Epstein-Barr virus (EBV) is a human herpesvirus implicated in cancer and autoimmune disorders. Little is known concerning the roles of RNA structure in this important human pathogen. This study provides the first comprehensive genome-wide survey of RNA and RNA structure in EBV.Novel EBV RNAs and RNA structures were identified by computational modeling and RNA-Seq analyses of EBV. Scans of the genomic sequences of four EBV strains  and of the closely related Macacine herpesvirus 4 using the RNAz program discovered 265 regions with high probability of forming conserved RNA structures. Secondary structure models are proposed for these regions based on a combination of free energy minimization and comparative sequence analysis. The analysis of RNA-Seq data uncovered the first observation of a stable intronic sequence RNA (sisRNA) in EBV. The abundance of this sisRNA rivals that of the well-known and highly expressed EBV-encoded non-coding RNAs (EBERs).This work identifies regions of the EBV genome likely to generate functional RNAs and RNA structures, provides structural models for these regions, and discusses potential functions suggested by the modeled structures. Enhanced understanding of the EBV transcriptome will guide future experimental analyses of the discovered RNAs and RNA structures. Epstein-Barr virus (EBV) is widely disseminated in the human population. Upwards of 95% of the adult human population is infected with EBV . EBV is The ~170000 bp genome of EBV is a linear double-stranded (ds)DNA that circularizes to form an episome in the host cell nucleus. Infection occurs by entry of the EBV virion into human host epithelial cells and initially proceeds via an aggressive lytic phase. The virus migrates to B cells where it causes persistent lifelong infections marked by extended periods of latency with interspersed lytic reactivation . EBV lat7 copies per cell [The EBV transcriptome is complex, consisting of multiple pre-mRNA transcripts that undergo extensive alternative splicing events and can, at various states of infection, yield different sets of gene products . Two welper cell ). Roles per cell  and the per cell . Both EBper cell -26, inclper cell . Except RNA secondary structure plays important roles in many viruses, mediating functions as diverse as translation initiation , catalysRefSeq genomes (~170 kbp long) from four EBV strains  and one from the closely-related  also faIn addition to the regions corresponding to EBER1, the v-snoRNA, and 14 loci specifying EBV pre-miRNAs, there are 249 additional EBV regions with predicted conserved RNA structure . The number of W repeats can vary, and the optimal number appears to be five to eight . The W rSix EBNA proteins are expressed from mRNAs produced by extensive alternative splicing. Transcription of the EBNA RNA can occur from an upstream promoter, the C promoter (Cp), or a downstream W promoter (Wp) . Early iWithin loci 18, 23, 28, 33, 38, 43, 48, and 53 is a highly unusual modeled RNA structure, a very long (586 nt) tetraloop hairpin . The MHV4 and CeHV12 hairpins are most similar to EBV in sequence  and structure  was constructed using nuclear RNA isolated from cultured human B lymphocytes stably infected with EBV BJAB-B1 cells ). These  cells . Stable intronic sequence (sis)RNAs have been described in Xenopus oocytes  and, intIntrons homologous to ebv-sisRNA-1 were found in other lymphocryptoviruses Figure D. The ebAlthough ebv-sisRNA-1 did not overlap an RNAz-predicted region, the secondary structure for this RNA was modeled on the basis of the eight sequences shown in Figure Other than the W repeat sisRNAs, EBV possesses 18 additional short introns Table  interspeParticularly interesting is the sequence from nt 105929 to 106056, which has the most favorable predicted folding free energy and z-score of any EBV intron Table B. RatherFour EBV short introns have significant numbers of mapped reads in the small RNA library Table ; howeverThe importance of RNA structure in the regulation of splicing has been highlighted in two excellent reviews ,87. StruIn EBV, there are seven predicted structured regions that include splice sites  predictions overlap EBV coding regions, in some cases including a significant portion of an ORF. In the lytically-expressed EBV FGAM-synthase gene, for example, predictions cover 40% the coding nt , which is involved in the regulation of host B-cell signaling and is implicated in EBV-associated tumorigenesis . The firBioinformatics analyses of the EBV genome revealed 265 regions with putative conserved RNA structure. The computational screen successfully identified most of the known EBV ncRNAs: EBER1, a virally-encoded snoRNA, and 42 miRNAs. Other predicted structured regions of the EBV genome occur at or near sites that suggest potential functions for RNA structures. A very long hairpin structure in the W repeat region that is conserved in lymphocryptoviruses contains at least one A-to-I editing site, with possible implications for splicing. Additional regions span splice sites or start and stop codons. These structure predictions may prove useful since RNA and RNA structures are attractive targets for chemotherapy -111. TheRNA-Seq data identified several putative sisRNAs, including one in the W repeat region (ebv-sisRNA-1) that is abundant and localizes in the nucleus. These results lay the groundwork for future experiments to further characterize the proposed structures and to understand the functional significance of the discovered RNAs and RNA structures in EBV. All structural data are available in the Additional files. In addition, data for the W repeat long hairpin and ebv-sisRNA-1 will be deposited in the Rfam database.EBV RefSeq genomes from four different viral strains, EBV-1 (NC_007605.1), EBV-2 (NC_009334.1), GD1 (AY961628.3), and GD2 (HQ020558.1), plus one closely-related genome from Macacine herpesvirus 4 (NC_006146.1), were obtained from the NCBI nt database. Genome sequences were aligned using the MAFFT program ,50 impleFrom each predicted locus the EBV-2 sequence was extracted and used in a BLAST  search a2D renderings of modeled RNAs were generated using the PseudoViewer program  and manu2 in RPMI media supplemented with FBS (10%) and penicillin [EBV-positive BJAB-B1 cells were maintained at 37°C and 5% COnicillin . Cells tnicillin  (10 mM Knicillin  (data gi2O). The membrane was then incubated overnight at 37°C with 5′-radiolabeled DNA probe complementary to nts 37 to 66 of the 106 nt human U6 snRNA (NR_046494.1), probe sequence: 5′-GCAGGGGCCATGCTAATCTTCTCTGTATCG-3′. The blot was washed 3× in wash buffer (0.005% SDS and 3× SSC), wrapped in plastic film, exposed to a phosphor screen for 16 hrs and imaged on a Storm phosphorimager. The blot was rehybridized with 5′-radiolabeled DNA probe complementary to nt 19 to 46 of ebv-sisRNA-1, probe sequence: 5′- TGTGGTGGAGTGTTGGGCTTAGCAGAAA -3′.20 μg of total RNA from EBV-positive BJAB-B1 and EBV-negative (but otherwise isogenic) BJAB cells were fractionated by denaturing 10% PAGE. RNA was transferred to an Amersham Hybond-N + positively charged nylon membrane then cross-linked to the membrane using UV radiation. The membrane was incubated at 37°C for 1 hr in pre-hybridization buffer (GE) and washed 3× in double-distilled water  initial denaturation at 90°C for 10 min, (ii) melt at 90°C for 15 s, (iii) anneal at 53°C for 30 s, (iv) extend at 60°C for 30 s, (v) cycle back to step ii 40×. Amplification products were analyzed by denaturing 10% PAGE and stained with ethidium bromide. Amplicon sizes were determined by comparison to a 10 bp ladder (Invitrogen).For the RT-PCR and RT-qPCR analyses, 2 μg nuclear or cytoplasmic RNA were used to generate cDNA with the High Capacity cDNA synthesis kit (Applied Biosystems). 5 μl 100× diluted cDNA was used for PCR with EBER1 primers complementary to the transcript’s 5′ and 3′ ends: FWD 5′-AGGACCTACGCTGCCCTAGA-3′ REV 5′-AAAACATGCGGACCACCAGCTGG-3′, and ebv-sisRNA-1 primers complementary to the 5′ end and nt 52 to 71: FWD 5′-GTAAGTGGACTTTAATTTTTTCTGCTAAGCCC-3′ REV 5′-TGGGTGTGTGTAGTGTGTGC-3′. Template was combined with 5 μl 10× PCR buffer (NEB), 1 μl 10 mM dNTPs, 1 μl each of 10 μM primer and 0.25 μl Taq (NEB) and ddH2O and each primer . 45 μl aliquots were added to a 96-well plate before adding 5 μl 100× dilute cDNA . In addition to the ebv-sisRNA-1 primers, amplification of nt 8 to 104 of the nuclear-enriched U2 snRNA (NR_002716.3) used primers: FWD 5′-CTCGGCCTTTTGGCTAAGAT-3′ REV 5′-TATTCCATCTCCCTGCTCCA-3′; and nt 994 to 1071 of the cytoplasmically-enriched 18S rRNA (NR_003286.2) used primers: FWD 5′-CGAAAGCATTTGCCAAGAAT-3′ REV 5′-GCATCGTTTATGGTCGGAAC-3′. The same thermocycler protocol used for PCR was used for the qPCR run, and data was collected on an Applied Biosystems StepOnePlus Real-Time PCR system. Data were exported to Microsoft Excel and analyzed using the Delta Ct method to find the ratio of nuclear to cytoplasmic abundance.For RT-qPCR a master mix was made of FastStart Universal SYBR Green Master Mix (Roche), ddHAPSI: Average pairwise sequence identity; CaHV3: Callitrichine herpesvirus 3; CeHV12: Cercopithecine herpesvirus 12; EBV: Epstein-Barr virus; MHV4: Macacine herpesvirus 4; miRNA: MicroRNA; ncRNA: Non-coding RNA; nt: Nucleotide(s); sisRNA: Stable intronic sequence RNA; snoRNA: Small nucleolar RNA; v-snoRNA: Viral snoRNA; EBNA: Epstein-Barr nuclear antigen; Cp: C promoter; Wp: W promoter; ORF: Open reading frame; ADAR: Adenosine deaminase acting on RNA; LAT: Latency associated transcript; EBER: Epstein-Barr virus encoded RNA.The authors declare that they have no competing interests.WNM: design and execution of experiments, data analysis/interpretation, and manuscript preparation. JAS: data analysis/interpretation, manuscript preparation, and final approval of the version to be published. All authors read and approved the final manuscript.Table showing RNAz-predicted regions with likely stable and conserved RNA structure. Annotated regions (e.g. splice sites and miRNAs) are highlighted in different colors, which match color-annotated structure models provided in Additional file Click here for fileBED file with locations of RNAz-predicted structured regions.Click here for fileSpreadsheet showing structure models for predicted EBV structured regions. The sequence shown is for the EBV-2 strain (NC_009334.1). In EBV, transcription can occur from either DNA strand and results for the forward and reverse sense genome sequences are presented in separate worksheets (FWD_STR and REV_STR). RNA structures are in “dot-bracket” notation, with paired sites indicated with matched brackets and unpaired with dots. Colored regions correspond to annotated RNAs in Additional file Click here for fileStructure models and predictions of possible A-to-I editing sites (using the Inosine Predict program [) for lymphocryptovirus repeat long hairpin RNAs. Each virus has a separate listing with nt position numbered for just the hairpin, followed by the sequence, the secondary structure in dot-bracket notation, the calculated percent editing for four ADAR specificities, and the maximum predicted value of all ADAR specificities. A reported A-to-I editing site [ program ) for lyming site  is colorClick here for fileBED file with aligned RNA-Seq reads from BJAB-B1 nuclear small RNA sample.Click here for file


Secondary mutations restoring growth to a ridA mutant in the presence of serine were in dapA (encoding dihydrodipicolinate synthase) and thrA (encoding homoserine dehydrogenase). These mutations suppressed multiple ridA mutant phenotypes by increasing the synthesis of threonine. The ability of threonine to suppress the metabolic defects of a ridA mutant is discussed in the context of recent biochemical data and in vivo results presented here.The RidA (YjgF/YER057c/UK114) family of proteins is broadly conserved in the three domains of life yet the functional understanding of these proteins is at an early stage. Physiological studies of  Salmonella enterica lacking RidA display several characteristic phenotypes, including: synthesis of thiamine biosynthetic intermediate phosphoribosylamine (PRA), inability to grow on pyruvate as a sole carbon and energy source or in the presence of serine Salmonella enterica led to a general model in which RidA eliminated reactive products that were generated in normal metabolic reactions involving IlvA The RidA (formerly YjgF/YER057c/UK114) family of proteins is well conserved throughout the three domains of life. Members of this protein family have been implicated in a diverse number of phenotypes in a variety of organisms In vitro studies, which were informed by the phenotypic analyses, identified a biochemical function for the RidA protein family. RidA deaminated reactive enamine/imine metabolites generated by IlvA in vitro by this mechanism, which was consistent with the phenotype observed only in a ridA mutant.in vivo consequences of a ridA mutation are not understood in the context of the biochemical activity of RidA. The in vitro biochemical work characterizing RidA did not address the significance of the enamine deaminase activity in vivo or relate the previously observed phenotypes to the in vitro activity. Herein suppressor analyses dissected the basis of the other phenotypes caused by the loss of RidA in vivo. The data showed that threonine reversed many of the phenotypes of a ridA mutant of S. enterica. We propose that threonine outcompetes serine for the active site of threonine dehydratase (IlvA) thus preventing the formation of a deleterious serine-derived reactive intermediate that is normally removed by RidA.Aside from the IlvA-, TrpD-dependent formation of PRA, the S. enterica serovar Typhimurium LT2 and are listed with their respective genotypes in Strains used in this study are derivatives of 4No-carbon E medium (NCE), supplemented with 1 mM MgSO−1) were determined as µ =  ln(X/X0)/T where X =  OD at 650 nm and T =  time in hours during logarithmic growth.Cells from overnight cultures in NB medium were pelleted and resuspended in an equal volume of saline (0.85% NaCl), and an aliquot (0.2 mL) was used to inoculate 5 mL of the appropriate minimal medium. Cell growth was monitored as optical density (OD) at 650 nm over time at 37°C with shaking. Growth rates (in hint-201) Transductional crosses were performed using the high-frequency general transducing mutant of bacteriophage P22  were grown overnight in NB, centrifuged, and resuspended in the same volume of saline. 107 cells were spread on solid minimal glucose medium with 5 mM serine. Spontaneously arising mutations (∼10−7) that allowed ridA mutants to grow on serine were isolated after 36 hours at 37°C. A transposon (Tn10d(Tc)) genetically linked to the causative mutation in one strain was isolated by standard genetic techniques and used to reconstruct the mutant for phenotypic confirmation. The chromosomal location of relevant insertions was determined by sequencing using a PCR-based protocol 10d(Tc) insertion sequence and sequenced at the University of Wisconsin Biotechnology Center. Strains carrying suppressor mutations were reconstructed by transducing the relevant allele into dapA::cat (DM10460) and selecting for growth without diaminopimelic acid.To isolate mutants, independent cultures of dapA genes from strains DM3480, DM7604, DM7606, and DM11019 were amplified by PCR using Herculase II Fusion DNA Polymerase (Stratagene) and primers 5′ DapANdeI (GGGGCATATGTTCACGGGAAGTATTC) and 3′DapAXhoI (GGGGCTCGAGTTACAGCAGGCCAGC) and cloned into the pET20b vector (Novagen) at NdeI and XhoI restriction sites. Sequence analysis of each clone confirmed the presence of the N-terminal hexahistidine tag and the relevant lesion. The construct carrying the wild-type allele (pLD-dapA) complemented a dapA mutant (DM10460), indicating that the gene was expressed in this construct (data not shown).The E. coli BL21(AI) according to the manufacturer’s protocol (Invitrogen). Cells from the resulting cultures were broken at 15,000 psi in a French Pressure cell at 4°C. Cell debris was removed by centrifugation  for 30 min at 4°C. Proteins were purified using a column containing Ni-NTA superflow resin (QIAGEN) according to manufacturer’s protocol. Fractions containing DapA were concentrated at 30 psi under Argon gas using a 10,000 Da molecular weight cut-off membrane (Amicon). The protein was dialyzed in 0.5 M NaCl, 20 mM Tris-HCl, 5 mM imidazole, pH 7.9 and stored at −80°C. DapB was purified according to standard protocol using a hexahistidine-tagged dapB clone from the ASKA collection The wild-type and variant DapA proteins were overexpressed in DapA activity was measured in a coupled assay with DapB  following a published protocol 14C]-2-ketobutyrate (2-KB) formed from [14C(U)]-L-threonine. 200 µL reactions containing 100 mM Tris pH 8.0, 50 µM pyridoxal-5′-phosphate, 20 mM ammonium chloride, 1 mM dithiothreitol (DTT), and 2 µg purified IlvA were initiated with a final concentration of 40 mM [14C(U)]-L-Threonine (12.5 µCi mmol−1), incubated for 12 minutes at 37°C, and stopped with 0.5 mL 0.1% 2,4-dinitrophenylhydrazine in 2 N HCl. Derivatized [14C]-2-KB was extracted with 0.5 mL toluene and radioactivity from 200 µL toluene phase, representing quantity of [14C]-2-KB generated, was counted in 5 mL scintillation fluid using a scintillation counter (Packard).IlvA was assayed as previously described The transaminase B activity assay was based on previously described protocols 650 nm of 0.8, pelleted, and resuspended in 0.5 mL 100 mM HEPES pH 8.0 with 0.125 mM DTT. Cells were disrupted by sonication, extract was clarified by centrifugation, and total protein concentration was estimated by the method of Bradford The homoserine dehydrogenase activity assay was adapted from a previously described protocol ridA null mutant (DM3480) cannot grow on minimal glucose medium in the presence of 5 mM serine ridA that grew in the presence of serine were isolated. Using Tn10d(Tc) insertions to map the location of the mutations, each of the causative mutations was subsequently found to affect the dapA locus, encoding dihydrodipicolinate synthase (EC 4.2.1.52). ridA mutant in the presence of serine. Four lesions generated variant DapA proteins (DapAA563G was isolated twice), one affected the Shine-Dalgarno sequence and one was in the dapA promoter. Strains with each of the mutant alleles were reconstructed (DM11635–40) and were analyzed in liquid media for growth in the presence of serine. ridA mutant strains containing alleles dapA356, dapA357, or dapA358 grew similar to a wild-type strain in the presence of serine and are represented by strain DM11637 in ridA strain (DM3480) failed to grow after 12 hours as expected. The strain carrying a lesion 36 nucleotides upstream of dapA (DM11640) had limited growth with serine and was concluded to decrease transcription of the dapA gene. (The promoter of dapA from E. coli resides within a 70-base region upstream of dapA containing an extended −10 and −35 site ridA dapA359 (DM11639), was indistinguishable from the parental strain on minimal glucose medium (data not shown). The dapA359 allele encoded a variant with two deleted amino acid residues and despite growth on solid medium with serine, growth was not detected in liquid media after 24 hours in the absence of exogenous diaminopimelic acid (DAP).A dapA were cloned into the pET20b vector to generate C-terminal hexahistidine tagged proteins, creating pLD-dapA, pLD-dapAD188G, pLD-dapAS48F and pLD-dapAΔ84–85. The recombinant proteins were purified by affinity chromatography. Wild-type and variant proteins were assayed for dihydrodipicolinate synthase activity using a coupled assay The wild-type gene and each of three suppressor alleles of ridA mutant in the presence of serine. Complementation analysis eliminated the formal possibility that an altered function of DapA was responsible for allowing growth of a ridA mutant. When provided in trans, wild-type dapA eliminated growth of the ridA dapA356 mutant strain in the presence of serine and did not affect growth of a ridA mutant (data not shown).A simple interpretation of the above results was that decreased activity of DapA allowed growth of a dapA results in accumulation of ASA that allows a ridA mutant to grow in the presence of serine. ASA itself restored the growth of a ridA mutant in the presence of serine, supporting a role for this molecule in suppression of the ridA phenotype. As little as 0.5 mM ASA in the medium allowed a ridA mutant to reach full density in medium with 5 mM serine. Growth rate (µ) of ridA (DM3480) in the presence of serine (µ = 0.06±0.01) was restored by 1 mM ASA (µ = 0.55±0.03) and was the same as the growth rate of the same strain grown on minimal medium without serine (µ = 0.54±0.03). The nutritional requirements of an asd mutant , which cannot make ASA, DapA functions in the synthesis of some aspartate-derived amino acids and uses aspartate 4-semialdehyde (ASA) as a substrate . In one dapA alleles restored IlvE activity in a ridA mutant. The IlvE activity in the ridA strain carrying the dapA356 allele (230±7 nmol/min/mg) was restored to an intermediate level between the wild-type (303±13 nmol/min/mg) and ridA mutant strain (140±7 nmol/min/mg). This result suggested intracellular accumulation of ASA could impact the activity of IlvE in a ridA mutant. No evidence of a direct role for ASA in mediating phenotypic suppression was found. The activity of purified IlvE was not significantly affected by 10 min incubation with 10 mM ASA . Further, ASA had no detectable effect on the activity of threonine deaminase (IlvA) in vitro. While as little as 500 µM isoleucine inhibited IlvA, ASA failed to inhibit IlvA in vitro at a range of concentrations (0.1 µM –1.0 mM) (data not shown). These data showed that the effect of ASA was not due to mimicking the effect of isoleucine as a feedback inhibitor In addition to suppressing serine sensitivity, the dapA described above, a mutation in thrA (thrA1371), encoding aspartokinase I/homoserine dehydrogenase I, previously reported to suppress serine sensitivity of a ridA mutant G403D. The homoserine dehydrogenase activity in a strain with the ThrAG403D variant was indistinguishable from the wild-type parental strain. The location of the G403D substitution suggested the variant could be altered in allosteric interaction properties G403D variant was resistant to inhibition by threonine. Significantly, this effect was evident at a low of concentration of threonine, as would be expected under in vivo conditions where the threonine concentration was reported to be 0.2 mM G403D variant could increase conversion of ASA to homoserine in vivo, consistent with the above conclusion that metabolism of ASA is required for suppression.In addition to the alleles of ridA mutant to grow in the presence of serine ilvA alleles caused a detectable growth defect on minimal glucose medium (ilv operon ridA ilvA3210 (DM10009) and ridA ilvA3211 (DM11558) had the same growth rates as a ridA mutant (DM10010)  when grown in a minimal medium containing 5 mM serine and 1 mM ASA. These data suggested that ASA did not correct growth by increasing intracellular isoleucine levels.ASA is a biosynthetic precursor to isoleucine, which is known to allow a e medium . HoweverridA mutant with serine. Nutritional tests showed qualitative suppression of multiple phenotypes with both homoserine and threonine. Addition of exogenous threonine to the growth medium of a ridA mutant restored growth on serine , growth on pyruvate , and IlvE activity .Other metabolites in the pathway from ASA to the branch chain amino acids were considered and tested for their ability to suppress growth of a ridA mutant dapA and thrA generated increased cellular threonine levels. If the dapA and thrA mutations acted by increasing flux to threonine, they would be expected to increase the PRA formed in a ridA mutant. A purF mutant strain background was used to detect PRA, as it requires PRA to make thiamine and allow growth. The data in thrA and dapA suppressors increased growth of a purF ridA strain, and exogenous threonine further increased growth. These results supported the conclusion that flux to threonine was increased by these mutations. Additionally, since isoleucine has been shown to have the opposite effect and inhibit PRA synthesis in a ridA mutant dapA mutations were not increasing the synthesis of isoleucine. Considering the results of nutritional and suppressor analyses in total, threonine was identified as the metabolite that had a direct effect in suppressing the phenotypes caused by lack of RidA.Threonine is a precursor in PRA formation in a in vitro that RidA family members deaminate reactive enamine/imine intermediates generated by threonine dehydratases  in vivo phenotypes observed in a ridA mutant in S. enterica. Suppressor analyses identified an important role for threonine in attenuating multiple phenotypes of a ridA strain, including sensitivity to exogenous serine, lack of growth on pyruvate, and a decreased specific activity of IlvE.The RidA (YjgF/YER057c/UK114) family of proteins is highly conserved, but the diverse cellular defects caused by its absence are not understood ridA mutant by outcompeting serine in the IlvA active site. Threonine dehydratase (IlvA) was required for a number of ridA phenotypes mK for serine than for threonine  alleles and the feedback-resistant thrA (homoserine dehydrogenase) allele described here. These mutants not only demonstrated that increased flux to threonine was key to reversing the serine-sensitivity of a ridA mutant, but they also suggested that the primary control of threonine levels in the cell occurs at the homoserine dehydrogenase step and can be affected by increasing substrate (ASA) or decreasing the allosteric control of ThrA. This finding has important implications for metabolic engineering and groups endeavoring to generate organisms that overproduce threonine or downstream metabolites.The significance of threonine as a key metabolite that can modulate the ridA mutants. It has been shown that IlvA generates reactive enamine/imines that are removed by RidA in vivo in the absence of RidA can have either deleterious or productive consequences, depending on the substrate used . Together these results suggest a complex role for IlvA in the in vivo phenotypes of ridA mutants. Continued studies are needed to identify the diversity of both the reactive metabolites eliminated by RidA and the targets of these reactive intermediates to better understand the breadth of metabolic consequences that result from the lack of the conserved RidA protein.The findings herein emphasized the central role of threonine in compensating for the lack of RidA. In combination with past results, these data refine a model to explain the phenotypes of


Establishment of an infection cycle requires mechanisms to allocate the genomes of (+)-stranded RNA viruses in a balanced ratio to translation, replication, encapsidation, and movement, as well as mechanisms to prevent translocation of viral RNA (vRNA) to cellular RNA degradation pathways. The ratio of vRNA allocated to various functions is likely balanced by the availability of regulatory proteins or competition of the interaction sites within regulatory ribonucleoprotein complexes. Due to the transient nature of viral processes and the interdependency between vRNA pathways, it is technically demanding to work out the exact molecular mechanisms underlying vRNA regulation. A substantial number of viral and host proteins have been identified that facilitate the steps that lead to the assembly of a functional potyviral RNA replication complex and their fusion with chloroplasts. Simultaneously with on-going viral replication, part of the replicated potyviral RNA enters movement pathways. Although not much is known about the processes of potyviral RNA release from viral replication complexes, the molecular interactions involved in these processes determine the fate of the replicated vRNA. Some viral and host cell proteins have been described that direct replicated potyviral RNA to translation to enable potyviral gene expression and productive infection. The antiviral defense of the cell causes vRNA degradation by RNA silencing. We hypothesize that also plant pathways involved in mRNA decay may have a role in the coordination of potyviral RNA expression. In this review, we discuss the roles of different potyviral and host proteins in the coordination of various potyviral RNA functions. Figure 1), consist of several substeps and a complex regulatory interaction network. In addition to viral proteins, each viral process engages several cellular proteins with either pro- or antiviral functions and it locates to a certain subcellular structure into which the viral RNA (vRNA) and the viral and host proteins involved need to be transported. This review will look at recent developments in understanding the trafficking and functional coordination of potyviral genomes to various pathways in an infected cell . Potyviruses comprise a very large group of (+)RNA viruses that infect cultivated plants all over the world. Similarly to many other (+)RNA plant viruses, potyvirus infection exploits the protein synthesis machinery of the host in the production of viral proteins; it exploits the endomembrane and cellular secretion systems in the formation of viral replication complexes (VRCs) and plasmodesmata (PD) to enable the spread of the viral genome to other cells (reviewed in The replication cycle of positive-stranded (+)RNA viruses involves a chain of several partially overlapping events. The main steps of the replication cycle, namely entry, translation, replication, cell-to-cell movement, antiviral defense/counterdefense, and encapsidation . Its subsequent release in the cytoplasm leads to initiation of the replication cycle. Second, the virus may enter a neighboring healthy cell from a previously infected cell, allowing initiation of translation. The establishment of infection within a cell requires a sufficient amount of vRNA to enter the translational machinery without being degraded. Encapsidated potyviral RNA is covalently linked to the viral protein genome-linked VPg; , which mTobacco etch virus  RNA proceeds to a certain position in the coat protein (CP) gene, which is followed by a cis-active replication element RNA serves as a template for both translation and replication. These two functions of vRNA are mutually exclusive, although the exact determinants responsible for the shift from translation to replication have yet to be identified for potyviruses. Interestingly, successful genome amplification and infection require that the translation of the  element . The aut element . NIb is  element . In addi element . A genom element . TherefoTurnip mosaic virus -induced vesicles have been shown to contain or associate with several host proteins, many of which function in translation, namely eukaryotic initiation factor 4E (eIF4E), eukaryotic elongation factor 1A (eEF1A), RNA helicase-like protein RH8, poly(A) binding-protein (PABP), and heat shock protein 70 (HSP70) ((+)RNA viruses, including potyviruses, induce host membrane rearrangements connected to the recruitment of vRNA and replication-associated proteins to assemble VRCs . The end (HSP70) . At a la (HSP70) . The latAgrobacterium infiltration  replication was tracked by quantitating viral gene expression with Renilla luciferase activity and RNA amounts by qRT-PCR a difference in these between wild type and replication-deficient PVA could be detected starting from day 2 after initiation of infection by Agrobacterium infiltration. By day 3 the virus had already spread and it had formed infection foci consisting of a substantial number of cells  that takes place in a narrow cell layer at the infection front  infectious cDNA (icDNA) showed that PPV (-)RNA accumulation reached the maximum at 12 h post-transfection and that RNA amounts decreased to less than 50% of the maximum by 24 h RNA is released to the cytoplasm, whereas viral (-)RNA remains in the VRCs . The spaytoplasm . To be acisterna . Similarcisterna . This suPlasmodesmatas are channels that connect the cytoplasm and the ER of two adjacent cells. Plant viruses take advantage of these channels in their cell-to-cell movement, utilizing specialized virus-encoded movement proteins to modify the PD and to target and move vRNA through the PD. Potyviruses encode several proteins, which have a direct role in movement, namely P3N-PIPO, CI, and CP . These pPepper vein banding virus  and PVA virions are asymmetric and contain a tip structure at the VPg-containing virion end  VRCs gather and dock to the site of PDs , which is deficient in its RNA-binding and particle-formation capacity  belonging to the family of heat shock protein 40 (HSP40) chaperones RNA to new rounds of protein synthesis/replication. Similar to host mRNAs, vRNAs can be assumed to be associated with RBPs, which are required to protect the integrity of RNA, to suppress RNA degradation pathways, and to coordinate vRNA functions. Assuming that potyviral RNA is transported from VRCs in a similar manner to other (+)RNAs, one possible site for attachment of host proteins to viral RNP complexes is the moment when vRNA or its 5′end enters the cytoplasm. Interestingly, several host proteins that function in translation and/or mRNA regulation associate with (+)RNA VRCs. In the case of potyviruses, these include eIF(iso)4E, PABP, eEF1A, and RH8 . A limitThe determinants of efficient targeting of potyviral RNA in translation have long been discussed. At the heart of these discussions is the VPg -eIF4E/eIF(iso)4E interaction, which was discovered more than 15 years ago . This inin vitro and in vivo translation has been observed in the presences of ectopically expressed VPg with monocistronic template RNAs containing potyviral 5′UTRs, whereas those containing non-homologous UTRs lead to inhibition of translation . Increasing levels of VPg pull vRNA to translation, leading to concomitant down-regulation of cell-to-cell movement.Potyviral VPg appears to have a dual role in translation. Slight up-regulation of nslation . The inht manner . The trat manner . Therefoting RNA . This isting RNA . We propThe host factors eIF4E/eIF(iso)4E and P0, a ribosomal protein, are involved in VPg-mediated up-regulation of PVA gene expression . The aciIn vitro interactions between the Lettuce mosaic virus (LMV) VPg and helper-component- proteinase (HC-Pro), as well as LMV VPg and eIF4E of lettuce, have been identified (The mechanism of recessive resistance to potyviruses caused by incompatibility between eIF4E and eIF(iso)4E and potyviral VPg proteins has long been a puzzle. The resistance mechanism may be dependent on successful formation of an essential RNP complex because many potyviral proteins have the potential to overcome resistance conferred by translation initiation factors in various plants. In addition to the potyviral VPg , amino aentified . Researcentified  and inteentified . The intentified . Interacentified . Among tentified . Other pFigure 3). Such a suggestion is feasible, as conditions to support VPg-mediated translation pathway develop during infection. The more vRNA is translated: the more potyviral VPg is available. Some is transported to the nucleus in the form of NIa  aggregate in cytoplasmic bodies referred to as siRNA bodies . The RNAProcessing bodies (P-bodies) and stress granules (SGs) represent other types of RNA bodies formed in the cytoplasm of plant cells . NumerouPapaya ringspot virus (PRSV) increased when the 20S proteasomal activity in the host plant, papaya, was inhibited , PBB (β2), and PBE (β5) subunits  subunit  may affect potyviral RNA amounts in infected cells . UPS hasnhibited , indicatnhibited . Interessubunits  of the A subunit . The int subunit . The RNaV HC-Pro .Arabidopsis plants that carried a mutation in DCP2 was reverted upon TuMV infection . Previous work suggested that perinuclear globular structures that develop gradually during potyvirus infection provide a site for viral assembly . This coViral RNA trafficking within an infected cell is a tightly coordinated process, which includes many pathways, such as movement, translation/replication, RNA degradation, and encapsidation that compete for the vRNA produced in VRCs. The site of vRNA release from VRCs to the cytoplasm is likely the location of the RNP complexes which allocate functions to vRNA. The varying concentrations of viral and host proteins in different infection phases may be a key factor in determining the fate of vRNA. Coordination of vRNA may be achieved through competition between various RBPs or RNP complexes for the same regulatory elements. Many interesting links have been discovered between the potyviral RNA pathways . A sophisticated combination of research methods needs to be exploited to dissect the exact roles of various viral and host proteins in these interdependent pathways and to understand the timing and cellular location of each process. Investigations are required to better understand the molecular mechanisms underlying potyvirus infection as a whole and to identify host factors as potential targets for engineering potyvirus-resistant plants.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Quantification of microRNAs in specific cell populations microdissected from tissues can be used to define their biological roles, and to develop and deploy biomarker assays. In this study, a number of variables were examined for their effect on the yield of microRNAs in samples obtained from formalin-fixed paraffin-embedded tissues by laser microdissection.MicroRNA yield was improved by using cresyl violet instead of hematoxylin-eosin to stain tissue sections in preparation for microdissection, silicon carbide instead of glass fiber as matrix in RNA-binding columns, and overnight digestion of dissected samples with proteinase K. Storage of slides carrying stained tissue sections at room temperature for up to a week before microdissection, and storage of the microdissectates at room temperature for up to a day before RNA extraction did not adversely affect microRNA yield.These observations should be of value for the efficient isolation of microRNAs from microdissected formalin-fixed tissues with a flexible workflow. Laser microdissection (LMD)  is commo® LMD6000 system. For some experiments, areas of tissue sections were dissected out along with PEN membrane by hand using a surgical blade. To obtain replicate samples, morphologically identical quadrants of stained serial sections were cut. Dissectates were lysed with proteinase K and total RNA was extracted by affinity chromatography using the Ambion® RecoverAll™ Total Nucleic Acid Isolation, or Norgen Biotek® FFPE RNA Purification kits that respectively use silica or glass fiber (GF), or carborundum or silicon carbide (SiC) as the RNA-binding matrix. Total RNA, with microRNA in an amount expected to be a constant proportion of that of total RNA, was eluted from columns using identical volumes of water, and quantified using RiboGreen dye in a fluorescence assay [® TaqMan™ microRNA assays, based on reverse transcription-PCR (RT-PCR) [miR-16, an abundant and ubiquitous microRNA , a 45 base-long, housekeeping nucleolar RNA. Inter-group differences were analyzed using t tests assuming equal variances. P values determined in different statistical tests were two-tailed and a cut-off of 0.05 was used to appraise significance.We obtained FFPE tissues of human lung cancers or their xenografts grown in mice for this work. Tissues were cut into 8 μm-thick sections, which were then placed on glass slides covered with polyethylene naphthalate (PEN) membrane. The sections were deparaffinized and stained with either hematoxylin and eosin (H&E), or cresyl violet (CV), and used for LMD within a day with a pulsed ultraviolet laser on a Leicace assay , or by m(RT-PCR) , for micA , and RNRNU6-2 levels correlated well with total RNA estimations by RiboGreen assay with a Pearson coefficient of 0.91  whereas there was no significant correlation with total RNA quantifications by absorbance at 260 nm .An analysis of RNA preparations from 23 different dissectates from xenografts showed that E Figure . With SiRNU6-2 and miR-16 levels respectively were an average of 3.9 and 7.0 times higher with SiC columns than GF columns. When H&E was the stain, RNU6-2 and miR-16 levels respectively were on average 3.7 and 7.9 times higher with SiC columns than GF columns. These improvements in RNU6-2 and miR-16 yields, significant in paired t tests disregarding the histologic stain , could be because of differences in column design and not necessarily because of a better efficacy of the SiC matrix per se. Because of convenience during the staining step and with assessment of histological morphology, we decided to use H&E stain for the rest of the experiments of this study. As the goal of the experiments was to assess improvements in yield, the influence of downstream variables such as those during poteinase K digestion or storage of the microdissectates was expected to not deny superiority of CV stain over H&E.The efficacies of the two types of RNA-binding columns that used wither GF or SiC as the RNA-binding matrix were also compared. For this, proteinase K lysates were prepared from dissectates from three xenografts and divided into two equal portions, each of which was used for the two types of columns. As shown in Figure miR-16 assays of the RNA preparations showed that RNA yields were not reduced at day 4 compared to day 0, or at day 7 compared to day 3  the slides were prepared or after a period of 3-7 days. RiboGreen and 3 Figure . This ob3 Figure . There wRNU6-2 and miR-16 measurements respectively were on average 1.5, 2.3 and 1.3 times higher when the duration was increased to 3 h at 55°C from 15 min at 55°C followed by 15 min at 80°C . Extending treatment time from 3 h to 20 h resulted in 1.7, 3.8 and 1.8 times higher RiboGreen, RNU6-2 and miR-16 measurements, respectively .As expected from previous studies on RNA extraction from FFPE tissues , RNA yi®. As shown in Figure 2 area. The average RNA yields from the 17 tumors of adenocarcinoma histology (78 ng/mm2) and from the 10 tumors of squamous cell carcinoma histology (92 ng/mm2) were not statistically different from each other (P = 0.24). RiboGreen assay of four different RNA preparations that were treated with DNAse I, RNAse A or neither at 37°C for 1 h showed that 37%-39% of the nucleic acids in the RNA preparations was DNA and not RNA  microarrays. With both 250 and 400 ng input, about 56% of the 1291 microRNAs detectable by the microarrays were identified as expressed. However, microarray signals were stronger with higher RNA input . Informed consent specifically for this study was not obtained from the participants as such a requirement was waived under the protocol.®, Durham, NC) into 8 μm-thick sections, which were placed on glass slides covered with a PEN membrane . Slides were dried overnight, de-paraffinized with xylene and rehydrated using a graded ethanol series  for staining with either CV , or H&E using Harris hematoxylin  followed by eosin Y  according to protocols provided by the manufacturers. Slides were then dehydrated using a reverse graded ethanol series and xylene, and used for LMD within a day. LMD was performed with a pulsed ultraviolet laser on an LMD6000 system (Leica®) at 50×-200× magnification with laser power, speed and specimen-balance settings of 98, 2 and 11, respectively, in a room with > 35% humidity. Dissectates were collected in 0.5 ml polypropylene tubes. The duration of LMD to obtain a dissectate sample varied from 15 to 120 min. Dissectates were also obtained by manually excising tissue sections along with the PEN membrane with a scalpel blade. Morphologically identical quadrants of serial sections were cut for replicate samples. All work was done with precautions to maintain an RNAse-free environment.FFPE tissues of human non-small cell lung cancer and their xenografts in immunodeficient mice were kindly provided by, respectively, the core pathology facility of RPCI, and Dr. Bonnie Hylander of the Department of Immunology, RPCI. Tissue blocks were cut on a CUT4055 rotary microtome , miRCURY™ Cell and Plant Tissue RNA Isolation , and FFPE RNA Purification  kits. All three kits contain spin columns with an RNA-binding matrix: ~0.01 g silica in case of RecoverAll™, and ~0.1 g SiC powder in the other two. The columns provided with the kits of Exiqon® and Norgen Biotek® are identical as Exiqon® procures the columns from Norgen Biotek®. Lysis of tissues and treatment with proteinase K at 55°C before a lysate was loaded on columns were done using reagents and instructions provided with the FFPE RNA Purification or the High Pure™ miRNA Isolation  kits. The concentration of proteinase K in the reactions set up as per the methods recommended for the two kits were 0.65 and 5.7 μg/μl respectively. Loading of lysates on a column and column washes were done using solutions and protocols supplied with the kit for that column. RNA was eluted from a column using either 50 or 100 μl water with the same volume used for all elutions in any given experiment.Total RNA was isolated using protocols and reagents supplied with the RecoverAll™ Total Nucleic Acid Isolation , with identification number 391, was used to measure microRNA miR-16. A similar assay was designed as per principles outlined in previous studies [RNU6-2 . Sequences  of the RT, and forward and reverse PCR primers, and the TaqMan™ probe were, respectively, GTCGTA TCCAGT GCAGGG TCCGAG GTATTC GCACTG GATACG ACAAAA ATAT (50 nM), GTGCAG GGTCCG AGGT (1 μM), GCAAGG ATGACA CGCAAA T (1 μM) and TATGGA ACGCTT CACGA (200 nM). For the RT-PCR assays, 5 μl each of RNA preparations were reverse transcribed using RNA-specific primers and reagents provided with the TaqMan™ MicroRNA Reverse Transcription Kit (Applied Biosystems®). RT reactions were used as templates in 40 cycle-PCR reactions on a 7900HT real-time PCR machine (Applied Biosystems®). Quantification cycle (Cq) values, approximately inversely proportional to log2 values of analyte RNA concentrations, were obtained with SDS™ software . The average of Cq values of triplicate PCR reactions was used for analysis. Cq values were > 40 for negative controls, for which RT reactions were performed without RNA. Cq values were subtracted from 40 to obtain measurements directly proportional to log2 values of analyte RNA concentrations.TaqMan™ MicroRNA RT-PCR assay  as per the method suggested by the manufacturer. Yeast tRNA (Ambion®) was used to prepare standards of known RNA concentration. RNA samples (1-4 μl) were diluted to 100 μl using 10 mM tris hydrochloride with 1 mM ethylenediaminetetraacetic acid at pH 7.5 , and mixed with 100 μl of the buffer with 200- or 2000-fold diluted RiboGreen . Fluorescence at 535 nm following excitation at 485 nm was measured for 0.1 s on a Victor Wallac™ 1420 plate reader . Unknown RNA concentrations were extrapolated from standard curves generated for yeast tRNA.Nucleic acid concentration in RNA preparations was quantified in duplicate with Quant-it™ RiboGreen RNA reagent . Ten μl of nuclease reactions were set up at 37°C for 1 h using 1 U of either enzyme, buffer provided by Fermentas® for use with DNAse I, and 8 μl of RNA preparation containing < 0.1 μg RNA as per RiboGreen assay. Control reactions using yeast tRNA (0.1-0.2 μg) confirmed completeness of the RNAse reactions and absence of RNAse activity in the DNAse I stock.Bovine pancreas RNAse A (DNAse- and proteinase-free) and recombinant DNAse I (RNAse-free) were obtained from Fermentas®  using their 6th generation miRCURY™ LNA™ microarrays. Each array had more than 2383 LNA capture probes for multiple RNAs of human, mouse, rat, and some viruses printed in quadruplicate on randomly distributed spots of 100 μm diameter with an inter-spot distance of 210 μm. A total of 1304 probes targeted 1291 human microRNAs, including 66 proprietary ones , and 23 non-microRNA human small RNAs with < 200 nucleotides, including the 5S ribosomal RNA and the RNU6-2 small nucleolar RNA (U6B). Every microRNA was recognized by only one of the 1276 probes for microRNAs. Eight probes recognized two microRNAs each, and three and six microRNAs were recognized by one probe each. For simplicity, the signals from such probes were considered as representing single microRNAs. Before hybridization to a microarray, 0.25 or 0.4 μg of an RNA sample, reduced in volume at room temperature in a speed-vacuum apparatus, and a human 'universal reference' total RNA preparation made by mixing the RNA pools provided in the FirstChoice® Human Total RNA Survey Panel  were 3'- or 5'-end-labeled with Cy3-like Hy3™ or Cy5-like Hy5™ (Exiqon®) dyes, respectively, using miRCURY™ LNA™ microRNA Hi-Power Labeling kits (Exiqon®). Microarrays were scanned for analysis using ImaGene® software . Examinations of the scans and analyses of microarray signal values for 52 spiked-in synthetic, small RNAs showed that all labeling reactions and hybridizations were of good quality. Hy3™ and Hy5™ signal values were processed with the limma [This work was performed as a commercial service by Exiqonhe limma . Microar®, La Jolla, CA), P value of 0.05 was the cut-off for deciding significance, and t tests were two-tailed, assumed equal variances, and used paired samples when possible.Unless specified otherwise, statistical analyses and graphical plotting were done in Prism™ software  were labeled with Hy5™ dye and the same amounts of RNA prepared from laser microdissected tissue using FFPE RNA Purification kit (Norgen Biotek®) were labeled with the Hy3™ dye, and co-hybridized to a locked nucleic acid microarray (Exiqon®). Fifty-two different synthetic artificial microRNAs were exogenously added to the RNAs before labeling. Scatter-plots of the Hy5™ and Hy3™ microarray signal values for the 52 spike-ins, and their linear regression lines (ordinary least squares method) are shown. The slopes of the lines are 0.70 and 0.81 for 250 and 400 ng RNA input, respectively, suggesting that the method used to isolate RNA from dissectates did not negatively affect the labeling and hybridization of the RNA.Click here for fileFigure S2. Validation of a custom reverse transcription (RT)-PCR assay for RNU6-2. A. Quantification cycle (Cq) values were determined for 40, 15 or 5 ng total RNA isolated from cells derived from the A549 human lung cancer cell-line. The TaqMan™ microRNA RT-PCR assay with ID 1093 from Applied Biosystems® (ABI) or a similar but custom assay for the RNU6-2 nucleolar RNA were used. The two assays were different for only the primers and probes. The linear regression line (ordinary least squares method) for the scatter-plot is also shown. The Pearson correlation coefficient is > 0.99 (P = 0.02). B. An ethidium bromide-stained agarose gel (2%) after electrophoresis of the RT-PCR products for the assays with 40 ng RNA input was transilluminated with ultraviolet light and photographed. Sizes of DNA molecular weight markers  in base-pairs (bp) are shown. The RT-PCR product expected in the custom assay has a size of 75 bp.Click here for file


The identification of A-to-I RNA editing sites has been dramatically accelerated in the past few years by high-throughput RNA sequencing studies. RADAR includes a comprehensive collection of A-to-I RNA editing sites identified in humans (Homo sapiens), mice (Mus musculus) and flies (Drosophila melanogaster), together with extensive manually curated annotations for each editing site. RADAR also includes an expandable listing of tissue-specific editing levels for each editing site, which will facilitate the assignment of biological functions to specific editing sites.We present RADAR—a rigorously annotated database of A-to-I RNA editing (available at  RNA editing is the post- or co-transcriptional modification of RNA nucleotides from their genome-encoded sequence. The most common type of editing in metazoans is the deamination of adenosine into inosine (A-to-I) catalyzed by the adenosine deaminase acting on RNA (ADAR) family of enzymes . ADAR enHistorically, the identification of A-to-I editing sites has been dependent on the sequencing technologies available at the time. When DNA sequencing technologies were first being developed and automated, the identification of editing sites was slow and often occurred serendipitously. The development and growth of nucleotide databases facilitated the identification of additional editing sites. In recent years, the advent of high-throughout RNA sequencing (RNA-seq) has enabled transcriptome-wide identification of RNA editing sites and has greatly accelerated the discovery of A-to-I editing sites.http://darned.ucc.ie)  (.ucc.ie) . Althoug.ucc.ie) , and to et al.  using the liftOver tool from the University of California, Santa Cruz (UCSC) genome browser . For eacWe designed a user-friendly web interface to query the database. The search page is displayed in An example results page is displayed in Tissue-specific editing levels from RNA-seq data ,29–31 arhttp://RNAedit.com.RADAR was built using the Django web framework coupled with a backend MySQL database. The web page was published using an Apache server hosted by Amazon Web Services. RADAR is freely accessible at The recent boom in A-to-I editing site identification has necessitated the development of RNA editing databases to help elucidate the biological functions of specific editing sites. The major advantages of RADAR over DARNED are the comprehensive compilation of A-to-I editing sites, the curation of extensive annotations and the gathering of tissue-specific editing level measurements for each editing site. RADAR contains ∼1.4 million human editing sites, which is a substantial increase over the ∼600 000 editing sites in DARNED. Furthermore, RADAR allows users to search for specific subsets of editing sites using any combination of five annotations: genomic location, gene, genic location, repetitive elements and/or editing conservation, whereas DARNED searches are restricted to sequence context or any combination of three annotations: genomic location, gene and genic location. Finally, the catalog of tissue-specific editing levels will help shed light on which biological contexts each editing site may be involved in. The major advantages of DARNED over RADAR are implementation of sequence-based searches, dbSNP identifiers and links to Wikipedia annotations. We are open to implementing similar features in RADAR if so requested by users.We anticipate that the continued development of high-throughput sequencing technologies will result in numerous new investigations into A-to-I editing in various physiological and pathological contexts. Recent evidence has already linked dysfunction of A-to-I editing with a myriad of human diseases such as cancer  and autoStanford Genome Training Program and Stanford Graduate Fellowship (to G.R.). The U.S. National Institutes of Health [GM102484 to J.B.L.]. Funding for open access charge: National Institutes of Health.Conflict of interest statement. None declared.


We found that two APE1 variants, L104R and E126D, exhibit a unique RNase inhibitor-resistant endoribonuclease activity, where the proteins cleave c-myc RNA 3′ of specific single-stranded guanosine residues. Expression of L104R and E126D APE1 variants in bacterial Origami cells leads to a 60–80% reduction in colony formation and a 1.5-fold increase in cell doubling time, whereas the other variants, which exhibit diminished endoribonuclease activity, had no effect. These data indicate that two human APE1 variants exhibit a unique endoribonuclease activity, which correlates with their ability to induce cytotoxicity or slow down growth in bacterial cells and supports the notion of their biological functionality.Apurinic/apyrimidinic endonuclease 1 (APE1) is the major mammalian enzyme in the DNA base excision repair pathway and cleaves the DNA phosphodiester backbone immediately 5′ to abasic sites. APE1 also has 3′-5′ DNA exonuclease and 3′ DNA phosphodiesterase activities, and regulates transcription factor DNA binding through its redox regulatory function. The human APE1 has recently been shown to endonucleolytically cleave single-stranded regions of RNA. Towards understanding the biological significance of the endoribonuclease activity of APE1, we examined eight different amino acid substitution variants of APE1 previously identified in the human population. Our study shows that six APE1 variants, D148E, Q51H, I64V, G241R, R237A, and G306A, exhibit a 76–85% reduction in endoribonuclease activity against a specific coding region of the c- Apurinic/apyrimidinic endonuclease 1 (APE1) is a multi-functional protein with established roles in DNA base excision repair and in activation of the DNA-binding capacity of several transcription factors Given the significant biological role played by APE1, it is of no surprise that investigations into single nucleotide polymorphisms (SNPs) and amino acid substitution variants have been conducted. To date, several APE1 human population variants have been identified, with the Asp148Glu (D148E) substitution being the most commonly observed, at a frequency of ∼45% Several human APE1 population variant proteins have been expressed and purified using recombinant techniques, and their AP-DNA endonuclease activity characterized myc mRNA in vitro, and to regulate c-myc mRNA levels and half-life in cells We have described the ability of APE1 to preferentially cleave at specific single-stranded regions of the c-myc RNA substrates. Moreover, both L104R and E126D variants exhibited distinct RNA-cleaving specificity, and when over-expressed, had a cytotoxic effect in a bacterial model system.We sought herein to analyze the RNA-cleaving activity of amino acid substitution population variants of APE1, specifically Q51H, I64V, L104R, E126D, D148E, R237A, G241R, and G306A. We show that most APE1 variants, including D148E, have a significantly diminished capacity to cleave single-stranded c-NdeI and EcoRI restriction sites of pET15b. The following primer pairs, synthesized by Integrated DNA Technologies (IDT) Inc. , were used for PCR site-directed mutagenesis to generate all the other pET15b APE1 variants: L104R, 5′-TCA GAG AAC AAA CGA CCA GCT GAA CTT-3′, 5′-AAG TTC AGC TGG TCG TTT GTT CTC TGA-3′; E126D, 5′-CCT TCG GAC AAG GAC GGG TAC AGT GGC-3′, 5′-GCC ACT GTA CCC GTC CTT GTC CGA AGG-3′; G306A, 5′-TCC AAG GCC CTC GCC AGT GAT CAC TGT-3′, 5′-ACA GTG ATC ACT GGC GAG GGC CTT GGA-3′; D148E, 5′-TAC GGC ATA GGC GAA GAG GAG CAT GAT-3′, 5′-ATC ATG CTC CTC TTC GCC TAT GCC GTA; I64V, 5′ GCC ACA CTC AAG GTC TGC TCT TGG AAT-3′, 5′-ATT CCA AGA GCA GAC CTT GAG TGT GGC-3′. Plasmid sequences were confirmed by DNA sequencing performed at Macrogen Inc. . Plasmids used for generation of recombinant L104R, E126D, D148E, R237A, G241R, and G306A, have been previously described pCMV6-XL5-APE1 plasmid was purchased from OriGene Technologies Inc.  and was used to generate plasmids pCMV6-XL5-L104R, pCMV6-XL5-D148E, pCMV6-XL5-E126D using PCR-based site-directed mutagenesis. pET15b-APE1 was used to generate pET15b-I64V plasmid. For generation of pET15b-Q51H, we sub-cloned a cDNA into The following recombinant APE1 variants, including the WT APE1 used for comparative studies, were over-expressed in bacteria and purified as described myc CRD RNA corresponding to nts 1705-1792, the plasmid pGEM4Z-myc 1705-1792 was linearized and in vitro transcribed as described in vitro endoribonuclease assay, the RNA was 5′-end radiolabeled with -[32P]-ATP using T4 polynucleotide kinase. For electrophoretic mobility shift assay (EMSA), internal labeling of c-myc CRD RNA nts 1705-1886 with α-[32P]-UTP  was performed during in-vitro transcription To synthesize human c-32P -radiolabeled RNA (∼5×104 cpm). Reactions were incubated for 25 min at 37°C unless otherwise indicated. Forty µl of loading dye  were added to the reaction samples, and then 10 µl of reaction mixtures were subjected to electrophoresis in 8% polyacrylamide, 7 M urea gel. Gels were then dried and subjected to phosphorImaging using a Cyclone PhosphorImager.The standard 20 µl-reaction mixture used for this assay included 2 mM DTT, 2 mM magnesium acetate, 1.0 unit of RNasin, 10 mM Tris-HCl, pH 7.4, and 25 nM (unless otherwise stated) of 5′-5′-GAGTCGTAACACGGTGAC-3′ were synthesized by IDT Inc. The oligonucleotide containing the AP site was 5′-end radiolabeled with -[32P]-ATP using T4 polynucleotide kinase. The reaction was stopped by heating at 95°C for 2 mins followed by hybridization to 7-fold molar excess of the anti-sense strand at room temperature for 60 mins and at 4°C overnight. The AP-DNA incision assay contains 15 µl reaction mixture consisting of 80,000 cpm (0.1 pmoles) of AP-DNA, 25 pg (0.7 fmoles) of APE1, 50 mM Tris–HCl (pH 8), 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 2 mM MgCl2 and 100 µg/mL bovine serum albumin. The reaction was carried out at 37°C for 3 mins. Thirty µl of loading dye  were added to the reaction samples, and then subjected to electrophoresis in 8% polyacrylamide, 7 M urea gel. Gels were then dried and subjected to phosphorImaging.The previously established protocol for APE1 AP-DNA endonuclease assay was used with minor modifications 32P] RNA sample was heated to 50°C for 5 min and cooled to room temperature before adding to the EMSA-binding buffer. EMSA-binding buffer containing radiolabeled RNA was then incubated with purified recombinant protein in a 20-µl reaction volume at 35°C for 15 min. A total of 2 µl EMSA loading dye  was added to each reaction and 10 µl of the EMSA reaction was loaded onto an 8% native polyacrylamide gel and resolved at 25 mA for 2 hrs. Gels were then dried and subjected to phosphorImaging.EMSA-binding buffers  were prepared on ice prior to each experiment. In order to facilitate RNA denaturation and renaturation, 30 nM of [E.coli Origami cell growth assay was successfully used to indirectly assess the ribonuclease activity of RNase A and angiogenin in bacterial cells in vivo ribonuclease activity of RNase A and angiogenin The E. coli cell density at OD600. All plasmid transformed cells were then equalized to a common cell count using LB-Ampicillin broth and subsequently transferred to 96-well tissue culture plates in triplicates. The initial OD600 was measured for each plasmid transformed cell type and recorded as the initial cell count at time point zero. The culture plates were then placed in an Innova40 incubator shaker set at 37°C and 200 rpm to facilitate cell growth. The micro-plates were removed from the incubator and the OD600 measured every hour after the initial reading for up to 12–15 hours. The data obtained was plotted in a growth curve using Kaleidagraph 4.0. Subsequently, the doubling time for each transformation was also determined using KaleidaGraph 4.0 using the equation fit parameter (e((0.693147/m1)*m0)*m2;m1 = 5;m2 = 0.05) which describes doubling time of bacterial cells during the exponential growth phase. The data collected from five biological replicates were then pooled and One-way ANOVA statistical analysis was performed.For growth assay in broth, the following procedures were undertaken. Individual Origami (DE3) colonies containing the respective empty pET15-b vector or pET15b-APE1 constructs were prepared. Single 100 µL aliquots of cells were transformed with 100 ng plasmid DNA as indicated above. Upon transformation, cells were plated on LB-Ampicillin plates and grown overnight. Individual colonies of each plasmid-transformed bacteria were then selected and picked using a sterile loop and transferred to 1 ml of LB-Ampicillin broth. The cultures were then grown for one hour at 37°C. The cell counts for each of the plasmid transformed cell types were then determined using a previously established standard curve for 32P-end-labeled 18-bp DNA duplex containing a model analog of an AP site, tetrahydrofuran (F)  containing 50 mM KCl, 10 mM MgCl2, 10% glycerol, 0.05% Triton X-100, and 100 µg/ml BSA As previously described, recombinant wild-type (WT) APE1 and specific APE1 variants found in the human population, specifically Q51H, I64V, L104R, E126D, D148E, R237A, G241R, and G306A, were purified to greater than 95% homogeneity uran (F) . In agreuran (F) . R237A suran (F) , which iuran (F) , which i32P-c-myc coding region determinant (CRD) RNA substrate relative to WT APE1 myc CRD RNA substrate, which was either 5′-radiolabeled with 32P-γ-ATP or uniformly labeled with 32P-α-UTP, we have specifically demonstrated the endoribonuclease activity of the recombinant WT APE1 protein in vitro is not unusual, as this has been shown for other endoribonucleases including PMR-1, IRE1, ARD-1, angiogenin, SMG6, and Rrp44 myc CRD RNA at the following sites: 1727CA, 1730UG, 1742CA, 1747UA, 1751UA, 1757UA, 1768CA, 1771CA. However, most variants, particularly D148E, R237A, G241R, G306A, I64V, and Q51H, exhibited a significant reduction (76–85%) in their ability to cleave c-myc CRD RNA as compared to the WT APE1 protein . Surprisingly, both L104R and E126D variants had significantly enhanced ability to cleave at an abasic site in RNA compared to the WT protein.We have previously demonstrated that WT APE1 also preferentially cleaves at UA, UG, and CA sites on additional RNA substrates, including CD44, orf1b, orf3, spike, miR-21, and miR-10b myc CRD RNA, we performed electrophoretic mobility shift assays (EMSA). We have previously optimized the conditions for binding of WT APE1 to c-myc CRD RNA and found that at a concentration range from 0 to 1412 nM, WT APE1 exhibits essentially linear binding myc CRD RNA in the EMSA buffer, despite the presence of the RNase inhibitor, RNasin.The reduced endoribonuclease activity of the APE1 variants could be due to a reduced ability to bind RNA and/or a deficiency in catalysis. To assess the capacity of the APE1 variants to bind c-myc CRD RNA endonucleolytic assay as performed above in myc CRD RNA were gradually reduced in the presence of increasing RNasin (0.25–1.0 unit) . Hence, in the experiments described below, protein production was not induced and was due to leaky expression. RNase A was used as a positive control, and We next compared the colony formation ability of Origami cells transformed with pET15b plasmids harboring a cDNA for each of the APE1 variants. We found that 300 ng of pET15b-Q51H or pET15b-I64V  Recombinant WT APE1 (lane 4) at 1.4 µM, and variant L104R (lanes 5–7), and variant E126D (lanes 8–10), each at 1.4, 3, and 6 µM, were incubated with 350 fmoles of 5′-32P-radiolabeled 34F AP-RNA for 30 minutes at 37°C in a total volume of 20 µl. Samples were run on a 12% polyacrylamide/7 M urea gel. For reference, an alkaline hydrolysis ladder was generated (lane 1) and RNase T1 (lane 12) and RNase A (lane 11) digest of the 34F AP-RNA were performed. Numbering on the left indicates guanosine residue sites cleaved by RNase T1 under denaturing conditions, as well as sites cleaved by RNase A. (B) Secondary structure of 34F AP-RNA and the cleavage sites generated by WT APE1 and APE1 variants L104R (left panel) and E126D (right panel). The box at the bottom of the figure indicates the strong and weak cleavage sites generated by APE1 and its variants. The oligonucleotide contains the model analog of an AP site, tetrahydrofuran (F). The asterisk in both (A) and (B) indicates the abasic cleavage site.(TIF)Click here for additional data file.


Saccharomyces cerevisiae presence of intact Sm binding site is required for the exosome-mediated processing of telomerase RNA from a polyadenylated precursor into its mature form and is essential for its function in elongating telomeres. Additionally, we demonstrate that the same pathway is involved in the maturation of snRNAs. Furthermore, the insertion of an Sm binding site into an unstable RNA that is normally completely destroyed by the exosome, leads to its partial stabilization. We also show that telomerase RNA accumulates in Schizosaccharomyces pombe exosome mutants, suggesting a conserved role for the exosome in processing and degradation of telomerase RNA. In summary, our data provide important mechanistic insight into the regulation of exosome dependent RNA processing as well as telomerase RNA biogenesis.A key question in the field of RNA regulation is how some exosome substrates, such as spliceosomal snRNAs and telomerase RNA, evade degradation and are processed into stable, functional RNA molecules. Typical feature of these non-coding RNAs is presence of the Sm complex at the 3′end of the mature RNA molecule. Here, we report that in  Small Nucleolar)RNAs) and mRNA splicing RNAs) are produced as precursor molecules that are trimmed from their 3′-ends S. cerevisiae and S. pombe, respectively).Once synthesized, RNA undergoes multiple processing reactions that often account for the diverse biological functions performed by RNA in the cell. Many cellular RNAs that play key roles in important cellular processes such as translation , the catalytic subunit responsible for both 3′→5′ exonucleolytic and endonucleolytic activities of the complex in both yeast and human 2,2,7mG-cap (Tri-Methyl-Guanosine-cap (TMG-cap) and an Sm binding site. This site provides a platform for the assembly of the heteroheptameric Sm complex, comprised of Sm protein products of SMB1, SMD1, SMD2, SMD3, SME1, SMX2 and SMX3 genes Ever-Shorter-Telomeres). The RNA binding subunit Est1, together with Est3 and Est4 (Cdc13) are also components of the telomerase complex. Telomerase RNA (TCL1) was reported to exist in two forms in S. cerevisiae: a shorter form representing the majority of TLC1 in the cell and a less abundant longer form. The longer form was hypothesized to be a precursor for the shorter (mature) form of TLC1Distinctive and conserved features of many snRNAs are both the presence of a 5′-Cryptic Unstable Transcripts (CUTs) in vivo cross-linking approach has revealed that Nrd1 is bound to 3′-extended TLC1 RNA suggesting that it might be processed by the exosome complex similar to other ncRNAs that are terminated by Nrd1 It is well established that the Nrd1-Nab3-Sen1 complex terminates RNA polymerase II (pol II) transcription of nc transcripts, such as snRNAs, snoRNAs and in vitro experiments using fractionated extract from mammalian cells demonstrated that the presence of an Sm site may contribute to 3′end formation of U1 RNA TLC1 RNA is processed by the exosome complex from polyadenylated (poly(A)+) precursor. Our data show that simultaneous disruption of the exosome function and the Sm binding site leads to stabilization of the unprocessed precursor telomerase RNA and snRNAs molecules suggesting that the Sm and exosome complexes might function in the same pathway. Interestingly, unprocessed telomerase RNA is not functional and its accumulation in cells leads to telomere length shortening. Furthermore, we demonstrate that an inserted Sm site leads to stabilization of Cryptic Unstable Transcripts (CUTs). We therefore hypothesize, that binding of the Sm complex leads to restriction of RNA degradation beyond the Sm site and imposes the processing mode of the exosome. We also show that the exosome regulates poly(A)+ telomerase RNA in S. pombe.Previous reports imply that binding of the Sm complex to ncRNA may play an important role for ncRNA biogenesis. In yeast an accumulation of unprocessed U1 precursor was observed upon mutation of the Sm site S. cerevisiae and S. pombe strains used in this study are listed in TLC1 and pRS316-TLC1, NotI/PstI and BamHI/EcoRI fragments of pSD107 were subcloned into the same sites of pRS315 or pRS316 respectively. Plasmids pAS501 (tlc1-s), pAS502 (tlc1-sm2T) and pAS503 (tlc1-sm4C5C) were kind gifts of Tom Cech NotI/PstI fragments were subcloned into the same sites of pRS315 to generate pRS315-tlc1-s, pRS315-tlc1-sm2T and pRS315-tlc1-sm4C5C.NTS1. This plasmid was digested with EcoRV and SacI and the NTS1 containing fragment was subcloned into the same sites of pRS423 to generate pRS423-NTS1. A 9 nucleotide sequence containing the Sm site was introduced by PCR-directed mutagenesis using oligos 2369 and 2370 to create pRS423-NTS1::Sm. Similarly, a mutated Sm site was introduced using oligos 2369 and 2373.The NTS1 CUT encoding region was amplified using primers rDNA#13_up and rDNA#17_down from YF336 genomic DNA and cloned initially into pTOPO vector (Invitrogen) to create pTOPO-32P] dATP using the Prime It II Random Primer Labelling system (Agilent). For Southern Blotting a 3′-end-digoxigenin-labeled 5′-TGTGGGTGTGGGTGTGGGTGTGGG-3′ oligonucleotide was used as a probe. Genomic DNA was resolved on 0.8% agarose gel.Northern blot experiments were performed as previously described TLC1 plasmid. A single 5-FOA resistant colony was propagated by 3 successive single-colony streak-outs (10–70 generations) on synthetic medium lacking leucine that maintained selection for pRS315-TLC1 and pRS315-tlc1-sm4C5C plasmids. Colonies that grew after every streak-out were inoculated into 10 ml of liquid synthetic medium and grown to saturation. Genomic DNA was prepared by bead-beating and phenol-chloroform extraction followed by RNAse A digestion according to the standard protocol XhoI cutting internal to the telomere in chromosomes containing Y′ elements, producing ∼1–1.3 kb diffuse band. Restriction digests were run on a 0.8% agarose gel and telomere length was examined by Southern blot analysis For telomere length analyses cells were plated on 5-fluoroorotic acid (5-FOA)-containing medium to counterselect for loss of the pRS316-TLC1 and tlc1-sm4C5C, rrp47Δ strains expressing ProteinA-Est2. The bead bound material was analyzed by western blotting and RT PCR using TLC1_up and TLC1_down (#2492 and #2493) oligos (5′-TGTGGGTGTGGGTGTGGGTGTGGG-3′), 0.05 µM 5′-[γ32-P]-labeled oligonucleotide, 100 µM dTTP, 1.5 µl [α32-P]dGTP (3000 Ci/mmol), 20 mM Tris-HCL, pH 8.0, 5 mM MgCl2, 50 mM NaCl, 5% glycerol, 0.5 mM DTT, 1 mM Spermidine, and 1 µl RNAsin (40 U/µl). The reactions were incubated for 1 h at 30°C, then were stopped by the addition of 250 µl Stop buffer  and incubated for 20 minutes with 10 µg of proteinase K at 30°C. Reaction products were extracted with phenol/chloroform/isoamyl alcohol (25∶24∶1), then ethanol precipitated overnight at −80°C with 2.5M NH4OAc, 10 mM MgCl2 and 1 µg of glycogen and resolved on a 15% acrylamide/7M urea sequencing gel.Telomerase complexes were isolated using IgG beads from ) oligos . TelomerS. pombe exosome complexes with and without the Rrp6 subunit were purified from 16L of yeast culture by consecutive two step affinity chromatography essentially as described in 32P] labeled synthetic AU-rich RNA oligonucleotide (AAUUAUUUAU UAUUU AUUUA UUAUUUAUUU AUUUAUUAUU UAUUUAUUA) and telomerase RNA isolated together with associated proteins as described above. In vitro RNA degradation reaction mixtures containing 10 mM Tris-HCl (pH 8.0), 1 mM DTT, 50 mM KCl, 1 U/µl RNasin (NEB), 5 or 0.05 mM MgCl2, 5 mM KHPO4, and 50 nM 5′[γ-32P] AU-rich RNA oligonucleotide were incubated with 8.2 nM of the purified exosome at 30°C for 5–60 min. The reactions were quenched by the addition of stop buffer containing 10 mM Tris-HCl (pH 8.0), 10 mM EDTA, 300 mM NaCl, 1% SDS and incubated with Proteinase K (10 µg) 37°C for 20 min. The reaction products were extracted with phenol/chloroform/isoamylalcohol (25∶24∶1) and ethanol precipitated. Reaction products were separated on 20% acrylamide, 7 M urea, 1×TBE gels and visualized by autoradiography and phosphorimaging. The telomerase RNA processing reaction was performed at 30°C for 90 min, reaction products were extracted as described above and analyzed by RT-PCR using primers: 2492, 2493 and 2489.TAP-tagged TLC1 in wild type (WT) cells and exosome mutants by northern blotting. In agreement with published data TLC1 RNA in WT cells but is enriched upon oligo-dT purification, consistent with previous report that it is polyadenylated rrp6Δ and dis3 exo- ) an accumulation of the poly(A)+ form of TLC1 is observed  that function in addition to the exosome complex A levels , lane 8.of Rrp47 , lane 7.TER1) accumulation in S. pombe exosome mutants. An interesting feature of TER1 RNA is that its 3′end is generated by an unusual one-step spliceosomal cleavage at a 5′ splice site that produces an active form of the RNA but is not followed by conventional exon ligation S. cerevisiae, the poly(A)+ form of TER1 RNA accumulates four- to three-fold in rrp6Δ and dis3-54 strains + TER1 RNA represents an un-spliced TER1 precursor or a processing intermediate resulting from the spliceosomal cleavage reaction that is normally degraded by the exosome complex. Alternatively, the exosome might be involved in TER1 3′end trimming and provide another processing pathway for TER1 maturation. Taken together, the above data indicate an evolutionarily conserved role for the exonucleolytic activity of the nuclear exosome in maintaining the levels of the poly(A)+ form of telomerase RNA, either through its degradation or processing into the mature form.To investigate whether the role of the exosome in maintaining proper levels of telomerase RNA is evolutionarily conserved we assessed the levels of telomerase RNA (TLC1 poly(A)+ RNA is also regulated by the cytoplasmic 5′–3′ RNA degradation machinery. Northern blot analysis of RNA from cells lacking Dhh1, involved in mRNA decapping demonstrated no significant change in the TLC1 poly(A)+ levels compared to the WT strain  in the temperature sensitive rat1-1 strain, perhaps reflecting a transcription termination defect caused by this mutation .Similarly, no effect on poly(A)+ RNA levels was observed upon mutation of Lsm2, a component of the Lsm1-7 complex that was previously implicated in stimulating decapping and 5′–3′ degradation of mRNAs in the cytoplasm TLC1 levels in the absence of Kap122, a nuclear importin known to be required for the cytoplasmic accumulation of TLC1TLC1 in the kap122Δ mutant, suggesting that re-import of TLC1 to the nucleus is not important to regulate TLC1 levels and processing.To further assess the significance of the cytoplasmic stage in telomerase RNA biogenesis we analyzed TLC1 RNA we reasoned that they may exist in a precursor-product relationship as initially proposed by Chapon and colleagues (4–6)GPu sequence known as Sm site positioned approximately 7–10 nt upstream of the mature 3′end of the RNA + form of telomerase RNA was recovered to the level observed in the single rrp47Δ mutant (TLC1 levels to the same extent (TLC1 supports a view that the poly(A)+ form functions as a precursor for mature telomerase RNA rather than being an independently transcribed form distinguished by alternative transcription termination. Disappearance of the shorter form upon accumulation of the longer (poly(A)+) form in the double mutant also implies that both the Sm complex and the exosome are needed to produce mature TLC1 and support a previously suggested view that the longer form is a precursor for the mature RNA. To test whether the two TLC1 forms observed in the exosome and the Sm site mutants are identical to the TLC1 species seen in WT, we performed RNase H experiments on total RNA from WT, rrp47Δ and sm4C5C cells. RNase H cleavage directed by oligonucleotides #2008 and oligo(dT) resulted in the formation of two major bands, a ∼450 nt product (RNase H product1) observed in WT and rrp47Δ (rrp47Δ and sm4C5C cells (TLC1 and a longer transcript To understand the relationship between the longer poly(A)+ and shorter ‘mature’ form of  the RNA . This se Sm site  in WT an the RNA , lane 5.e extent , lane 4.TLC1 RNA observed in Sm mutants is due to the absence of the modified cap. We analyzed TLC1 RNA in a tgs1Δ mutant that lacks the nuclear methyltransferase involved in TMG formation TLC1 RNA observed in Sm mutants.Another conserved feature of telomerase RNA, also present in snRNAs is a TMG-cap structure TLC1 processing upon metabolic depletion of the Sm complex component, Smd1 (TLC1 and U1 RNAs and on other non-Sm-containing cellular RNAs such as ADH1 mRNA and rRNA (TLC1 RNA was observed that is reflected by the appearance of the precursor band shifting the poly(A)+/poly(A)− ratio from 0.1∶1 to 0.8∶1 + and – RNAs remained the same + precursor form of TLC1 is competent of binding to Est2. This implies that neither Sm site nor TMG structure are essential for telomerase complex formation. It was previously shown that in WT cells poly(A) − RNA assembles with Est proteins and based on this data it was concluded that only mature molecule can form telomerase complex TLC1 in WT cells represents poly(A) − RNA and does not contradict with our data that poly(A)+ RNA can also bind to Est proteins. Incubation of the bead-bound telomerase complex isolated from the WT strain with a telomeric oligonucleotide, in the presence of dTTP and radioactively labeled [α32P]dGTP (the two nucleotides required for telomere extension) resulted in the appearance of bands characteristic of the extended oligonucleotide, that correspond to the products of the telomerase reaction + form of blotting . Telomer compare igure 2B.gG beads . Interesreaction , lane 2.lomerase , lane 1.TLC1 RNA into the shorter form we attempted to reconstitute the RNA processing reaction in vitro. The exosome core was purified from an S. pombe strain expressing a TAP-tagged Dis3 subunit. A mock purification from the non-tagged strain was performed in parallel  suggesting that like its S. cerevisiae and human counterparts Dis3 is a hydrolytic enzyme. The activity of the exosome core in the presence of Rrp6 was studied using conditions optimized for the exosome core. The RNA degradation assay performed in the presence of 0.05 mM MgCl2 with the Rrp6-containing exosome showed comparable levels of RNA degradation as the exosome core (2+ concentration to 5 mM in the presence of Rrp6 did not abolish the RNA degrading activity of the complex (data not shown). Therefore, we conclude that both of the hydrolytic enzymes Dis3 and Rrp6 are active as a part of the purified Dis3-TAP exosome complex. No activity was detected when reactions were performed with the mock purification.To further support the hypothesis that the exosome is involved in converting the poly(A)+ form of parallel . Mass spactivity . In the ome core . In contTLC1 RNA was purified from rrp47Δ, tlc1-sm4C5C rrp47Δ and WT strains and incubated with the purified exosome . Thus primer pair F/R2 amplifies both forms and can be used to measure the total levels of TLC1 RNA. While total levels of TLC1 associated with Est2 are the same for all three purifications (TLC1 form significantly vary depending on which strain the telomerase complex was purified from (TLC1 form associated with Est2 reflecting the low abundance of this form of RNA in the cell (rrp47Δ), much higher levels of the long form are detected to be a part of the telomerase complex  allowing for distinction from full size WT U1 RNA  compared to rrp47Δ alone  . Since UT U1 RNA  [20]. InΔ mutant , lane 4.observed , lane 2.RP51A pre-mRNA in U1Δ192–507sm and U1Δ192–507 sm, rrp47Δ strains. Cells were grown in galactose or glucose media to control the expression of WT U1 RNA. Northern blot analysis of RNA isolated from these mutants showed that in the presence of WT U1 nearly no un-spliced RP51A RNA is detected  region located in the repetitive rDNA loci rrp47Δ strains. No increase in NTS1 RNA was detected in cells expressing exogenous NTS1 in addition to endogenously produced transcripts  reporting the in vivo kinetics of TLC1 RNA accumulation nicely demonstrate a precursor-product relationship initially suggested in the earlier work of others using an inducible system for TLC1 expression TLC1 7 nucleotides downstream of the Sm binding site. Recent biochemical and structural studies support the view that the exosome substrates pass through the exosome channel to reach the Dis3 catalytic site TLC1 maturation. However, further analysis is required to test whether Rrp6 or/and other exonucleolytic activities are required for subsequent trimming to produce the fully mature form.Previous studies have implicated the exosome complex to function in multiple RNA processing reactions raising an important question: how does the exosome trim these RNAs to produce precise 3′ ends rather than degrading RNA to completion? A likely explanation is that the composition of RNP particles contributes to RNA fate determination by regulating access to the RNA by the exosome complex. We have presented evidence supporting the previously proposed hypothesis that yeast telomerase RNA is processed from the precursor RNA. Indeed, if the processing is blocked by mutating exosome activity there is an accumulation of the longer poly(A)+ ese RNAs . The higTLC1 sequence is dispensable for function The RNA moiety of the telomerase complex in yeast is much longer than its mammalian counterpart TERT RNA. Indeed, much of the rrp47Δ cells did not rescue the growth phenotype of U1Δ192–507 sm, demonstrating that the U1 precursor is not functionally active and must be processed by the exosome complex to produce a functional molecule. Additionally, the accumulation of unprocessed TLC1 poly(A)+ RNA in the cell resulted in shorter telomeres, an effect that was further enhanced in the rad52Δ background. Indeed, further analysis using a biochemical assay confirmed that telomerase complexes assembled with the longer form of telomerase RNA have a much lower activity in elongating telomeres than those associated with the mature RNA. Thus, the role of the Sm-exosome pathway in TLC1 processing appears to be of functional importance similar to its role in regulating the function of U1 RNA during mRNA splicing. It is not clear whether association of the Sm complex with ncRNA can regulate RNA function independently of its role in RNA processing. Binding of the Sm complex to RNA has been linked to TMG-cap formation in vivoTLC1 RNA is affected neither by absence of the Sm site nor of the associated TMG structure. We also show that TLC1 levels and processing are not affected in tgs1Δ cells, suggesting that telomerase function must be compromised at a different level. One possible reason for defective telomerase function in tgs1Δ could be the sequestering of RNAs lacking a TMG-cap in the nucleoli S. pombe has demonstrated a significant decrease in RNA levels as well as defective processing of the telomerase RNA by the spliceosome upon TGS1 deletion TER1) in S. pombe is produced as a longer polyadenylated precursor, similar to TLC1TER1 RNA is generated by a cleavage event that employs the spliceosome, a mechanism so far thought to be unique for TER1 RNA in S. pombeTER1 3′end formation via spliceosomal cleavage The accumulation of unprocessed U1 precursor in TLC1 in S. cerevisiae, the abundance of poly(A)+ TER1 RNA is increased in rrp6Δ and dis3–54 mutants. This raises the possibility that the exosome could act to process a minor fraction of TER1 RNA by trimming in a pathway parallel to spliceosomal cleavage to generate the 3′end of the mature RNA. In another, more likely scenario, the exosome could be involved in degrading the splicing intermediate or the functionally inactive fully spliced form of TER1. In this scenario, the exosome could act in concert with the spliceosome, a prospect previously suggested to be involved in the suppression of RNAs produced from centromeric heterochromatin in S. pombeDrosophila that demonstrated a direct role for the exosome in processing a micro RNA precursor following cleavage by the spliceosome Intriguingly, our data show that as it the case for TLC1 abundance TLC1 levels observed in the rrp47Δ mutant does not greatly affect telomere length. This could be explained if other components of the complex are not present in sufficient amounts in the cell to form functional telomerase complexes when TLC1 RNA is over-expressed.Is it important for cells to maintain appropriate levels of telomerase RNA? We and others have shown that in yeast the dramatic decrease in telomerase RNA levels observed upon mutation of its Sm site results in telomere shortening TLC1 and snRNAs that Tgs1 methyltransferase catalyses this reaction in the nucleolus prior to telomerase export to the cytoplasm TLC1 processing or of its levels. Moreover, we demonstrate that if re-import of TLC1 RNA is blocked by a kap122Δ mutation, the processing of poly(A)+ RNA is not affected. Finally, levels of the poly(A)+ RNA precursor are affected in rrp47 mutants but not in mutants of the cytoplasmic 5′–3′ RNA decay machinery suggesting that the nuclear exosome processes TLC1 RNA into poly(A) − RNA in the nucleus before it migrates to the cytoplasm. Perhaps shuttling of the telomerase to the cytoplasm is necessary to titrate down the amounts that are engaged in telomere extension and to eliminate “old” or damaged molecules.It has been well documented that the telomerase travels to the cytoplasm, however the biological significance of the cytoplasmic phase on the regulation of telomerase RNA remains unclear TLC1, co-purifies with Dyskerin, another RNA-binding protein, which is also found associated with H/ACA snoRNAs. Similar to the Sm site, the H/ACA box is situated just upstream of the 3′-end of the mature RNA. Mutating Dyskerin in murine embryonic stem cells was shown to cause severe destabilization of telomerase RNA, coupled with a reduction in telomerase activity. Interestingly, when hTR RNA is expressed in yeast, it co-purifies with Nhp2, Nop10 and Cbf5 (yeast counterpart of Dyskerin) proteins known to be a part of the core H/ACA RNPs and importantly, these proteins are also required for the accumulation of the mature hTR RNA cis-acting elements (sequences) and the trans-acting protein factors are required for H/ACA snoRNA processing and accumulation Our study provides the first mechanistic insight into exosome mediated processing of non-coding RNAs such as telomerase and sn/snoRNA in yeast. Given some evolutionary conservation of snRNA-like features in telomerase RNAs, it is probable that a similar mechanism operates in higher eukaryotes. Interestingly, hTR RNA, the human counterpart of In light of our data it is possible that H/ACA box proteins have a role in protecting snoRNAs from exosome mediated degradation similar to that of the Sm complex for snRNAs. We speculate that 3′end protection based on the presence of specific features such as secondary structures or sequences bound by RNA binding proteins represents a common and evolutionary conserved strategy adopted by RNAs that undergo 3′ exonucleolytic processing.Figure S1TLC1 RNA from rat1-1, Pgal::NRD1 (YLV115) and rat1-1, Pgal::NRD1, rrp47Δ(YLV154) cells. In this experiment, Nrd1 is expressed from a galactose-inducible promoter, and depletion occurs upon shifting to glucose media. Cells were grown in galactose containing synthetic medium to OD = 0.5 at a permissive temperature (23°C), and then maintained for an additional 2 h either on glucose or galactose followed by a 1 h temperature shift to 37°C to inactivate Rat1.Northern blot analysis of (PDF)Click here for additional data file.Figure S2dhh1Δ (YF1186), xrn1Δ (YF1694), ccr4Δ (YF1064), lsm2 (YF1926), and rrp47Δ (YF1465) mutant strains, alongside the WT (YF336) strain and analysed by northern blotting as described in lsm2 mutant strain and its WT control (YF336) that were grown at a permissive temperature (23°C) to OD = 0.4 and then shifted to 37°C for 1 hour. The radioactive RNA bands were visualised using a Fujifilm FLA-7000 phosphorimager, and quantified with AIDA software. Band intensities were normalised to Methylene-Blue stained 18 s rRNA that was quantified from a JPEG image using ImageJ software . Quantification of TLC1 RNA levels was done with the WT ratio set to 1. Values were calculated from three independent experiments, and error bars correspond to standard deviations.Poly(A)+ is not regulated by the cytoplasmic 5′–3′ RNA degradation pathway. Total RNA was isolated from (PDF)Click here for additional data file.Figure S3TLC1 RNA are not affected in kap122 Δ and tgs1Δ mutants. RNA was resolved on a 5% polyacrylamide gel and TLC1, U1 and ADH1 RNAs were visualized with the probes shown in Levels of (PDF)Click here for additional data file.Figure S4TLC1Saccharomyces cerevisiae (S.c.), together with TER1Schizosaccharomyces pombe (S.p.) were aligned manually. These RNAs each have an Sm site (boxed) close to the 3′ termini. The Sm site consensus sequence Comparison of the 3′ terminal sequences of mature snRNAs and telomerase RNAs from yeast. U1 (PDF)Click here for additional data file.Figure S5SMD1 construct. Transcription of SMD1 is driven from a galactose inducible promoter (GALp) and therefore when glucose is used as a carbon source SMD1 is not expressed. B) Schematic diagram describing the positions of ADH1 and U1 probes. U1 RNA is encoded by the SNR19 gene. Probes are depicted as black bars. C) Analysis of TLC1 processing upon Smd1 depletion. Northern blot analysis of total RNA from GALp::SMD1, rrp47Δ (YLV34), lanes 1 and 3; or GALp::SMD1 (YF182), lanes 2 and 4 was performed as described in SMD1 has been deleted and replaced with a plasmid borne-copy under the control of a galactose inducible promoter. Cells were grown in galactose containing synthetic medium to OD = 0.5, and maintained in logarithmic phase for a further 10 hours in media containing galactose (lanes 1 and 2), or glucose (lanes 3 and 4) A) Schematic diagram describing the plasmid encoded galactose inducible (PDF)Click here for additional data file.Figure S6TLC1 (URA3) together with either pRS315-TLC1 (LEU2) (YLV22) or pRS315-tlc1-sm4C5C (LEU2) in the context of a chromosomal TLC1 deletion were grown on media containing 5-fluoroorotic acid (5-FOA) to shuffle out the URA3 marked plasmid. Cells were passaged for 3 further restreaks on synthetic media lacking leucine (-LEU) to maintain selection for the LEU2 containing plasmid. For telomere length analysis genomic DNA was prepared from each restreak, corresponding to 10–15 generations, 40–50 generations and 60–70 generations respectively.Schematic illustration describing the analysis of telomere length. Strains carrying pRS316-(PDF)Click here for additional data file.Figure S7In vitro activity of the exosome complex. 50 nM of 5′[γ-32P] AU-rich RNA oligonucleotide was incubated with 8.2 nM of the purified exosome or mock purified sample at 30°C. Aliquots were taken at the time points indicated and reaction products were analysed by electrophoresis on a 20% acrylamide, 7M urea gel. C) S. pombe exosome core activity is Mg2+ dependent. Dis3-TAP exosome was purified by two-step affinity chromatography from an rrp6Δ strain (YP54). In vitro activity of the purified exosome complex (8.2 nM) was assayed with 50 nM of 5′[γ-32P] AU-rich RNA oligonucleotide in the presence of 0.05 (lanes 1–6) and 5 mM MgCl2 (lanes 8–12). RNA was incubated with the purified exosome at 30°C and aliquots were taken at the time points indicated. Reaction products were analysed by electrophoresis on 20% acrylamide, 7M urea gels.Purification of the exosome complex. A) Analysis of the exosome complex purification using tandem affinity chromatography (TAP). Elution fractions of the exosome purification from Dis3-TAP strain (YP53). Each step of the purification  was analyzed by 4–12% gradient SDS-PAGE alongside a mock purification from the WT strain (YP22). The position of the Dis3 protein attached to the remaining 3×HA-calmodulin binding module is shown with an arrow. Asterisks indicate contaminating bands common with the mock purification. Molecular weight markers were run in parallel (lane Mr). B) (PDF)Click here for additional data file.Figure S8rrp47Δ (YF1465) strains. B) Sm site insertion inhibits the exosome mediated degradation of NTS1 derived non-coding RNA. Northern blot analysis of NTS1 expression in WT (YF336); rrp47Δ (YF1465); NTS1 (YLV46); NTS1, rrp47Δ (YLV47); NTS1::Sm (YLV81); NTS1::Sm, rrp47Δ (YLV82); NTS1::sm4C5C (YLV93) and NTS1::sm4C5C, rrp47Δ (YLV94) (lanes 1–8 respectively). NTS1 RNA appears as a diffuse band, containing both plasmid derived (exogenous) and endogenous NTS1 transcripts. ADH1 and 18S rRNA bands are shown as loading controls.Insertion of an Sm site into an unstable RNA (NTS1 CUT) prevents its degradation by the exosome complex. A) Inserting an Sm binding site to the NTS1 CUT. Schematic representation of an rDNA unit showing NTS1 and NTS2, together with 5S (arrowhead) and 35S rRNA genes. A region of NTS1 encoding for NTS1 CUT (broken arrow) was cloned into a plasmid (shown in grey). A 9 nucleotide sequence corresponding to a WT Sm site (nts::sm) or mutated variant (nts::sm(4C5C) was inserted into the plasmid-borne copy at the position shown. Uppercase nucleotides indicate the inserted sequence; lowercase nucleotides show the flanking sequences in NTS1. The position of the NTS1 probe is depicted as a black bar. NTS1-encoding plasmids were transformed into WT (YF336) or (PDF)Click here for additional data file.Table S1Strains used in this study.(PDF)Click here for additional data file.Table S2Oligonucleotides used in these analyses.(PDF)Click here for additional data file.


Standard RNA sequencing approaches generally require double-stranded cDNA synthesis, which erases RNA strand information.Synthesis of a randomly primed double-stranded cDNA followed by addition of adaptors for next-generation sequencing leads to the loss of information about which strand was present in the original mRNA template. The polarity of the transcript is important for correct annotation of novel genes, identification of antisense transcripts with potential regulatory roles, and for correct determination of gene expression levels in the presence of antisense transcripts. Different strand-specific RNA-seq approaches have been developed to preserve information about strand polarity with different level of performances.Using Illumina Deep Sequencing Technology, this work investigates the performance of two different directional RNA-Seq (strand-specific RNA-seq) strategies. One is based on direct ligation of adaptors to the RNA ends and the other is based on the labeling and excision of the second strand cDNA. The RNA-seq workflows present in this work have been improved over current more laborious RNA-seq methods. Their low RNA input and streamlined workflows make them compatible with high throughput and automation. We also analyze the effect of different RNA fragmentation methods .We will provide a comparative full data analysis of different strand-specific RNA methods .Our results show improved methods for high-quality strand-specific RNA-seq library construction amenable to large-scale library construction and automation.