Finally, elongation factors known to regulate messengers RNA production by RNA polymerase II are also involved in rRNA production and work cooperatively with Rpa49 in vivo.Ribosomal RNA (rRNA) production represents the most active transcription in the cell. Synthesis of the large rRNA precursors (35–47S) can be achieved by up to 150 RNA polymerase I (Pol I) enzymes simultaneously transcribing each rRNA gene. In this paper, we present recent advances made in understanding the regulatory mechanisms that control elongation. Built-in Pol I elongation factors, such as Rpa34/Rpa49 in budding yeast and PAF53/CAST in humans, are instrumental to the extremely high rate of rRNA production per gene. rRNA elongation mechanisms are intrinsically linked to chromatin structure and to the higher-order organization of the rRNA genes (rDNA). Factors such as Hmo1 in yeast and UBF1 in humans are key players in rDNA chromatin structure  The most importance is placed on RNA polymerase II (Pol II), which is responsible for synthesizing mRNA and a large variety of noncoding RNAs. The vast majority of RNA production in growing cells is carried out by RNA polymerase I (Pol I), which transcribes the precursor of large rRNA, and by RNA polymerase III (Pol III), which transcribes 5S rRNA, tRNA, and some noncoding RNAs. Observation of cryofixed cryosubstituted other sections analyzed by electron microscopy reveals that exponentially growing budding yeast cells contain up to 10 per μm3   associates with the Pol I promoter, and this binding is stabilized via TBP by an upstream-associated factor, or UAF –27. CF at rpa49Δ . TherefoSurprisingly, both human and yeast Pol I enzymes are unable to initiate productive RNA synthesis with only promoter-bound factors , 30. Onlin vivo. The target of rapamycin complex 1 (TORC1) regulates ribosome production in response to nutriment availability [in vitro system, this fusion, called CARA, led to a constitutively active Pol I even during stress, showing that Pol I complexed with Rrn3 is initiation competent even under conditions known to inhibit ribosome production [in vivo. However, other findings are now challenging this initial regulatory model based on the availability of an Rrn3-Pol I complex. Rrn3 function is not restricted to initiation only, and it is also involved in a postinitiation step of the Pol I transcription cycle. Rrn3 is released from Pol I during postinitiation, and this process requires Rpa49, another Pol I-specific subunit [in vitro in a partially purified extract. Nevertheless, in this background, rRNA synthesis was downregulated in vivo upon nutrient depletion [in vivo [bona fide Pol I transcription factors, also bind most RPG promoters [Is Pol I initiation the only regulated step in rRNA production in vivo? Although the association of Rrn3 with Pol I is a very important regulatory step, it is only one of the numerous pathways that regulate Pol I activity. In this paper, we will extensively describe how the rRNA elongation step might be regulated to integrate all the complex processes necessary to achieve this early step of ribosome assembly.The amount of Pol I-Rrn3p complexes represents a limiting step in transcription initiation, but how this association is achieved and regulated remains a major research topic . Numeroulability . Upon inlability , 38, 39. subunit . In the  subunit . Therefo subunit , which s subunit , 41. A m subunit . Recent  subunit , 44. A n[in vivo . Indeed,[in vivo , 47. Rrn[in vivo . Along tromoters . Stochioromoters . Is Pol RNA synthesis in the nucleus is invariably coupled with the recruitment of specific proteins shortly after synthesis and leads to the formation of large ribonucleoproteins (RNPs). Strikingly, the fate of the RNA depends on the RNA polymerase synthesizing the transcript. Through their association with the transcribing polymerase appropriate RNA-interacting proteins are driven into the local proximity of the newly synthesized RNA. The COOH-terminal domain (CTD) of the largest subunit of Pol II is the best example of this mechanism . The CTDnucleolus organizer regions) occurs independently of transcription but depends on the protein UBF [E. coli [Dyctiostelium [in vivo labeling approaches [Pol I transcription provided the first example of cotranscriptional RNP particle assembly . Paradoxtein UBF . Miller [E. coli , Dyctiosostelium , and budostelium . This coproaches . Followiproaches  that utiproaches .When driven by a strong Pol II promoter, ribosomal DNA can be transcribed by Pol II . Functiorpa135-D784G) in the second largest subunit of Pol I, near the active center of the enzyme, rRNA processing was affected with the greatest defect occurring in the 60S assembly pathway [Biochemical purification of Pol I activity copurified a large fraction of the rRNA maturation machinery . Early m pathway . This suWhat are the consequences of this clustering? By opening the DNA duplex, DNA in front of RNA polymerase becomes overwound, or positively supercoiled; the DNA behind the polymerase becomes underwound, or negatively supercoiled [rpa49Δ mutant should lead to massive rDNA supercoiling. Indeed, Rpa49 deletion has a tight genetic relationship with the two type I yeast topoisomerases: Top1 and Top3 [in vivo and is genetically linked to Rpa49 [The structural analysis comparing the three nuclear RNA polymerases revealed one feature that distinguished Pol I from the other two (Pol II and Pol III): the presence of the heterodimer Rpa49/Rpa34 . Rpa49 aercoiled . Importaand Top3 , 74. Topto Rpa49 . Top1 isto Rpa49  and seemto Rpa49 .  Is topological stress a selective feature of Pol I transcription? A good answer to this question came from the study using actinomycin D, a DNA-intercalating agent widely used in vivo as a Pol I inhibitor in metazoan cells. The transcription of rRNA genes in mammalian cells is about 50–100-fold more sensitive to actinomycin D than the synthesis of small RNAs and heterogeneous nuclear RNA [in vitro or in a transfected reporter system [in vivo; Pol I elongation, however, is strongly inhibited [in vivo. The extremely high transcription rate of Pol I should lead to extensive torsional stress on the rDNA template.lear RNA . Howeverr system . In factnhibited . Such sunhibited . The spetop1 deletion mutant led to the formation of R-loops (DNA-RNA hybrids) within transcribed rRNA genes [rpa135-D784G and rpa49Δ . Ad spreads . Top2 iscription  but in cby Pol I . The difby Pol I , 86. In by Pol I , have beIn eukaryotes, rRNA genes are either organized in a closed chromatin state in which they are transcriptionally inactive in transcription or are in an open chromatin state . In mammActive rDNA with an open chromatin structure can be distinguished from inactive rRNA genes by psoralen crosslinking. Psoralen is an intercalating reagent which crosslinks to DNA under UV irradiation. The nucleosomes associated with inactive DNA lock the DNA topology and prevent psoralens from crosslinking to DNA. When rDNA is subjected to psoralen treatment and crosslinking, two types of bands are detected: a slow- and a fast-migrating band , 88. TheDuring the cell cycle, the ratio of open to closed rDNA changes. Newly replicated rDNA becomes psoralen inaccessible and shows nucleosome assembly on both strands after the passage of the replication forks . FollowiIn sharp contrast to ChEC data, chromatin immunoprecipitation (ChIP) analyses targeting H3 and H2B histones in mutant strains harboring most rRNA genes in an open chromatin state (42 or 25 rDNA copies) strongly suggested that significant quantities of histone molecules are present on active rRNA genes   [spt5-C292R) suppresses the growth defect of the rpa49Δ Pol I mutant [spt4 deletion mutant led to decreased rDNA copy numbers, and an rRNA processing defect [in vivo. In addition, the FACT complex stimulates Pol I activity in human cells [rpa49Δ [Spt5 copurifies with Pol I and has been shown to be associated with rDNA in vivo . Spt5 isa (RpoE) , 102. Mua (RpoE) . Spt5 inI mutant . PhenotyI mutant . Other fg defect . The Pafg defect  and is ig defect . Spt6 isan cells . FACT coan cells , and is  [rpa49Δ . Another [rpa49Δ . The growing list of Pol II elongation factors that also regulate Pol I activity is interesting, but some mechanistic insights are still lacking. The elongation mechanisms of Pol II and Pol I are very different. Pol II carries regulators via interactions with CTD . With febona fide Pol I elongation factor, the nucleolin, called Nsr1 in budding yeast and Gar2 in fission yeast. Following nucleolin's first identification as an abundant nucleolar protein, it has been implicated in numerous cellular processes [One factor can now be defined as a rocesses , 113. Amrocesses  and for rocesses . Nucleolrocesses . NucleolIn this paper, we tried to focus on the unanswered questions of rRNA production, rather than make an exhaustive review of the large body of work addressing regulation of this complex multistep process. We still know little about how cells adjust the production of each ribosomal constituent in time and space to allow cotranscriptional assembly of preribosomal particles. The answer to this question probably lies in the existence of highly redundant pathways, which are all designed to coregulate rRNA production by Pol I, ribosomal protein production, and the availability of the ~200 transacting factors required to assemble eukaryotic ribosomes. The discovery of redundant pathways has clearly resulted from the extensive study of budding yeast. Most regulatory pathways affecting rRNA production are not essential for cell growth. Out of 14 Pol I subunits, four are not required for cell growth. However, when double inactivation is performed, their functions can be revealed and studied . We have


Caenorhabditis elegans. We found that expressing the orthologous E466K mutation (unc-18 E465K) enhanced alcohol sensitivity. This enhancement in sensitivity was surprisingly independent of rab-3. In contrast unc-18 R39C, which decreases syntaxin binding, enhanced sensitivity to alcohol in a manner requiring rab-3. Finally, overexpression of R39C could suppress partially the reduction in neurotransmitter release in rab-3 mutant worms, whereas wild-type or E465K mutants showed no rescue. These data indicate that the epistatic interactions between unc-18 and rab-3 in modulating sensitivity to alcohol are distinct from interactions affecting neurotransmitter release.The molecular mechanisms underlying sensitivity to alcohol are incompletely understood. Recent research has highlighted the involvement of two presynaptic proteins, Munc18 and Rab3. We have previously characterised biochemically a number of specific Munc18 point mutations including an E466K mutation that augments a direct Rab3 interaction. Here the phenotypes of this and other Munc18 mutations were assessed in alcohol sensitivity and exocytosis using  Drug addiction is one of the leading causes of preventable death, generating a considerable financial burden to society. Indeed alcohol use and abuse can lead to increased incidence of liver disease, cardiovascular disease, cancer and other debilitating illnesses . AlthougCaenorhabditis elegans is an excellent platform for the genetic dissection of alcohol sensitivity as it has a similar dose-dependent response to exogenous alcohol to mammals [C. elegans has determined a role in alcohol sensitivity for proteins central to the exocytotic machinery, yet distinct from synaptic transmission efficacy. Loss-of-function (lof) mutations in the GTPase rab-3 reduces sensitivity to alcohol in C. elegans [C. elegans [ Current models of alcohol action within the nervous system predict low-affinity interactions of alcohol with specific target proteins or protein complexes . Genetic mammals . Recent  elegans . Similar elegans . Both mu elegans ,11 emphaunc-18 (e81) alleles display strong behavioural phenotypes including paralysed locomotion and resistance to inhibitors of acetylcholinesterase [lof rab-3 mutants exhibit loopy, mildly slower locomotion and are also resistant to inhibitors of acetylcholinesterase [ Munc18 is an essential protein in presynaptic vesicle exocytosis whose precise function remains somewhat enigmatic ,13. Biocesterase . Rab3 isesterase  and vesiesterase  have alsesterase .gain-of-function (gof) mutation affecting direct binding to Rab3 [unc-18, the nematode orthologue of Munc18-1, in both a wild-type and lof rab-3 genetic background. A mutation that interferes with closed-conformation syntaxin binding (unc-18 R39C) was hypersensitive to alcohol as was the orthologous mutation that enhances the Munc18-Rab3 interaction (unc-18 E465K). In addition overexpression of the R39C mutation partially compensated for lof rab-3 in neurotransmitter release; yet, was recessive to lof rab-3 in alcohol sensitivity. Conversely, the E465K mutation was dominant to lof rab-3 in alcohol sensitivity, but recessive in neurotransmitter release. We conclude that the specific interactions between unc-18 and rab-3 that govern exocytosis are functionally distinct from sensitivity to alcohol. We have previously investigated a number of point mutations of mammalian Munc18 that alter protein interactions , includi to Rab3 . In thisC. elegans unc-18 (D214N) reduces sensitivity to both low and high concentrations of exogenous ethanol [unc-18 in a null (unc-18 e81) background  in Munc18 acts biochemically by reducing binding to the assembled SNARE complex and that the orthologous mutation in  ethanol . Previou ethanol ,24, P2426N in Mun ethanol . To asseckground  and asseon rates . Similarild-type  althoughC. elegans cuticle, the internal ethanol concentrations are estimated to be substantially reduced and approximate that seen in intoxicated humans [unc-18 point mutations had effects on sensitivity to exogenous ethanol at either the stimulatory or depressive concentrations. In contrast to the previously characterised D214N mutation, the R39C and E465K mutations enhanced sensitivity to alcohol at both the stimulatory and the depressive concentrations  causes a depression in locomotion ,25. In ad humans , althougtrations . There wunc-18 R39C/E465K). Although the single mutants each had small inhibitory effects on basal thrashing rate, locomotion of the double mutant was in fact enhanced to a greater level than wild-type  produced no additive effect on alcohol sensitivity  or null (unc-18) genetic background and found similar phenotypic effects either in the presence or absence of endogenous unc-18 [unc-18 (e81) allele, expression of R39C in lof rab-3 (y250) caused a significant decrease in basal locomotor rate in comparison to expression of wild-type unc-18 (rab-3 y250), 25 (Wt), 30 (R39C) and 30 (E465K)). The enhancement in locomotor rate by the R39C/E465K double mutant in comparison to the single R39C mutation was also apparent in the lof rab-3 background , 30 (E465K) and 30 (R39C/E465K)). Rab3 is a GTPase involved in the trafficking of synaptic vesicles and various aspects of exocytosis . Lof rab alcohol . The E46 alcohol  without  alcohol . We thers unc-18 . Similarlof rab-3 worms exhibited a normal stimulation of locomotion  worms were less sensitive than Bristol N2 wild-types  worms were resistant to aldicarb in comparison to Bristol N2 wild-types  that bypasses the requirement for a functional Rab protein during ER to Golgi vesicle trafficking [lof rab-3 genetic background eliminates the rab-3 phenotype. What then are these unc-18 mutations or lof rab-3 doing to alter ethanol sensitivity? Previous work has excluded the interpretation that ethanol sensitivity is a simple reflection of alterations in signalling strength [unc-18 and rab-3 are characterised primarily as exocytotic proteins involved potentially in docking, priming and fusion itself [lof rab-3 or the unc-18 mutations are altering the trafficking of postsynaptic receptors whose function is modulated by ethanol. Indeed, ethanol can affect many neurotransmitter receptors including GABA (γ-aminobutyric acid), glutamate and serotonin [unc-18 E465K mutation acts independently and can circumvent the requirement of functional rab-3 in ethanol sensitivity. The results of these mutations in the fficking . This mustrength ; yet, bon itself ,18. It rerotonin . The preunc-18 and rab-3 that determine ethanol sensitivity stand in direct contrast to those for signalling strength. At the worm neuromuscular junction, the R39C mutation induced resistance to aldicarb implying a reduction in signalling strength. The R39C mutation has been previously shown to increase evoked postsynaptic currents in Drososphila [C. elegans. Contrary to ethanol sensitivity, R39C unc-18 is partially dominant to lof rab-3. Indeed as the R39C mutation is itself resistant to the effects of aldicarb in comparison to wild-type unc-18, it is possible that R39C is completely dominant to lof rab-3 for aldicarb sensitivity. It is most likely that this mutation overcomes the loss of functional rab-3 in exocytosis via changes to vesicle recruitment. Null unc-18 worms have a reduction in docked vesicles [lof rab-3 alleles also reduce both the total number of synaptic vesicles and their trafficking [ The epistatic interactions between sosphila  which masosphila . The totsosphila  which wovesicles  that is fficking . Indeed fficking . The datunc-18 E465K) in the lof rab-3 genetic background could be relatively easy to rationalise. Indeed the rab-3 (y250) allele produces no detectable RAB-3 protein [lof rab-3 genetic background suggestive of an additional functional role of the E465K mutation. At present, no other biochemical effects of the E465K mutation are known [rab-3, which itself is potentially downstream of E465K. The Munc18 E466K mutation acts to increase Rab3 binding and the number of fusion events from bovine adrenal chromaffin cells . Therefo protein . The phere known ,22. NoneC. elegans, as the vast majority of exocytotic proteins have orthologues in nematodes [unc-18 R39C mutation can overcome the secretory defects associated with lof rab-3. In addition, genetic interactions between unc-18 and rab-3 in alcohol sensitivity determined that, for this phenotype, the unc-18 E465K mutation eliminated a requirement of rab-3. Most surprisingly, we demonstrate that the epistatic interactions between mutants of unc-18 and rab-3 are distinct depending on the phenotypic context such that the R39C mutation acts downstream of Rab3 in exocytosis whereas it acts upstream of Rab3 in ethanol sensitivity. Finally, our data emphasises that simple modulation of synaptic strength is unrelated to sensitivity to ethanol and that the functional actions of alcohol are a complex cellular mechanism involving a large spectrum of neuronal proteins.  The phenotypic effects presented here are likely to be consistent with phenotypic effects in mammals. Indeed, the pleiotropic action of alcohol in mammals is conserved for many phenotypes in nematodes . Mutatioematodes . The intematodes ,40,49 anunc-18 rescuing construct were introduced by site-directed mutagenesis using either the GeneTailor (Invitrogen) or QuikChange (Stratagene) methods as described previously [ All point mutations of the eviously ,23.C. elegans strains were grown and maintained on nematode growth medium (NGM) plates at 20°C with Escherichia coli OP 50 as a food source as previously described[unc-18 (e81) and rab-3 (y250). Transgenic worms were generated by germline injection as previously described [unc-18 cDNA, either wild-type or the indicated point mutations, under the control of its own genomic flanking regions. Successful transgenic expression was verified by co-injection with a sur-5::GFP marker (pTG96) . The concentration of injected DNA was made up to 100 ng/µl with empty pBlue Script SK+ vector for all injections. For each transgenic construct, 3-5 individual independently-derived lines were generated and analysed. Results presented here were consistent for all generated lines.described,23. Straescribed ,23. Tran2, 1 mM MgCl2 and 5 mM HEPES; pH 7.4; with bovine serum albumin at 0.1 mg/ml) over a 1 minute period as described previously [ All behavioural assays were performed in a temperature controlled room at 20°C using young adult hermaphrodite animals from sparsely populated plates. Locomotion rate was quantified by measuring thrashing in 200 ul Dent’s solution (140 mM NaCl, 6 mM KCl, 1 mM CaCleviously ,23. A theviously ,10,25. A


Retroviruses exploit nuclear trafficking machinery at several distinct stages in their replication cycles. In this review, we will focus primarily on nucleocytoplasmic trafficking events that occur after the completion of reverse transcription and proviral integration. First, we will discuss nuclear export of unspliced viral RNA transcripts, which serves two essential roles: as the mRNA template for the translation of viral structural proteins and as the genome for encapsidation into virions. These full-length viral RNAs must overcome the cell’s quality control measures to leave the nucleus by co-opting host factors or encoding viral proteins to mediate nuclear export of unspliced viral RNAs. Next, we will summarize the most recent findings on the mechanisms of Gag nuclear trafficking and discuss potential roles for nuclear localization of Gag proteins in retrovirus replication. Retroviruses interact with nuclear trafficking machinery during several different phases of their replication cycles . Retrovitrans  tail, like cellular mRNAs. A fraction of the viral RNA is spliced and exported out of the nucleus to serve as mRNA for translation into viral proteins. The remainder of the viral RNA remains unspliced and must be transported from the nucleus into the cytoplasm where it serves two functions: (i) as the template for translation of the viral structural proteins, Gag and Gag-Pol; and (ii) as the viral genome, which is packaged into virus particles. Because export of unspliced and incompletely spliced cellular RNAs is prevented by host machinery to prevent translation of abnormal proteins (reviewed in ), retroviewed in ). The geThe Gag protein, which directs the assembly and budding of virus particles from the plasma membrane, is localized primarily in the cytoplasm of infected cells. However, the Gag proteins of Rous sarcoma virus (RSV), feline immunodeficiency virus (FIV), mouse mammary tumor virus (MMTV), prototype foamy virus (PFV), murine leukemia virus (MLV), Mason-Pfizer monkey virus (MPMV), HIV-1, and several retrotransposons undergo nuclear localization under certain conditions . The RSVtrans-acting viral proteins that export their intron-containing viral RNAs from the nucleus. HIV-1 Rev was the first member of this family to be discovered; however, Rev-like proteins have been described in the Lentivirus  [Deltaretrovirus [ and bovine leukemia virus (BLV)], and Betaretrovirus  genera  ,91,92,93] genera ,94,95,96] genera ,99,100. cis-acting Rev-responsive element (RRE) in HIV-1 RNAs [HIV-1 Rev recognizes and binds to the highly structured V-1 RNAs ,103,104 V-1 RNAs ). MultimV-1 RNAs . HoweverV-1 RNAs  and smalV-1 RNAs ,109. TogIn the nucleoplasm, Rev co-localizes with the SR-domain splicing factor SC-35 in nuclear speckles ,110, intHIV-1 Rev mediates export of unspliced viral RNAs through an interaction between the NES of Rev and the CRM1/RanGTP export complex. However, many other cellular proteins also interact with Rev, indicating that nuclear export of unspliced HIV-1 RNA depends upon a complex network of interactions ,126,127.trans-acting factors to facilitate nuclear export of unspliced RNA, simple retroviruses have evolved cis-elements to circumvent the blockade to export of unspliced transcripts from the nucleus. MPMV and other type D retroviruses, including simian retrovirus-1 and 2 (SRV-1 and SRV-2), contain a small cis element, the constitutive transport element (CTE), which is required for nuclear export of unspliced viral RNA [In contrast to complex retroviruses that encode iral RNA ,138. Wheiral RNA . Thus, RXenopus oocyte nuclei expressing TAP/NXF1 and an intron containing the CTE resulted in nuclear export of the RNA in the absence of splicing [in vivo [.Insight into the mechanism by which CTE-containing RNAs are exported from the nucleus was provided by proteomic studies that identified the host nuclear export protein Tip-associating protein/Nuclear RNA export factor 1 (TAP/NXF1) as a binding partner of CTE complexes ,140. Micsplicing ,142. Thesplicing ,144,145.splicing . Mutatio[in vivo .cis-acting unspliced RNA transport element was also identified in RSV, which lies within 115 nucleotide direct repeat (DR) sequences flanking the v-src oncogene [src sequence maintain at least a single DR element to remain replication-competent [A putative oncogene . DR elemoncogene , and strompetent ,150. Theompetent ,152,153.et al. used a subviral reporter construct containing either the DR sequence or the ψ sequence to examine whether Gag could enhance translation by promoting nuclear export of unspliced RNA [RSV RNAs containing the DR elements are exported by the cellular mRNA export factor TAP/NXF1 and the RNA helicase Dbp5 ,154,155.Taking the available data into account, we postulate that there is a temporal switch in RSV replication, such that viral transcripts produced early after integration are exported using DR-mediated interactions with Tap/NXF1 and Dbp5 to initiate the synthesis of Gag and GagPol proteins. We hypothesize that as the levels of these viral structural proteins increase, the Gag protein enters the nucleus where it may bind unspliced viral RNA and export it into the cytoplasm for encapsidation into virions ,156. It Spumavirus, share similarities with both simple and complex retroviruses, yet they have several unique characteristics . Uniewed in ,160,161.iewed in . Similariewed in . FVs do iewed in , therefocis-acting RSV and MPMV RNA transport elements [cis packaging signal are present only on unspliced viral RNA [Among retroviruses, PFV utilizes a unique set of cellular factors that bind to its viral RNA for nucleocytoplasmic transport. Rather than using TAP/NXF1, like the elements ,155, splelements . HuR asselements ,166,167.elements , althougelements . Althougelements . It is niral RNA .Historically, the Gag proteins of orthoretroviruses were thought to exist only in the cytoplasm of infected cells. However, it has recently been shown that Gag proteins of diverse retroviruses localize to the nucleus under specific conditions ,24,30,72in vitro evidence demonstrated that binding of Gag to nucleic acids facilitates the association with the CRM1-RanGTP export complex, suggesting that upon binding to nucleic acids in the nucleus, Gag is primed for export [The first retroviral Gag protein discovered to undergo active nucleocytoplasmic trafficking was RSV Gag, which has a well-understood mechanism of nuclear entry and egress. Whereas under steady-state conditions RSV Gag is detected primarily in the cytoplasm and along the plasma membrane, treatment of RSV-infected or Gag-expressing cells with the CRM1-inhibitor leptomycin B (LMB) dramatically concentrates Gag in the nucleus ,170. To r export . OngoingTo dissect the mechanism by which RSV Gag undergoes transient nuclear trafficking, a series of experiments were performed to identify nuclear import and nuclear export signals (NES) in the RSV Gag polyprotein ,26,170. env sequence, and the virus was pseudotyped with the vesicular stomatitis virus G envelope protein, which may have affected the infectivity results [The MA domain does appear to be a critical determinant of RSV Gag function, as replacement of the RSV MA sequence with HIV-1 MA abrogates nuclear trafficking of the RSV/HIV chimera . The RSV results ,171,172.cis-acting ψ-packaging sequence. Experiments to test this hypothesis are underway in our laboratory. Why does RSV Gag contain two NLSs that interact with three different import factors? One possibility is that Gag encodes redundant signals to ensure it enters the nucleus. Alternatively, each NLS might be used selectively: the NLS that mediates entry of the Gag precursor during assembly could use one or more sets of import factors whereas the MA and NC NLSs in the mature proteins could interact with different importins during early infection. In support of this idea, insertion of a canonical NLS into MA  interferes with infectivity . The repAlthough the mechanism of nuclear trafficking is the best understood for RSV Gag, other orthoretroviral Gag proteins also localize to the nucleus or at the nuclear rim . The MLVet al. raised the possibility that FIV Gag may encapsidate its RNA genome in the nucleus, but because the efficiency of export exceeds import, both Gag and the viral RNA genome appear to accumulate at the cytoplasmic leaflet of the nuclear membrane [The lentiviral FIV Gag protein has a substantial degree of nuclear localization under steady-state conditions in feline cells, its natural host  Figure . Nearly membrane . In the same study, HIV-1 genomic RNA also accumulated at the nuclear envelope  Figure . HoweverLike FIV, a link may exist between MPMV Gag accumulation at the nuclear pore complex (NPC) and genomic RNA packaging . The MPMgag initiation codon causes Ty1 RNA to accumulate in the nucleus, where it is degraded, suggesting that Gag stabilizes the RNA. Expression of Gag in trans restores Ty1 RNA nuclear export and RNA stability [mex67-temperature sensitive strain, Gag accumulates at the nuclear envelope in a subset of cells [The yeast long terminal repeat (LTR) retrotransposons Ty1 and Ty3 have some trafficking properties in common with retroviruses. Ty1 and Ty3 encode Gag proteins that bind unspliced genomic RNA to form virus-like particles. Mex67p, the yeast ortholog of TAP/NXF1, is required for the export of Ty1 RNA from the nucleus . In the tability   are important to pursue. Is Gag nuclear trafficking involved in genomic RNA packaging? To date, studies of RSV, MPMV, and Ty1 support this idea. PFV Gag associates with chromatin and plays a role in integration, although it might also function in genome encapsidation of nuclear transcripts ,83. In sFinding that several retroviral Gag proteins undergo nucleolar localization raises intriguing questions about whether there is a unifying function of the nucleolus in virus replication. One possibility is that retroviral Gag proteins interact with a host protein or RNA in the nucleolus involved in genome packaging or virus assembly. It is intriguing that several RNA pol III transcripts are enriched in retroviral particles, including RNAs that are processed in the nucleolus such as tRNA, U6 small nuclear RNA, 7SL ,209, and


Towards this end, we have developed a novel approach that combines a cell-based selection method . Several rounds of positive (VSMCs) and negative  selection were performed to enrich for aptamer sequences that preferentially internalize into VSMCs. To identify candidate RNA aptamer sequences, HTS data from each round of selection were analyzed using bioinformatics methods: (1) metrics of selection enrichment; and (2) pairwise comparisons of sequence and structural similarity, termed edit and tree distance, respectively. Correlation analyses of experimentally validated aptamers or rounds revealed that the best cell-specific, internalizing aptamers are enriched as a result of the negative selection step performed against ECs.We describe a novel approach that combines cell-internalization SELEX with HTS and bioinformatics analysis to identify cell-specific, cell-internalizing RNA aptamers. Our data highlight the importance of performing a pre-clear step against a non-target cell in order to select for cell-specific aptamers. We expect the extended use of this approach to enable the identification of aptamers to a multitude of different cell types, thereby facilitating the broad development of targeted cell therapies. Some properties of aptamers that make them promising therapeutic reagents include the intermediate size of aptamers, ease of development, and absence of synthesis constraints associated with small molecule inhibitors and protein-based drugs . Aptamers routinely achieve binding affinities and specificities comparable to therapeutic antibodies. In addition, due to their amenability to modification by medicinal chemistry, aptamers avoid the immunogenicity concerns of protein-based drugs and can be engineered to have optimized pharmacokinetic (PK) and pharmacodynamic (PD) profiles for via a process termed SELEX  15 sequences) is incubated with a protein target. The protein-bound aptamers are then specifically recovered and amplified by PCR. Single stranded DNA or RNA sequences are then generated from the amplified product and used in a subsequent round of selection. Since the invention of the SELEX process around 1990, many high affinity aptamers that target a wide-range of proteins including transcription factors Isolation of aptamers with affinity and specificity for a target of interest involves iterative rounds of affinity purification and amplification in vitro SELEX approach is that it facilitates the identification of aptamer sequences that recognize the target (e.g. membrane receptor) in its native milieu; that is, in the context of the cellular membrane. Importantly, this approach overcomes the difficulties in obtaining purified preparations of recombinant membrane proteins Although the traditional SELEX method utilizes a soluble, pure form of the target protein , different methods have also been developed that target aptamers to membrane-associated cell surface proteins, termed whole-cell SELEX) Aptamers that bind to extracellular targets have high potential for diagnostic and therapeutic applications. For diagnostics, aptamers are used to differentiate among different cell types, such as normal and tumor cells cell-internalization SELEXin vitro SELEX approach: (1) it favors the isolation of RNAs that bind to receptors in their native state; and (2) it enriches for RNAs that are internalized by the target cell. Using the cell-internalization SELEX approach, we enriched for aptamers that selectively bind to and internalize into HER2+-breast cancer cells for delivering therapeutic siRNAs to the cancer cells While whole cell-SELEX has increased the repertoire of aptamers that bind to membrane receptors in vitro SELEX is that cells are much more complex targets than single recombinant proteins. Two major challenges for cell-based aptamer selections are 1) insufficient methods to monitor the progression of a cell-based selection, and 2) the complexity of aptamer sequences derived from this type of selection. We address these issues by applying a combination of HTS and bioinformatics analysis to the cell-internalization SELEX approach. In this study, we demonstrate the usefulness of this approach by identifying vascular smooth muscle cell (VSMC)-specific internalizing aptamers. We also demonstrate the importance of categorizing aptamers based on structural similarity (structure families), in addition to sequence similarity (sequence families), in order to encourage the identification of functional sequences. In summary, these studies demonstrate the utility of HTS and bioinformatics analysis for facilitating the rapid identification of ‘winner’ sequences from an aptamer selection performed against a complex target. Furthermore, these studies have resulted in several VSMC-specific internalizing RNA sequences that could be used to deliver siRNAs or other small molecule drugs specifically to VSMCs.A potential downside to the cell-based SELEX approaches as compared to traditional cell-internalization SELEX protocolnon-target cell (negative selection) and a target cell (positive selection) . To enable the identification of VSMC-specific sequences, a negative selection step against endothelial cells  was introduced at rounds 4 through 8 of selection  were enriched using the andWe monitored the progression of the selection by measuring the complexity of the RNA pools at each round of selection using a modified DiStRO DNA melt assay We next verified cell-specific internalization of the RNA pools at each round of selection (Table S1). These raw reads were filtered based on the RNA library constant region sequences. After filtering, 2,546,770 total reads were obtained. Of the total filtered reads, 1,425,964 were unique reads (Table S1). After normalizing to total reads (white squares) at each round of selection, the number of unique reads (black squares) was decreased with each subsequent round  (data not shown). Sequence Enrichment (% Enrichment) was then determined by taking the complement of % sequence complexity   We hypothesized that the observed decrease in minimum free energy  in later selection rounds may have resulted from loss of structural diversity. Therefore, structural diversity was assessed by determining the probability (ensemble probability) of the most likely structure for a given sequence (p<0.001) higher in round 8 (black bars) compared to round 0 (gray bars)  deletions or insertions within the variable region of a DNA or RNA SELEX library can occur during the course of a selection Table S1). Importantly, thousands of unique reads from rounds 1 through 8 where found in two or more rounds, with many unique reads found in as many as six rounds  .Next, we reasoned that an additional measure of a true selected sequence would be its number of rounds in which a given sequence is found , representing 1,123,533 total reads, were analyzed for edit distance ( output for edit distance = 1 shown) by the program process.seqs . All of the predicted structures connected with tree distances ranging from 0  to 22 (red node). LocARNA multiple sequence/structure alignments of the predicted RNA structures at the various tree distances were used to establish structure families  was predicted using RNAfold. Each unique read was assumed to have only a single structure, which is supported by the data in fold enrichmentrising’, defined by the increasing trend in read number over progressive rounds of selection, (3) read number, defined by total reads in a given round and (4) rate enrichment, defined by the change in read number over change in round number. These parameters were applied to the following rounds: rounds 1–8, 1–3, 3 through 8 and 6 through 8, based on the selection conditions described in orphan’ . Together, these data highlight the importance of performing a tree distance analysis in addition to categorizing aptamers based on sequence similarity. Importantly, these data also suggest that aptamers that do not fit into either edit distance or tree distance families are likely to be ‘junk’ sequences and should not be included in further analyses.To evaluate aptamer internalization, the 33 individual RNA aptamers identified in Materials S1). We assessed binding/internalization of two independent fluorescently-labeled aptamers (#51 and #420). As observed with the RT-qPCR assay, aptamer #51 is a strong cell-specific internalizer, while aptamer #420 is a weak cell-specific internalizer . Together, these data confirm that the aptamers selectively internalize into VSMCs vs.ECs.Next, we performed a fluorescent-based ‘plate reader’ assay to confirm specific binding and internalization of our selected aptamers into VSMCs (rising (trend of increasing read number across progressive rounds), (3) rate enrichment and (4) read number  of the relationship between two variables as described by the correlation coefficient.We used correlation analysis to determine the dependence of fold internalization (into VSMCs) and the following parameters: (1) fold enrichment ber  and 3. The p<0.05) were obtained when comparing fold internalization to fold enrichment for rounds 1 through 8 and rounds 3 through 8 but not for rounds 1 through 3 and rounds 6 through 8. Similar results were obtained when comparing fold internalization to rate enrichment  was introduced  that include the isolation of RNA aptamers that recognize African trypanosomes Mycobacterium tuberculosisMycobacterium tuberculosis are examples of simple, unicellular organisms, complex in vivo-selections have also been attempted in order to isolate aptamers against intrahepatic carcinomas in mice The approach described herein, is of importance in light of the rise in complex target SELEX, in which aptamers are directly selected against complex protein mixtures, cells, or even whole organisms fold enrichment during the course of the selection fold enrichment. Interestingly, although we also observed a positive correlation between fold internalization and read number, this association occurred only after the introduction of a negative selection step. Together, these studies highlight the importance of performing HTS on all rounds of selection in order to assess fold enrichment of specific sequences over the course of a selection. In addition, our data identify the negative-selection step as a key criterion to successfully enrich for cell-specific sequences Despite the complex nature of published aptamer selections, the resulting ‘winner’ aptamer sequences are typically few in number and, in most cases, only one sequence is identified and further characterized. The likely reason for the “one selection-one aptamer” phenomenon is that only the most highly represented sequences are sequenced using the traditional chain terminator method, i.e., Sanger sequencing read number, to identify aptamers sequences that are subsequently analyzed experimentally edit distance and tree distance analyses to identify sequences that are related based on sequence or structure. These analyses resulted in the identification of 27 sequences (out of 32) that were among the best cell-specific internalizing aptamers . Of particular importance, the aptamer with the highest fold internalization (#51) was not among the aptamers with the highest read number. Aptamer #51 was identified only through the tree distance analysis, highlighting that RNA structure should also be considered when choosing sequences for experimental validations.To date, bioinformatics analyses performed on aptamer selections have relied predominantly on an arbitrary cutoff, typically based on tree distance analysis is that it is based on the assumption that each sequence has only a single predicted structure. While RNA molecules can assume multiple breathing dynamics, our data seem to indicate that structural diversity decreases over the course of a selection. However, the inclusion of all possible RNA structures may improve the outcome of current tree distance analyses, though this type of analysis is currently hindered by the existing algorithms for structure prediction. Indeed, we anticipate these multi-structure analyses are likely to become more feasible as predictive RNA structural algorithms evolve and as computational algorithms become more complex.A potential current limitation of the In conclusion, our studies highlight the utility of combining HTS with bioinformatics analysis for the identification of ‘winner’ sequences from an aptamer selection performed against a complex target. These efforts have yielded (1) predictive tools that are broadly applicable to all aptamer selections for target sequence identification; and (2) several VSMC-specific RNA aptamers that can subsequently be used for targeted delivery. Thus, as the aptamer field enters an exciting new chapter in which HTS and bioinformatics tools become a critical component of aptamer discovery, methodologies such as the one described herein both complement and add to existing approaches for sifting through millions of sequence reads generated from high-throughput sequencing data of aptamer selections.2. The A7r5  cell line was cultured in DMEM  supplemented with 10% heat inactivated FBS  and the YPEN-1  cell line was cultured in MEM  supplemented with 5% heat inactivated FBS , 1.5 g/L Na bicarbonate , 0.1 mM MEM NEAA , 1.0 mM Na pyruvate  and 0.03 mg/mL heparin . All cell lines were screened for contamination by mycoplasma, which is known to degrade 2′-fluoro pyrimidine modified RNA aptamers All cell lines were cultured at 37°C under 5% CO5′-TCGGGCGAGTCGTCTG-N20-CCGCATCGTCCTCCC-3′  was extended using Choice Taq Polymerase  in the presence of Sel2 5′ primer, 5′-TAATACGACTCACTATAGGGAGGACGATGCGG-3′. The extension reaction was performed in a thermocyler by heating the Sel2N20 ssDNA template oligo and the primer at 95°C for 3 min, annealing at 25°C for 10 min and extending at 72°C for 30 min, followed by a 10 min incubation at 25°C. The duplex DNA library was in vitro transcribed for 16 hrs at 37°C using Y639F mutant T7 RNAP The duplex DNA library was generated as follows. The Sel2N20 single stranded DNA (ssDNA) template oligo, We applied a modified cell-based SELEX 5′-TCGGGCGAGTCGTCTG-3′. The RT protocol is as follows: 55°C for 60 min followed by a 15 min incubation at 72°C. The RT product was PCR-amplified using Choice Taq DNA polymerase  in the presence of the Sel2 5′ and Sel2 3′ primers. The dNTP concentration in the PCR reaction was 2.5 mM . The PCR amplification protocol is as follows. 95°C for 2 min, followed by 25 cycles of heating to 95°C for 30 sec, 55°C for 30 sec and 72°C 30 sec. A final extension step was performed for 5 min at 72°C. The DNA duplex library was in vitro transcribed as described above.Following incubation of the RNA library, A7r5 cells were washed 3 times with ice cold DPBS to remove unbound RNA. Cell-surface bound RNA was removed by washing cells once with ice cold 0.5 M NaCl DPBS, incubating cells 5 min at 4°C with ice cold 0.5 M NaCl DPBS and washing once with ice cold DPBS. Internalized RNA was recovered by TRIzol  extraction by following the manufacturer's instructions. The recovered RNA aptamers were reverse transcribed (RT)  using the Sel2 3′ primer: 2) using a DNA melt assay protocol . The raw DNA melt assay data (performed in triplicate) was averaged and plotted as fluorescence intensity (SYBR) vs. temperature (C°). Higher library complexity was indicated by a relative shift of the DNA melt curve towards lower temperatures, whereas, lower library complexity was a shift to higher temperatures.We used a modified DiStRO method 2. Unbound and cell-surface bound RNA was removed by washing with ice-cold 0.5 M NaCl DPBS. Internalized RNA was recovered by TRIzol containing 0.5 pmoles/mL M12–23 aptamer 5′- GGGGGGATCCAGTACTATCGACCTCTGGGTTATG -3′). The RT protocol is as follows. The recovered RNA and the primers were heated at 65°C for 5 min, annealed at 22°C for 5 min and extended at 42°C for 30 min followed by and extension at 48°C for 30 min. In Step two, the product from the RT reaction was PCR amplified with iQ SYBR Green Supermix  using a Eppendorf Mastercycler epgradient S with realplex2. The qPCR protocol is as follows. The RT product was heated at 95°C for 2 min, followed by 50 cycles of heating at 95°C for 30 sec, annealing at 55°C for 30 sec, and extending at 72°C for 30 sec. A melt curve was performed by heating at 60°C–95°C for 20 min. Reactions were all done in 50 µL volume in triplicate with either the Sel2 5′ and 3′ primers or M12–23 aptamer reference control 5′ (5′- GGGGGAATTCTAATACGACTCACTATAGGGAGAGAGGAAGAGGGATGGG -3′) and 3′ primers. Data were normalized to the M12–23 reference control  and to cell number as determined by counting cells cultured in conjunction with each experiment.We applied similar RT-qPCR methods to detect internalized RNA as described previously 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-8 nt Barcode-GGGAGGACGATGCGG-3′; 5′-CAAGCAGAAGACGGCATACGAGCTCTTCC GATCTTCGGGCGAGTCGTCTG-3′). The PCR Protocol is as follows. The RT product was heated at 95°C for 2 min, followed by 10 cycles of heating at 95°C for 30 sec, annealing at 55°C for 30 sec and extending at 72°C for 30 sec and a final extension step at 72°C for 5 min. The PCR product was run on a 2.5% agarose gel and the appropriate band (∼151 bp) was excised, gel purified and quantitated using a UV spectrophotometer (OD 260). Samples were combined at equal molar amounts and submitted for Illumina sequencing .The RNA pools for selection rounds 0 (in duplicate), 1, 3, 5, 6, 7 and 8 were reverse transcribed using Superscript III  in the presence of the Sel2 3′ primer. The RT protocol is as follows. The round RNA was heated at 55°C for 60 min and extended at 72°C for 15 min. The product from the RT reaction was PCR amplified using Choice Taq DNA Polymerase  in the presence of Illumina primers (read number (cluster size) associated with each sequence. The unique sequences and total sequences were then converted from DNA code to RNA code and the 5′/3′ constant regions were added to give the full length RNA aptamer sequence. Table S1).The Illumina base calls were pre-processed and filtered to identify the variable region sequence as previously described Sequence complexity was determined using the equation; sequence complexity = unique sequences/total sequences. Sequence enrichment was calculated by taking the complement of % sequence complexity using the equation; sequence enrichment = 1-. Unique sequences and total sequences refer to the data obtained during Illumina high-throughput sequencing data pre-processing.read number for each unique sequence.The set of unique sequences found within each round were analyzed with RNAfold read number (cluster size) of the sequence . Using this database; the average, SEM, mode, maximum, minimum and frequency of the variable region nucleotide length was determined for each round; the frequency of the number of rounds sequences were found was determined for round 0 and rounds 1 through 8; the frequency of cluster sizes was determined for round 0 and rounds 1 through 8.A non-redundant database was created of all unique sequences found within the rounds of selection . For each sequence, this database tracked the variable region nucleotide (nt) length , the number of rounds the sequence was identified in  and the Figure S3; data output for edit distance = 1) or tree distance . Process.seqs was run using increasing values of edit/tree distance to determine the maximum edit/tree distance where all sequences/structures connected. Clusters of sequences/structures interconnected at each edit/tree distance were determined and separated using the program Cytoscape (v 2.8.1). Using these data, a dendrogram of sequence/structure relatedness by edit/tree distance was created and the resulting dendrogram was evaluated using Cytoscape. The edit/tree distance dendrogram indicates the edit/tree distance that the unique sequences interconnect. Sequence/structure families were evaluated at each edit/tree distance using the multiple sequence alignment program ClustalX (v 2.1) or multiple RNA structure alignment program LocARNA (webserver).A program (process.seqs) was created that used RNAfold and RNAdistance from the Vienna Package (v 2.0.0) read number of each sequence from a given round to the total reads from all rounds. To avoid dividing by 0 and simplify calculations, a read number of 1 was assumed with sequences with a read number of 0. The rising, fold enrichment and rate enrichment was determined for all unique sequence between rounds 1 through 3, 1 through 8, 3 through 8 and 6 through 8. ‘Rising’ was determined by calculating the correlation coefficient (−1.0 through 1.0) for each sequence using the read number and round number. Fold enrichment was determined by dividing the number of reads in a given rounds with the read number in a previous round. Rate enrichment was calculated by dividing the change in number of reads by the change in round number.The previously described non-redundant database was used to normalize the p value of <0.05. Correlation coefficients were determined using GraphPad Prism 5 by first determining normality and then the Spearman's correlation coefficient. Significance of the correlation coefficient was determined at a two-tailed p-value of <0.05.Data were plotted using either Microsoft Excel 2010 or GraphPad Prism 5. Average, SEM, mode and correlation were determined using Microsoft Excel 2010. Curve fitting was done using GraphPad Prism 5. Student's t-test was done using Microsoft Excel 2010 with significance set at a Figure S1Amount of RNA aptamer library (Round 0) internalized into VSMCs over time. Round 0 RNA was incubated with VSMCs (A7r5) for 15, 30, 60, 90 or 120 minutes. Unbound RNA or RNA bound to the surface of cells was removed with a stringent salt wash. Internalized RNA was extracted by TRIzol extraction and measured using RT-qPCR.(TIF)Click here for additional data file.Figure S2Recovery of RNA processing control. The M12–23 reference control RNA was added to TRIzol prior to cell lysis as a control for processing of internalized RNA. The reference control M12–23 RNA was measured by RT-qPCR. Internalized RNA data was normalized to each paired processing M12–23 reference control RNA.(TIF)Click here for additional data file.Figure S3Edit distance = 1 output from process.seqs program. Unique sequences (black nodes) interconnect by edges (lines) at edit distance 1 (blue lines). This analysis resulted in several sequence clusters as well as sequences that did not fall within a sequence cluster . Different unique sequences within a sequence cluster may have edit distances greater than 1, but are linked by intervening unique sequences separated by no more than 1 edit distance (blue lines). Cytoscape was used to assign the unique sequences of each separate cluster a number. This data was used to generate the dendrogram in (TIF)Click here for additional data file.Figure S4Unique sequences interconnected by a tree distance = 3. Structures of unique sequences (black nodes) interconnect by edges (lines) of tree distances 0–3 . This analysis resulted in several structure clusters as well as structures of unique sequences that did not fall within a structure cluster . Different unique sequences within a structure cluster may have tree distances greater than 3, but are linked by intervening structures separated by no more than 3 tree distances . Cytoscape was used to assign the unique sequences of each separate structure cluster a number. This data was used to generate the dendrogram in (TIF)Click here for additional data file.Figure S5Binding and internalization of Fam-GTP labeled aptamers. RNA aptamers #51 and #420 were labeled during in vitro transcription with a Fam-GTP. Binding of aptamers #51 and #420 was determined by fluorescence after several PBS washes without high salt. Internalization of aptamer #51 was determined at 150 nM and 300 nM by fluorescence following several PBS washes that included a high salt wash.(TIF)Click here for additional data file.Table S1Summary of high-throughput sequence data.(DOC)Click here for additional data file.Materials S1Supplementary Information. Methods are presented for the binding and internalization assays performed using fluorescent-labeled RNA aptamers (FAM-G-aptamers). Corresponding reference citations for this assay are also presented.(DOCX)Click here for additional data file.Database S1Comma separated values (.CSV) database includes all unique selected sequences . Column label identifiers after importing into excel: A (#) = identification number. B (Representatives) = selected RNA aptamer representatives (X). C (HTS read variable region) = variable region identified by HTS. D (Length) = nucleotide length of the variable region. E (1) = number of reads in round 1. F (3) = number of reads in round 3. G (5) = number of reads in round 5. H (6) = number of reads in round 6. I (7) = number of reads in round 7. J (8) = number of reads in round 8. K  = total number of reads in rounds 1–8. L (Rounds) = number of rounds. M (RNA) = RNA aptamer sequence (variable region with 5′ and 3′ constant regions). N (Structure) = predicted secondary structure of the RNA aptamer sequence. O (Sequence) = sequence family. P (Structure) = structure family.(CSV)Click here for additional data file.


Deep sequencing is a powerful tool for novel small RNA discovery. Illumina small RNA sequencing library preparation requires a pre-adenylated 3’ end adapter containing a 5’,5’-adenyl pyrophosphoryl moiety. In the absence of ATP, this adapter can be ligated to the 3’ hydroxyl group of small RNA, while RNA self-ligation and concatenation are repressed. Pre-adenylated adapters are one of the most essential and costly components required for library preparation, and few are commercially available.Solanum lycopersicum) using the T4 RNA ligase 1 adenylated adapter.We demonstrate that DNA oligo with 5’ phosphate and 3’ amine groups can be enzymatically adenylated by T4 RNA ligase 1 to generate customized pre-adenylated adapters. We have constructed and sequenced a small RNA library for tomato (We provide an efficient and low-cost method for small RNA sequencing library preparation, which takes two days to complete and costs around $20 per library. This protocol has been tested in several plant species for small RNA sequencing including sweet potato, pepper, watermelon, and cowpea, and could be readily applied to any RNA samples. The rapidly increasing NextGen sequencing capacity is enabling researchers to combine more samples for multiplexed sequencing, and the sequencing cost itself could be less than that of the library preparation . We haveIt has been reported that T4 DNA ligase can convert DNA oligos to pre-adenylated adapters . We reasIn this report, we demonstrate that T4 RNA ligase 1 can efficiently adenylate DNA oligos with 5’ phosphate and 3’ blocking group, and we have optimized the reaction condition for large-scale adapter production. Using the enzymatically adenylated adapters, we developed a rapid and cost-effective protocol to generate small RNA sequencing libraries.Agarose gel electrophoresis systemPolyacrylamide gel electrophoresis systemThermocyclerQubit Fluorometer (Invitrogen)T4 RNA liagse 2, truncated (NEB)50% PEG8000 (NEB)10 mM ATP (NEB)RNase Inhibitor (Promega)T4 RNA ligase 1 (Enzymatics)SuperScriptIII Reverse Transcriptase (Invitrogen)Phusion Hotstart DNA Polymerase (NEB)MinElute Gel Extraction kit (Qiagen)Quant-iT HS DNA assay kit (Inivtrogen)5’ adapter: GUUCAGAGUUCUACAGUCCGACGAUC3’ non-multiplex adapter (BL1): Phosphate-CTGTAGGCACCATCAAT-amine.3’ multiplex adapter: Phosphate-CAGATCGGAAGAGCACACGT-amine.Substrate dA: Phosphate-ACTTCGTATGCCGTCTTCTGCTT-amineSubstrate dT: Phosphate-TAGTCGTATGCCGTCTTCTGCTT-amine.Substrate dC: Phosphate-CTGTCGTATGCCGTCTTCTGCTT-amine.Non-multiplex RT primer: amine-ATTGATGGTGCCTACAG.Multiplex RT primer: amine-ACGTGTGCTCTTCCGATCTG.Universal forward PCR primer (* indicates phosphothio modification): AATGATACGGCGACCACCGAGATCTACACGACAG GTTCAGAGTTCTACAGTCCGACGAT*C.Non-multiplex reverse PCR primer: CAAGCAGAAGACGGCATACGAGATTGATGGTGCCTACA*G.Multiplex reverse PCR primers with 6 nt barcode (NNNNNN indicates the location of TruSeq index sequences):CAAGCAGAAGACGGCATACGAGAT-(NNNNNN)-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T.1. Dissolve the 5’ phosphorylated and 3’ blocked DNA oligo in RNase-free water to a final concentration of 100 uM.2. Setup a reaction in 0.2 ml PCR tube containing 10 uL oligo, 10 uL 10x T4 RNA ligase 1 buffer, 40 uL 50% PEG8000, 35 uL RNase-free water and 5 uL T4 RNA ligase 1, and incubate overnight at room temperature.3. Optional: setup multiple reactions if more adenylated adapters are required.4. Purify the adenylated adapter using a Sephadex G-25 or silica-based spin column, e.g., Qiagen Nucleotide Removal Kit. Adjust the adapter concentration to 10 uM with RNase-free water. Note: In this reaction, T4 RNA ligase 1 will first adenylate the 5’ phosphorylated DNA oligo and attempt to join it with a suitable acceptor molecule with a 3’ hydroxyl group. The 3’ end of the oligo is block with amine and could not take part in the ligation reaction, leading to accumulation of the adenylated intermediate.5. Denature the sRNA sample at 65°C for 30 sec, immediately chill on ice.6. Denature the adenylated 3’ adapter at 65°C for 30 sec, immediately chill on ice.7. Setup the ligation reaction with 8 uL small RNA, 1 uL adapter (10 uM), 2 uL 10x T4 RNA ligase buffer without ATP, 0.5 uL RNase inhibitor, 5 uL PEG8000 and 0.5 uL T4 RNA ligase 2, truncated. The reaction is incubated at 25°C for 4 h or 18°C overnight. Note: ligation reaction must be performed in the absence of ATP to prevent self-ligation of the small RNA that has a 5’ phosphate. Successfully ligated small RNA could be visualized on Urea-PAGE  to the ligation reaction, heat inactivate the reaction at 65°C for 15 min and incubate the reaction at 25°C for 10 min.9. Optional: to minimize adapter dimer formation in the subsequent PCR enrichment, the ligated sRNA could be PAGE purify before proceeding to the following 5’ adapter ligation steps.10. Denature the 5’ adapter  at 65°C for 30 sec, immediately chill on ice.11. Setup the 5’ ligation reaction with 20.5 uL 3’ ligation product, 1 uL 5’ adapter (10 uM), 1 uL 10x T4 RNA ligase 1 buffer with ATP, 0.5 uL RNase Inhibitor, 2 uL 10 mM ATP, 4 uL RNase-free water and 1 uL T4 RNA ligase 1. The reaction is incubated at 25°C for 4 h or 18°C overnight. Note: the 3’ end of the small RNA has already been ligated to the 3’ adapter that has an amine group at the 3’ end, and could no longer take part in the ligation reaction; thus its 5’ end could be ligated to an RNA oligo in the presence of ATP.12. Add 2 uL dNTP (10 mM each), 3 uL DTT (100 mM), 9 uL 5xSuperScriptIII Frist Strand reaction buffer, and 1 uL SuperScriptIII reverse transcriptase to the 5’ ligation reaction.13. Incubate the reaction at 50°C for 1 h, and heat inactivate the reverse transcriptase at 75°C for 15 min.14. Assemble the PCR reaction with 10 uL RT product, 1 uL forward primer, 1 uL reverse primer, 1 uL dNTP, 6 uL 5x Phusion HF buffer, 10.5 uL water and 0.5 uL Phusion DNA polymerase. The remaining RT product could be stored at −20°C.15. Perform 12 to 15 cycles of PCR amplification as followed: 94°C 2 min, 12–15 cycles of 98°C for 12 sec, 65°C for 30 sec and 72°C for 30 sec, and a final extension at 72°C for 2 min.16. Seperate the PCR product on a 2% agarose gel. Note: The multiplex sRNA-Seq library and adapter dimer are 150 and 120 bp respectively  of each libraries, concentrate using Qiagen MinElute kit, and use 10 uL library with a final concentration no less than 2 ng/ul for each lane of Illumina sequencing.It has been shown that T4 RNA ligase 1 could be used to adenylated RNA oligos ,8. To exThe authors declare no competing interests.ZF and SZ devise the project and wrote the manuscript. YC performed the experiment and YZ, BL and JJG performed the analysis and contributed to the manuscript writing. All authors approved the final manuscript.


Most genes have multiple polyadenylation sites (PAS), which are often selected in a tissue-specific manner, altering protein products and affecting mRNA stability, subcellular localization and/or translability. Here we studied the polyadenylation mechanisms associated to the beta-adducin gene (Add2). We have previously shown that the Add2 gene has a very tight regulation of alternative polyadenylation, using proximal PAS in erythroid tissues, and a distal one in brain. Using chimeric minigenes and cell transfections we identified the core elements responsible for polyadenylation at the distal PAS. Deletion of either the hexanucleotide motif (Hm) or the downstream element (DSE) resulted in reduction of mature mRNA levels and activation of cryptic PAS, suggesting an important role for the DSE in polyadenylation of the distal Add2 PAS. Point mutation of the UG repeats present in the DSE, located immediately after the cleavage site, resulted in a reduction of processed mRNA and in the activation of the same cryptic site. RNA-EMSA showed that this region is active in forming RNA-protein complexes. Competition experiments showed that RNA lacking the DSE was not able to compete the RNA-protein complexes, supporting the hypothesis of an essential important role for the DSE. Next, using a RNA-pull down approach we identified some of the proteins bound to the DSE. Among these proteins we found PTB, TDP-43, FBP1 and FBP2, nucleolin, RNA helicase A and vigilin. All these proteins have a role in RNA metabolism, but only PTB has a reported function in polyadenylation. Additional experiments are needed to determine the precise functional role of these proteins in Add2 polyadenylation. Most eukaryotic mRNAs acquire an untemplated polyA tail at their 3′ end by a process named polyadenylation. The addition of the polyA tail is the result of a highly coordinated two-step process, in which the pre-mRNA is first cleaved at the CA dinucleotide of the cleavage site (CS) and then the polyA tail is subsequently added. This process requires the formation of protein-RNA complexes at the core polyadenylation elements: the hexanucleotide motif , and the U/UG-rich element located downstream of the CS . The Hm is the binding site of the cleavage and polyadenylation specificity factor (CPSF) complex, while the DSE is bound by the cleavage stimulation factor (CstF). Recent genome- and transcriptome-wide analyses suggest that the consensus motif of CstF-64 corresponds to UGUGU Interestingly, about 70% of mammalian genes contain multiple polyadenylation sites (PAS), generating transcripts differing in the 3′UTR or coding regions Adducins are membrane skeletal proteins encoded in mammalian genomes by three closely related genes: α-, ß-, and γ-adducins . Adducins are present in the membrane skeleton of cells as homo- and heterodimers where they cap the fast growing end of actin filaments We have previously predicted the putative core polyadenylation elements of the brain-specific PAS The minigenes used for the transfection experiments were generated by inserting the fragments containing the tissue-specific polyadenylation sites of ß-adducin and their flanking sequences , 1X reaction buffer, 200 µM dNTP mix). An extension reaction was performed and the amplification product was digested with DpnI to eliminate the parental DNA template allowing the selection of mutation-containing synthesized DNA. The DpnI-resistant DNA was then transformed into competent bacterial cells. This method was used to create the deletions and mutations constructs of ß-adducin chimeric minigenes.The plasmid containing the target sequence (50 ng) and the oligonucleotides primers containing the desiderate mutations (complementary to opposite strands of the target sequence) were added to the reaction mix (2.5 U/µl Liposome-mediated transfections of plasmid DNA into HeLa and HEK293 cell lines were performed using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. Along with the test minigene construct, each plasmid DNA mix contained the plasmid expressing SV40 T-antigen to stimulate SV40 promoter activity. pEGFP-C2 (Clontech) was used as transfection efficiency control. The plasmid DNA used for transfections was purified with JetStar columns (Genomed).Total cellular RNA was isolated with TRI REAGENT (Sigma) following manufacturer’s instructions. cDNA was prepared from total RNA  by using M-MLV reverse transcriptase (RT) (Invitrogen) following manufacturer’s protocols.The polymerase chain reaction (PCR) was performed as described by the Taq polymerase manufacturers (Roche or NEB). The oligonucleotide primers used to perform the different amplifications are shown below.3′-RACE-PCR. After the cDNA synthesis using an oligo dT primer containing an anchor sequence at the 5′ end, a first PCR was performed using as forward primer an internal oligonucleotide complementary to the cDNA and a primer complementary to the anchor sequence (3′RACE 1st reaction). To give further specificity to the reaction a second couple of primers was used to amplify the first PCR product (3′RACE 2nd reaction). 3′RACE PCR products were agarose gel purified and sequenced (Macrogen).32P) dCTP. The DNA fragments used as probes were obtained by PCR from genomic DNA (mß-Add 16 ex Probe) and pEGFP-C2 vector (GFP Probe), labeled with Rediprime II Random Prime Labeling System (Amersham Bioscience) and then purified from unincorporated nucleotides by Nick columns (Amersham bioscience).The Northern Blot analysis was performed as described 32P) UTP was added to the transcription mix. The cold reaction was phenol-chloroform purified while the hot one through nick-columns (Amersham bioscience). Denaturing agarose (cold RNA) or polyacrylamide gels (hot RNA) were run to check quality and size of the synthesized RNA.For RNA-EMSA and RNA-pull down, RNA was in vitro synthesized with T7 RNA polymerase (Stratagene) from a DNA template (PCR amplified) containing the T7 RNA polymerase promoter sequence (oligonucleotides are indicated below) as described by the manufacturer. In order to radioactively label RNAs (hot RNAs),  with HeLa nuclear extract (Cilbiotech). To modulate the stringency of the binding we used different final concentrations of heparin (5 µg/µl and 15 µg/µl) and nuclear extract (50 µg and 2 µg), as described in the Legends to the Figures. The RNA-proteins mix was incubated at room temperature for 20 minutes before running on native polyacrylamide gels (5%). When cold RNA fragments were used as competitors, a pre-incubation between cold RNA and the nuclear extract was done before adding the radiolabeled RNA. Gels were dried before X-OMAT film or Cyclone (Packard) exposure.RNA-EMSAs were conducted according to procedures already described (Buratti 2011) with minor modifications. Radioactively labeled RNA fragments were incubated in EMSA binding buffer . Cold RNAs (50 µg)-beads were incubated with 0.8 mg of HeLa cell nuclear (Cilbiotech). After the washing steps, proteins bound to RNA-beads were separated on a gradient SDS–PAGE gel (gradient 8–15% or 10%). After running, gels were either stained with colloidal Coomassie G-250 or transferred to nitrocellulose membrane for western blot analysis. Sequence analysis from the Coomassie-stained bands excised from the SDS–PAGE gel was performed by Protein Network Group from ICGEB, Trieste, Italy.Western blot analyses were performed as previously described (Porro 2010). For western blot the following primary antibodies were used: anti TDP-43 (ProteinTech), PTB . The results were quantified with Quantity One software (Bio-Rad Laboratories).AGGAGGAGGAGCCGAGCGTA 3′) and m ß-Add 16 ex rev 1 (5′ TCCTCCTTCCCATTCACCAC 3′) for synthesis of the mß-Add16 ex Probe; GFP813FW (5′ CGGCGTGCAGTGCTTCAGCCGCTAC 3′) and GFPendRV (5′ CTTGTACAGCTCGTCCATGCCGAGAG 3′) for the GFP Probe; BR A4 dir 1 tail (5′ CGGCGTGCAGTGCTTCAGCCGCTAC 3′), dT-anchor 1 (5′ TAGGAATTCTCGAGCGGCCGCT(17)A/C/G 3′), mXhoI dir1 (5′ CCGCTCGAGTCTGCCTTTCTCTATGCTA 3′) and anchor 1 (5′ CCGCTCGAGTCTGCCTTTCTCTATGCTA 3′) for the 3′ RACE reactions; T7 mBRA4 dir 1 (5′ TAATACGACTCACTATAGGTGTTTGGGGTGGACTCTG 3′) and mBR A4 rev a (5′ CTCCCACCTCCCCAGGAG 3′) for the generation of the DNA template for T7-directed in vitro transcription.Primers: m ß-Add 16 ex dir 1 . We generated the A1-A23-A4 mutDSE, A1-A23-A4 mutUSE and A1-A23-A4 mutUSE/DSE constructs which contained point mutations in the GU repeats of the Add2 USE, DSE and a combination of both sites mutated, respectively see . When thTo determine the role of the non-canonical hexamer AGUAAA and to elucidate the interplay of the canonical hexamer with the Add2 A4 PAS DSE we reverted the AGUAAA sequence to a canonical AAUAAA hexamer. Therefore, we constructed an additional series of plasmids containing the optimized AAUAAA PAS  in combination with the natural Add2 DSE (A1-A23-A4 CHm), a deletion of the DSE (A1-A23-A4 CHm/ΔDSE) or point mutations in the GU repeats of the Add2 DSE (A1-A23-A4 CHm/mutDSE) . After tWe then performed EMSA with HeLa nuclear protein extracts and radioactive RNA probes of the wt or deleted A4 PAS . All proWhen we performed the EMSA experiments in more stringent conditions, we observed that the ΔDSE probe was not able to form stable complexes when incubated with HeLa nuclear extracts , Lane 4,To identify the proteins participating in Add2 polyadenylation at the A4 PAS, we implemented a pull-down approach using the same RNAs probes and HeLa nuclear protein extract used above. Proteins were allowed to bind to the different versions of the A4 PAS. Bound proteins were eluted and separated in a SDS-PAGE . SurprisIn the present report we used chimeric minigenes to study the mechanisms of polyadenylation of the mouse Add2 gene. We accurately reproduced cleavage and polyadenylation at the distal PAS of the Add2 pre-mRNA. We have shown that the Hm and DSE promote efficient use of this polyadenylation signal, as their deletion or point mutation resulted in the activation of cryptic PASs. This region actively interacts with nuclear proteins. We have identified some of the putative proteins recognizing the USE and DSE of Add2 pre-mRNA. Among them we found proteins known to participate in RNA metabolism and processing, such as PTB, TDP-43, FBP1, FBP2 and RNA Helicase A.Many different Hm variants are present in genes, being the “classical” AAUAAA together with the AUUAAA variant the most common versions, found in about 70–80% of genes We observed that deletion or point mutation of the A4 PAS USE results in an increase in the amounts of mRNA suggesting that this region negatively regulates mRNA processing or stability. However, we did not observe any difference in the pull down analysis respect to the wt probe. Bioinformatic search of seed sites for miRNAs overlapping with the USE showed that miRNAs could potentially bind that region. Some of these miRNAs are expressed in HeLa and HEK293 cells with, thus, the potentiality to bind the Add2 3′ UTR. However, only the experimental validation by point mutation of the binding site together with miRNA depletion or overexpression will tell us whether the increase observed after USE deletion could be related to the elimination/mutation of a putative miRNA binding site.Add2 pre-mRNA undergoes tissue-specific regulation of APA. Erythroid tissues use proximal PASs while a distal PAS is preferentially used in brain tissues As mentioned above, we focused our attention on the proteins that bind to the distal Add2 A4 PAS and may have a potential role in 3′ end processing. Surprisingly, among the proteins identified in the pull down experiments we did not detect any protein of the core polyadenylation machinery, such as CPSF-160 and CstF-64, known to bind to the hexanucleotide motif and DSE, respectively. Among the identified bands we found Polypyrimidine Tract Binding Protein (PTB), a well-known RNA binding protein in the splicing field that preferentially binds to UCUU(C) and UUCU/C sequences in a pyrimidine-rich context Another interesting protein detected in the pull-down analysis was TAR-DNA binding protein-43 (TDP-43), a protein with DNA and RNA binding properties. Reported TDP-43 nuclear functions are multiple, ranging from the regulation of HIV transcription FAR-upstream Binding protein 1 (FBP1), originally identified as a DNA binding protein regulating c-Myc gene transcription, was shown in recent years to act as an RNA-binding protein and to regulate mRNA translation or stability of genes by binding to A/U rich sequences (ARE), competing with members of the HuR family c-fms mRNA 3′UTR destabilizing the transcript and negatively regulating its translation All of the other identified proteins, except Septin-9 To summarize, we have characterized the core polyadenylation elements of the distal PAS of Add2. We have shown that nuclear proteins bind the A4 PAS RNA, and that the formation of protein-RNA complexes depends on the presence of the DSE. We identified some of the proteins that bind to the DSE element and among them we found proteins known to participate in polyadenylation, such as PTB and FBP2. We also found proteins reported to be involved in splicing and stability of mRNAs, such as PTB-43, vigilin, FBP1 and FBP2, nucleolin. Future experiments will be necessary to determine their functional role in Add2 polyadenylation.Figure S1Alignment of the distal PAS of the Add2 gene (A4 PAS). Panel A: The sequence of the distal A4 PAS of the Add2 gene is shown (234 bp), together with the reported ESTs for that region (from the UCSC browser). The USE, Hm, cleavage site and DSE are indicated (red and orange rectangles). The homology among vertebrates, mammals and Euarchontoglires is shown (bottom). Panel B: A lower magnification scheme of the same region is shown, corresponding to 869 bp.(TIF)Click here for additional data file.Figure S2Alignment of the proximal PAS of the Add2 gene (A1 PAS): Scheme showing the A1 PAS and the other annotated transcripts for that region. The USE, Hm, cleavage site and DSE are indicated (red and orange rectangles). The homology among vertebrates, mammals and Euarchontoglires is shown (bottom).(TIF)Click here for additional data file.Figure S3Alignment of the A23 PAS of the Add2 gene: The scheme shows the annotated transcripts in other species for that region. The canonical polyadenylation elements were not detected, as they highly differ from the consensus ones. The homology among vertebrates, mammals and Euarchontoglires is shown (bottom).(TIF)Click here for additional data file.Figure S4Sequence of the A4 PAS region in the deleted and mutated constructs: The scheme shows the sequence of the A4 PAS region of the RNAs used for the cell-transfection experiments, and for the band shift and pull down analysis. The mutations introduced are indicated in red (underlined). The USE, hexanucleotide motif and DSE are indicated.(TIF)Click here for additional data file.


Recent technological developments have brought forth a new era of RNA research in which large sets of data are collected rapidly using the high-throughput next generation sequencing technology. Growing evidence suggests that only around 5% of nucleotides in the mammalian genomes are transcribed into protein-coding RNA, and large amount of transcripts are non-protein-coding RNA (ncRNA). During the last decade, much information has been generated from the studies of one type of ncRNA, namely microRNA . miRNA modulates the expression of target genes through repression of mRNA translation or mRNA degradation. Its dysregulation has been implicated in various biological disorders and human diseases. Meanwhile, the long-non-coding RNA  has recently emerged to catch significant attention. lncRNA is involved in chromatin modification, epigenetic regulation, transcription control, and pre- and post-translational mRNA processing. The functions of lncRNA are believed to be associated with development, imprinting, mental and psychiatric disorders, and tumor growth.Bioinformatics is a pivotal component of this new RNA research revolution. It utilizes mathematical models and computer simulations to form, extract and analyze RNA data, and to search new ncRNA gene sequences and predict their targets. Assumptions in this computational modeling are derived from the observations that ncRNAs are produced following step-wise processes from precursors to functional end products. Based on miRNA biogenesis, criteria in searching for new miRNAs from sequencing data include that the precursors fold into a stable stem-loop structure, mature miRNAs are found on one arm of the stem, and these sequences are usually evolutionarily conserved  have offered much improved avenue for ncRNA discovery  accompanied by a quality score for each nucleotide in each sequence. Because of the high sensitivity of the technique, the “raw” data will also contain sequencing primers and contaminants which can potentially produce sequence bias that requires more sophisticated computational approaches to sieve out miRNA transcripts (Mendes et al., Computational approaches developed so far make extensive use of evolutionary conservation information either to predict ncRNA genes or ncRNA-target associations, sometimes ignoring the subtle rules presiding ncRNA biogenesis and target specificity. Thus, approaches combining high-throughput sequencing biochemical techniques and bioinformatic analyses that emphasizes the synergy of genome-wide approaches are essential (Mendes et al.,


Since the first identification of the virus in 1971, the disease caused by canine coronavirus (CCoV) has not been adequately investigated, and the role that the virus plays in canine enteric illness has not been well established. Only after the emergence in 2002 of SARS in human has new attention been focused on coronaviruses. As a consequence of the relatively high mutation frequency of RNA-positive stranded viruses, CCoV has evolved and, with the biomolecular techniques developed over the last two decades, new virus strains, serotypes, and subtypes have been identified in infected dogs. Considering the widespread nature of CCoV infections among dog populations, several studies have been carried out, focusing upon the epidemiological relevance of these viruses and underlining the need for further investigation into the biology of CCoVs and into the pathogenetic role of the infections. This paper reports the evolutionary processes of CCoVs with a note onto recent diagnostic methods. Coronaviridae family, order Nidovirales, are large, enveloped, RNA viruses that cause highly prevalent diseases in humans and domestic animals. CoVs are spherical enveloped particles about 100–120 nm in diameter with a capped, polyadenylated single-stranded, positive-sense genomic RNA 27.6 to 31 kb in length, the largest known RNA virus genome. The 5′ end of the genome consists of a 65 to 98 nt sequence, termed the leader RNA, that is also present at the 5′ end of all subgenomic mRNAs. An untranslated region (UTR) of 200 to 400 nts follows this leader sequence. At the 3′ end of the RNA genome is another UTR of 200 to 500 nts followed by a poly(A) sequence of variable length. Both 5′- and 3′-UTRs are important for RNA replication and transcription. The remaining genomic sequence includes different open reading frames (ORFs) which differ markedly among coronaviruses in number, nt sequence, genes order, and in method of expression. At the 5′ end of each gene, all CoVs have a common intergenic sequence of about 7 bases which is essential for the formation of subgenomic RNAs [Coronaviruses (CoVs), a genus in the The first two-thirds of the genome consists of two partially overlapping ORFs, ORF1a and ORF1b. These ORFs are translated into a polyprotein which is the precursor both of the viral RNA-dependent RNA polymerase and of proteases. The one-third in the 3′ end of the genome contains ORFs encoding for the major structural proteins, spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. These ORFs are interspersed with several ORFs encoding for different nonstructural proteins, most of which are of unknown function .Betacoronavirus, see below), the 180 kDa S protein is cleaved during or after virus maturation by a cellular protease enhancing the cell fusion activity or viral infectivity. The S protein of phylogroup 1 CoVs  is not cleaved even though some of these viruses can induce cell-to-cell fusion [The S glycoprotein, which forms the large spikes on the surface, is the major inducer of virus-neutralizing antibodies and plays an important role in the biology and pathogenesis of CCoV infections, inducing both fusion of the viral envelope with host cell membranes and cell-to-cell fusion. In most phylogroup 2 CoVs  type 1 and type 2, transmissible gastroenteritis virus (TGEV) of swine, porcine respiratory coronavirus (PRCoV), porcine epidemic diarrhea virus (PEDV) and canine coronaviruses (CCoVs). Recently, a ferret coronavirus has been identified as a member of phylogroup 1 [Presently, the ogroup 1 . Phylogrogroup 1  and caniogroup 1 . Soon afogroup 1 . Howeverogroup 1 , 15. A tintergroup pairwise amino acid identity scores for the true coronaviruses are comparable to those calculated for structural and nonstructural proteins of different genera in other RNA virus families  [Coronaviridae and the organization of the still-to-be-established subfamily Coronavirinae are based upon rooted phylogeny and pairwise comparisons using Coronaviridae-wide conserved domains in replicase polyprotein (ORF1ab), as well as the structural proteins S, E, M, and N [de facto criterion, the CSG has formulated a taxonomic proposal to the ICTV Executive Committee, which would intuitively follow the unofficial (but widely accepted) nomenclature, to convert phylogroups 1 through 3 into genera designated Alpha-, Beta- and Gammacoronavirus, respectively,  have developed a framework for consistent classification and have established definite genus- and species-demarcation criteria. The viridae) . The proM, and N –18. Basectively, . The virCCoV was first described during an epizootic in a canine military unit in Germany in 1971 –26. Whereas fatal infections are unusual unless mixed with infections by other pathogens or with overcrowding and unsanitary conditions, CCoV alone is an important canine pathogen responsible for epizootics . NeverthIn 2001, by sequence analysis of CCoVs detected in fecal samples collected from diarrheic dogs in the South of Italy, multiple nt substitutions accumulating over a fragment of the M gene were observed . The poiThese preliminary observations gave a meaningful impulse to studying the genetic evolution of CCoVs. Extensive sequence analysis on multiple regions  of the viral genome from CCoV-positive faecal samples, provided strong evidence for the existence of two separate genetic clusters of CCoV. The first cluster includes CCoVs intermingled with reference CCoV strains, such as Insavc-1 and K378, while the second cluster segregates separately from CCoVs and, presumably, represents a genetic outlier referred to as FCoV-like CCoVs . A possibetacoronaviruses and gammacoronaviruses identified and classified to date. Recently, Lorusso et al. [Taking into account the genetic drift toward FCoV observed in the M gene of the FCoV-like CCoVs, the genetic differences between these divergent strains and the typical reference CCoV strains were evaluated in the ORF2 sequence, characterized by a more evident variability. The nt sequence of a region encompassing about 80% of the S gene of one of these FCoV-like CCoVs, strain Elmo/02, clearly indicates that a novel CCoV type, highly divergent from the reference CCoV strains and more closely related to FCoV type 1, circulates among dogs. Comparison of the inferred amino acid sequences revealed about 81% identity to FCoVs type 1 and about 54% identity to both FCoVs type 2 and CCoV reference strains. On the basis of the evident and significant identities between Elmo/02 strain and FCoVs type 1, the new strain Elmo/02 was designated as the prototype of the newly recognised genotype 1 (CCoV type 1), and the reference strains were designated as CCoV type 2 . The higo et al.  also hasThe significance of all these data is still unclear, but it raises several questions regarding the biology of CCoVs. The literature on CCoVs-induced disease offers little or no useful information, and although in more recent years additional important data have shed light on the more obscure aspects of the infection, several studies need to be performed to extend knowledge of these two CCoV genotypes. The data acquired on the genome of the enteric CCoVs and on their evolution focus upon important epidemiological outcomes in the field, in terms of both prophylaxis and virus evolution. Hence, the serological correlation between the two viruses requires further study, and several questions regarding the pathobiology of CCoV type 1 and the efficacy of currently available CCoV vaccines that contain only CCoV type 2 remain to be answered.The factors regulating the course of the natural diseases caused by enteric CCoVs are not well understood. CCoVs are responsible for enteritis in dogs, and signs of infections may vary from mild to moderate, but they are more severe in young pups or in combination with other pathogens. Common signs include soft faeces or fluid diarrhoea, vomiting, dehydration, loss of appetite, and, occasionally, death. Dual infections by CCoV and canine parvovirus type 2 (CPV2) are especially severe when infections occur simultaneously , but CCoThe natural route of transmission is faecal-oral, and virus in faeces is the major source of infection. In neonatal dogs, the virus appears to replicate primarily in the villus tips of the enterocytes of the small intestine causing a lytic infection followed by desquamation and shortening of the villi and resulting in diarrhoea 18–72 h post infection . ProductRecent extensive biomolecular analysis of faecal samples collected from infected dogs in Italy revealed that CCoVs infection is widespread and often characterized by the occurrence of both genotypes simultaneously , 40. CCoin vitro [These data raise several questions, and more indepth investigations into the pathobiology of CCoVs type 1 and type 2 are required. Therefore, failure to isolate CCoV type 1 in vitro  hinders CCoVs, like other CoVs, show relatively high mutation frequency, and, in recent decades divergent strains have been described. One of the first observations was from Wesley,  who demoThe identification of a novel CCoV, strain UWSMN-1, from a fatal case of gastroenteritis in pups from breeding colonies was reported in Australia in 2001 . SequencIn 2002, an epizootic outbreak of diarrhea occurred in a Beagle breeding colony in United Kingdom. A new CCoV, strain BGF10, was isolated and characterized. The virus revealed a highly divergent region at the amino-terminal domain of the M protein and a long nonstructural protein 3b of 250 amino acids associated with virulence in other CoVs .In 2005, a fatal disease in puppies was described in Italy, and a pathogenic variant of CCoV, strain CCoV type 2-CB/05, was isolated from all tissues examined except brain . The seqRecently, CCoVs with a potential double recombinant origin through partial S-gene exchange with TGEV were identified in the gastrointestinal tract and internal organs of pups which had died of acute gastroenteritis . A TGEV-betacoronavirus in the polymerase and S genes, with the highest amino acid identity with the corresponding BCoV proteins and proved to be only distantly related to enteric CCoVs. By sequence comparison of cDNA polymerase in the analyzed 251 bp sequence, the identity was 98.8% for BCoV and 98.4% for HCoV polymerase gene, whereas it was only 68.5% for CCoV, strain 1–71. When comparing the amino acid sequence obtained by translation of the cDNA sequence from CRCoV to the amino acid sequence of BCoV, HCoV-OC43, and enteric CCoV spike proteins in an overlap of 1093 amino acids, the identities were 96%, 95.2%, and 21.2%, respectively , virus isolation (VI) in cell cultures, and biomolecular analysis. EM examination of negatively stained fecal suspensions and immune electron microscopy are rapid procedures for detecting coronavirus and appear to be valuable diagnostic tools . Howeveraviruses , and EM aviruses , but is aviruses  reduces In the past decade, several PCR-based methods have been developed for detecting CCoV RNA in the feces of dogs, allowing the detection limits of virus isolation to be overcome. PCR has been identified as the gold standard because of the improvement in both sensitivity and specificity when compared to conventional methods , 61, 62.1 to 108 copies of standard RNA. The genotype-specific fluorogenic assay can be useful to detect and measure viral loads in fecal samples collected from dogs naturally or experimentally infected with type 1, type 2, or both genotypes [Recently, two genotype-specific fluorogenic RT-PCR assays were developed for the detection, discrimination, and quantitation of CCoV type 1 and CCoV type 2 RNAs in the feces of dogs with diarrhea. The assay showed high specificity, sensitivity, and reproducibility, allowing a precise quantitation of CCoVs RNA over a linear range of about eight orders of magnitude, from 10enotypes .alphacoronavirus is accounted for by linear evolution as well as by sudden dramatic shifts generated by RNA deletions or recombination [One of RNA's most intriguing features is its ability to carry genetic information despite its labile nature. CoVs are unique among RNA viruses in many aspects of their biology. They are characterized by a extremely large genome, by a nested set of subgenomic mRNAs, by a discontinuous transcription mechanism, and by a high frequency of RNA recombination events because of the high error frequencies of RNA polymerase . Geneticbination .betacoronaviruses [RNA recombination has been developed into a potent genetic tool to introduce desired RNA sequences into CoV genomes, revealing itself as an important mechanism in the natural evolution of CoVs. For example, new strains of IBV in poultry flocks are the results of natural recombination between different field strains. FCoVs type 2 have arisen by recombination events between FCoV type 1, wholly feline, and CCoV, resulting in a FCoV genome consisting of the spike gene and part of the adjacent gene from CCoV . Recombiaviruses , while taviruses . SARS emaviruses . As a consequence of the relatively high mutation frequency, the RNA viruses have the potential to rapidly adjust to certain negative pressures, such as those presented by the immune system. Recombination events affecting CCoVs could clarify the evolutionary processes leading to the proliferation of new virus strains, serotypes, and subtype, as happened for SARS-CoV and for strain CCoV type 2-CB/05. Notwithstanding the several studies carried out on CCoVs, there are a lot of aspects yet to be clarified: the meaning of simultaneous infection by CCoV type 1 and CCoV type 2, the real pathogenetic role of these two viruses, the immune response against CCoV type 1 and CCoV type 2, and the assessment of CCoV type 2 CB/05-like virus distribution among dog populations.