The ATP binding cassette containing transporters are a superfamily of integral membrane proteins that translocate a wide range of substrates. The subfamily B members include the biologically important multidrug resistant (MDR) protein and the transporter associated with antigen processing (TAP) complex. Substrates translocated by this subfamily include drugs, lipids, peptides and iron. We have constructed a comprehensive set of comparative models for the transporters from eukaryotes and used these to study the effects of sequence divergence on the substrate translocation pathway. Notably, there is very little structural divergence between the bacterial template structure and the more distantly related eukaryotic proteins illustrating a need to conserve transporter structure. By contrast different properties have been adopted for the translocation pathway depending on the substrate type. A greater level of divergence in electrostatic properties is seen with transporters that have a broad substrate range both within and between species, while a high level of conservation is observed when the substrate range is narrow. This study represents the first effort towards understanding effect of evolution on subfamily B ABC transporters in the context of protein structure and biophysical properties. The ATP-binding cassette (ABC) superfamily of transporters translocate a wide range of compounds across cellular membranes using the energy released through ATP hydrolysis. They are integral membrane proteins that have an all helical transmembrane domain (TMD) consisting of between 6 and 11 helices and an intracellular domain containing the ATP-binding cassette . The NBD has a Walker A and Walker B motif separated by the ABC signature, LSGGQ. These proteins have a common domain arrangement of two TMDs and two NBDs and are either produced as a single polypeptide (full transporter) or as half transporters containing one NBD and one TMD (for reviews see ).Multiple ABC transporter genes exist in the genomes of bacteria and eukaryotes  and, in The ABC subfamily B consists of full and half transporters that have a diverse range of biological roles. Members of the subfamily include MDR1, initially identified in drug-resistant cancer cell lines , and TAPS. aureus, was reported . Clusters of homologous protein sequences for all 12 members of the ABC subfamily B were obtained (Homologene cluster identifiers are: tap1(B2), 495; tap2(B3), 37323; B1a, 55496; B1b, 69084; B4, 56421; B5, 83488; B6, 83488; B7, 3175; B8, 5203; B9, 10491; B10, 6474; B11, 74509.A total of 116 amino acid sequences including the eukaryotic ABC transporter subfamily B and bacterial orthologs of the Sav1886 protein from http://img.jgi.doe.gov/)   of pre-cSequence alignment and structural comparison of gene products within the subfamily B of ABC transporters is a complex problem that has necessitated some approximations. The most significant of these, is the alignment of single polypeptides that form a functional full transporter against single polypeptides that form half transporter units that are only functional as dimers. To address this, the half transporter sequences were duplicated to represent a “pseudo-full-transporter” which allowed comprehensive alignment over the full transporter sequences. As there is substantial sequence difference between eukaryotic and bacterial ABC transporter sequences, the multiple sequence alignment was constructed in two phases. Initially, separate multiple sequence alignments were computed for eukaryotes and bacteria, 102 and 14 sequence sets respectively. The alignments were then combined using a profile approach to avoid misalignment resulting from the construction of pseudo-full-transporter sequence. Due to its efficiency with this sized data set, the guide tree directed neighbour joining method as implemented in CLUSTALW  was emplAtomic models were computed using MOD-ELLER8.2  with defElectrostatic surfaces were generated by solving the non-linear Poisson-Boltzmann equation using the APBS package  and visuhttp://foo.maths.uq.edu.au/~huber/ABC/abcB.html.All data including sequences, multiple sequence alignment and model coordinates for all ABC transporters in this study are freely available from our web site S. aureus transporter, Sav1866, for which the full structure was recently determined. Overall, the higher eukaryotes used in this study have a larger number of full transporters, likely the result of gene duplication and fusion that, along with divergence in amino acid sequence has allowed diversification in functions and substrates of this gene family.Evolutionary analysis of ABC transporters is an extremely challenging problem, with factors such as different architectures and differing degrees of sequence divergence between domains, a likely necessity for development of biological function, requiring consideration. It is further complicated by integration into contexts of protein structure and biochemical function. The sequence identity between Sav1866 and members of the eukaryotic ABC subfamily B is in the range 25 to 35% which constitutes a remote sequence relationship but allows template-based comparative modelling of these transporters. Structural models have been constructed for the products of all eukaryotic genes in this study . The halInsertions and deletions were observed both in the extracellular and intracellular loops of the TMD and some minor length variations were also seen in the surface regions of the NDB. This indicates that the functional regions of the protein, the translocation pathway and the ATP binding site impose strong structural constraints on the evolution of these proteins. This may suggest that amino acid type distribution in the translocation pathway and resulting surface properties are likely to influence the distinct subfamily B substrate selectivity.http://www.ncbi.nlm.nih.gov/Taxonomy). To understand more the functional influence of evolution on translocation pathways of subfamily B transporters in higher eukaryotes, and its relationship to substrate specificity, electrostatic potential was computed for a range of related sequences from different organisms, mapped to the solvent accessible surface of the translocation pathway, and interpreted within the taxonomic lineage described by the NCBI Taxamonic database  transported. Substrate specialisation within the subfamily is also supported by a weak similarity in charge properties observed between the plant B1 a protein from A. thaliana and human B1 gene product.Human B1, well-characterised as a drug transporter, has a concentrated strong positive electrostatic potential, >10kT toward the substrate entrance of the translocation pathway. This positive charge decays along the pathway, giving a more neutral electrostatic character closer to the substrate exit site. The bile salt transporter B11 has similar surface properties (not shown). In comparison, B5, also associated with drug transport, has a positive electrostatic potential of >8kT covering the full length of the translocation pathway (not shown). In contrast to the drug transporters, B4, which has phosphatidyl choline as a substrate, has a concentrated negative electrostatic potential (<–8kT) that decays to neutral toward the substrate exit. The mouse and rat transporters display similar electrostatic properties to human B1, B4, and B5. Notably, the rodent B5 gene product unlike its human homolog, has a predominantly strong negative surface throughout the pathway, and a concentrated patch of positive potential around the substrate entrance site. This particular pathway electrostatic surface is specific to rodents. The more divergent D. melanogaster) and plant  (not shown). This is consistent with their clustering as distant relatives to the B9 and TAP gene products , while the mouse TAP translocation pathway surface differs in that it has concentrated patches of strong positive potential at both ends, decaying to a centrally located band of neutral to weak negative electrostatic potential (not shown). The lysosomal peptide transporter B9, which is more closely related to the TAP gene products rather than the mitochondrial peptide transporters, also possesses a strong negative surface potential throughout the translocation pathway, a property also conserved in the rodent B9 gene product (not shown). In contrast, the inner mitochondrial membrane peptide transporters, B8 and B10, have strong positive electrostatic potential throughout the pathway, which is also conserved in related proteins from fly (products . Taken tD. melanogaster and A. thaliana.The human iron transporters, B6 and B7, both have electrostatic potential surfaces similar to the human drug transporter B5 in that they have a positive charge throughout the translocation pathway. This is clearly important in the biological function of iron transport as it has also been conserved in the gene products from We have developed a comprehensive set of structural models for eukaryotic members of the ABC transporter subfamily B, and used these to better understand the effects of evolution on electrostatic properties of the translocation pathway within the subfamily. Comparison of these models indicates there are no substantial structural changes in the transporter core, implying evolutionary pressure to maintain a pathway through the lipid bilayer for the ATP dependent translocation of substrates. As the surface of the translocation pathway is important for substrate selection and movement, we analysed the electrostatic properties of a number of subfamily B representatives, from different species, covering the three clusters into which this subfamily partitions. In general, each cluster is associated with a distinct substrate class, which is reflected in the different electrostatic potential properties adopted by the pathway surface. Furthermore, there is divergence of electrostatic properties within the clusters between members within a species and between species likely reflecting the range or origin of substrates recognised and translocated. This divergence is most clearly seen in the cluster of transporters that translocate diverse substrates such as drugs, lipids, bile salts, and peptides, while the more restricted substrate range of the iron transporters is reflected in the conservation of their electrostatic properties. Interestingly, the electrostatic surface for human B1 reflects the physicochemical properties of substrates for this transporter, which are lipophilic compounds possessing a tertiary basic amine, giving a positive centre, while the negative surface of the phosphotidyl choline transporter B4 reflects the negative charge associated with the phosphate and acidic groups on its phospholipid substrate. This apparent mirroring of electrostatic properties is also observed in the iron transporters, in that they have a positive electrostatic surface and transport a positively charged substrate, this similarity is unexpected as is counterintuitive, but may indicate that iron is transported in complex. Furthermore, it is possible that these properties change along with conformational rearrangements associated with ATP binding.


La Peste, by Albert Camus: “Everyone knows that pestilences have a way of recurring in the world; yet somehow we find it hard to believe in ones that crash down on our heads from a blue sky.” This apt quotation might lead the reader to believe that the evolution and emergence of RNA viruses in causing new diseases would be discussed, but in fact the book, as its title suggests, concentrates on how RNA viruses evolve and emerge at the molecular level, not how they cause disease.This impressive monograph by Edward Holmes opens with a quotation from In addition to explaining what is currently known about the origins of RNA viruses, the book describes the mechanisms of RNA virus evolution, RNA virus quasispecies, and comparative genomics, as well as interesting new concepts, such as phylogeography. This term refers to the spatial movement of a phylogenetic species, which can be described in various ways (Holmes lists 5), two of which are the gravity model and the strong spatial subdivision model. In the former, patterns of transmission are driven by major population centers before moving out to smaller populations (influenza virus). In the spatial subdivision model, no clear evidence of migration among populations is presented (hepatitis C virus), and genomic diversity is partitioned into a series of clades (types and subtypes).Holmes argues persuasively that research in this area is limited by the size and detail of genome databases, combined with relevant epidemiologic and clinical information, such as precise geographic location, exact date of sampling, and transmission dynamics of the disease. The book is fully referenced and has a useful index, and I recommend it to those who have knowledge of and interest in the molecular biology of RNA viruses.


A novel intrinsic HIV-1 antisense gene was previously described with RNA initiating from the region of an HIV-1 antisense initiator promoter element (HIVaINR). The antisense RNA is exactly complementary to HIV-1 sense RNA and capable of forming ~400 base-pair (bp) duplex RNA in the region of the long terminal repeat (LTR) spanning the beginning portion of TAR in the repeat (R) region and extending through the U3 region. Duplex or double-stranded RNA of several hundred nucleotides in length is a key initiating element of RNA interference (RNAi) in several species. This HIVaINR antisense RNA is also capable of forming multiple stem-loop or hairpin-like secondary structures by M-fold analysis, with at least one that perfectly fits the criteria for a microRNA (miRNA) precursor. MicroRNAs (miRNAs) interact in a sequence-specific manner with target messenger RNAs (mRNAs) to induce either cleavage of the message or impede translation. Human mRNA targets of the predicted HIVaINR antisense RNA (HAA) microRNAs include mRNA for the human interleukin-2 receptor gamma chain (IL-2RG), also called the common gamma (γc) receptor chain, because it is an integral part of 6 receptors mediating interleukin signalling . Other potential human mRNA targets include interleukin-15 (IL-15) mRNA, the fragile × mental retardation protein (FMRP) mRNA, and the IL-1 receptor-associated kinase 1 (IRAK1) mRNA, amongst others. Thus the proposed intrinsic HIVaINR antisense RNA microRNAs (HAAmiRNAs) of the human immunodeficiency virus form complementary targets with mRNAs of a key human gene in adaptive immunity, the IL-2Rγc, in which genetic defects are known to cause an X-linked severe combined immunodeficiency syndrome (X-SCID), as well as mRNAs of genes important in innate immunity. A new model of intrinsic RNA silencing induced by the HIVaINR antisense RNA in the absence of Tat is proposed, with elements suggestive of both small interfering RNA (siRNA) and miRNA. Ma. Ma77]. It is of particular interest that human miR-146 has been shown to functionally interact with human mRNA 3'UTR sites for IRAK1 and thereby downregulate protein expression. ExpressWhile RNA silencing triggered by double-stranded RNA [dsRNA] precursors occurs in a wide variety of eukaryotic organisms as a mechanism to regulate gene expression, early eIn this model, I propose that HIV-1 employs limited genetic space to best effect by producing a primary HIVaINR antisense RNA with multiple functions Figure 14]. The. The14].The endogenous HIVaINR antisense RNA further has the intrinsic capacity for forming multiple dsRNA hairpin structures with complementary or near-complementary base-pairing Figure . PrimaryWhy would a virus utilize host cell enzymes Drosha and Dicer to dice up its own messages? Here, we must return to the initial concept of this paper, where timing of gene expression is so important to viral survival. The early skirmish in the battle between the virus and the host cell might require some sacrifice of intact viral messages for a time. By generating HAA miRNAs that incorporate into the host cell RISC, the virus can impede mRNAs from the host genes critical in enabling host innate as well as adaptive immune responses. The virus thereby employs the host's own anti-viral RNA silencing defence against the host cell. It can't be accidental that HAAmiRNA 1 has sequence that could target multiple complementary sites in human mRNA for the human IL-2R γ or common gamma (γc) chain, a requisite component in the receptors for all known T-cell growth factors (interleukins (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21). The same HAAmiRNA 1 sequence can also target multiple sites in the interleukin-15 mRNA, a key cytokine involved in NK cell functioning.In addition, until conditions are appropriate in the host cell for intact HIV RNA and protein production, dicing up the early transcripts Figure  or usingThe particular HIV-1 genetic regions encompassing HAAmiRNA sites 1–3 are well conserved in the B clade, and even more remarkably, particularly conserve miRNA sequence required for mRNA target recognition in most of the clades of group M , with the exception of the O group strains (underlined, additional file RNA silencing for regulation of gene expression is now recognized as an important tool for many species, including humans, to control how and when proteins are made. Viruses have undoubtedly already developed mechanisms that allow them to survive in their host mammalian cell, including subversion of the host cell machinery for RNA silencing. HIV encodes, within its long terminal repeat, an antisense gene responsible for RNA and protein products. The antThe author declares that they have no competing interests.LBL accepts full responsibility for the observations/opinions stated herein.HIVaINR antisense RNA  analyzedClick here for fileHIVaINR antisense RNA  and predClick here for file


Here, in order to determine whether the efficacy of chemotherapy can be enhanced by changing the regimen, we evaluated the effect of the varied timing and dosage of chemotherapy in combination with androgen withdrawal on time to androgen-independent (AI) progression in the human androgen-dependent LNCaP tumour model. We first demonstrated the synergistic effect of mitoxantrone on induction of apoptosis in LNCaP cells maintained in the steroid hormone-depleted charcoal-stripped media (CSM) compared to those in the standard media. In addition, this synergy was most remarkable during the simultaneous treatment of LNCaP cells with mitoxantrone and CSM compared to the pre- or post-use of mitoxantrone with CSM. LNCaP tumour growth in athymic nude mice and their increase in serum PSA levels were significantly inhibited by the simultaneous injection of mitoxantrone with castration, compared to the administration of mitoxantrone 2 weeks before or after castration. The TUNEL staining revealed that apoptotic cell death was extensively induced in LNCaP tumours treated with simultaneous castration and mitoxantrone compared to tumours treated with the other schedules. Furthermore, nude mice bearing LNCaP tumours were injected with a total of 0.5 mg mitoxantrone divided into 2, 5 and 10 days, with the most significant therapeutic effect and delayed AI progression observed in mice given injection of mitoxantrone for 2 days. These findings suggest that this might be the optimal way to perform cytotoxic chemotherapy and androgen withdrawal simultaneously in patients with advanced prostate cancer and to administer chemotherapeutic agents at high dosage within short intervals. © 2001 Cancer Research Campaign


Human hepatitis delta virus (HDV) is the smallest RNA virus in genome. HDV genome is divided into a viroid-like sequence and a protein-coding sequence which could have originated from different resources and the HDV genome was eventually constituted through RNA recombination. The genome subsequently diversified through accumulation of mutations selected by interactions between the mutated RNA and proteins with host factors to successfully form the infectious virions. Therefore, we propose that the conservation of HDV nucleotide sequence is highly related with its functionality. Genome analysis of known HDV isolates shows that the C-terminal coding sequences of large delta antigen (LDAg) are the highest diversity than other regions of protein-coding sequences but they still retain biological functionality to interact with the heavy chain of clathrin can be selected and maintained. Since viruses interact with many host factors, including escaping the host immune response, how to design a program to predict RNA genome evolution is a great challenging work. Viruses are aheterogeneous class of agents that parasitize every form of life including not only animals and plants but also bacteria, archaea, and fungi. Viruses vary greatly in particle size and morphology and in genetic complexity and host range. There are the DNA and the RNA viruses defined by the type of nucleic acid present in the mature virion particles. The genome size of the virus, be it a DNA or RNA virus, determines the number of proteins encoded by the viral genome. The minimal set of viral proteins includes capsid and envelope structural proteins required for the assembly of the virion particles and DNA or RNA polymerase for replication of the viral genome. A subclass of viruses, containing only few members, has been called satellite viruses since their genome can be encapsidated by their own coat proteins but require helper viruses for providing envelopes for assembling mature virions.The length of the genome of a DNA virus varies from a few kilo-bases (kb) to several hundred kb. The smallest known DNA virus is the human hepatitis B virus (HBV) which is 3.2 kb long and contains four open reading frames (ORFs) that encode the surface antigens, the core proteins, a polymerase, and an X protein , 2. TogeThe origin of viruses remains elusive and debatable because there are no fossils of viruses. Currently, there are two popular hypotheses to explain possible origin of viruses, viz the “regressive” or “degeneracy hypothesis” and the “cellular origin” or “vagrancy hypothesis” , 11. TheThe “cellular origin hypothesis” is based on the finding of the presence of plasmids and various types of mobile elements, such as transposons and retrotransposon, in the current prokaryotes and eukaryotes. When present in ancient cells, these elements could evolve to become viruses when they had gained new properties through evolutionary divergence to enable them to escape from the entrapping host cells to gain entry to and propagate in new hosts. Retroviruses and HBV, despite being different in the RNA or DNA content, respectively, might have originated from the same family of ancestral retrotransposons since replication of both viral genomes involves reverse transcription , 13. HenHowever, no single hypothesis can satisfactorily explain the origin all known viruses. In this paper, we use the smallest human RNA virus, HDV, to illustrate how this virus could have evolved and diverged. The evolution of larger genome size of RNA viruses, such as retroviruses, flaviruses, picornavirus, and corona viruses, is not a subject in this review.HDV was first discovered in HBV-infected patients by an Italian physician, Mario Rizzetto, in 1977. It was originally thought to be a new nuclear antigen associated with HBV . It was Most RNA viruses encode their own replicases or RNA-dependent RNA polymerases (RdRp) essential for viral genome replication. However, the HDV genome does not carry a replicase gene rendering HDV totally dependent on the host replication machinery for its propagation . It has isoforms of hepatitis delta antigen (HDAg) during replication. The small hepatitis delta antigen, SDAg, is a 24-kDa protein composed of 195 amino acid residues; the large hepatitis delta antigen, LDAg, is 27 kDa and consists of 214 residues [The HDV genome has only one ORF to encode two residues , 27. Theresidues –34. In tresidues –37. In sresidues .Based on the percentage of nucleotide identity of the genome, HDV was initially classified into three genotypes, designated genotypes I-III . The disRecently, an increasing number of HDV isolates have been identified and sequenced. The classification of HDV thus changes from the three genotypes into the eight clades, HDV1 to HDV8 , 43. In Analysis of the nucleotide sequences of 43 HDV isolates mined from databases in the public domain has revealed that the percentage nucleotide sequence identity of the complete HDV genome ranges from 64.1% to 76.4% in comparison of each out-group . The perThe current hypothesis of the origin of HDV favors the “cellular origin” concept because numerous studies have found similarities in structures and sequences between the nucleotide and protein sequences of cellular genes and the coding and viroid-like sequences of HDV. CPEB3 gene encodes the cytoplasmic polyadenylation element-binding protein 3 [CPEB3 but is dissimilar in the primary nucleotide sequence to that of the HDV ribozyme. However, the secondary structure of the CPEB3 ribozyme is similar to that of the HDV ribozyme both of which serve the function of self-cleavage of the multimeric precursor. These findings provide a clue that the viroid-like sequence of HDV could have arisen from an ancestral CPEB3 ribozyme in the mammal. Two other HDV viroid-like sequences with sequence similarity to a cellular RNA come from the findings that the HDV genomic sequence from nucleotide (nt) 683–724 is complementary to nt 10–55 of the 7SL RNA sequence and the antigneomic sequence from nt 858–899 is complementary to nt 188–233 also of the 7SL RNA [In a genomewide search for ribozyme sequences, it has been found that an HDV-like sequence in the human rotein 3 . CPEB3 i 7SL RNA , 46. Bot 7SL RNA . DIPA gene is a homolog of HDV and that the capture of some DIPA transcripts by a viroid-like sequence could have initiated the evolution of the very first HDV. Findings of RNA recombination between different HDV genotypes in patients or in cell cultures support the idea that DIPA transcripts and viroid-like sequence could join together [A cellular protein, termed delta interacting protein A (DIPA), is reported to interact with HDAg affecting HDV replication . Alignmetogether , 48, 49.together , 51. This “cellular origin hypothesis” then raises the interesting questions of when and in what animal host(s) such RNA recombination first occurred. Since HDV has to coexist with HBV for propagation, a first guess would be that HDV should have evolved in animals that were susceptible to infection by hepadnaviruses. The most primitive HBV is thought to DHBV found in ducks, the viral genome of which contains three ORFs but lacks the X-protein ORF that is found in the human HBV , 52. HowMutations frequently occur as a result of nucleotide mismatches during DNA or RNA replication. In general, the mutation rate of RNA viruses is higher than that of DNA viruses because the fidelity of base-pairing is lower during RNA replication than DNA replication. Diversity of the RNA genome of HDV should, therefore, be derived primarily from accumulation of mutations. Mutations that had occurred in essential sequences that could lead to impairment of virion formation could have been eliminated during viral replication. In contrast, mutations in nonessential sequences could have been preserved.cis-elements important for HDV replication. An essential cis-element is the promoter sequence that is recognized by cellular DdRps for transcription. Another important cis sequence is recognized by ADARs for RNA editing to result in LDAg production. In addition to the ribozyme sequence in the viroid-like region and the HDAg coding sequence, there are several 198ILFPADPPFSPQSCCRPQ214 in HDV1 (as Group I), 198GPSPPQQRLPLLECTPQ214 in HDV2 and HDV4-8 (as Group II), and 198FTPPPPGYYWVPGCTQQ214 in HDV3 (as group III)  is 211CTPQ214, and HDV3 (Group III) is 211CTQQ214. The original sequence encoding the CaaX box may yet be unknown but one could envisage that any nucleotide variations leading to the coding of a varied CaaX box would be maintained as has, indeed, been observed in various HDV sequences.Among the essential sequences for HDV replication and maturation, the sequence encoding the C-terminal peptide of LDAg is highly variable among the HDV1-8; oup III) . The ideoup III) . There aoup III) . The low199LFPAD203 and 206LPLLE210, respectively, while the HDV3 (Group III) does not have such a sequence  has shown that the CHC is one of interacting proteins of the viral capsid protein . Althougd to WNV . AnotherAs compared with many other satellite viruses which derived from a deletion of helper viruses, HDV lacks any similarity of nucleotide sequences of helper virus, the HBV, and evolved from two distinct nucleotide components through a recombination. In addition to discussing the possible origin of HDV, in this review, we proposed that the HDV nucleotide diversity could be resulted from the selection of interacting with host factors. By using the programs of PIST and PLATO, Anisimova and Yang analyzed all three codon positions of HDAg from 33 HDV isolates and explained that the HDV sequence diversity could be resulted from the positive selection force by escaping host immune response . Neverth


Most significantly, methods for following the movement of mRNA in living cells are now in widespread use. These include the introduction of labeled transcripts by microinjection, hybridization based methods using labeled antisense probes and complementary transgenic methods for tagging endogenous mRNAs using bacteriophage components. These technical innovations are now being coupled with super-resolution light microscopy methods and promise to revolutionize our understanding of the dynamics and complexity of the molecular mechanism of mRNA localization.Localized mRNA provides spatial and temporal protein expression essential to cell development and physiology. To explore the mechanisms involved, considerable effort has been spent in establishing new and improved methods for visualizing mRNA. Here, we discuss how these techniques have extended our understanding of intracellular mRNA localization in a variety of organisms. In addition to increased ease and specificity of detection in fixed tissue,  More recently, technical advances have allowed the visualization and quantitation of mRNA movement in living cells, enabling more effective analysis of the molecular mechanisms involved. In this review, we discuss key advances in mRNA labeling and detection approaches, imaging instrumentation, post acquisition analysis and the impact this has made on the field.The question of when and where genes are expressed has been of major interest in biology for at least 50 years. Although the study of the spatial positioning of transcripts initially focused on differences in expression levels between tissues, approximately 30 years ago it was realized that transcripts can also localize asymmetrically within cells. Intracellular localization of mRNA is now thought to be a very common mechanism to target protein function, occurring in most eukaryotic model organisms and for a very wide range of transcripts in the genome. mRNA starts its life as nascent transcripts that are first processed and then exported from the nucleus into the cytoplasm. Such transcripts associate in the nucleus with RNA binding proteins to form ribonucleoprotein complexes (RNPs), whose composition is then thought to be extensively remodeled during export from the nucleus and over the subsequent life cycle of the mRNAs in the cytoplasm. Specific RNA binding proteins within RNPs play essential roles in mRNA localization, translational regulation and degradation When intracellular mRNA distribution was being established as a mechanism for creating embryonic asymmetry trans-acting factors could be investigated with a high degree of accuracy while co-visualizing the RNA Drosophila blastoderm embryo The advent of fluorescent methods for detecting transcripts enabled higher quality three-dimensional imaging, multiplexing different RNA species and co-visualization of RNA with proteins Caenorhabditis elegans, improvements in direct labeling techniques have recently solved the long standing difficulties with ISH in this tissue, allowing the successful mapping of RNA distributions  Today, ISH is still a commonly used technique for examining the cellular distribution of mRNA in fixed tissue. However, to further explore mRNA transport dynamics, maintenance mechanisms and localization machinery, observation of mRNA in live tissue is required.in vitro and subsequent chemical coupling to fluorescent derivatives. The resulting fluorescently labeled RNA can be introduced into living cells by microinjection. In Xenopus, injections into oocytes have been a key assay for elucidating the mechanism of Vg1 mRNA localization Drosophila, RNA labeled in this way was used to show that oskar (osk) mRNA can at least partly localize if injected near its final destination at the posterior of the oocyte One of the biggest challenges to performing live cell imaging is how to label the RNA in the living cell. Some of the first attempts to image RNA in living cells relied on feeding cells fluorescent labeled metabolites that are subsequently incorporated into transcripts in vitro. Injecting RNA labeled in this way into live cells led to the formation of bright RNP particles   the MS2 RNA coat protein (MCP) fused to an FP expressed either globally or in chosen tissue. With the MS2 system, the fusion protein has been shown to bind to the RNA motif in the modified mRNA with a dissociation constant of 5 nM, which is essentially irreversible in the lifetime of an experiment. This means that, once associated, the components are effectively permanently coupled MS2-tagged mRNA, physiological levels of mRNA are produced that follow the complete mRNA localization pathway.Imaging of mRNA P system a, which ns (FPs) b–e [39].MS2 tagging has proven to be particularly valuable in situations where ISH or injection of labeled RNA has proven impractical Drosophila, the MS2 system has been indispensable for studying mRNA localization in late oogenesis when ISH is unreliable owing to the follicle cells laying down the chorion and vitelline membrane The same technology has been applied to other systems with great success, facilitating the analysis of the mechanism of mRNA localization in a variety of organisms MS2-MCP system has been used widely, the drawbacks of this method include the necessity of making transgenic animals that express the constructs and the fact that there are no clear rules for choosing where best to place RNA stem–loops in the endogenous transcript. Another difficulty that can arise is that the MCP-FP fusion itself can become localized in the cytoplasm in a tissue dependent manner or aggregate into particles without the RNA being expressed. There is also a limit on the number of loops that can be introduced into the endogenous mRNA because of the difficulty in cloning repeats and the effect of increasing the size of the RNA considerably. This limits the brightness of the MCP tagged mRNA. Another difficulty that can arise is nonspecific fluorescence in the cytoplasm or the formation of fluorescent aggregates in tissue where the MCP-FP fusion is expressed without the RNA. Despite these drawbacks, MS2–MCP has become an invaluable method for following transcripts in vivo.Although the Labeling RNA is only the first challenge to be met when studying RNA localization, because it is also crucial to consider cell viability, the most appropriate imaging approach and the use of post acquisition analysis. Quantitative live cell imaging enables the detection of subtle differences in movement and phenotypes, which is particularly powerful when used in combination with transport inhibitors and in mutant backgrounds.trans-acting factors or cytoskeletal components. Multi-channel imaging of rapidly moving objects presents some considerable technical challenges. Most obvious is the requirement to minimize crosstalk between detection channels. To resolve the need to maximize photon collection while imaging multiple channels simultaneously, multi-detector systems coupled with spectrally well-separated probes and appropriate filter sets are utilized. The optical microscope experimental (OMX) system, with fast live multichannel imaging, designed in the Sedat lab, is one such multi-detector system Matching the experimental approach to the imaging method is essential for all live-cell imaging situations Tracking sub-micron mRNA transport particles moving at speeds on the order of 1–3 μm/s requires objective and precise data analysis. Image analysis can be separated into three aspects: (i) pre-processing to improve the quality of data – this includes deconvolving, denoising and contrasting images MS2–MCP based method is not sufficient to address all questions, in all circumstances. For example, it is often necessary to follow several mRNA species simultaneously, image mRNA movement within thick specimens or resolve images where considerable autofluorescence is present. One emerging approach is the development of alternative labeling systems  can be determined at precisions that greatly exceed the resolution limits of light microscopy This is a true optical technique that works by ‘sculpturing’ the emission of light from a laser-scanned specimen Currently implemented on the OMX system  [58], thOne of the obstacles to studying RNA localization is being able to follow the fate of localizing transcripts from transcription through translation to degradation. A major limiting factor with long term imaging at high spatial and temporal resolution is dye bleaching. Recent developments in improved sensitivity of imaging, denoising algorithms and dyes with improved brightness and stability hold the promise that long term RNA imaging, to follow the whole lifecycle of the RNA, is achievable.in situFluorescence nanoparticles, quantum dots, are now commercially available and have successfully been used for long term imaging of single molecules in cells These methods have also been making a considerable contribution, for example, ‘denoising’, to improve the quality of images acquired with very low excitation times mRNA localization remains an intense area of investigation and with the tools being developed the field is poised to tackle long standing and new questions. The ongoing development of bacteriophage based tagging systems will allow the simultaneous imaging of multiple RNAs together with proteins and cytoskeletal components in living cells. Improvements in image processing and RNA tracking methods coupled with new generation of fast simultaneous three-dimensional multi-color imaging systems will allow a quantitative evaluation of the entire path of movement of transcripts in cell. Single particle live imaging in synergy with super-resolution methods and electron microscopy will allow researchers to elucidate the changes in RNP composition that occur during the life cycle of single RNA molecules. We await the application of these new methods with great interest.


Molecular characterization of breast and other cancers by gene expression profiling has corroborated existing classifications and revealed novel subtypes. Most profiling studies are based on fresh frozen (FF) tumor material which is available only for a limited number of samples while thousands of tumor samples exist as formalin-fixed, paraffin-embedded (FFPE) blocks. Unfortunately, RNA derived of FFPE material is fragmented and chemically modified impairing expression measurements by standard procedures. Robust protocols for isolation of RNA from FFPE material suitable for stable and reproducible measurement of gene expression  remain a major challenge.We present a simple procedure for RNA isolation from FFPE material of diagnostic samples. The RNA is suitable for expression measurement by QPCR when used in combination with an optimized cDNA synthesis protocol and TaqMan assays specific for short amplicons. The FFPE derived RNA was compared to intact RNA isolated from the same tumors. Preliminary scores were computed from genes related to the ER response, HER2 signaling and proliferation. Correlation coefficients between intact and partially fragmented RNA from FFPE material were 0.83 to 0.97.We developed a simple and robust method for isolating RNA from FFPE material. The RNA can be used for gene expression profiling. Expression measurements from several genes can be combined to robust scores representing the hormonal or the proliferation status of the tumor. Breast cancer has been widely studied in the past and molecular characterization has increased the understanding of biological pathways that are altered during neoplastic transformation of cells -4. HowevDNA chip studies are based on measuring gene expression for many genes in parallel ,4,7,8. MObviously, procedures based on formalin-fixed, paraffin-embedded (FFPE) material would greatly facilitate and speed up research in this area as large amounts of highly valuable material and clinical data have already been collected. In many cases, FFPE blocks are still available and they could be used for a molecular analysis. Especially material from clinical trials would allow investigating distinct clinical questions with existing material rather than material from newly designed studies.Many efforts are currently made to individualize diagnosis of breast cancer by including molecular parameters into diagnosis. Fresh frozen material would obviously be ideal for a molecular analysis by gene expression measurements but it may be difficult to implement novel procedures which complicate current workflows of daily routine. Procedures based on FFPE material would be more feasible as they do not interfere with current protocols and they do not affect routine diagnosis as material for molecular analysis could be collected after standard diagnosis has been terminated. Only relatively few molecular approaches have been described which are based on FFPE material. For example, Paik and co-workers have established a recurrence score , it allows to quantify the likelihood of distant recurrence and to predict the magnitude of chemotherapy benefit ,12.It is generally accepted that molecular profiles which reflect primarily biological characteristics of tumor cells, may complement clinical and histopathological diagnosis, resulting in a more detailed characterization of individual tumors, a pre-requisite for better treatment decisions. In this study we present the development of a novel procedure for RNA isolation from FFPE material and an optimized workflow for expression measurements by QPCR.Human breast cancer specimens were divided into two aliquots, one of which was processed for histological diagnosis by fixation with formalin and embedding in paraffin. FFPE material was obtained from the Institute of Pathology (University of Bern) and the Pathology Länggasse, Bern. Tissue (3–5 mm thick slices of tumor) was fixed over night in buffered formalin and processed for paraffin embedding in a Tissue Processing Center TPC 15 . The second aliquot was frozen on dry ice and stored at -80°C. Fresh frozen material was obtained from the Tumorbank Bern. Both, FF and FFPE samples were checked by hematoxylin and eosin staining and only samples with more than 50% tumor cells were used for this study. An informed consent to use the material for research was obtained from all the patients.2. The tissue was homogenized in 420 μl lysis buffer , 8.0,1 using a TissueLyser  at 15 Hz for 3 min. Total RNA was bound to silica-based columns , treated with DNase I , washed once with lysis buffer (containing 30% ethanol) and once with 20 mM NaCl (containing 20% ethanol) and eluted in 50 μl 10 mM Tris pH 7.4, 0.1 mM EDTA and stored at -20°C. RNA quantity was measured on an ND-1000 spectrophotometer  and quality assessed by capillary electrophoresis with an Agilent 2100 Bioanalyzer  using Agilent RNA 6000 Series Nano kits.Intact RNA was isolated from four 25 μm thick kryo-sections of approximately 0.5 cm4Cl) and incubating at 94°C for 20 min or as described in the text. RNA was bound to silica-based columns and digested with DNase I as described for fresh-frozen tissue samples. The reproducibility of our own procedure was tested by isolating several independent RNAs from consecutive sections of the same tissue block. About 10 μg of total RNA could be isolated from 5 to 10 FFPE sections (0.5–1 cm2/section). RNA was isolated from closely matched sections using the RNeasy FFPE kit (Qiagen) or the ncLysis system (Applied Biosystems) according to the protocols included with the kits. In both cases, the RNA was purified on silica-based columns. 22 samples were available. In 14 cases sufficient RNA was obtained from all 4 parallel isolations. In 2 cases of FF material (samples 4 and 11) and in 6 cases of FFPE material  less than 1.5 μg RNA could be isolated with the ncLysis protocol. These samples were excluded from further analysis.RNA was isolated from ten 10 μm thick FFPE sections according to the RNeasy FFPE protocol of Qiagen  and random or gene-specific primers. Reverse primers were kindly provided by Applied Biosystems, they were used at 1 μM each, cDNAs were made in the presence of 3, 10 or 22 reverse primers as 3-plex, 10-plex or 22-plex, respectively. Regular Assays on Demand (Applied Biosystems) were used for QPCR  values represent logbe the new value for each measured gene. The cut off value was set empirically to 35.0 as any higher raw Ct value was deemed unreliable. This cut off was fixed a priori and kept constant throughout all the experiments reported here. Then, the final value of each target gene was taken to bewhere R represents the reference value and was taken as the mean of Ct' values of 5 selected reference genes . The approach guarantees that all ΔCt values are positive and upper bounded by max_val .We used the scores associated with three of the gene groups listed in Table where the gene symbols stand for the corresponding ΔCt values.Finally, for each tumor a Total score was computed asThe Total score, together with the group scores as computed above, are used in all subsequent discussions.Total RNA was isolated from FF human breast cancer specimen which resulted in intact RNA in all samples Fig. , panel DSeveral parameters were systematically optimized to improve the protocol for RNA isolation from FFPE-derived sections. For example, QPCR made in the presence of primers specific for large amplicons  was used for normalizing expression values of all the genes in each RNA. Normalized expression values were compared between intact and FFPE-derived RNA for each gene and each tumor [see Additional File Normalized expression values were also used to compute scores representing ER-related genes , HER2-related genes (HER2 and GRB7), genes related to proliferation  and a Total score representing all the genes of the three scores (for details see Methods). The computation of biologically meaningful scores with multiple genes instead of relying on just one has the scope to reduce noise variation. Module scores and Total scores were computed separately from normalized expression values of intact RNAs (circles) and of RNAs isolated according to our own protocol (triangles) and Total scores are depicted separately for each tumor Fig.  and 8B. -8, CI = 0.893; 0.989), 0.856  and 0.833  for ER, HER2 and Proliferation scores, respectively. The corresponding Spearman correlations were 0.938 (p < 2.2*10-16), 0.851 (p = 1.167*10-4) and 0.867 (p = 2.048*10-5), respectively.The similarity between the results generated from intact and partially fragmented RNA was also assessed by calculating Pearson correlation coefficients between the scores of both RNAs. Correlation coefficients  were 0.966 , it also improves the recovery of RNA from FFPE sections. RNA yield and quality can be further improved by extensive digestion of FFPE material with protease in a buffer containing guanidinium thiocyanate. Reverse transcription in the presence of gene-specific primers prevents the initiation of cDNA synthesis inside amplicons and therefore, cDNA made in the presence of gene-specific primers is a better template for QPCR than cDNA made from random primers Fig. . SeveralFinally, normalization of raw data is used to eliminate or at least reduce the effect of poorer quality of starting RNA. Various approaches of normalization were proposed in the literature ,14,38,32We also applied GeNorm  to charaOur own RNA isolation protocol was compared to RNA that was isolated from the same material but according to commercial protocols and products (Qiagen RNeasy FFPE and ncLysis system of Applied Biosystems). Additional products for FFPE material from commercial providers  were tested and the results obtained with our own protocol were superior to all tested commercial products (data not shown).We determined module scores for each of the 14 tumors in this study. The limited number of samples does not allow statements about the clinical significance of module scores but they can be used to compare scores computed from intact RNA from FF material and RNA isolated from FFPE according to our own protocol. Pearson correlations between these RNAs in the 14 tumors were 0.966, 0.856 and 0.833 for ER, HER2 and Proliferation scores, respectively. As kryo-preserved RNA and RNA from FFPE material always originated from different portions of the same tumor, a certain variation of gene expression cannot be excluded and, as a consequence, part of the observed variability between kryo and FFPE material may be attributed to biological heterogeneity in the tumors. The three module scores were combined to a Total score. The Total score is similar to the recurrence score described by Paik , with hiThe data generated from FF and FFPE material were also compared to ER and HER2 levels assessed by IHC results from the same tumors. Three tumors #15, #18, #20) were ER-negative and one was strongly HER2-positive (#6)  declare that they have no competing interests.AO and AB developed the procedure, performed validation studies and were involved in all the experiments presented here. HJA and SA contributed clinical and pathological information. JA and SM participated in the design of the study and VP and MD performed the statistical analysis and developed the scoring system. RJ conceived the study, participated in its design and coordination and drafted the manuscript. All the authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Comparison of normalized expression for each gene in FF and FFPE material. Expression was determined by QPCR from RNA derived of FF and FFPE material (own protocol). Normalized expression levels (see Methods for details) are shown for each gene and the 14 tumors as polygonal plots.Click here for filePolygonal representation of ER, HER2, Proliferation and Total scores. Gene expression was measured from RNA derived of FF and FFPE material (own protocol) and ER, HER2, proliferation and Total scores were computed for each RNA of the 14 tumors and results are shown as polygonal plots. Parallel lines indicate good correlations and crossing lines are indicative for discrepancies between scores computed from FF and FFPE-derived RNAClick here for file


It is widely accepted that the highly error prone replication process of influenza A virus (IAV), together with viral genome assortment, facilitates the efficient evolutionary capacity of IAV. Therefore, it has been logically assumed that the enzyme responsible for viral RNA replication process, influenza virus type A RNA polymerase (IAV Pol), is a highly error-prone polymerase which provides the genomic mutations necessary for viral evolution and host adaptation. Importantly, however, the actual enzyme fidelity of IAV RNA polymerase has never been characterized.++ with Mn++, IAV Pol displayed increased polymerase activity, but with significantly reduced processivity, and misincorporation was slightly elevated in the presence of Mn++. Finally, when the IAV nucleoprotein (NP) was included in the reactions, the IAV Pol complex exhibited enhanced polymerase activity with increased fidelity.Here we established new biochemical assay conditions that enabled us to assess both polymerase activity with physiological NTP pools and enzyme fidelity of IAV Pol. We report that IAV Pol displays highly active RNA-dependent RNA polymerase activity at unbiased physiological NTP substrate concentrations. With this robust enzyme activity, for the first time, we were able to compare the enzyme fidelity of IAV Pol complex with that of bacterial phage T7 RNA polymerase and the reverse transcriptases (RT) of human immunodeficiency virus (HIV-1) and murine leukemia virus (MuLV), which are known to be low and high fidelity enzymes, respectively. We observed that IAV Pol displayed significantly higher fidelity than HIV-1 RT and T7 RNA polymerase and equivalent or higher fidelity than MuLV RT. In addition, the IAV Pol complex showed increased fidelity at lower temperatures. Moreover, upon replacement of MgOur study indicates that IAV Pol is a high fidelity enzyme. We envision that the high fidelity nature of IAV Pol may be important to counter-balance the multiple rounds of IAV genome amplification per infection cycle, which provides IAV Pol with ample opportunities to generate and amplify genomic founder mutations, and thus achieve optimal viral mutagenesis for its evolution. The high genetic diversity of influenza A virus (IAV), together with gene reassortment, forms viral quasispecies which enable IAV to acquire its efficient host adaptability. It is widely accepted that the efficient evolutionary capacity of IAV is facilitated by its highly error-prone replication process IAV Pol has been qualitatively studied for its RNA-dependent RNA polymerase activity using various biochemical settings 32P-labeled radioactive NTP (i.e. α-32P-GTP), which aids in the visualization of the extended products, and the other three NTPs are nonradioactive , 32P-GTP) is relatively very low (0.08 to 50 µM), simply because the highly concentrated radioactive NTP is not readily available, and use of a large quantity of radioactivity is technically challenging. Indeed, many recent reports that studied the steady state kinetic NTP incorporation by IAV Pol still used approximately 1/100 to 1/1000 biased nucleotide pools for the radioactive NTP (i.e. GTP) to the other three nonradioactive NTPs  Most frequently used polymerization assays of IAV Pol in current studies are gel-based standing start primer extension reactions that incorporate NTP substrates using ApG or cellular mRNAs as primers. In typical IAV Pol assays, one of the four NTPs is an α-In this report, we established new assay conditions that use unbiased and biologically relevant NTP substrate concentrations and avoid the kinetic bias found in the currently available gel-based assays. Importantly, this adjustment of the assay conditions not only revealed the robust polymerase activity of IAV Pol complex, but also enabled us to investigate the enzyme fidelity of IAV Pol. Also, with this new assay condition, we were able to conduct a series of comprehensive biochemical analysis to study the impact of various reactions parameters such as temperature, metals and viral NP on the IAV Pol fidelity. Overall, our results suggest that IAV Pol is least error-prone when compared to two reverse-transcriptases, HIV-1 RT and MuLV RT and also to bacterial phage T7 RNA polymerase. Importantly, the biochemical finding of the high fidelity nature of IAV Pol introduces a new concept of the mechanistic relationship between IAV Pol fidelity and the IAV replication strategy which uniquely provides IAV Pol with ample opportunities to generate genomic mutations during the multiple rounds of cRNA and vRNA amplification in a single infection cycle.BglII and XbaI sites. The PA gene was tagged at the C-terminus with the tandem affinity purification (TAP) tag, which consisted of a thrombin cleavage site followed by 6X His tag, tobacco etch virus (TEV) cleavage site and finally an IgG-binding domain. Each of the three pVL1392 plasmids were individually transfected into Sf9 insect cells for the production of recombinant viruses in SF900II SFM media (GIBCO), following the protocols provided by the vendor (BD Biosciences Pharmingen). To prepare the purified trimeric IAV polymerase, the three recombinant viruses were used for coinfecting Tni insect cells grown in Express Five SFM media supplemented with L-Glutamine (GIBCO). Cells were harvested 72 hours post infection and the lysates obtained were purified by the TAP purification technique as described previously For IAV polymerase, the PA, PB1 and PB2 genes  were amplified by reverse-transcription PCR from the H3N2 IAV strain (A/chicken/Nanchang/3-120/01), obtained from Dr. R. Webster, St. Jude Children's Research Hospital, Memphis, TN, USA. They were then cloned to the baculovirus expression vector pVL1392 (Invitrogen) between the ++ column chromatography following the protocol provided by the vendor (Novagen). Fractions containing NP were pooled and dialyzed to a low salt buffer (50 mM Tris-Cl (pH 7.5), 1 mM EDTA, 200 mM NaCl, 10% glycerol, 1 mM DTT). The dialyzed protein was further purified using a second DEAE-Sephacel column to minimize any RNase contamination. The RNA binding of the purified NP was examined by a filter binding assay employing both protein and nucleic acid binding filters (data not shown).The H3N2 Nanchang NP gene (A/Chicken/Nanchang/3-120/01) was cloned to pET28a (Novagene), expressing NP protein fused to the six histidine tag at its N-terminal end. NP was purified using Ni5′ AGUAGAAACAAGGCC 3′) and the 14-nucleotide (nt) 3′ vRNA which serves as a template (5′ GGCCUGCUUUUGCU 3′) from the PA gene of influenza A virus were purchased from Dharmacon. The other two templates (50-nt and 137-nt) used encode the first 50-nt and 137-nt sequences from the 3′ end of the Nanchang PA gene. The 50-nt template was purchased from Integrated DNA Technologies. The 137-nt RNA template was prepared by in vitro transcription using a double stranded (ds) PCR product amplified by a 37-mer forward primer encoding T7 promoter sequence and 19-nt of the viral sequence and a 16-mer reverse primer encoding 3′ sequence of the PA gene. The PCR product was used for synthesizing the 137-mer RNA template using in vitro transcription kit (Ambion), which was purified as described 5′ vRNA (5′ GCAUUGUCGCAAUCAGUACCUGCUUUCGCU 3′) encoding the first 30-nt sequence from the 3′end of the PA gene was used (Integrated DNA Technologies). The T7 template sequence (GCGAAAGCAGGTACTGATTGCGACAATGC 3′5′ TAATACGACTCACTATAG) contains the T7 promoter region which is the first 18 nucleotides and the remaining 29 nucleotides (see the sequence underlined) are the same sequence as that of the flu template. The first A nucleotide is deleted because T7 pol requires G at the +1 site. This also explains why the T7 bands run one nucleotide lower than the IAV Pol reaction bands on the PAGE. For the HIV-1 RT and MuLV RT reactions, a 48-nt RNA template is used. This 48-mer RNA template (GCAUUGUCGCAAUCAGUACCUGCUUUCGCUCUGGCUUAAGGGCGAUCG 3′5′ ) encodes not only the same first 30-nt sequence of the PA gene as the 30-mer Flu Pol template, but also the 18-nt non-PA gene which is annealed to a 18-mer DNA primer containing the A–G sequence at its 3′ end.For the IAV Pol misincorporation reactions, a 30-nt RNA template , IAV Pol complex, 0.25 U/µl RNasin, 0.3 mM ApG, 1.6 µM 14nt 3′vRNA, 1.6 µM 5′vRNA, 0.08 µM radioactive α-32P-GTP , and varying concentrations of nonradioactive NTPs  as described in figure legends. Transcription reactions were stopped after 1 h by the addition of 5 µl trichloroacetic acid (TCA) dye, denatured at 95°C for 3 min and transcription products were separated by 8 M urea denaturing 18–20% polyacrylamide gel electrophoresis (PAGE). The dried gels were analyzed by autoradiography using a PhosphorImager (Molecular Dynamics) and the intensity of the fully extended products was quantitated by OptiQuant . In order to validate the linearity of the radioactive signals in this assay, we determined the signal intensity of the samples diluted by known ratios , and used that for all subsequent analysis of the signals. In the reactions described in 32P-GTP and 1 mM of other three nonradioactive NTPs .We tested the transcriptional activity of the purified polymerase complex using the ApG transcription assay in which the 3′ and 5′ vRNA and the dinucleotide primer, ApG (Biosynthesis) are provided. ApG was extended using the 3′ vRNA template (14-nt) by the IAV polymerase (200 ng), generating a 14-nt long RNA fragment product. The ApG transcription reactions were performed as described elsewhere 32P-NTPs or dNTPs  and nonradioactive NTPs or dNTPs (500 µM). For IAV Pol, the ApG-transcription reactions were performed with the 30-nt 3′ RNA template as described  were incubated with RT for 1 hour at 37°C under conditions described previously (16). In the (−) A and (−) T reactions, dATP and dTTP were left out, respectively.In the misincorporation assays with all four polymerases, an equal concentration of all the nucleotides (NTPs or dNTPs) was provided using a mix of four α-escribed , and theescribed  in the pescribed , 500 µM ++, Mg(OAC)2 (5 mM) in the buffer was replaced with varying concentrations of MnCl2 . Further, the reactions were performed with and without different amounts of purified NP. For reactions with NP, NP was preincubated with the RNA template prior to the addition of IAV Pol in the reaction mixture. Note that to minimize any loading variations between reactions, which can affect the total activity measurement, we added an equal amount of a 5′ end 32P-labeled non specific primer to each reaction, and used this as the loading control . For testing the effect of Mn32P-GTP and 1 mM of other three nonradioactive NTPs . The polymerase was expressed and purified from the baculovirus system as we described previously First, in order to address the potential kinetic bias induced by the biased NTP pools that have been typically used in the gel-based IAV Pol assays, we conducted the ApG-initiated IAV RNA polymerase reaction using gradually increasing concentrations of the limited NTP (GTP) in the reaction, which abolishes the NTP pool bias . The ApG32P-GTP proportionally decreases upon mixing with every 10-fold increase in concentration of the nonradioactive GTP  are provided. The ratio of radioactive NTP to nonradioactive NTP is kept constant for all the NTPs and is approximately 1/1600, allowing us to use a low concentration of radioactive NTPs. The 30-nt template also encodes the 3′ end 30-nt sequence of the viral PA gene template . As showUnlike the UTP stop sites, it is difficult to observe the first stop sites induced by the deletion of other three NTPs  because the first 5 nucleotides in the 3′ end conserved sequence of the IAV genomes contain A, G and C. Thus, their first stop sites generate only 3–5 nucleotide long products which are technically difficult to separate even with 8 M urea high percentage denaturing PAGE. However, as shown in the reactions with (−) ATP pool , the fulNext, we compared the enzyme fidelity of IAV Pol complex with three other polymerases, bacterial phage T7 DNA-dependent RNA polymerase as well as two retroviral RNA-dependent DNA polymerases which are well-characterized for their fidelity 32P-NTP and α-32P-dNTP, which were used in the IAV Pol and RT reactions, respectively, were identical (3000 Ci/mmole), and thus the same band intensity of the fully extended products in the reactions with different polymerases indicates the same level of nucleotide incorporation and nucleic acid synthesis.Next, we conducted the (−) dTTP and (−) dATP misincorporation assay with low fidelity HIV-1 and high fidelity MuLV RT proteins. The RT reactions employed a 48-nt RNA template encoding the 3′ end 30-nt sequence of the PA gene and 18-nt non-viral sequence annealed to an 18-nt DNA primer . Thus, t32P-labeled DNA primer and four different biased dNTP pools (data not shown).Next, we repeated the same RT reactions with the equal activity of low fidelity HIV-1 and high fidelity MuLV RTs but with the (−) TTP or (−) dATP biased pools. Indeed, HIV-1 RT displayed much more extension beyond the first stop site  strategy that amplifies their genomes many times per infection by using exclusively their own polymerase complex without any assistance from host RNA polymerases. This is contrasted to retroviruses (i.e. HIV-1) that replicate their single stranded RNA genomes into double-stranded proviral DNA in two ways. First, HIV-1 replicate their RNA genome to proviral double stranded DNA only twice per infection (conversion of (+) single strand RNA genomes to (−) single strand DNA and then conversion of the synthesized (−) DNA to double stranded proviral DNAs) by using their own polymerases. Second, the RNA genomes of every HIV-1 progenies are synthesized by host RNA polymerase II by transcribing the proviral DNA integrated into the host chromosomes. It is a reasonable assumption that the viruses, which require a high level of genomic mutations for their evolution and escape but have only a few chances to replicate their genomes by their own polymerases (i.e. HIV-1), might have evolved to harbor highly error-prone polymerases (i.e. HIV-1 RT) to generate sufficient mutations in really limited chances. In contrast, due to numerous rounds of the amplification of single viral RNA genomes to viral RNAs only by the viral polymerase, IAV Pol has ample chances to make and accumulate genomic mutations in each viral infection cycle. It has been estimated that the viral burst size in case of influenza, in one cycle of replication, is about 1000 The similar reasoning of the high fidelity viral RNA polymerases envisioned above for IAV Pol can be applied to polioviruses which also amplify their RNA genomes multiple times per infection solely by their own high fidelity RNA polymerase. In addition, it was demonstrated that the elevation of genomic mutation rates of poliovirus, which is promoted by an antiviral agent (ribavirin), results in the catastrophic mutagenesis It is also important to address other potential cellular events that may generate IAV genomic mutations during viral life cycle. One potential mechanism well established is host enzymes that can chemically alter the structures of the nucleotide substrates. APOBEC3G is a host cytidine deaminase which can convert to C to U in the proviral DNA of HIV-1, and this event induce the G to A mutation in viral genomic DNA post reverse transcription, leading to lethal mutagenesis


Superoxide Dismutase 1 (Sod1) mRNA with high affinity through a novel RNA motif, SoSLIP (Sod1 mRNA Stem Loops Interacting with FMRP), which is folded as three independent stem-loop structures. FMRP induces a structural modification of the SoSLIP motif upon its interaction with it. SoSLIP also behaves as a translational activator whose action is potentiated by the interaction with FMRP. The absence of FMRP results in decreased expression of Sod1. Because it has been observed that brain metabolism of FMR1 null mice is more sensitive to oxidative stress, we propose that the deregulation of Sod1 expression may be at the basis of several traits of the physiopathology of the Fragile X syndrome, such as anxiety, sleep troubles, and autism.Fragile X syndrome, the most frequent form of inherited mental retardation, is due to the absence of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein involved in several steps of RNA metabolism. To date, two RNA motifs have been found to mediate FMRP/RNA interaction, the G-quartet and the “kissing complex,” which both induce translational repression in the presence of FMRP. We show here a new role for FMRP as a positive modulator of translation. FMRP specifically binds  The most common form of inherited mental retardation, Fragile X syndrome, is caused by the absence of the Fragile X Mental Retardation Protein (FMRP), which can bind mRNA. In mice devoid of FMRP, several hundreds of mRNAs with altered expression and localization have been reported. The impact of these abnormalities is particularly important in the brain, where the absence of FMRP affects the structure and function of connections between neurons, resulting in reduced cognitive abilities. In recent years, scientists studying Fragile X syndrome have focused on identifying the RNAs that are specifically bound by FMRP. We and others have identified two specific RNA structures bound by FMRP/RNA. Importantly, when FMRP binds these structures, the translation of the RNA into its protein product is inhibited. In this new study we have identified a third FMRP-binding RNA structure that is found in the mRNA that encodes superoxide dismutase, an oxidative-stress-mitigating enzyme. Most significantly, FMRP enhances translation of the superoxide dismutase mRNA when it interacts with the structure. These findings suggest that absence of FMRP might result in reduced levels of superoxide dismutase, which in turn leads to increased oxidative stress in the brain. Interestingly, oxidative stress in the brain has already been linked to anxiety, sleeping difficulties, and autism, all of which typically affect individuals with Fragile X syndrome. Sod1 mRNA, suggesting that increased oxidative stress may be involved in the pathophysiology of Fragile X syndrome..Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein mostly involved in translational repression, may also act as a translational activator of An incr2+ ,4. This  (PP2Ac) –5. FMRP  (PP2Ac) ,7. Moreo (PP2Ac) .MAP1B mRNA expression regulation [PP2Ac mRNA [Post Synaptic Density 95 (PSD95) mRNA, by interacting with its 3′ UTR, was recently reported [Superoxide Dismutase 1 (Sod1) mRNA with high specificity and affinity via a novel RNA structure that we named SoSLIP (Sod1Stem Loop Interacting with FMRP). SoSLIP is organized in a triple stem-loop structure and acts as an FMRP-dependent translational enhancer and as a mild internal ribosome binding site (IRES) in an FMRP-independent manner. The characterization of this novel RNA motif interacting with FMRP sheds new light on the ability of this protein to bind RNA and to improve the translation of SoSLIP-containing mRNAs. Our results, taken together with other recent findings [Sod1 expression may have an important role in the pathogenetic mechanism of Fragile X syndrome.Taking into consideration the results obtained from different laboratories, several mechanisms of action of FMRP have been proposed suggesting: (i) polysomal stalling for gulation ; (ii) regulation ; (iii) i2Ac mRNA . Moreovereported . Here wefindings ,12, sugg+, K+, or Mg2+. Indeed, K+ ions stabilize the G-quartet RNA structure, leading to a robust interaction with FMRP [2+ favors FMRP/kissing complex RNA interaction [Sod1 interaction, which takes place in the presence of K+ (+ (+ affects the binding of FMRP to the N19 sequence that contains the G-quartet present in FMR1 mRNA (nucleotide (nt) 1470–1496) [Sod1 mRNA, we performed a reverse transcriptase (RT) elongation reaction assay, as previously described [+, G-quartet RNA is very stable, blocking RT progression at its 3′ edge and resulting in a truncated transcription product. Conversely, in the presence of Na+, G-quartet structures are destabilized, and the RT can proceed to the end of the RNA [Sod1 mRNA did not reveal any K+-dependent stop of the polymerase  [Sod1 mRNA via a novel sequence/structure. We continued the characterization of the FMRP/Sod1 mRNA interaction by testing the ability of Sod1 mRNA to compete for the binding of the FMRP/G-quartet RNA structure [Sod1 mRNA competed very efficiently (65%) with the previously identified N19 FMRP binding site in a gel-shift assay, whereas a negative control, N8 RNA (corresponding to nt 1–654 of FMR1 mRNA that does not contain the G-quartet), was not able to compete for the same interaction , a central part of the coding region (Sod1-mid region), and a fragment overlapping the end of the coding region and the 3′ UTR (Sod1–3′ region) (Sod1–5′ region (spanning nt −70 to +148 of Sod1 mRNA) competed with N19 binding to FMRP with the same affinity as the full-length Sod1 mRNA (3 nM concentrations of both cold probes compete for 50% of FMRP/N19 binding) -FMRP, as previously described [Sod1 AUG start codon that is protected by FMRP. We subcloned this sequence, and we synthesized its corresponding RNA, generating the Sod1–64 RNA. This RNA was bound specifically by FMRP, because it was able to compete for the FMRP/Sod1 full-length mRNA interaction (Sod1–64 interaction is competed for by the N19 G-quartet-containing RNA to the same extent (unpublished data).With the goal to find novel mRNA structures specifically recognized by FMRP, we performed a systematic analysis of known FMRP mRNA targets, focusing on those that have been shown to interact in vivo with FMRP by the antibody-positioned RNA amplification (APRA) technique . First, ith FMRP , whereasce of K+ A and is of K+  A. RNA sebinding) B. To ideeraction C. IntereSod1 mRNA, we produced protein fragments of the different RNA-binding domains of FMRP  as recombinant proteins in a bacterial system [Sod1–64 RNA. Interestingly, we observed that Sod1–64 RNA interacts only with the C-terminal domain of FMRP encompassing the RGG box  isoforms to bind ructures . We usedSod1–64 interaction in translational control, we decided to determine Sod1–64 RNA structure in the absence and in the presence of FMRP. To determine the secondary structure of Sod1–64 RNA, we probed the structure of this 64-base region in solution, using a panel of chemical and enzymatic modifications, as described [Sod1–5′ region), which were subjected to random digestion with RNases T1, T2, and V1 or chemical modifications with dimethyl sulfate (DMS) and a carbodiimide derivative (CMCT). RNase T1 cuts after guanine residues in single-stranded regions, RNase T2 cleaves after all single-stranded residues, but preferentially after adenines, whereas RNase V1 cuts at double-stranded or stacked bases. DMS alkylates the N1 position of adenines and the N3 position of cytosines, whereas CMCT modifies the N1 position of guanines and the N3 position of uridines. The sites of cleavage or modification were then identified by primer extension with reverse transcriptase, using a radiolabeled primer complementary to the Sod1-5′ region. Analysis of the resulting cDNAs was performed on sequencing polyacrylamide gels that were run together with the corresponding RNA sequencing ladder to allow identification of the modified residues (http://helix.nih.gov/apps/bioinfo/mfold.html). The structure of Sod1–64 RNA appears as a succession of three independent stem-loop structures that are separated by short single-stranded regions , as well as the second stem and the more 3' part)  B. In conSod1 mRNA. To explore Sod1 mRNA decay, we blocked transcription in primary cultured hippocampal neurons and in STEK cell lines by actinomycin D treatment. We did not observe any significant difference in Sod1 mRNA levels of neurons obtained from wild-type or of Fmr1 null mice  RT-PCR, the amount of Sod1 mRNA was found to be equivalent in both wild-type and Fmr1 knockout cells and tissues when normalized to the level of Hprt mRNA  obtained from fibroblasts  is observed in medium and heavy fractions where the amount of FMRP is most abundant, and nonsignificant differences are observed in the light fractions where the amount of FMRP is less abundant. These results suggest that the absence of FMRP plays a key role in Sod1 mRNA incorporation in the translating machinery.We investigated the impact that the absence of FMRP has on the stability and translatability of ull mice A or fibrreatment B. Then, EK cells , total bprt mRNA C. All ofin brain –19 and ain brain ,21, we sroblasts D, as welroblasts E. IndeedSod1 mRNA with polyribosomes impairs the expression of the Sod1 protein in the absence of FMRP, we analyzed total protein extracts obtained from STEK cells expressing or not expressing a FMR1 transgene [Fmr1 null cells, as compared to that of cells expressing FMRP and after normalization of its expression to that of β-tubulin   E. We theFMR1-expressing STEK cells  (Sod1 RNA as the cold competitor). All three mutations affect the SoSLIP translational enhancer properties, reducing the level of luciferase activity  to evaluate the effect of the presence of SoSLIP on the expression of a reporter protein. We transfected primary cultured hippocampal neurons with the SoSLIP-luciferase vector (SoSLIP-Luc) or with the Luc vector, and we tested luciferase activity, showing on average an 8-fold increase when SoSLIP is placed upstream of the reporter A. A simiEK cells A. AnalysEK cells A and S4Bndition) B. These ndition) C. Using ndition) D but didactivity E, if compe cells E  and enhanced green fluorescent protein (eGFP) are under the control of the same promoter. The DsRed is translated in a cap-dependent manner, whereas eGFP is translated only if an IRES sequence is cloned in front of it, as described [. The same result was observed following transfection of the neuroblastoma NG108 cell line, neurons, COS cells, and STEK cells expressing FMR1 (unpublished data). No differences were observed in the intensity level of GFP fluorescence from the SoSLI-PRIG vector in cells expressing or not expressing FMRP, suggesting that this protein is not required for the mild IRES-like activity of SoSLIP  (probably binding to SL1) to carry out its function as an enhancer of translation. Due to the conformational changes of L2 and L3 stems induced by FMRP, we therefore propose that FMRP would facilitate ribosome scanning by participating in the remodelling of the SoSLIP structure, promoting in particular the exposure of the AUG of Sod1 mRNA. This function is possibly cooperating with factor(s) binding the SL1 stem loop. Furthermore, our data suggest that the mechanism of action of FMRP is dependent on the type of RNA structure to which it binds. FMRP binds both G-quartet and SoSLIP RNAs through its C-terminal region containing the RGG box, even if in different ion concentrations. For this reason, it is tempting to speculate that in vivo the local ionic environment modulates the RNA binding properties for FMRP and favors the binding of either G-quartet or SoSLIP-containing mRNAs. This possibility might be particularly relevant in the synaptic compartment, because the binding of FMRP to its different mRNA targets might be directly modulated by depolarization.In this study, we define a new function of FMRP by dissecting the mechanism of binding of FMRP to the  neurons . Here weIn conclusion, SoSLIP can be considered as a “bipartite” translational activator: one domain (SL2 and SL3) acts in an FMRP-dependent-manner; the other (SL1) is independent of the presence of the Fragile X protein. Moreover, we also observed that SoSLIP may act as an IRES-like sequence. We observe that FMRP does not promote and/or influence this additional function of SoSLIP, suggesting that a specific mechanism of translational regulation is probably activated to translate SoSLIP-containing uncapped RNA. These data confirm the complexity of the translational regulation mediated by SoSLIP, and further studies will be necessary to fully understand its mechanism of action. Most important for our study, in this context, the function of FMRP is relevant to positively modulate SoSLIP-containing mRNA synergizing with other factor(s).FMR1 null flies, because changes in the expression of proteins involved in redox reactions have been observed (1-cys peroxiredoxin in brain and peroxiredoxin and thioredoxin peroxidase in testis) [Fmr1 knockout mice has been recently described [Sod1 expression and the fact that the two FXR1P isoforms most expressed in the brain (Isoa and Isod) are also able to bind SoSLIP, even if with a lower affinity if compared with that of FMRP, suggesting that they can partially rescue FMRP function in Fmr1 null cells. In addition, the FXR1P muscular isoform (Isoe) could functionally replace FMRP in muscle cells where this protein is absent. Moreover, modifications of oxidative stress have been linked to anxiety [Fmr1 knockout mice [Fmr1 null mice [Fmr1 null synapses. In conclusion, our study suggests a role for Sod1 in the physiopathology of Fragile X syndrome and proposes a new function and novel mechanism of action for FMRP.Sod1 is a well-known protein with antioxidant properties. Alterations of oxidative stress have been proposed to occur in  testis) ,30, and escribed . This mo anxiety , sleep t anxiety , and aut anxiety , all phe anxiety . Interesout mice . At the out mice . In thisull mice , suggestSod1 and FMR1 cDNAs are summarized in Primer sequences used to amplify Sod1 (BC002066) and two of its deletion constructs (Sod1-mid region and Sod1–64/SoSLIP) were subcloned into the pGEM-T vector (Promega), and the Sod1–3′ UTR construct was subcloned into the pCR2.1-TOPO vector (Invitrogen). SL1, SL2, and SL3 mutants were generated starting from the Sod1–64 pGEM-T vector and using the QuickChange site-directed mutagenesis kit (Stratagene) and the oligonucleotides described in Mouse full-length Sod1–64/SoSLIP HindIII primers described in SoSLIP and its three mutants were cloned into the pcDNA3.1 zeo vector (Invitrogen) (Luc) using FMR1 ISO7 cDNA [Sequences coding for KH1, KH2, and FMRP C-terminal domains were amplified by PCR from obtained SO7 cDNA  using thFor pPRIG-empty, the IRES sequence was removed by digestion of pPRIG-HA-red  with SalProtein extraction and immunoblot analysis were performed as previously described . The antRecombinant protein expression and purification were performed as previously described . In vitrSod1–5′ region was obtained by digesting pGEM-T Sod1 full-length with BstXI. Restriction enzymes were purchased from New England Biolabs. RNAs were purified on NucAway spin columns (Ambion), and their qualities were verified on an polyacrylamide/urea gel after staining with Stains-All (Sigma). Protein/RNA interactions were analyzed either by electromobility shift assay or by filter binding assay, as previously described [All RNAs were produced using T7 RNA polymerase (Promega), according to the manufacturer's protocol by starting from linearized plasmids. The pGEM-T and pTL1 vectors were linearized using PstI, and the pCR2.1 TOPO vector was linearized with BamH1. The escribed . All of Sod1 mRNA was tested both by binding assay and by RT with different primers along the Sod1 mRNA, as previously described, in the presence of Na+ or K+ in both experiments [32ATP 3′-end-labeled primers [The presence of a G-quartet structure in the eriments . For the primers :Primer I 5′-CTCTTCAGATTACAGTTT-3′; primer II 5′-GTACGGCCAATGATGGAATG-3′; primer III 5′-GGATTAAAATGAGGTCCTGC-3′; primer IV 5′-CTTCTGCTCGAAGTGGATG-3′; primer V 5′-CTTCAGCACGCACGC-3′.Sod1–64 RNA boundaries were determined as previously described [The FMR1 null mouse embryos at 18 days of gestation. Primary cultures of cortical neurones were grown into 24-well format plates in 500 μl of Neurobasal Medium (Gibco) supplemented with 1× B27 (Gibco) and 0.5 mM l-glutamine in the presence of 100 IU/ml penicillin/streptomycin at 37 °C in 5% CO2. STEK FMR1 null cells and its derivative stably transfected with FMR1 cDNA [-firefly luciferase) and 5 ng of the plasmid coding for the Renilla luciferase used as a normalizer for each well.Primary cultures of hippocampal neurons were obtained from wild-type and Renilla and firefly luciferase activities were determined using the Dual-Luciferase Reporter Assay System (Promega) and following the manufacturer's protocol. Luciferase activities were measured using a Luminoskan Ascent luminometer. AUs of luciferase activity were calculated accordingly the protocol of the luminometer manufacturer (Thermo Labsystem). In each transfection, firefly luciferase values were normalized with Renilla luciferase values.Assays were performed 12 h after transfection. Starting from cell monolayers in 24-well cluster dishes, Sod1 RNA (5 pmol) was renatured at 40 °C for 15 min in the appropriate native buffer . Chemical modifications were performed in a final volume of 20 μl using either 1 μl of DMS diluted 1:2 (v/v) in ethanol or 60 μg of CMCT (carbodiimide) at 20 °C for 5 and 15 min, respectively, and in the presence of 2 μg of Escherichia coli tRNA. Enzymatic modifications were performed with V1 (0.0002 and 0.001 U), T1 (0.05 and 0.1 U), and T2 (0.05 and 0.1 U) nucleases, followed by a phenol/chloroform extraction. After ethanol precipitation and solubilization in the appropriate buffer, modified RNAs were reverse-transcribed using labeled primer III, and sequencing reactions and gel analysis were carried out as previously described [The 5′ UTR of escribed . In the Sod1 and the control Hprt are indicated in The RT reactions were performed with 2 μg of RNA using the ThermoScript RT-PCR system (Invitrogen). All mRNAs extracted from transfected cells were treated with DNase I (4 U for each RT reaction) for 1 h at 37 °C before RT. DNase was removed by phenol/chloroform treatments, and the mRNA was recovered after ethanol precipitation. The PCR reactions were carried out with the qPCR Core kit for Syber Green I (Eurogentec) in an ABI PRISM 7000 instrument (Applied BioSystems). Primers used to amplify Sod1 mRNA level quantification was performed with the STEK cells and neurons cultured in vitro for 10 days were treated with 5 μM actinomycin D (Sigma) for 2, 4, 6, and 12 h. Total RNA was purified from actinomycin-treated cells using the Qiagen RNeasy Mini Kit, and the RNA quality was verified on a 1% agarose gel and by optical density (OD) measurement. The  method .2, 0.5% NP40, 1 U/ml RNAsin). Resuspended polyribosomes were analyzed by 15–45% sucrose gradients composed of 25 mM Tris HCl, pH 7.4, 100 mM KCl, 5 mM MgCl2, and 1 U/ml RNAsin. After centrifugation in a Sorvall TH-641 rotor for 2 h at 34,000 rpm and 4 °C, gradients were fractionated by upward displacement using an ISCO UA-5 flow-through spectrophotometer set at 254 nm and connected to a gradient collector. Fifteen fractions of 800 μl each were collected from the sucrose gradient. Approximately 100 μl of each fraction was ethanol-precipitated, resuspended in 50 μl of Laemmli buffer, and analyzed by immunoblot. The remaining 700 μl of each fraction was treated with Trizol (Invitrogen) to purify RNA. The quality of RNA was verified on a 1% agarose gel and by OD measurement. Three independent polyribosomal purifications were carried out for brain extracts and for STEK cell extracts.Polyribosome purification and analysis were performed following our previously described protocol  with minCells were transfected for 48 h and after washing in phosphate-buffered saline were analyzed with a FACScalibur instrument (Becton Dickinson BD system). Cells expressing DsRed, with a constant intensity, were selected, and the mean of the eGFP intensity (expressed as AUs) of each sample was measured with the instrument software. The experiment was repeated three times in HeLa cells, COS cells, STEK cells expressing or not expressing FMRP, NG108 cells, and hippocampal primary neurons.Figure S132P- 5′-end-labeled primer I that hybridizes at positions +486; +464 (A), primer III  (B), or primer IV  (C). The resulting cDNA was separated on an 8% polyacrylamide/8 M urea sequencing gel and analyzed by autoradiography. RNA sequencing reactions were run in parallel. No specific RT stops were detected in the presence of potassium ion, thus excluding the presence of any G-quartet.RT reactions were performed in the presence of 100 mM KCl or 100 mM NaCl, using the (1.5 MB TIF)Click here for additional data file.Figure S232P-labeled Sod1 RNA, and competition was performed using the same unlabeled RNA.(A) Filter binding assay using FMRP and FXR1P Isoe, Isod, and Isoa. The RNA probe used is (B) The same experiment was repeated using as a competitor the N8 RNA sequence, which we have previously shown  to be un(4.2 MB TIF)Click here for additional data file.Figure S332P- 5′-end-labeled primer IV. The resulting cDNA was separated on an 8% polyacrylamide/8 M urea sequencing gel and analyzed by autoradiography. RNA sequencing reactions were run in parallel. The nature and positions of different loops and stems are indicated at right. Increasing concentrations of RNase V1 (V1), RNase T1 (T1) (right), or chemical agents (DMS or CMCT) (left) were added before the reverse transcription step. The en-dash indicates the lanes where the untreated RNA was loaded.Cleavage and modification sites were detected by primer extension using the (4.5 MB TIF)Click here for additional data file.Figure S4Hprt in each sample. As shown in the diagram, luciferase mRNA levels were not affected by the presence of SoSLIP or its mutants or by the presence or the absence of FMRP. No statistically significant differences have been observed for luciferase mRNA levels obtained from different constructs if compared with luciferase expressed from Luc. No statistically significant differences have been observed for luciferase mRNA levels in tissues and cell lines expressing or not expressing FMRP.(A) Cytoplasmic RNA was extracted from STEK cells expressing or not expressing FMRP and transfected with Luc, SoSLIP-Luc, SL1-Luc, SL2-Luc, and SL3-Luc, respectively. Luciferase mRNA levels were analyzed by qRT-PCR and normalized to Sod1 mRNA levels were quantified at different times  after the treatment. Values obtained are shown in (B) STEK cells expressing FMRP were transfected with vectors Luc, SoSLIP-Luc, SL1-Luc, SL2-Luc, and SL3-Luc. Twelve hours after transfection, cells were incubated with 5 μM actinomycin D. Fmr1 null STEK cells were transfected with vectors Luc, SoSLIP-Luc, SL1-Luc, SL2-Luc, and SL3-Luc. Twelve hours after transfection, cells were incubated with 5 μM actinomycin D. Total RNA was extracted, and Sod1 mRNA levels were quantified at different times  after the treatment. Values obtained are shown in (C) (6.2 MB TIF)Click here for additional data file.Table S1(27 KB PDF)Click here for additional data file.Table S2(37 KB PDF)Click here for additional data file.


Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the gold standard technique for mRNA quantification, but appropriate normalization is required to obtain reliable data. Normalization to accurately quantitated RNA has been proposed as the most reliable method for in vivo biopsies. However, this approach does not correct differences in RNA integrity.18S, ACTB, ATUB, B2M, GAPDH, HPRT, POLR2L, PSMB6 and RPLP0) that cover a wide expression spectrum. Our results show that RNA degradation could introduce up to 100% error in gene expression measurements when RT-qPCR data were normalized to total RNA. To achieve greater resolution of small differences in transcript levels in degraded samples, we improved this normalization method by developing a corrective algorithm that compensates for the loss of RNA integrity. This approach allowed us to achieve higher accuracy, since the average error for quantitative measurements was reduced to 8%. Finally, we applied our normalization strategy to the quantification of EGFR, HER2 and HER3 in 104 rectal cancer biopsies. Taken together, our data show that normalization of gene expression measurements by taking into account also RNA degradation allows much more reliable sample comparison.In this study, we evaluated the effect of RNA degradation on the quantification of the relative expression of nine genes (We developed a new normalization method of RT-qPCR data that compensates for loss of RNA integrity and therefore allows accurate gene expression quantification in human biopsies. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most sensitive method for mRNA quantification -4 as it In molecular oncology, pre-therapeutic biopsies are interesting material for gene expression studies that aim at identifying prognostic or predictive molecular markers. However, it has been suggested that housekeeping genes should not be used for normalization when studies involve biopsies, since they exhibit large expression variability between individuals . As an a18S, ACTB, ATUB, B2M, GAPDH, HPRT, POLR2L, PSMB6 and RPLP0) that cover a wide expression spectrum. Our results show that RNA degradation could introduce large errors in gene expression measurements when data were normalized to total RNA. Therefore, to avoid unspecific variations due to RNA degradation, we developed a corrective algorithm that take into account the RNA integrity of each sample and we validated the proposed model through the quantification of EGFR, HER2 and HER3 mRNA in colon and breast cancer cell lines. Finally, we applied this strategy for the quantification of EGFR, HER2 and HER3 in rectal cancer biopsies.The aim of this work was to evaluate the limits of normalization to accurately quantitated total RNA when using degraded samples and to improve this method by introducing a normalization factor that compensates for the loss of RNA integrity. For this purpose, using cell lines we first assessed the influence of RNA degradation on the quantification of the relative expression of nine genes  ranged between 0.86 and 1.00. The mean slope value was 0.086 ± 0.025 (95% CI 0.076–0.096). The relatively low standard deviation observed indicates that all genes had comparable degradation profiles. Obviously, the lowest expression ratios were obtained for the most degraded samples. The minimum ratio observed was 0.48 for the RPLP0 gene in the HCT116 sample with RIN = 4.7 and it corresponded to a 2.08-fold difference between the intact sample ratio and the measured expression ratio. In other words, there was an error of 108% in the reported expression level of RPLP0 in this sample. Similarly, the maximum errors observed for samples with 5 ≤ RIN < 6, 6 ≤ RIN < 7, 7 ≤ RIN < 8 and RIN ≥ 8 reached 104%, 92%, 75% and 47%, respectively.To evaluate the limits of normalization to total RNA when using samples with impaired RNA integrity, we studied the effect of RNA degradation on gene relative expression. For that purpose, we aliquoted intact total RNA from HCT116, BxPC-3 and A427 cell lines and we gradually degraded each aliquot by hydrolysis at 70°C for different length of time ranging between 0 and 165 min. We monitored the degree of degradation with an Agilent 2100 bioanalyzer following the RNA integrity number (RIN) classification . For eac18S, ACTB, ATUB, B2M, GAPDH, HPRT, POLR2L, PSMB6 and RPLP0 in the increasingly degraded HCT116, BxPC-3 and A427 RNA samples  could then be calculated as follow:To set up a normalizing factor that could compensate for the loss of RNA integrity, we first determined the average gene degradation profile based on the data we obtained for EGFR, HER2 and HER3 expression in LS174T (colon adenocarcinoma) and SKBr3 (breast carcinoma) samples displaying variable RNA integrity  with and without the application of our RIN-based normalization factor. The mean fold-difference between non-normalized and RIN-normalized values was 1.52 ± 0.17 (95% CI 1.47–1.55) and ranged from 1.13 to 2.10. Moreover, our RIN-based algorithm allowed the exposure of some differences in gene expression levels among samples that could not have been seen otherwise. For instance, without normalization, sample 48 and 50 exhibited EGFR expression ratios of 4.59 and 4.61, respectively, which would lead to the conclusion that EGFR was expressed at similar levels in both samples. After normalization of variations due to RNA degradation using our corrective RIN-based algorithm, EGFR was more strongly expressed in sample 48 (ratio = 9.60) than in sample 50 (ratio = 6.91). Furthermore, variations in RNA integrity may generate misleading differences in gene expression measurements. Indeed, the non-normalized ratios of sample 95 (ratio = 7.71) and sample 100 (ratio = 9.07) suggested that the former exhibited a lower EGFR expression level, while the RIN-normalized ratios led to the opposite conclusion. EGFR expression was actually higher in sample 95 (ratio = 15.10) than in sample 100 (ratio = 11.51).To evaluate the accuracy of normalization to total RNA of RT-qPCR data obtained from in vivo biopsies, we determined the RIN of 112 RNA samples isolated from 56 paired normal/tumor rectal tissues Figure . The majNormalization of gene expression levels to total RNA requires precise quantification of the RNA template. Several methods exist for measuring RNA concentrations, and we have previously discussed their respective advantages and drawbacks . In thisCAB mRNA control , and they ranged from 90% to 100%. To exclude between-run variations, all cDNA samples were tested in duplicate in the same analytical run along with a calibrator. A value of 1 was attributed to the calibrator and all gene expression levels were expressed as an n-fold difference relative to the calibrator, according to the relative standard curve method [We developed quantitative SYBR green PCR assays for the 12 genes involved in this study and the spiked plant mRNA control .qPCR: quantitative polymerase chain reaction; RIN: RNA integrity number; RT: reverse transcription.AH designed the study, performed the experiments, and wrote the manuscript. CB performed the statistical analyses and contributed to the content and writing of the paper. EA performed the endoscopic biopsies and provided the clinical samples. FBo and FBi collected and validated the biopsies. DC and MY managed and coordinated the project. EL supervised the design and execution of experiments and participated in the writing of the manuscript. All authors have read and approved the final manuscript.