This review describes the viral and cellular partners that retroviral RNA encounters during its maturation that begins in the nucleus, focusing on important events including splicing, 3' end-processing, RNA trafficking from the nucleus to the cytoplasm and finally, mechanisms that lead to its compartmentalization into progeny virions.Post-transcriptional events in the life of an RNA including RNA processing, transport, translation and metabolism are characterized by the regulated assembly of multiple ribonucleoprotein (RNP) complexes. At each of these steps, there is the engagement and disengagement of RNA-binding proteins until the RNA reaches its final destination. For retroviral genomic RNA, the final destination is the capsid. Numerous studies have provided crucial information about these processes and serve as the basis for studies on the intracellular fate of retroviral RNA. Retroviral RNAs are like cellular mRNAs but their processing is more tightly regulated by multiple  In this review, we summarize the current understanding of the processes affecting retroviral RNA metabolism as the RNA moves from its site of synthesis within the nucleus to its encapsidation into viral particles that emerge from the plasma membrane. The field has not only illuminated the cellular processes regulating RNA fate in general but also provided insights into potential strategies to impair replication of these viral pathogens.The life of an mRNA is directed by the protein components of ribonucleoprotein particles (RNP) whose roles include nuclear processing reactions, transport, translation and degradation. Retroviral replication depends on many of the same processes to form viral mRNA and genomic RNA providing an experimentally tractable system to study the env 3'ss is controlled by a suboptimal branchpoint (bpt) sequence and a nearby exonic splicing enhancer (ESE) [src 3' ss has a suboptimal pyrimidine tract (ppt) [src 3' ss [The expression of viral proteins from unspliced, incompletely spliced and fully spliced transcripts has necessitated that retroviruses evolve strategies to control the extent of RNA splicing. Extensive studies of avian sarcoma virus (ASV) splicing revealed three mechanisms of splicing control. The first involves the maintenance of suboptimal 3' splice site (ss) signals. Use of the er (ESE) ,2 wherearc 3' ss ,5. Whethrc 3' ss ,7. The Ngag intron deletion mutations that increased splicing in RSV [gag intron approximately 300 nt from the 5'ss and more than 4000 nt from the first of two alternative 3' splice sites (env and src) [cis and trans factors that govern U1 and U11 binding was important.The NRS was originally identified from g in RSV -8. The ag in RSV . Unlike and src) . Mutagenand src) -12. Overand src) . Bindingand src) ,11,14. TThe upstream, purine-rich region of the NRS was shown to have potent splicing enhancer activity and to bind members of the SR protein family of splicing factors and hnRNP H ,15,16. OThe determinants for U11 binding are largely distinct from U1. U11 is at a competitive disadvantage for NRS binding, being 100-fold less abundant than U1 . It was In contrast to murine leukemia and avian sarcoma viruses, the increased coding capacity of HIV-1 has necessitated the evolution of a more complex splicing regimen. In addition to structural proteins, HIV-1 expresses six additional proteins that regulate various facets of the virus lifecycle . To accoSubsequent research determined that the suboptimal nature of the 3'ss of HIV-1 was not the only point of regulation. It was established that exon sequences also influence the use of individual 3'ss. These exon regulatory sequences fall into two groups; exon splicing enhancers (ESEs) that act to enhance recognition and use of the adjacent splice site, and exon splicing silencers (ESSs) that suppress the use of adjacent 3'ss. To date, four ESSs have been mapped and control the use of the 3'ss for Vpr (ESS-V), Tat , and the terminal 3'ss (ESS3) ,32-37. Fenv results in generation of aberrant spliced products due to the creation of a splicing enhancer that activates a cryptic 3'ss (SA6) [Countering the inhibitory signals of the ESSs are the three ESEs present within the first  and second (ESE3) coding exon of Tat ,37,46-50ss (SA6) ,57.cis and trans modulators of RNA processing. Examination of EIAV splice sites revealed that both the 5'ss and bpt sequences do not deviate significantly from consensus. In contrast, the polypyrimidine tracts are interrupted by purines, which may reduce splice site usage by decreasing binding of the splicing factor U2AF [While RNA processing has been most extensively studied in ASV and HIV-1, work in other systems has also illuminated patterns of splicing regulation. Studies of equine infectious anemia virus (EIAV) have identified both tor U2AF . The purtor U2AF ,60. Deletor U2AF . This obtor U2AF . Howevertor U2AF .In contrast to HIV-1, there is only a limited understanding of splicing control in human T cell leukemia virus type 1 (HTLV-1). Like HIV-1, HTLV-1 produces several factors, in addition to the structural proteins, by alternative splicing. Little is known of the cis-acting elements controlling splice site use but evidence of regulation is provided by the marked differences in abundance of the various spliced RNA isoforms in different infected cell lines ,63. OverSeveral cis-acting elements that affect RNA stability and processing in Moloney murine leukemia virus (MoMLV) have also been identified. Analysis of sequences adjacent to the 3'ss revealed several elements that control splicing . DeletioIn summary, it would appear that retroviruses have used a common set of tools  to regulate the extent of viral RNA processing and achieve a balanced level of unspliced and spliced RNAs compatible with virus replication. The use of cellular factors  to regulate splicing suggests that the extent of RNA processing and hence, the capacity of the virus to replicate, is also dictated by the required mix of host cell splicing regulatory factors and determine the host range of the virus. Supporting this conclusion are observations from studies using MLV and HIV-1 based vectors. Lee et al.  noted thPolyadenylation plays a key role in the life of an mRNA, regulating its transport, translation and turnover. Controlling where in the retroviral genome polyadenylation occurs is critical for replication. For several retroviruses , choice of polyadenylation site use is straightforward since the major signal for the reaction (AAUAAA) occurs only once in the transcript. For other retroviruses , the situation is rendered more complex by the duplication of the polyadenylation signals (AAUAAA and the 3' G/U-rich sequence) at the 5' and 3' ends of the transcript. Successful replication has necessitated that these viruses evolve mechanisms to suppress recognition of the first polyadenylation signals. Although research has shown that sequences within U3  can enhance use of the downstream polyadenylation signal, this finding does not readily explain its almost exclusive use.In the case of HIV-1, it was initially believed that the proximity of the first polyadenylation site to the start site of transcription reduced its recognition by the host polyadenylation machinery, possibly as a result of a secondary structure that masks the AAUAAA polyadenylation signal ,73. HoweIn addition to the cis-acting signals that control use of the first polyadenylation site, ESE3 and ESS3 play a role in modulating use of the second polyadenylation signal. Deletion of ESE3 not only results in decreased use of SA7 but also an inhibition of Rev-dependent viral gene expression ,78 that Cellular and viral proteins have been implicated in regulating HIV-1 RNA polyadenylation. Experiments with Sam68, a member of the STAR family of RNA binding proteins, revealed that its overexpression dramatically enhanced Rev function -83, whicAlthough both HIV-1 and Moloney murine leukemia (MoMLV) virus share the same problem of suppressing use of the 5' proximal polyadenylation signal, they have evolved different mechanisms to solve the problem. In contrast to HIV-1, mutation of the 5'ss in MoMLV has little effect on the use of the first polyadenylation signal. Rather than regulating use of the signal, MoMLV appears to have adopted the use of inefficient signals, resulting in a significant proportion of viral transcripts failing to use either of the two viral encoded polyadenylation signals and the RNA terminates in the adjacent cellular sequences .In the avian RSV, the NRS appears to play an important role in modulating polyadenylation efficiency. Avian retroviral 3'-end formation is inherently inefficient with ~15% of RNA representing read-through transcripts where poly(A) addition occurs at downstream cellular sites . Miller Once the challenges of manipulating the splicing apparatus to preserve pools of unspliced RNA have been met, retroviruses face the task of exporting these molecules in a cell that normally restricts unspliced RNA to the nucleus. This could involve overcoming nuclear retention signals and/or recruiting export factors that otherwise would have little attraction for genome-length viral RNA. Export of bulk mRNA is thought to be facilitated by the recruitment of the general export factor TAP/NXF1:p15 to the RNA through different adaptor proteins, including REF/Aly and SR proteins . Adaptorenv reading frame for Rev function [Early mutagenesis studies of HIV-1 revealed that loss of Rev expression resulted in a complete loss of viral structural protein expression without significantly affecting levels of the various viral RNAs. Subsequent fractionation studies determined that the absence of HIV-1 structural protein production upon inactivation of the Rev reading frame was due to sequestration of the unspliced 9 kb and singly spliced 4 kb viral RNAs in the nucleus -96. Onlyfunction ,96. The Intense investigation of HIV-1 Rev function has resulted in it being one of the most thoroughly characterized export systems and readers are referred to more extensive reviews on its function that are briefly summarized here ,96. MultAlthough it is possible that Rev interacts with all RRE-containing HIV-1 RNAs in the nucleus (the 9 and 4 kb class of RNAs), studies have indicated that several parameters dictate which RNA will be exported to the cytoplasm. First was the demonstration that Rev function was dependent upon the continued transcription of the target RNA despite the presence of significant levels of RRE-containing RNA in the nucleus . This fiOnce the Rev/Crm-1/RanGTP complex assembles on the appropriate RNA, its journey from the site of synthesis to the cytoplasm begins. Most of the details of this process remain unclear but recent experiments have begun to identify host factors that play pivotal roles in the process. At least two members of the DEAD box RNA helicase family, DDX3 and DDX1, play essential roles in mediating Rev-dependent RNA export ,114. DepAlthough the study of the HIV-1/lentivirus systems clearly demonstrated a role for Rev-like proteins as adaptors to facilitate the export of viral RNAs to the cytoplasm, parallel work indicated that other viruses evolved alternative solutions to the export problem. An element at the 3' end of the MPMV designated the constitutive transport element (CTE) was shown to support Rev-independent HIV structural protein expression -119. An src gene (DR2) [src shows similar activity, and at least one DR is required for RSV replication. The DR sequence is conserved between avian retroviruses and a similar activity was ascribed to the DR element in RAV-2 ALV [Identification of the CTE export element in MPMV implied that similar export elements would be found in other simple retroviruses. One such element required for cytoplasmic accumulation of Pr-C RSV unspliced RNA was localized to the direct repeat region downstream of the ne (DR2) . A seconAV-2 ALV . DespiteAV-2 ALV ,126,127.As discussed above, HIV-1 Rev serves as an adaptor to target HIV RNA to the CRM1 export pathway, whereas the MPMV CTE directly binds NXF1 for export via the mRNA route. There is no obvious sequence similarity between the MPMV CTE and the avian DR elements, but it is perhaps reasonable to speculate that other simple retroviruses evolved to exploit the NXF1 pathway for export. Two studies took advantage of reagents to block the CRM1 pathway and found that, like the MPMV CTE, export of DR reporter RNAs was unaffected under conditions that blocked Rev export ,128. ThuIt is well established that the nuclear history of an mRNA can influence its fate in the cytoplasm. This property can be attributed to the nature of the mRNP that assembles on the RNA. One well-studied example is the affect that mRNA splicing has on mRNP composition through the deposition of the exon junction complex (EJC) and its consequences to downstream events such as export, translation, and decay . The posMammalian cells are nonpermissive for ALV infection due to defects in RNA processing, RNA export, Gag cleavage and particle assembly ,130,131.A recent report by Swanson et al. demonstrated a similar link between HIV-1 unspliced RNA export and Gag assembly . Gag proOnce delivered into the cytoplasm, two fates exist for the unspliced, genomic viral RNA: translation to produce the structural proteins and selection for encapsidation into the forming virions. The majority of unspliced retroviral RNA is not captured for encapsidation but serves other roles in generating viral structural proteins and enzymes or as a cofactor for assembly  to the sites of assembly remains a considerable challenge requiring targeting signals in the viral RNA, viral proteins, and/or a host cell targeting machinery . While Rpsi RNA packaging signal within the affected RNA and an intact NC domain in Gag, consistent with their demonstrated requirements for viral RNA packaging. The vesicles were comprised of both endosomal and lysosomal vesicles as evidenced by co-trafficking of the labeled RNA on transferrin- and lysotracker-positive vesicles in cells. Results of experiments in which monensin was used, a drug that prevents acidification of endosomal and organellar compartments, indicated that trafficking was achieved on vesicles that likely emanate from a steady-state endosomal compartment and not rapidly recycling vesicles that contain Rab11. Gag protein is recruited from late endosomal/lysosomal compartments to these endosomal membranes by the viral glycoprotein, Env, demonstrating important contributions of Env to this process. Not only could cellular RNAs replace viral RNA on vesicles in their system when psi was mutated, but some evidence suggests that the psi RNA sequence that is comprised of four stem loops in MLV, harbours an endosomal trafficking signal [The movement of MLV RNA to sites of virion assembly was investigated by monitoring MLV genomic RNA movement in live cells using a bacteriophage MS2 tethering system. In this study , it was g signal . Other sg signal , althougg signal ) the simg signal . Our owng signal . While ig signal . MPMV RNThe directed movement of viral RNA within the cytoplasm relies on its interaction with multiple host proteins generating an RNA transport granule (RTG). The concept of an RTG derives from the studies in neuronal cells, which have both specialized functions and extended morphologies -152. RTGPublished reports provide ample evidence that components of the RTG play roles in multiple steps of retroviral replication including transcription, RNA splicing, nucleocytoplasmic transport, translation as well as genomic RNA encapsidation . The recent identification of these proteins in RTGs in the cytoplasm [As described above, nuclear export of retroviral RNA involves several cellular RNA helicases. Recent observations have identified roles for RNA helicase A (RHA) and two DEAD-box proteins, DDX1 and DDX3 in the nuclear export of retroviral RNAs ,156-158.ytoplasm , albeit ytoplasm ,162.vpr RNA has been shown to be active in RNA trafficking [hnRNPs in general are believed to function at many post-transcriptional levels (reviewed in ). Particfficking . Subsequfficking . Kinesinfficking  and is pfficking . This obfficking ,169. Thebona fide RNA trafficking protein whose function is conserved from lower to higher eukaryotes. It plays a critical role in the localization of several mRNAs in Drosophila, likely via its direct interaction with target RNAs or in the context of RNP complexes [Drosophia Staufen preferentially binds RNA homodimers [While many of the proteins within the RTG have functions in several cellular compartments, Staufen1 works mainly in the cytoplasm. Staufen1 represents a omplexes ,171. Sevomplexes . Second,modimers , the formodimers . Fourth,modimers . Fifth amodimers ,175; L. modimers . Likely,Several functional and physical links between the transcription, RNA processing, nucleocytoplasmic and cytoplasmic transport machineries as well as the metabolic machineries for RNA in the cytoplasm are made by RNA-binding proteins, some of which mark the RNA before their exit from the nucleus and modulate RNA fate in the cytoplasm. The journey of retroviral RNA from the nucleus to its ultimate destination in the capsid is likely to be characterized by similar phenomena, yet up until now the details of these events have been scant. Many details of how the RNA processing machinery is manipulated to produce the appropriate spectrum of mRNAs and to allow genome-length RNA to escape splicing have been revealed in the last few years and illuminated potential targets for arresting virus replication. Recent work has already exploited our understanding of retroviral RNA processing to develop small molecule inhibitors of HIV-1 RNA splicing . AnotherThe author(s) declare that they have no competing interests.All authors contributed equally to the inception and writing of the manuscript.


A PCR product was amplified from RT reactions that were carried out without addition of RT-primer. This finding jeopardizes the accuracy of RT-PCR when analyzing RNA that is expressed in both orientations. Current literature findings suggest that naturally occurring antisense expression is widespread in the mammalian transcriptome and consists of both coding and non-coding regulatory RNA. The primary purpose of this present study was to investigate the occurrence of primer-independent cDNA synthesis and how it may influence the accuracy of detection of sense-antisense RNA pairs.The ability to accurately measure patterns of gene expression is essential in studying gene function. The reverse transcription polymerase chain reaction (RT-PCR) has become the method of choice for the detection and measurement of RNA expression patterns in both cells and small quantities of tissue. Our previous results show that there is a significant production of primer-independent cDNA synthesis using a popular RNase Hin vitro synthesized RNA suggest that these products are likely the results of RNA self-priming to generate random cDNA products, which contributes to the loss of strand specificity. The use of RNase H+ RT enzyme and carrying the RT reaction at high temperature (50°C) greatly improved the strand specificity of the RT-PCR detection.Our findings on cellular RNA and While RT PCR is a basic method used for the detection and quantification of RNA expression in cells, primer-independent cDNA synthesis can interfere with RT specificity, and may lead to misinterpretation of the results, especially when both sense and antisense RNA are expressed. For accurate interpretation of the results, it is essential to carry out the appropriate negative controls. The ability to accurately measure patterns of gene expression is essential in studying gene function. RNA expression patterns can be determined using northern blots or RNase protection assays. However, these methods lack sensitivity and require large amounts of RNA sample. Therefore, they are not suitable in most cases especially when analyzing low expression levels of RNA. The reverse transcription-polymerase chain reaction (RT-PCR) has become the method of choice for the detection and measurement of RNA expression patterns in both cells and small quantities of tissue. The first step in this process consists of the cDNA synthesis in which a DNA complementary to the RNA is synthesized with the help of a reverse transcriptase enzyme in the presence of dNTP and DNA primers. An oligo dT primer is used to make cDNA of poly A-RNA such as mRNA. In contrast, random primers are used for making cDNA of all the RNA population including poly A-RNAs and ribosomal RNAs. In addition, gene specific primers that are complementary to a specific sequence of the RNA are often used for gene-specific RT. This latter method is commonly used for studying expression of RNAs that are thought to be expressed in both orientations. Recently, we found that cDNA synthesis can occur in the absence of primers in the RT-reaction . The occRecent studies show that bidirectional transcription is more widespread in the mammalian transcriptome than initially thought -17, whicMyosin heavy chain (MHC) is a key striated muscle motor protein which plays a major role in the regulation of the contraction process. MHC is expressed in several isoforms which are the products of distinct genes. These isoforms differ in their biochemical and functional properties. Two isoforms are expressed in heart muscle, namely α (MYH6) and β (MYH7). MYH6 and MYH7 genes are located in tandem on the same chromosome , and each gene is 25 kb separated by 4.5 kb of intergenic region. These genes' expression in cardiac myocytes is highly coordinated in an antithetical fashion, and a natural antisense RNA is involved in their coordinated regulation. In a recent study, we have encountered technical problems while analyzing sense and antisense RNA and pre-mRNA across the cardiac MYH genes , the Hoxin vitro synthesized RNA led us to believe that these products  are likely the results of RNA secondary structure associated with self-priming to generate random cDNA products. The reverse transcriptase enzyme characteristics and reaction conditions affect primer-independent cDNA synthesis and this can be minimized or avoided with proper analytical conditions. These self-primed products may be a major source of the confounding error in the detection and quantification of RNA expression when the antisense RNA strand is also expressed.The primary purpose of this present study was to investigate the basis of amplification of PCR products following reverse transcription (RT) of the RNA in the absence of primers in the RT reaction. Our investigation using cellular RNA and - reverse transcriptase in the absence of RT-primers, PCR resulted in detection of a product only in reactions from tissue that expresses the target RNA [see Additional file When reverse transcription of total RNA used RNase H- RT may not be stringent enough to demonstrate expression of a specific RNA strand if the complementary strand is also expressed. One way to insure strand specific expression is to use the net PCR signal between specifically primed cDNA and primer-independent cDNA synthesis under identical RT-PCR conditions.If a gene locus is transcribed on only a single strand, RNA detection without primers will only add to the signal, but is not problematic since it retains its sequence specificity. In contrast, if a gene locus is transcribed on both strand, thereby producing sense and antisense RNA pairs, RT reactions using RNase H- reverse transcriptase (Superscript II) according to the recommended protocol (Invitrogen). Results show that the strand-specific RT-PCR reactions resulted in the formation of products. Sense MYH7 pre-mRNA was detected in the NC heart, and this was upregulated in the PTU-treated heart , and this ratio decreased in the PTU heart Fig . In contin vitro, and examine their behaviors in RT-PCR in pure forms or in mixtures of known composition of sense and antisense RNA templates. A plasmid DNA vector was constructed so that after restricted digestions and in vitro RNA transcription one can obtain RNA strands that are complementary to each other as that encountered for overlapping genes [Additional file When we initially observed the formation of no-primer RT-PCR products during analyses of cardiac and skeletal muscle MYH genes' RNA, we thought that the primer-independent cDNA synthesis was caused by the presence of sense and antisense RNA pairs in solution. It was believed that the sense RNAs prime the antisense RNAs in the reverse transcription reaction, and vice versa. In order to test this hypothesis, we decided to synthesize sense and antisense RNA pairs in vitro RNA were tested: pure SP6, pure T7, and a mix of Sp6 and T7 in equal amounts. Reverse transcriptions were performed under two conditions: 1) in the absence of any primer (-p), and 2) in presence of a specific primer to Sp6 RNA (+p)  Fig . Our res(+p) Fig , and thi(+p) Fig . In cont(+p) Fig . In a mi(+p) Fig . An incr(+p) Fig . Therefo-) at the recommended temperature by the manufacturer, 44°C. To answer the question on how the RNase H activity of the reverse transcriptase or the reaction temperature may affect the RNA strand-specificity of cDNA synthesis, we compared RNase H positive (+) RT  vs. RNase H- RT  specificity at two different temperatures, 44°C and 50°C, basing the comparison on RT-PCR results when the RT is carried out in the absence of primers. Under both temperature conditions, RNase H- RT was associated with formation of more no primer product than the RNase H+ RT , or with inclusion of non-specific primers that are not related to the target gene's sequence. If these RT reactions (no primer and non-specific primer RT) result in amplifiable PCR-products  extension product of the primer. The PCR reaction amplifies the cDNA to produce a DNA fragment, the intensity of which correlates with the initial amount of specific target RNA in the analyzed sample. Lack of strand specificity does not appear to be a concern when a gene locus is transcribed on only one strand, because the no primer RT-PCR product is sequence specific [Additional file ucts Fig , these pin vitro synthesized and DNase treated, was also associated with this "background" no primer RT-PCR product  is more important in establishing specificity than the initial heat denaturing of the RNA. The limiting factor under these conditions would be the thermostability of the RT enzyme. In this context, it is worth mentioning that self priming of the RNA was recognized previously as a potential source of interference with reverse transcription reaction efficiency, and carrying the RT reaction at higher temperature was recommended as a way to improve the reaction efficiency ,33.- RT was associated with more self primed cDNA products than when using RNase H+  and higher temperature were likely contributing to strand specificity of the RT in this one step-system. Other factors such as the reaction composition may have contributed to this increased specificity. Importantly, we tested four different commercially available kits , and all were found to be RNA strand specific under the conditions utilized Fig .in silico findings concerning overlapping transcripts via using a 2 Step RT PCR System with RNase H- RT. These verification reactions were performed without the negative control for strand specificity [- RT. Alternatively, their results could have been confirmed by performing an appropriate negative control such as RT in absence of RT primers, or in presence of a non specific primer, followed by PCR.In the literature, with the new modern advances in computational biology, several reports have emerged on the abundance of overlapping genes and the natural occurrence of antisense RNA ,9,17. Incificity ,36,37. BWhile RT PCR is a basic method used for the detection and quantification of RNA expression in cells, primer-independent cDNA synthesis can interfere with RT specificity, and may lead to misinterpretation of the results, especially when analyzing bidirectional RNA. Primer-independent cDNA synthesis occurs in most reverse transcriptase reactions, and this may interfere with strand specific cDNA synthesis when both sense and antisense RNA are expressed. Primer strand-specificity of the RT-PCR may be important in the new era of biology when new evidence is emerging rapidly supporting the expression of both sense and antisense strands of the genomic DNA. For accurate interpretation of the results, it is essential to carry out the appropriate negative controls.in vitro synthesized RNA.As templates for RT-PCR experiments, we used mammalian total cellular RNA and Total RNA from rat tissue was utilized. Young adult female Sprague Dawley rats  were utilized for this study (Taconic Farms). One group of animals (N = 6) was treated daily with propylthiouracyl . Another group (N = 6) was treated with triiodothyronine , in order to induce either a hypothyroid or a hyperthyroid state, respectively. These altered thyroid states are associated with a marked change in cardiac myosin heavy chain (MHC) gene expression . AnimalsTissue RNA was extracted using the tri-reagent procedure according to the manufacturer protocol (Molecular Research Center). Both, human brain and rat tissue total RNAs were treated with RQ1 DNase I (Promega) using 1 unit/μg total RNA at 37 degree C for 30 minutes. RNA extraction and DNase treatment were as described previously ,38. RNA In vitro RNA was transcribed from linearized plasmid DNA using the Mega Script RNA kit from Ambion with either SP6 or T7 RNA polymerase. At the completion of the in vitro transcription, the sample was treated with turbo DNase I (Ambion) which effectively removes traces of the original Plasmid DNA. After DNase treatment, the RNA was extracted using the tri-reagent LS (Molecular Research Center).To prepare a plasmid construct to be used as template for RNA synthesis, a 757 bp PCR fragment amplified from the rat genome was directly cloned into a TA cloning vector pGEM T easy (Promega) [see Additional file in vitro synthesized RNA is to be used for RT-PCR, it must be free of any precursor plasmid which can function effectively as template for the PCR. For all the tested PCR primers, the RNA preparation PCR reactions gave no product whereas the plasmids gave a strong signal of the expected size. These results ensured that the DNase treatment was effective, thus validating the use of these in vitro RNAs as templates for subsequent RT-PCR testing.The purified RNA was tested first for any plasmid contamination by PCR targeting specific regions and using 100 ng of pure RNA template. For the positive control for these tests and for each primer set, 50 ng plasmid (pGEM T easy containing the insert) was used as template. It is important to note that if the in vitro synthesized RNA was utilized, 5–10 ng of in vitro RNA were included per 20 μl reaction.Each RNA sample was subjected to reverse transcription under three different conditions [see Additional file -) from Invitrogen is a modified version of Moloney Murine Leukemia Virus (MMLV) in which RNase H activity is reduced. Omniscript (RNase H+) from Qiagen is a genetically engineered heterodimeric enzyme with an intrinsic RNase H activity. For these RT reaction procedures, the manufacturer recommendations were strictly followed. When a specific RT primer was included it was at 5 pmoles/20 μl reaction volume.For these analyses, we have tested two popular reverse transcriptases that differ with their RNase H activity. Superscript II  and compared to a modified condition whereby the temperature of the reaction was increased, while the reaction time was decreased [see Additional file A two-step RT-PCR system was utilized for most of these analyses. The cDNA products were amplified by either end point PCR (using a Stratagene Robocycler), or with real time quantitative PCR . For some RT-PCR analyses, a one step-RT-PCR system was utilized. PCR primer pairs were designed using Primer Select/DNA Star software. Sequence information on all RT and PCR primers used in this report is shown in Additional file Reactions were 25 μl in volume and included variable amounts of cDNA depending on target abundance along with 15 pmoles of each of the reverse (Rev) and forward (Fwd) primers and used Biolase thermostable Taq DNA polymerase as recommended by the manufacturer (Bioline). PCR started with an initial denaturing step of 3 min at 96°C, followed by a variable number of cycles (20–35 cycles) consisting of 1 minute at 96°C, 45 seconds at the annealing temperature, (variable depending on the primers Tm), 45 seconds at 72°C. When the different RT reactions of the same RNA samples were being compared, equal amounts of cDNA were included in the PCR reaction under the same reaction and cycling conditions.® Green QPCR master mix reagents from Stratagene . A PCR primer set targeting the 5'end of the β MHC gene was used for these experiments [see Additional file Real time PCR was performed in order to obtain a quantitative relationship between PCR product obtained from primer-independent cDNA and that obtained with specifically primed cDNA. As template for these analyses, we used control and PTU heart cDNA and we targeted the cardiac MHC RNA expression, a condition associated with significant change in MHC gene expression . QuantitIn addition to the above described two step RT-PCR reaction systems, a one step RT-PCR assay was also tested using four commercially available kits which were obtained from Qiagen, Promega, USB, and Clontech. In these reactions the RT and PCR were performed in a single tube in the following sequence: the RT reactions were performed at 50°C for 30 minutes under specified primer conditions, followed by 15 minutes heating at 95°C. At this stage, the missing PCR primers were added to the reaction, and PCR was conducted for a certain number of cycles depending on the target abundance. For each RNA sample four different reactions were carried out as follows which differed in their RT design: 1) no primers or non specific primers unrelated to the target RNA, were added to the RT reaction; 2) Either, the reverse PCR primer alone, or 3) the forward PCR primer alone were included in the RT reaction; 4) the RT enzyme was immediately denatured at 95°C for 15 minutes followed by adding the RNA template and PCR primers before performing the PCR. Under these last conditions, a product formation would be indicative of either genomic DNA contamination in the RNA sample or reagent contamination with the PCR product. For these One Step RT PCR reactions, each reaction was performed using 50–200 ng total RNA, and the PCR was carried out for 20–35 cycles depending on the abundance of the target RNA. Conditions to be compared were run on the same samples under similar conditions .in vitro RNA synthesis, and she participated in RT-PCR data generation and analyses. JG participated in the experimental design, data analyses, and interpretation of the results; as well as editing the manuscript. HG participated in the RNA extraction as well as real time PCR data generation and analyses. KMB contributed to the experimental design, interpretation of the results, and the writing of the manuscript. All authors read and approved the final manuscriptFH made the initial observation of primer-independent cDNA synthesis, and she contributed to the experimental design, analyses and interpretation of the data and drafted the manuscript. AXQ performed the plasmid construction for the Primer-independent cDNA synthesis and RT-PCR sequence specificity. (a) 2 μg total RNA were reverse transcribed in the absence of any primers in 20 μl reactions using RNase H- reverse transcriptase (Superscript II) and total RNA from rat tissue including heart (H), liver (L), white medial gastrocnemius (WMG), and soleus (S) muscle. PCR was performed targeting the18S rRNA, the cardiac beta MHC RNA (MYH7), the cardiac alpha MHC RNA (MYH6), and the alpha skeletal actin RNA (ACTA). PCR was carried out for 30 cycles on various amounts of cDNA for each PCR primer set. . Shown are ethidium bromide stained agarose gels depicting the product of such PCR reactions. Note that the selection of PCR primers was based on differential expression of their targets in the tested tissue RNA. For example, 18S rRNA is ubiquitously expressed, while the MYH7 RNA is expressed in the heart and soleus muscle but not in the liver and WMG muscle. The MYH6 RNA is expressed exclusively in the heart, while ACTA RNA is expressed only in WMG and soleus muscle. A small level of skeletal actin expression may also occur in the heart. (b) Human brain (HB) RNA analyses using primer-independent RT PCR. Representative images of ethidium bromide stained gels depicting the PCR products from primer-independent cDNA synthesis in a two-step RT-PCR system. In the RT, 2 μg total RNA was used in 20 μl reaction with RNase H- reverse transcriptase. PCR used 1 μl cDNA and was carried out for 30 cycles. Primers in b are based on Fig Click here for filein vitro RNA synthesis to generate complementary RNA. Schematic of the plasmid construct used for the A PCR product , was ligated to pGEM-T easy between T7 and Sp6 promoters using T/A cloning technique (Promega). The construct was linearized by digestion with restriction enzymes which cut at specific sites, e.g., NaeI at position 3465, or StuI at position 334 This was followed by RNA synthesis using T7 or Sp6 RNA polymerase (Mega Script RNA kit from Ambion). +1 is the start of T7 RNA transcription. MCS: multicloning site region of pGEM-T Easy vector.Click here for fileDescription of the different RT reaction conditions that we used for the two step RT-PCR systems.Click here for fileRT and PCR primer information. RT and PCR primer sequences, their specific target, PCR product size, and reference figure whereby these primers were used.Click here for file


Small, non-protein coding dendritic BC1 RNA is thought to regulate translation in dendritic microdomains. Following microinjections into cultured cells, BC1 RNA fused to larger mRNAs appeared to impart transport competence to these chimeras, and its 5′ ID region was proposed as the cis-acting dendritic targeting element. As these ID elements move around rodent genomes and, if transcribed, form a long RNA stem-loop, they might, thereby, lead to new localizations for targeted gene products. To test their targeting ability in vivo we created transgenic mice expressing various ID elements fused to the 3′ UTR of reporter mRNA for Enhanced Green Fluorescent Protein. In vivo, neither ID elements nor the BC1 RNA coding region were capable of transporting EGFP RNA to dendrites, although the 3′ UTR of α-CaMKII mRNA, an established cis-acting element did produce positive results. Other mRNAs containing naturally inserted ID elements are also not found in neuronal dendrites. We conclude that the 5′ ID domain from BC1 RNA is not a sufficient dendritic targeting element for mRNAs in vivo.Dendritic localization of mRNA/RNA involves interaction of  There are several hundred mRNAs known to be dendritically localized in neurons in vitro and in transfected cells Rodent BC1 RNA is a npcRNA expressed almost exclusively in neuronal tissue, where it is transported to neuronal dendrites BC1 RNA serves as a master gene for the amplification, via retroposition, of a subclass of short interspersed repetitive elements (SINEs), termed ID1 elements, in rodents in vitroin vivo, we created transgenic mice expressing the corresponding transgenes in neurons  harbors cis-acting element(s) sufficient for imparting extrasomatic localization to reporter mRNAs cis-acting dendritic targeting element in our system by generating transgenic mice expressing chimeric EGFP mRNA containing the 3′ UTR of α-CaMKII. In situ hybridization on coronal sections of the corresponding transgenic mouse brains revealed signal in cell bodies and throughout the full extent of the dendritic fields of hippocampal CA1, CA2, and CA3 regions as well as various layers of the cortex  on rat brain coronal sections revealed labeling only in the cell bodies of hippocampal CA1, CA2, and CA3 neurons in situ hybridization of synArfGEF mRNA (Accesion # AB057643) on adult rat brain sections revealed labeling only in the cell bodies of CA1 pyramidal cells in the hippocampus in situ hybridization on rat hippocampal primary cultures also revealed labeling in the cell bodies and to a much lesser extent only in the very proximal dendrites in situ hybridizations were available in databases (http://www.brain-map.org). We selected one example with an ID sequence similar to the ones we used in our transgenic mice. In mouse brain, in situ hybridization of Pex13 mRNA  within these 94 nucleotides seems sufficient to transport GFP reporter mRNA into dendrites In recent years, determining the subcellular localization of RNAs has emerged as an important field and dendritic if, for example, they became integrated within the UTR of the mRNA. To assess whether insertion of an ID element has the potential to impart dendritic localization, we generated transgenic mice with constructs that enabled us to test a somatic mRNA for potential acquisition of dendritic transport competence by inserting ID elements or the entire coding region of BC1 RNA into the 3′ UTR of the reporter mRNA. However, none of the chimeric reporter constructs harboring either the 5′ stem of BC1 RNA or other classes of ID elements  transgene and a recently discovered dimeric Alu-derived G22 RNA (300 nt) transgene in situ hybridization data are available. The three ID elements were in the correct orientation with respect to the mRNA and their stem structures closely resembled that of BC1 RNA  stem-loop of BC1 RNA were important for dendritic transport  BC1 RNA . None ofcis-acting elements for neuronal dendritic transport, as has been shown in microinjection experiments in cultured neurons. Even though the 3′ UTR of α-CaMKII mRNA was capable of transporting the chimeric EGFP mRNA into distal portions of dendrites, none of the ID or BC1 insertions was capable of targeting the EGFP reporter mRNA to dendrites.In conclusion, although some of the numerous retroposed SINE insertions add functionality to targeted genes, their RNA transcripts, or their protein products, we could not demonstrate under physiological conditions in transgenic mice that ID elements serve as potential Our experiments required a reporter mRNA that could be expressed in neurons where BC1 RNA had been shown to be dendritically localized, such as the hippocampal pyramidal cells XbaI and BclI restriction sites of a linker region (31 bp) downstream of the EGFP coding region, respectively. The inserts were generated using a PCR-based approach with appropriate restriction sites. The inserts for pNEID1 and pNEID2 were generated from pTubID1 and pTubID2 using the following primer pair: ID1/ID2for 5′-gcgtgctctagaGGGGTTGGGGATTTAGCTCAG-3′ and IDRev 5′-gcgcgtactagtCAAAACCAAGAAAAAAAGCCTCGAC; for pNEID4: ID4for 5′-gcgtgctctagaGGGGcTGGGGATTTAGCTCAG-3′ and IDRev, from pTubID4 template; for pNE1DI: BC1rev–for 5′-gcgtgctgatcaGGGGTTGGGGATTTAGCTCAG-3′ and BC1rev–rev 5′-gcgcgtagatctCAAAACCAAGAAAAAAAGCCTCGAC-3′ from pTubID1; for pNEBC1: 1D1/ID2for and BC1uni 5′-cgcgcaACTAGTTTTCCAACACACACGGTC-3′. Bases in upper case correspond to ID element or BC1 RNA sequences and those in lower case to flanking adapter sequences containing restriction sites for cloning. The plasmid pNECKu, containing part of the α-CaMKII mRNA ORF as well as the 3.1 kb of UTR PvuI and NruI/EcoRI restriction endonucleases, diluted to 3 ng/µl, and injected into the pronuclei of FVB oocytes Constructs for the various ID elements were generated in pβactEGFP2 . The vector transcribes an EGFP mRNA originally derived from pEGFP-N1 (Clontech Laboratories) driven by the chicken β-actin promoter from pβact-16 32P-labeled probe specific for EGFP DNA sequences as previously described To determine which mice were transgenic, genomic DNA from the tails of 3-week-old mice was extracted as previously described in situ hybridization. Total RNA from brain was extracted using TRIzol reagent (Gibco BRL) according to the manufacturer's instructions. Northern hybridization with an α-32P-labeled probe complementary to the EGFP region was carried out as previously described Transgenic and wild type mice were anaesthetized with halothane and transcardially perfused with PBS. The brains were divided into three parts by a coronal cuts through the optic chiasm and just in front of the cerebellum. The anterior and posterior portions were used for RNA isolation and the middle portion containing the hippocampus was placed in 4% paraformaldehyde to fix the tissue for in situ hybridization on transgenic mouse brain sections was carried out as previously described KpnI and SacI restriction sites of the multiple cloning sites. The probe specific for BC1 RNA was generated as previously described in vitro using T7 or T3 RNA polymerases, after linearization with KpnI and SacI restriction endonucleases, respectively, according to the manufacturer's instructions .To determine the subcellular localization of chimeric mRNAs, non-radioactive in situ data in the published literature.BLAST searches in NCBI Rat refseq_rna database were carried out using the sequence of the ID stem of BC1 RNA as the query sequence. Advanced BLAST options were used to limit these results to RNA entries that were expressed in brain. Presence and correct orientation of the repeats were then analyzed with the local RepeatMasker. Candidate mRNAs were then screened for available http://genome.cse.ucsc.edu/cgi-bin/hgTables and were analyzed for repeats utilizing the local RepeatMasker. . The output was separately analyzed for BC1- and ID-derived repeats. 3′ UTRs that contained ID- or BC1-derived SINE elements in the correct orientation were checked manually for the presence of Allen Brain Atlas entries via the BLAT server at http://genome.cse.ucsc.edu/cgi-bin/hgBlat?command = start. All cases for which the in situ hybridization probe included repetitive parts were excluded from further analysis. RNA structure predictions were performed using the MFOLD software at: http://bioweb.pasteur.fr/seqanal/interfaces/mfold-simple.htmlIn addition, mouse 3′UTRs of the known gene dataset were downloaded from table browser at: Figure S1The nucleotide sequence of a construct used to generate transgenic mice and deduced sequences of processed hybrid reporter mRNAs. (A) The basic construct, as well as relevant parts of the reporter mRNAs (B–G). Vector sequences in (A) flanking the sequence shown are in lower case. The chicken β-actin promoter sequence and the 5′ UTR are in upper case; the TATA box and transcriptional start site (A) are underlined. The intron is shown in lower case and the polylinker regions in upper case italics. The ORF of enhanced green fluorescent protein (EGFP) is shaded in grey. The various test sequences, which are potential cis-acting element(s)  are in bold and embedded between polylinker sequences . The subsequent UTR and polyadenylation signals (underlined) from SV40 virus are shown in upper case. In (B–G) the salient parts of the reporter mRNAs are shown beginning with the transcription start sites (underlined). The introns are removed and UTRs are depicted in upper case . The various inserts, full-length BC1 RNA (B), ID1 (C), ID2 (D), ID4 (E), ID1 in opposite orientation (F), and the 3′ UTR of α-CaMKII (G) are in bold type. The C-terminal portion of the α-CaMKII ORF is shaded in grey. The remaining portion of the 3′ UTR of the reporter mRNA is in regular upper case with polyadenylation signals underlined. The poly(A) tails  is probably terminated after the first polyadenylation signal.(0.06 MB DOC)Click here for additional data file.Figure S2Chimeric mRNAs are Expressed in the Brain Northern blot hybridization of total RNA extracted from brain tissue of transgenic and wild type mice. The extracted RNAs were separated on a 1.2% denaturing agarose gel and hybridized with 32P-labeled probe complementary to EGFP. Specific signals corresponding to the expected sizes was observed for chimeric RNAs. As a loading control the membrane was hybridized with a probe complimentary to α-tubulin mRNA.(7.73 MB DOC)Click here for additional data file.Figure S3Sequences of ID elements from our chimeric RNAs and from published examples found in the UTRs of other genes. Alignment of ID elements to the one found in the 5′ domain of dendritic BC1 RNA in rat  and mouse . Nucleotides identical to the ID domain of rat BC1 RNA are shown as dots, nucleotide replacements by the corresponding changes, and deletions by hyphens. The areas thought to be vital for dendritic transport (0.02 MB DOC)Click here for additional data file.Figure S4Secondary structures of ID elements. Secondary structures of the ID domains found in other genes and for which brain in situ hybridization data are available in the literature or in databases, respectively. Nucleotides corresponding to those thought to be vital for dendritic transport (see above) are highlighted in red and deviations from the ID element in BC1 RNA in blue. The corresponding nucleotides in BC1 RNA are indicated by arrows.(9.35 MB TIF)Click here for additional data file.


The hypothesis of an RNA-based origin of life, known as the "RNA world", is strongly affected by the hostile environmental conditions probably present in the early Earth. In particular, strong UV and X-ray radiations could have been a major obstacle to the formation and evolution of the first biomolecules. In 1951, J. D. Bernal first proposed that clay minerals could have served as the sites of accumulation and protection from degradation of the first biopolymers, providing the right physical setting for the evolution of more complex systems. Numerous subsequent experimental studies have reinforced this hypothesis.The ability of the possibly widespread prebiotic, clay mineral montmorillonite to protect the catalytic RNA molecule ADHR1 (Adenine Dependent Hairpin Ribozyme 1) from UV-induced damages was experimentally checked. In particular, the self-cleavage reaction of the ribozyme was evaluated after UV-irradiation of the molecule in the absence or presence of clay particles. Results obtained showed a three-fold retention of the self-cleavage activity of the montmorillonite-protected molecule, with respect to the same reaction performed by the ribozyme irradiated in the absence of the clay.These results provide a suggestion with which RNA, or RNA-like molecules, could have overcame the problem of protection from UV irradiation in the RNA world era, and suggest that a clay-rich environment could have favoured not only the formation of first genetic molecules, but also their evolution towards increasingly complex molecular organization. The hypothesis of an ancestral era during the evolution of life known as the "RNA world" , originaIn vitro selection experiments and observations concerning natural ribozymes have demonstrated that the range of catalytic capacities of RNA is larger and more promising than previously thought ·100, where I1 and I2 are the intensities of the bands corresponding to the full-length molecule and its 5' fragment of cleavage, respectively. The 15 nt 3' fragment was always runned out of the gel, to better separate the other two bands.Band intensities from ADHR1 self-cleavage reactions were analyzed by ethidium bromide or SybrGold (Invitrogen) staining with the software ImageJ, and percentages of self-cleavage calculated as follows: % = [It = F∞.The authors declare that they have no competing interests.EB designed and carried out the large part of the experimental work described on this paper. SB, M-CM, and EG equally contributed to the analysis and interpretation of the data, and to draft the manuscript. All authors read and approved the final manuscript.


Subacute sclerosing panencephalitis (SSPE) is a rare, slowly progressive neurological disorder caused by the persistent infection with measles virus (MV). Despite much research into SSPE, its pathology remains obscure. We examined autopsy tissues of eight SSPE patients by real time quantitative PCR, immunohistochemistry and immunoblotting to determine viral load. MV N, M and H gene RNA could be detected in the central nervous system (CNS) of all patients and in two non-CNS tissues of one patient. The viral burden between patients differed up to four-fold by quantitative PCR and corresponded with detection of MV protein. The level of both viral RNA and antigen in the brain may correlate with disease progression. Subacute sclerosing panencephalitis (SSPE) is a rare, slowly progressive neurological disorder of children and young adults, caused by the persistent infection with measles virus (MV). After acute infection with MV, typically at a very young age, the patients appear to recover normally. Following a period of asymptomatic persistence, usually 3 to 10 years, neural dysfunctions develop and progress over months to years, resulting eventually in coma and death. Although much effort has gone into researching SSPE, its pathology remains largely obscure. The progression of SSPE greatly differs in individual patients [Viral load has been correlated to disease development, progression and outcome in several infections, including HIV, SIV, cytomegalovirus ,3 and EpAs MV can usually be detected in brain tissue from biopsy or autopsy by RT-PCR and/or immunohistochemistry, it is thought to persist and spread within the CNS. Although the brain appears to be the main location of persistent MV in SSPE, it is not entirely clear if this is also the site of persistence and the only target during disease. Viral antigen or RNA have been detected outside the CNS in SSPE cases -10. To aThis study was carried out to determine whether differences in viral load caused diversity of disease progression. Autopsy tissues of brain and non-brain origin of eight confirmed cases of SSPE were analysed by quantitative PCR to determine amounts of the MV RNAs encoding the nucleoprotein (N), matrix protein (M) and haemagglutinin (H) and by immunological techniques to detect the N protein.Autopsy or biopsy tissues of eight SSPE patients were analysed, details are shown in Table 6 copies of N RNA per ng total RNA in occipital brain patient UK111 to 4 × 102 copies/ng RNA in temporal brain of case UK585, respectively. In addition to relating copy numbers to the amount of total RNA, they were standardised as a function of GAPDH mRNA. GAPDH  is a ubiquitously expressed message, which is frequently used to standardise across tissues. Although expression of GAPDH may not be uniform across all tissue types, this method was found acceptable in a comparison of nine CNS and non-CNS tissues from three SSPE patients. The maximum fold change between CNS tissues was <8-fold, between CNS and non-CNS tissues <11-fold  in all available CNS tissues from eight SSPE patients and in two non-CNS tissues of one patient. The concentration of MV N gene RNA in CNS tissues ranged from almost 2 × 106 copies of H RNA per ng total RNA. Again the weakest positive CNS sample was temporal brain of UK585 with just under 2 × 103 copies/ng RNA. This is approximately 9 × 103 %GAPDH and 3% GAPDH, respectively. The M gene signal was approximately ten-fold weaker, but the different cases were similar in relation to each other as with the N and H genes. Occipital brain of UK111 contained 4 × 105 copies M gene/ng RNA or 3 × 102 %GAPDH. This gene could not be detected in temporal brain tissue of patient UK585. In PCRs with MV N, M and H gene targets, occipital brain of UK111 was the strongest positive when looking at copies/ng RNA, while the brain of UK85 contained most MV RNA when expressed as a function of GAPDH. The efficiency of all three PCR reactions was found to be similar when titrations of p(+)MV, a MV genome containing plasmid, were used as template (data not shown), which indicates that the numbers obtained with the different PCRs can be compared. The M and H gene results confirm the relative findings with the N gene. All quantities are illustrated in Figure MV N RNA positive samples were also used for detection of MV M and H gene RNA. The H gene PCR lead to very similar results as the N gene PCR, both in copy numbers and %GAPDH. Occipital brain tissue of UK111 contained just under 2 × 10To determine whether the detected viral RNA is actively replicating, immunological methods were used to visualise MV protein in tissues from patients UK111, UK98 and UK585. In occipital brain tissue of UK111, a large number of MV N protein positive cells could be detected by immunohistochemistry, as seen in Figure To detect MV protein with higher sensitivity, tissue lysates were analysed by immunoblotting Figure . In agreA weak inverse correlation between viral loads measured by qPCR and SSPE disease duration was detected. Quantities of viral RNA were compared to durations of SSPE and of infection from primary MV acquisition to sample date. Results are shown in Figure 3 and 1 × 105 %GAPDH, respectively, was detected. The M gene of virus in case UK585, on the other hand, was strongly mutated and low virus levels of <1% GAPDH were measured. In case UK83, however, viral load was high (1 × 103 %GAPDH), despite presence of virus with a highly mutated M protein.To investigate a possible link between virus integrity and viral spread, MV loads detected here were compared with the extent of MV M gene and protein mutation reported previously . In caseBy Spearman's ranked correlation, no statistically significant links were found between viral load, duration of disease or infection and number of total or expressed mutations in the M gene of the isolated viruses.6 copies of MV N RNA could be detected per ng total RNA, approximately 100 times more than in the brain samples of UK98. The brain of UK111 was the only analysed tissue positive by immunohistology and was a much stronger positive by immunoblotting than specimens from UK98. In the brain of UK585, which was negative by immunoblotting, only 4 × 102 to 2 × 103 copies could be detected. Thymus and appendix of UK98 were the only non-CNS samples positive for the N gene by real time PCR. GAPDH normalisation of these samples may be less reliable due to greater heterogeneity of tissue origin. However, taking into account exclusively the copy numbers per total RNA, they were weak positives, contained fewer than 102 copies per ng RNA.Great variation in levels of MV RNA and protein were detected in different SSPE patients, while findings with quantitative PCR and immunological methods are in agreement. In the occipital brain of UK111, 2 × 10A weak correlation between the amount of MV RNA and the duration of SSPE was observed. As some CNS samples contained relatively little MV RNA, high viral load alone did not seem to be responsible for death, but may have accelerated destruction of tissue. MV specific interferon-gamma release by host immune cells may determine disease progression . It is fAs the MV genome is encoded in RNA, PCR detection does not distinguish between genomic and mRNA. It is therefore not known if the identified virus is actively transcribed, but presence of MV protein indicates that the virus is transcribed. However, it is not clear whether a larger number of MV RNA copies indicate a greater number of infected cells or greater number of MV RNA copies per infected cell. Increased MV RNA levels per cell may indicate a more actively replicating virus, which may causes faster progressing disease. Alternatively, infection of a greater number of cells may lead to destruction of more tissue, which may also lead to accelerated disease.Of patient UK98, tissues from five brain areas, frontal, parietal, occipital, temporal lobes and cerebellum, were available for analysis. Virus levels in the cerebrum – frontal, parietal, occipital and temporal lobes – were within a factor of 3.5, which may indicate that the virus did not favour any one particular area. Alternatively this may be an indication of very slow disease progression, as is the case in this patient, during which the virus had sufficient time to spread into all regions. In contrast, infection of the cerebellum was 30 to 100 times lower. The cerebellum is a very extensively connected part of the brain, which integrates information from the cerebrum as well as the periphery. Both efferent and afferent pathways link it to the cerebrum. Lack of axonal connection does therefore not cause this lower level of infection. It may rather be the different cell types present within this organ. The cerebellar neurons, the granule cells, may be less susceptible to MV infection.The detection of MV RNA in non-CNS tissues was very low, which indicated that these tissues were not reservoirs of infection. The detected RNA may have derived from infiltrating lymphocytes that were infected within the CNS. Peripheral lymphocytes of individuals with SSPE have been shown to contain MV . AlternaIn summary, the amount of MV protein and RNA differs greatly in different cases of SSPE, which may influence progression of the disease. Further studies of greater numbers of cases are required to confirm these findings.® SYBR® Green qPCR SuperMix UDG , 0.2 μM of each primer and 6.5 μl nuclease free water, using a Rotor-Gene 3000  with the following cycling conditions. Hold at 50°C for 2 min, hold at 95°C for 3 min, followed by 45 cycles of 95°C for 15 sec, 55°C for 30 sec, 72°C for 30 sec. Data was acquired at 72°C during each cycle with excitation wavelength of 470 nm and detection at 585 nm. Amplification was followed by melt analysis from 50–90°C. All samples were run as duplicates and analysed using Rotor-Gene 6.0.7 software. Integrity of all samples was demonstrated by amplification of GAPDH mRNA. DNA and RNA standards were included in each run and each reaction was performed in duplicate. For verification of correct amplification, PCR products were run on agarose gel and sequenced.Tissues were homogenised and total RNA extracted with RNeasy Mini kit . RNA was treated with DNase to avoid amplification of genomic DNA. The concentration of extracted RNA was determined by spectrophotometry and 100 ng RNA reverse transcribed using random primers. PCR primers were designed using Primer3 software  to yieldDNA and RNA standards were used for qPCR and RT-qPCR, respectively. The concentration of a plasmid containing the entire MV genome, p(+)MV , was determined by spectrophotometry. A ten-fold dilution series was made and used as DNA standard. From this plasmid, the whole N, M and H genes were amplified by PCR and cloned into pGEM . The pGEM plasmid was linearised with SpeI and the MV fragments transcribed in vitro using the T7 Riboprobe System  according to the manufacturer's instructions. The resulting RNA was purified and the concentration determined by spectrophotometry. Ten-fold dilution series were made and included in the RT reactions as RNA standards.In order to compare results from different tissues and correct for use of different numbers of cells in each sample, two normalisation methods were employed. First, the same concentration of total isolated RNA was used for each RT-PCR. This lead to results expressed as copies of MV RNA per ng total RNA input. Alternatively, a housekeeping gene was employed to compare RNA levels between different samples. To find an appropriate gene to normalise sample input, 5 ng RNA from nine samples were used for qPCR with primers against all three housekeeping genes, beta-actin, GAPDH and 18S rRNA. Results are shown in Table 2O2 in methanol. Tissues were washed in water and blocked in blocking solution ) for 10 min. Sections were incubated with a monoclonal mouse antibodies against the N protein  at a diTissues were homogenised in lysis buffer  and protein concentrations of whole lysates, without removal of cell debris, determined by spectrophotometry. Equal amounts of total protein of each sample were analysed by SDS-PAGE and immunoblotting. The 7C11 MV N specific antibody was used to probe the blots, followed by detection with an anti-mouse-HRP antibody  and ECL plus reagents .MKS conceived the study, carried out the laboratory work and drafted the manuscript. JL and DWGB participated in the design of the study and the revision of this manuscript. All authors read and approved the final manuscript.


Suffix has also been identified on the 3′ end of the Drosophila non-LTR F element, where it forms the last conserved domain of the reverse transcriptase (RT). In our current study, we show that the separate copies of suffix are far more actively transcribed than their counterparts on the F element. Transcripts from both strands of suffix are present in RNA preparations during all stages of Drosophila development, providing the potential for the formation of double-stranded RNA and the initiation of RNA interference (RNAi). Using in situ RNA hybridization analysis, we have detected the expression of both sense and antisense suffix transcripts in germinal cells. These sense and antisense transcripts are colocalized in the primary spermatocytes and in the cytoplasm of the nurse cells, suggesting that they form double-stranded RNA. We performed further analyses of suffix-specific small RNAs using northern blotting and SI nuclease protection assays. Among the total RNA preparations isolated from embryos, larvae, pupae and flies, suffix-specific small interfering RNAs (siRNAs) were detected only in pupae. In wild type ovaries, both the siRNAs and longer suffix-specific Piwi-interacting RNAs (piRNAs) were observed, whereas in ovaries of the Dicer-2 mutant, only piRNAs were detected. We further found by 3′ RACE that in pupae and ovaries, F element transcripts lacking the suffix sequence are also present. Our data provide direct evidence that suffix-specific RNAi leads to the silencing of the relative LINE (long interspersed element), F element, and suggests that SINE-specific RNA interference could potentially downregulate a set of genes possessing SINE stretches in their 5′ or 3′ non-coding regions. These data also suggest that double stranded RNAs possessing suffix are processed by both RNAi and an additional silencing mechanism.Separate conserved copies of  Retroelements are ancient components of the genome, and are potential participants in some RNA-related regulatory mechanisms in the cell. The recent discovery of RNAi has extended our knowledge of such processes by uncovering mechanisms in which short RNA molecules are used by protein complexes for the recognition of specific nucleotide sequences that are important for the regulation of gene expression and also the formation of chromosomal structures in vivo transcription of a SINE, and that some control signals are required from the insertion site Retroposition is an ancient genetic mechanism underlying the flow of information from RNA to DNA, resulting in the appearance of new copies of a corresponding sequence in the genome. Several classes of retroelements have now been detected during the last few decades: non-LTR mobile elements (or LINEs), LTR-elements that are closely related to retroviruses, and short retroelements (or SINEs). SINEs are too small to harbor a coding function, and for their transposition they use reverse transcriptases encoded by LINEs. Until now, the major portion of the SINEs described in different genomes are derived from either small structured RNA molecules of tRNAs or from 7SL RNA, which forms part of the ribosomal complex Suffix is an unusual example of a short retroelement. Although it has a poly(A) stretch and a size that is typical of a SINE, it lacks the usual RNA polymerase III promoter and possesses a short open reading frame. Previously, suffix was found as a separate repetitive element with different sequences around (separate copies), as well as on the extreme 3′ ends of some genes and also on the 3′ ends of F and Doc elements suffix and F elements led to the first demonstration that SINEs and LINEs share a common 3′ sequence, possessing a small region of coding sequence, a poly(A) signal and a poly(A) site Drosophila germline there are repeat-associated small interfering RNAs (rasiRNAs) that protect against retroelements by a novel RNA silencing mechanism It has recently been reported that in the suffix transcripts are present during all stages of Drosophila development and are co-localized in the germline. However, suffix-specific siRNAs, the putative RNAi products, are detectable only in pupae. It is of interest that in the wild type ovaries, two classes of small suffix-specific small RNAs are present, siRNAs and piRNAs, as this suggests that an additional silencing mechanism targeting the suffix-containing transcripts is involved in the germ line. F element transcripts lacking the suffix stretch can also be detected in pupae. These data indicate that the suffix element is involved in developmentally regulated RNA-interference, which leads to silencing of the F element. Our current data also suggest a hypothetical novel mechanism, whereby the concerted silencing of genes occurs by RNAi targeting of a SINE sequence in the non-coding regions of mRNA sequences.In our present study, we show that sense and antisense suffix elements found in genes are present in a reversed polarity, and that the poly(T)-containing strand (minus strand) forms the last, very short intron and exon suffix sequences within genes, from F elements and also from transcripts of separate individual suffix copies. In our preliminary experiments, we found that under such conditions, signals from divergent suffix copies and Doc-like versions of this element could not be detected. 32P]-RNA probes. For the preparation of these probes, we used suffix sequences and the upstream sequence of F elements subcloned into pGEM-vectors. The suffix-specific antisense [32P]-RNA probe would be expected to detect sense, poly(A)-containing transcripts generated from separate copies of suffix and the F element. We observed a major component of suffix-containing RNA bands (approximately 3500 nt) in both poly(A)+ and poly(A)− RNA samples during all developmental stages .Previously, we demonstrated that l stages Figure 2Alu sequences are also detectable in both poly(A)+ and poly(A)− RNA preparations Drosophila line under study (Oregon-Shostak), and not in several other lines that we tested throughout our analyses, did we observe a short poly(A)+ transcript of about 300 nt in length, which is the size expected for a suffix full-length transcript. Moreover, this transcript was detected only in pupae. There are weaker transcripts of different lengths containing the suffix sense strand in poly(A)+ RNA preparations in embryos, larvae, pupae and flies. Our data on the nature of suffix-containing transcripts obtained with RLM-RACE (Ambion), will be described in a separate report.It has been reported that 32P]-sense-RNA probe reveals the presence of suffix antisense transcripts that would be predicted to come mainly from copies of these elements within genes. As expected, these transcripts are more prominent in poly(A)+ samples and their pattern and intensity changes during development. Some bands may also correspond to antisense transcripts generated from F element copies.A [32P]-RNA probes after stripping of the previous probe. It has been believed for some time that the F element is transcribed for a short duration, and then only in embryos + bands were revealed by the use of an F element-specific antisense probe in embryos, larvae, pupae and flies + F element transcripts, but the corresponding bands are not visible against a background of much more highly abundant RNA molecules generated by the independent suffix copies -containing sense transcripts corresponding to F elements were also observed on a suffix-probed blot but only after a longer exposure (data not shown). Another difference is that, in the case of F elements, both sense and antisense transcripts are mainly polyadenylated. This again demonstrates that the essential portion of the suffix-specific transcripts is not generated from the F element.suffix sequences in embryos and flies, which probably come from regions of heterochromatin where the majority of these copies are found suffix transcripts are present during all stages of development, symmetrical polyadenylated transcripts from F elements are present mainly in embryos and flies. In the current Drosophila melanogaster databases, there are only 8 suffix-containing ESTs that correspond to F elements. Hence, our present data on transcription patterns of suffix clearly indicate that the databases are still poor in suffix-containing transcripts.There are known to be very long polyadenylated transcripts possessing F element and suffix and F element, we tested whether these transcripts are expressed in the same cells. We selected testes and ovaries for in situ hybridization analysis using the same RNA probes that were used in our Northern blots, but this time labeled with DIG , three unique cDNA clones binding only with an F element-specific probe were isolated by colony hybridization (suffix in F element mRNA (suffix sequence were obtained (not shown). We also used 3′ RACE to test whether in Drosophila ovaries, where both siRNAs and piRNAs are detectable, there are F element transcripts lacking the suffix region. We isolated two clones (cDNA-ov-15 and cDNA-ov-55) containing the same cut site at the 5′ end of the suffix region in the F element mRNA − RNA from ovaries of 414/ago2414ago2 mutant suffix sequences were obtained  and in the pOT2 (CG363) genes suffix regions possess a functional polyadenylation signal and site. It follows therefore that suffix-specific siRNAs could potentially target the corresponding transcripts and give rise to silencing of these genes in some cell types. It is possible also that other genes containing this element in their 5′ or 3′ non-coding sequences are regulated in the same way.In previous studies, suffix strands and the degradation of its transcripts by RNA silencing mechanisms are developmentally regulated. The former gives different patterns of element transcription in development, while the latter leads to siRNA formation, at least during the pupal stages of development, although suffix sense and antisense RNAs are even more abundant in embryos and during the imago stages. It is conceivable that RNAi mechanisms involving these sequences are downregulated in particular organs and tissues during development by as yet unknown factors. In this case, the formation of dsRNA alone is not sufficient to trigger RNAi. Recently, it was shown in Drosophila that Dicer-2 is not required for the formation of roo rasiRNA C. elegans enhances silencing, suggesting that some proteins are limiting for RNAi suffix-specific siRNAs were found to be between 21–25 nt, whereas LTR retroelements, such as roo, mdg1, and gypsy, non-LTR I element and Het-A rasiRNAs are about 24–29 nt long Su(Ste) rasiRNAs has revealed little or no periodicity in processing of its long dsRNA triggers suffix-specific RNA silencing mechanisms, as we have observed three excision sites in F element transcripts separated by distances of 4, 6, 9 and 13 nt  and by production of 26–31 nt piRNAs (Piwi-pathway). We thus speculate that the short suffix dsRNAs formed by sense and antisense transcripts of the element itself, and their hybrids with longer RNAs, can be processed in the germline in different ways. This difference between suffix RNA silencing and the silencing of other retroelements might be determined by the unusual behavior of suffix. Although suffix is mostly located in heterochromatic regions as a separate element or as a part of F element, it is also detectable in the opposite polarity at the very 3′ end of some genes suffix has also been found in the CAACA-microsatellite or amongst regions of different retroelements Drosophila to protect this organism from the expression of different retroelements.Only one class of small RNAs, 24–29 nt long rasiRNAs or piRNAs, corresponding to a number of suffix and the F element in the cytoplasm of the nurse cells in ovaries. The only evident difference between both elements is that the F element is also transcribed in the follicle cells located around the oocyte. Analysis of the expression of these elements in different RNA silencing mutants is now underway in our laboratory.We observed the coordinated expression of suffix and F element transcripts can be present in the same cells of the germline, it follows that mechanisms exist that control transcription from both strands of both elements in the same cells. Recently, it was reported that non-LTR retroelements can avoid accidental integration by interacting with a target-specific transcription factor to direct its integration Suffix possesses the 3′ end region required for the recognition of RT specified by the F element. Suffix also uses enzymes provided by F elements for its integration and its short transcripts likely integrate at the same sites as F element transcripts. In this case, the same pattern of distribution of transcripts would be expected for both elements. The integration of suffix copies in both orientations could give rise to sense and antisense transcripts in the same cells. Our current data on the distribution of suffix and F element sense and antisense transcripts in the germline support this contention.Because we observed that sense and antisense suffix-specific siRNAs and piRNAs is that during the pupal stages of Drosophila development or in the germline, abnormal suffix-containing transcripts are abolished, and mechanisms that protect against retroelement-containing RNAs are then switched on. Another potentially more interesting possibility, however, is that the dispersion of SINEs within transcripts provides a mechanism for concerted gene-silencing. Based on this speculation, SINEs could be considered not as selfish components of the genome, but as elements of biological significance that have important functions at the RNA level as components of RNAi. In this way, the degradation of the regions important for expression of a specific set of genes possessing particular SINE elements in 5′ or 3′ non-coding regions could be achieved .suffix copies could originate from the 3′ end of the F element. Recently, we identified a weak internal promoter spanning the junction between the F element and suffix that is active in cultured cells . Thus, the F element could be a source gene for suffix and provide it with 3′end sequence recognized by RT and other enzymes associated with retroposition. In return, suffix may downregulate F elements by RNA silencing mechanisms, which probably allow this LINE/SINE family to replicate without killing the host.Separate + or poly(A)− RNA samples isolated from the Oregon-Shostak line were electrophoresed in 2 mm thick, 1.2% agarose gels containing 25 mM NaPO4 (pH 7), 0.5 mM EDTA and 5% formaldehyde and blotted in 20×SSC onto Hybond-N+. Hybridization was performed in a solution containing 50% formamide, 5×SSC, ficoll, polyvinylpyrolidone, BSA, and SDS all at a concentration of 0.1%, denatured salmon DNA (50 µg/ml), tRNA (50 µg/ml) and 5-10×106 cpm of the appropriate probe. 109 cpm/µg of the corresponding RNA probes were synthesized with T7 RNA polymerase in vitro using adjacent fragments of F element-containing clones as shown in 2, 2 mM spermidine, 10 mM NaCl, 10 mM DTT, 1 u/µl RNasin, ATP, GTP, CTP (500 mM each), 20–40 µCi [α-32P] UTP  and 20 u of T7 RNA polymerase (Fermentas).Approximately 20 µg of poly(A)The RNA probes were added to the hybridization mixture following 24 h of pre-hybridization at 43°C. After further hybridization for 48 h at 43°C with the relevant probes, the membrane was washed twice (20 min each) in a 2×SSC, 0.1% SDS solution at 43°C, then 3 times at 65°C in the same solution and finally twice again at 65°C in 0.2×SSC, 0.1% SDS. 6000-200 nucleotide (nt) RNA markers were purchased from “Pequlab”, Erlangen, Germany.6 cpm of either sense or antisense suffix probes, which were added after 24 h of pre-hybridization. After hybridization for 48 h, the membrane was washed twice in 2×SSC-0.5% SDS at 50°C for 25 min and once in 0.5×SSC-0.5% SDS at 50°C for 15 min. [32P]-labeled run-off 25 and 14 nt long transcripts, synthesized by T7 RNA polymerase as described above using pGEM-1 DNA digested with SmaI or EcoRI, were used as the size markers. For partial degradation of the 25 nt RNA, treatment with 80 mM NaHCO3, 160 mM Na2CO3 at 60°C for 40 min was performed. The RNA content in each lane was tested by hybridization with a probe prepared by extension of the primer 5′ CAGCATGGACTGCGATATGCGTTC 3′ by AMV RT on 5.8S ribosomal RNA.Approximately 20 µg of a total RNA preparation was subjected to elecrophoresis on a 15% denaturing, 1 mm thick, polyacrylamide gel. The gel was then washed twice in distilled water for 5 min each (removing the urea), and then stained twice in 0.1 M ammonium acetate-ethidium bromide for 15 min each and photographed. The gel was placed in a 0.5×TBE solution and electroblotted in this solution onto Hybond N+ for 30 min (300 v/10 cm). Hybridization was performed in 25% formamide, 0.5 M NaCl, 0.1 M sodium-phosphate buffer, 25 mM EDTA; ficoll, polyvinylpyrolidone, and BSA (0.1% each), 0.1% SDS, denatured salmon DNA (150 µg/ml), tRNA (150 µg/ml) and 5-10×1032P]-labeled antisense RNA, corresponding to the 5′ region of suffix, was synthesized in 20 µl of a solution containing 1µg of DNA template, 40 mM Tris-HCl (pH 7.5), 6 mM MgCl2, 2 mM spermidine, 10 mM NaCl, 10 mM DTT, 1 u/µl RNasin, ATP, GTP, CTP (500 mM each), 1.25 µM [α-32P] UTP , 10 µM unlabelled UTP and 20 u of T7 RNA polymerase (Fermentas). The RNA was gel purified to remove shorter fragments. About 5 µg of the total Drosophila RNA preparation was hybridized at 50°C for 12 h in 20 µl of solution containing 0.7 M NaCl, 0.1 M Tris-HCl (pH 7.4), 0.1% SDS and 105 cpm of the RNA probe. After hybridization, 2 µl aliquots were mixed with 10 µl of solution containing 50 mM sodium acetate (pH 4.5), 0.28 M NaCl, 4.5 mM ZnSO4 and 0.1–0.8 u/µl SI nuclease (Promega). SI digestion was performed for 30 min at 20°C, followed by the addition of a 10 µl solution containing 90% formamide, 20 mM EDTA and dyes. The probes were separated in 12% denaturing, 0.2 mm, polyacrylamide gels.86 nt long ATP , 0.5 mM ATP and poly(A) polymerase (USB). After ethanol precipitation, the sample was used for reverse transcription with an oligo(dT)20 primer. This was followed by a 100 cycle PCR amplification in a solution containing 10 mM Tris-HCl (pH 8.3); 50 mM KCl; 2 mM MgCl2; 0.01% gelatin w/v; 1 mM dNTPs; 1 µg of oligo(dT)20 primer; 1 µg of a specific primer with an artificial EcoRI site; 1 u of Taq polymerase and 1 u of Tth polymerase. Amplification conditions were 90°C melting, 37°C annealing and 72°C for extension, for 1 min each. The specific primer, 5′ GAGCACAATCAAAGATTCTGAGAACCATCA 3′, corresponds to the region located about 120 bp upstream of suffix in the F element. The cloning was performed using an EcoRI-SmaI digested pUC12 vector. For colony hybridization, an F element specific oligonucleotide, corresponding to the region located about 80 bp upstream of suffix, and suffix-specific probes were used. The clones hybridizing only with the F element probe were selected and sequenced.For the cloning of 3′ ends of F element transcripts lacking