Vol 108 (2) ◽  
pp. 441-455
Author(s):  
M.R. White ◽  
M. Masuko ◽  
L. Amet ◽  
G. Elliott ◽  
M. Braddock ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Sodium Butyrate ◽  
Mammalian Cells ◽  
Viral Gene Expression ◽  
Luciferase Expression ◽  
Viral Gene ◽  
Time Analysis ◽  
Real Time Analysis ◽  
Viral Promoters

The regulation of human cytomegalovirus (hCMV) and human immunodeficiency virus (HIV) gene expression has been studied in single intact mammalian cells. Viral promoters were placed upstream of the firefly luciferase reporter gene and the resulting hybrid reporter constructs were stably integrated into the HeLa cell genome. A highly sensitive photon-counting camera system was used to study the level of gene expression in single intact cells. Luciferase expression was studied in the absence of activators of viral gene expression, in the presence of the HIV-1 TAT transactivator protein, or in the presence of sodium butyrate, a non-viral activator of gene expression. In the absence of any activator of gene expression, while expression was undetectable in most cells, significant levels of basal luciferase activity were observed in a few cells, indicating heterogeneity in gene expression in the cell population. In the presence of the general activator of viral gene expression, sodium butyrate, transcriptional activation from the viral promoters gave rise to significant and relatively homogeneous levels of luciferase expression in a majority of cells. The luciferase imaging technology was used for the real-time analysis of changes of gene expression within a single cell. This non-invasive reporter assay should become important for studies of the temporal regulation of gene expression in single cells.

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