Loss of ADAR1 protein induces changes in small RNA landscape in hepatocytes.

In recent years, numerous evidence has been accumulated about the extent of A-to-I editing in human RNAs and the key role ADAR1 plays in the cellular editing machinery. It has been shown that A-to-I editing occurrence and frequency are tissue specific and essential for some tissue development, such as liver. To study the effect of ADAR1 function in hepatocytes, we have created Huh7.5 ADAR1 KO cell lines. Upon IFN treatment, the Huh7.5 ADAR1 KO cells show rapid arrest of growth and translation, from which they do not recover. We analyzed translatome changes by employing a method based on sequencing of separate polysome profile RNA fractions. We found significant changes in transcriptome and translatome of the Huh7.5 ADAR1 KO cells. The most prominent changes include negatively affected transcription by RNA polymerase III and the deregulation of snoRNA and Y RNA levels. Furthermore, we observed that ADAR1 KO polysomes are enriched in mRNAs coding for proteins pivotal in a wide range of biological processes such as RNA localization and RNA processing, whereas the unbound fraction is enriched mainly in mRNAs coding for ribosomal proteins and translational factors. This indicates that ADAR1 plays more relevant role in small RNA metabolism and ribosome biogenesis.

Transcription apparatus of the yeast virus-like elements: Architecture, function, and evolutionary origin.

Extrachromosomal hereditary elements such as organelles, viruses, and plasmids are important for the cell fitness and survival. Their transcription is dependent on host cellular RNA polymerase (RNAP) or intrinsic RNAP encoded by these elements. The yeast Kluyveromyces lactis contains linear cytoplasmic DNA virus-like elements (VLEs, also known as linear plasmids) that bear genes encoding putative non-canonical two-subunit RNAP. Here, we describe the architecture and identify the evolutionary origin of this transcription machinery. We show that the two RNAP subunits interact in vivo, and this complex interacts with another two VLE-encoded proteins, namely the mRNA capping enzyme and a putative helicase. RNAP, mRNA capping enzyme and the helicase also interact with VLE-specific DNA in vivo. Further, we identify a promoter sequence element that causes 5' mRNA polyadenylation of VLE-specific transcripts via RNAP slippage at the transcription initiation site, and structural elements that precede the termination sites. As a result, we present a first model of the yeast virus-like element transcription initiation and intrinsic termination. Finally, we demonstrate that VLE RNAP and its promoters display high similarity to poxviral RNAP and promoters of early poxviral genes, respectively, thereby pointing to their evolutionary origin.

Co-localization of Interleukin-1α and Annexin A2 at the plasma membrane in response to oxidative stress.

Interleukin-1α (IL-1α) and Annexin A2 (AnxA2) are pleiotropic molecules with both intracellular and extracellular roles. They share several characteristics including unconventional secretion aided by S100 proteins, anchoring of the externalized proteins at the outer surface of the plasma membrane and response to oxidative stress. Although IL-1α and AnxA2 have been implicated in a variety of biological processes, including cancer, little is known about the mechanisms of their cellular release. In the present study, employing the non-cancerous breast epithelial MCF10A cells, we demonstrate that IL-1α and AnxA2 establish a close association in response to oxidative stress. Stress conditions lead to translocation of both proteins towards lamellipodia rich in vimentin and association of full-length IL-1α and Tyr23 phosphorylated AnxA2 with the plasma membrane at peripheral sites depleted of F-actin. Notably, membrane-associated IL-1α and AnxA2 preferentially localize to the outer edges of the MCF10A cell islands, suggesting that the two proteins participate in the communication of these epithelial cells with their neighboring cells. Similarly, in U2OS osteosarcoma cell line both endogenous IL-1α and transiently produced IL-1α/EGFP associate with the plasma membrane. While benign MFC10A cells present membrane-associated IL-1α and AnxA2 at the edges of their cell islands, the aggressive cancerous U2OS cells communicate in such manner also with distant cells.

Major splice variants and multiple polyadenylation site utilization in mRNAs encoding human translation initiation factors eIF4E1 and eIF4E3 regulate the translational regulators?

Alternative polyadenylation is an important and pervasive mechanism that generates heterogeneous 3'-termini of mRNA and is considered an important regulator of gene expression. We performed bioinformatics analyses of ESTs and the 3'-UTRs of the main transcript splice variants of the translational initiation factor eIF4E1 and its family members, eIF4E2 and eIF4E3. This systematic analysis led to the prediction of new polyadenylation signals. All identified polyadenylation sites were subsequently verified by 3'RACE of transcripts isolated from human lymphoblastic cell lines. This led to the observation that multiple simultaneous polyadenylation site utilization occurs in single cell population. Importantly, we described the use of new polyadenylation site in the eIF4E1 mRNA, which lacked any known polyadenylation signal. The proportion of eIF4E1 transcripts derived from the first two polyadenylation sites in eIF4E1 mRNA achieved 15% in a wide range of cell lines. This result demonstrates the ubiquitous presence of ARE-lacking transcripts, which escape HuR/Auf1-mediated control, the main mechanism of eIF4E1 gene expression regulation. We found many EST clones documenting the significant production of transcript variants 2-4 of eIF4E2 gene that encode proteins with C-termini that were distinct from the mainly studied prototypical isoform A. Similarly, eIF4E3 mRNAs are produced as two main variants with the same very long 3'-UTR with potential for heavy post-transcriptional regulation. We identified sparsely documented transcript variant 1 of eIF4E3 gene in human placenta. eIF4E3 truncated transcript variants were found mainly in brain. We propose to elucidate the minor splice variants of eIF4E2 and eIF4E3 in great detail because they might produce proteins with modified features that fulfill different cellular roles from their major counterparts.

HCVIVdb: The hepatitis-C IRES variation database.

BACKGROUND: Sequence variability in the hepatitis C virus (HCV) genome has led to the development and classification of six genotypes and a number of subtypes. The HCV 5' untranslated region mainly comprises an internal ribosomal entry site (IRES) responsible for cap-independent synthesis of the viral polyprotein and is conserved among all HCV genotypes. DESCRIPTION: Considering the possible high impact of variations in HCV IRES on viral protein production and thus virus replication, we decided to collect the available data on known nucleotide variants in the HCV IRES and their impact on IRES function in translation initiation. The HCV IRES variation database (HCVIVdb) is a collection of naturally occurring and engineered mutation entries for the HCV IRES. Each entry contains contextual information pertaining to the entry such as the HCV genotypic background and links to the original publication. Where available, quantitative data on the IRES efficiency in translation have been collated along with details on the reporter system used to generate the data. Data are displayed both in a tabular and graphical formats and allow direct comparison of results from different experiments. Together the data provide a central resource for researchers in the IRES and hepatitis C-oriented fields. CONCLUSION: The collation of over 1900 mutations enables systematic analysis of the HCV IRES. The database is mainly dedicated to detailed comparative and functional analysis of all the HCV IRES domains, which can further lead to the development of site-specific drug designs and provide a guide for future experiments. HCVIVdb is available at http://www.hcvivdb.org .

IRESite--a tool for the examination of viral and cellular internal ribosome entry sites.

The IRESite (http://www.iresite.org) presents carefully curated experimental evidence of many eukaryotic viral and cellular internal ribosome entry site (IRES) regions. At the time of submission, IRESite stored >600 records. The IRESite gradually evolved into a robust tool providing (i) biologically meaningful information regarding the IRESs and their experimental background (including annotation of IRES secondary structures and IRES trans-acting factors) as well as (ii) thorough concluding remarks to stored database entries and regularly updated evaluation of the reported IRES function. A substantial portion of the IRESite data results purely from in-house bioinformatic analyses of currently available sequences, in silico attempts to repeat published cloning experiments, DNA sequencing and restriction endonuclease verification of received plasmid DNA. We also present a newly implemented tool for displaying RNA secondary structures and for searching through the structures currently stored in the database. The supplementary material contains an updated list of reported IRESs.

Firefly luciferase gene contains a cryptic promoter.

A firefly luciferase (FLuc) counts among the most popular reporters of present-day molecular and cellular biology. In this study, we report a cryptic promoter activity in the luc+ gene, which is the most frequently used version of the firefly luciferase. The FLuc coding region displays cryptic promoter activity both in mammalian and yeast cells. In human CCL13 and Huh7 cells, cryptic transcription from the luc+ gene is 10-16 times weaker in comparison to the strong immediate-early cytomegalovirus promoter. Additionally, we discuss a possible impact of the FLuc gene cryptic promoter on experimental results especially in some fields of the RNA-oriented research, for example, in analysis of translation initiation or analysis of miRNA/siRNA function. Specifically, we propose how this newly described cryptic promoter activity within the FLuc gene might contribute to the previous determination of the strength of the cryptic promoter found in the cDNA corresponding to the hepatitis C virus internal ribosome entry site. Our findings should appeal to the researchers to be more careful when designing firefly luciferase-based assays as well as open the possibility of performing some experiments with the hepatitis C virus internal ribosome entry site, which could not be considered until now.

Ambiguous decoding of the CUG codon alters the functionality of the Candida albicans translation initiation factor 4E.

The eukaryotic translation initiation factor 4E is an essential and highly conserved protein. As a part of the translational machinery, it plays a key role in the recruitment of mRNA via binding to its m(7)GpppN 5' terminal cap structure. The opportunistic human pathogen Candida albicans is the only known eukaryotic organism with the ability to survive defects in mRNA capping, which suggests unique features of its eIF4E protein. Here, we provide the first experimental evidence of the function of the C. albicans putative gene orf19.7626 as an eIF4E protein. We also show that Ca4E(Leu116) and Ca4E(Ser116) protein variants, both of which occur naturally in C. albicans due to the ambiguous decoding of the CUG(116) codon, display different sensitivities to elevated temperature. Our results clearly point to the importance of the S4-H4 loop for the function of the Ca4E translation initiation factor, and suggest the possible regulatory role of the codon-reading ambiguity within this loop in C. albicans. We proved Saccharomyces cerevisiae as a useful tool organism for studies of C. albicans translation initiation apparatus.

Messenger RNAs of Yeast Virus-Like Elements Contain Non-templated 5' Poly(A) Leaders, and Their Expression Is Independent of eIF4E and Pab1.

We employed virus-like elements (VLEs) pGKL1,2 from Kluyveromyces lactis as a model to investigate the previously neglected transcriptome of the broader group of yeast cytoplasmic linear dsDNA VLEs. We performed 5' and 3' RACE analyses of all pGKL1,2 mRNAs and found them not 3' polyadenylated and containing frequently uncapped 5' poly(A) leaders that are not complementary to VLE genomic DNA. The degree of 5' capping and/or 5' mRNA polyadenylation is specific to each gene and is controlled by the corresponding promoter region. The expression of pGKL1,2 transcripts is independent of eIF4E and Pab1 and is enhanced in lsm1Δ and pab1Δ strains. We suggest a model of primitive pGKL1,2 gene expression regulation in which the degree of 5' mRNA capping and 5' non-template polyadenylation, together with the presence of negative regulators such as Pab1 and Lsm1, play important roles. Our data also support a hypothesis of a close relationship between yeast linear VLEs and poxviruses.

IRESite: the database of experimentally verified IRES structures (www.iresite.org).

IRESite is an exhaustive, manually annotated non-redundant relational database focused on the IRES elements (Internal Ribosome Entry Site) and containing information not available in the primary public databases. IRES elements were originally found in eukaryotic viruses hijacking initiation of translation of their host. Later on, they were also discovered in 5'-untranslated regions of some eukaryotic mRNA molecules. Currently, IRESite presents up to 92 biologically relevant aspects of every experiment, e.g. the nature of an IRES element, its functionality/defectivity, origin, size, sequence, structure, its relative position with respect to surrounding protein coding regions, positive/negative controls used in the experiment, the reporter genes used to monitor IRES activity, the measured reporter protein yields/activities, and references to original publications as well as cross-references to other databases, and also comments from submitters and our curators. Furthermore, the site presents the known similarities to rRNA sequences as well as RNA-protein interactions. Special care is given to the annotation of promoter-like regions. The annotated data in IRESite are bound to mostly complete, full-length mRNA, and whenever possible, accompanied by original plasmid vector sequences. New data can be submitted through the publicly available web-based interface at http://www.iresite.org and are curated by a team of lab-experienced biologists.

Characterization of Hepatitis C Virus IRES Quasispecies - From the Individual to the Pool.

Hepatitis C virus (HCV) is a single-stranded positive-sense RNA virus from the genus Hepacivirus. The viral genomic +RNA is 9.6 kb long and contains highly structured 5' and 3' untranslated regions (UTRs) and codes for a single large polyprotein, which is co- and post-translationally processed by viral and cellular proteases into at least 11 different polypeptides. Most of the 5' UTR and an initial part of the polyprotein gene are occupied by an internal ribosome entry site (IRES), which mediates cap-independent translation of the viral proteins and allows the virus to overcome cellular antiviral defense based on the overall reduction of the cap-dependent translation initiation. We reconsidered published results concerning a search for possible correlation between patient response to interferon-based antiviral therapy and accumulation of nucleotide changes within the HCV IRES. However, we were unable to identify any such correlation. Rather than searching for individual mutations, we suggest to focus on determination of individual and collective activities of the HCV IRESs found in patient specimens. We developed a combined, fast, and undemanding approach based on high-throughput cloning of the HCV IRES species to a bicistronic plasmid followed by determination of the HCV IRES activity by flow cytometry. This approach can be adjusted for measurement of the individual HCV IRES activity and for estimation of the aggregate ability of the whole HCV population present in the specimen to synthesize viral proteins. To detect nucleotide variations in the individual IRESs, we used denaturing gradient gel electrophoresis (DGGE) analysis that greatly improved identification and classification of HCV IRES variants in the sample. We suggest that determination of the collective activity of the majority of HCV IRES variants present in one patient specimen in a given time represents possible functional relations among variant sequences within the complex population of viral quasispecies better than bare information about their nucleotide sequences. A similar approach might be used for monitoring of sequence variations in quasispecies populations of other RNA viruses in all cases when changes in primary sequence represent changes in measurable and easily quantifiable phenotypes.

Multiple cathepsin B isoforms in schistosomula of Trichobilharzia regenti: identification, characterisation and putative role in migration and nutrition.

Among schistosomatids, Trichobilharzia regenti, displays an unusual migration through the peripheral and central nervous system prior to residence in the nasal cavity of the definitive avian host. Migration causes tissue degradation and neuromotor dysfunction both in birds and experimentally infected mice. Although schistosomula have a well-developed gut, the peptidases elaborated that might facilitate nutrition and migration are unknown. This is, in large part, due to the difficulty in isolating large numbers of migrating larvae. We have identified and characterised the major 33 kDa cathepsin B-like cysteine endopeptidase in extracts of migrating schistosomula using fluorogenic peptidyl substrates with high extinction coefficients and irreversible affinity-labels. From first strand schistosomula cDNA, degenerate PCR and Rapid Amplification of cDNA End protocols were used to identify peptidase isoforms termed TrCB1.1-TrCB1.6. Highest sequence homology is to the described Schistosoma mansoni and Schistosoma japonicum cathepsins B1. Two isoforms (TrCB1.5 and 1.6) encode putatively inactive enzymes as the catalytic cysteine is substituted by glycine. Two other isoforms, TrCB1.1 and 1.4, were functionally expressed as zymogens in Pichia pastoris. Specific polyclonal antibodies localised the peptidases exclusively in the gut of schistosomula and reacted with a 33kDa protein in worm extracts. TrCB1.1 zymogen was unable to catalyse its own activation, but was trans-processed and activated by S. mansoni asparaginyl endopeptidase (SmAE aka. S. mansoni legumain). In contrast, TrCB1.4 zymogen auto-activated, but was resistant to the action of SmAE. Both activated isoforms displayed different pH-dependent specificity profiles with peptidyl substrates. Also, both isoforms degraded myelin basic protein, the major protein component of nervous tissue, but were inefficient against hemoglobin, thus supporting the adaptation of T. regenti gut peptidases to parasitism of host nervous tissue.

Understanding the potential of hepatitis C virus internal ribosome entry site domains to modulate translation initiation via their structure and function.

Translation initiation in the hepatitis C virus (HCV) occurs through a cap-independent mechanism that involves an internal ribosome entry site (IRES) capable of interacting with and utilizing the eukaryotic translational machinery. In this review, we focus on the structural configuration of the different HCV IRES domains and the impact of IRES primary sequence variations on secondary structure conservation and function. In some cases, multiple mutations, even those scattered across different domains, led to restoration of the translational activity of the HCV IRES, although the individual occurrences of these mutations were found to be deleterious. We propose that such observation may be attributed to probable long-range inter- and/or intra-domain functional interactions. The precise functioning of the HCV IRES requires the specific interaction of its domains with ribosomal subunits and a subset of eukaryotic translation initiation factors (eIFs). The structural conformation, sequence preservation and variability, and translational machinery association with the HCV IRES regions are also thoroughly discussed, along with other factors that can affect and influence the formation of translation initiation complexes.

N-terminal domain of nuclear IL-1α shows structural similarity to the C-terminal domain of Snf1 and binds to the HAT/core module of the SAGA complex.

Interleukin-1α (IL-1α) is a proinflammatory cytokine and a key player in host immune responses in higher eukaryotes. IL-1α has pleiotropic effects on a wide range of cell types, and it has been extensively studied for its ability to contribute to various autoimmune and inflammation-linked disorders, including rheumatoid arthritis, Alzheimer's disease, systemic sclerosis and cardiovascular disorders. Interestingly, a significant proportion of IL-1α is translocated to the cell nucleus, in which it interacts with histone acetyltransferase complexes. Despite the importance of IL-1α, little is known regarding its binding targets and functions in the nucleus. We took advantage of the histone acetyltransferase (HAT) complexes being evolutionarily conserved from yeast to humans and the yeast SAGA complex serving as an epitome of the eukaryotic HAT complexes. Using gene knock-out technique and co-immunoprecipitation of the IL-1α precursor with TAP-tagged subunits of the yeast HAT complexes, we mapped the IL-1α-binding site to the HAT/Core module of the SAGA complex. We also predicted the 3-D structure of the IL-1α N-terminal domain, and by employing structure similarity searches, we found a similar structure in the C-terminal regulatory region of the catalytic subunit of the AMP-activated/Snf1 protein kinases, which interact with HAT complexes both in mammals and yeast, respectively. This finding is further supported with the ability of the IL-1α precursor to partially rescue growth defects of snf1Δ yeast strains on media containing 3-Amino-1,2,4-triazole (3-AT), a competitive inhibitor of His3. Finally, the careful evaluation of our data together with other published data in the field allows us to hypothesize a new function for the ADA complex in SAGA complex assembly.

Hepatitis C virus internal ribosome entry site initiates protein synthesis at the authentic initiation codon in yeast.

Hepatitis C virus (HCV) is an important pathogen causing both acute and chronic infections in humans. The HCV polyprotein is synthesized by cap-independent translation initiation after ribosome binding to the highly structured internal ribosome entry site (IRES). The HCV IRES has been shown to have a low requirement for translation initiation factors and the ability to bind directly to the 40S ribosomal subunit. A novel yeast bicistronic reporter system, suitable for sensitive and accurate analysis of IRES activity, has been developed. It employs signal amplification based on the Gal4p transcription factor-mediated activation of a variety of secondary reporter genes. The system has a broad dynamic range and, depending on the nature of the particular secondary reporter, can be used both for precise measurements of IRES activity and for selection and screening for novel IRES variants and IRES trans-acting factors. By using this novel bicistronic system, it was shown that the HCV IRES is functional in yeast cells. Mutational analysis of the IRES loop IV and the adjacent region revealed that, in yeast, as in mammalian cells, translation initiates preferentially at the authentic (342)AUG codon and that disruption of the HCV IRES loop IV abrogates its function, whilst minor positional changes or substitutions of the initiation codon within loop IV are largely tolerated. These findings bring more general insights to translation initiation, but also open the door for utilization of yeast and its sophisticated genetics for searching for new antiviral drugs and HCV IRES trans-acting proteins.

Denaturing RNA electrophoresis in TAE agarose gels.

Current methods of analytical RNA electrophoresis are based on the utilization of either complicated laboratory instrumentation or toxic, carcinogenic, or expensive chemicals. We suggest here the use of classical Tris-acetate-ethylenediamine tetraacetic acid (TAE) agarose gels combined with prior denaturation of RNA samples in hot formamide for the electrophoretic separation of RNA species. We present a brief comparison of the proposed TAE/formamide method with the most common 3-(N-morpholino)propanesulfonic acid/formaldehyde agarose gel protocol and show that both methods produce comparable results for size determination of RNA molecules and subsequent Northern blotting of gels. In addition to purified RNA samples, the robustness of the TAE/formamide protocol is demonstrated by its suitability for the analysis of RNA quality in crude yeast cell lysates containing large amounts of proteins, DNA, and other contaminating molecules. We therefore propose the TAE/formamide agarose electrophoresis as a rapid, simple, and cheaper alternative to current methods of RNA electrophoresis. Additionally, another benefit is the reduced exposure of laboratory personnel to hazardous chemicals.