Naturally occurring cellular RNAs contain an impressive number of chemically distinct modified residues which appear posttranscriptionally, as a result of specific action of the corresponding RNA modification enzymes. Over 100 different chemical modifications have been identified and characterized up to now. Identification of the chemical nature and exact position of these modifications is typically based on 2D-TLC analysis of nucleotide digests, on HPLC coupled with mass spectrometry, or on the use of primer extension by reverse transcriptase. However, many modified nucleotides are silent in reverse transcription, since the presence of additional chemical groups frequently does not change base-pairing properties. In this paper, we give a summary of various chemical approaches exploiting the specific reactivity of modified nucleotides in RNA for their detection. Native cellular RNAs contain numerous modified residues resulting from specific action of various RNA modification enzymes. These RNA modifications are ubiquitous in nature, but the specific modification profile varies depending on the organism. Over 100 chemically distinct modified nucleotides have been identified so far mostly in tRNAs, rRNAs, snRNAs and some snoRNAs. From the chemical point of view, these modifications are highly diverse and almost any position of the nucleobases as well as the 2′-OH of the ribose has been found to be a target of modification enzymes  leads to abasic sites in RNA which are susceptible to aniline cleavage. The wybutosine base is also acid-labile and will undergo depurination upon treatment with hydrochloric acid to leave an abasic site , 7271, 7reatment , 74. Indor 8 min . The rem2i6A present in E. coli tRNA also retains a CMCT derivative after acylation [Recent extensive studies on pseudouridine mapping by CMCT treatment and MALDI-TOF MS pointed out that modified nucleotide mscylation . However5U residues to hydrazinolysis compared to U and most other U-derived bases phenyl-4-mercuric chloride  and acp3U [2t6A, m6t6A, g6A, hn6A, and ms2hn6A, chm5U, cmo5U, cmnm5s2U, cmnm5Um, cmnm5U, m1acp3Ψ, τm5U, and τm5s2U and possibly others.A number of modifications typically occurring at positions 20, 34, and 37 of tRNAs consist in the incorporation of an amino acid via its amino function, resulting in the presence in the modified RNA of a free carboxy group. This can be selectively activated for conjugation with amine reagents by dilute water-soluble carbodiimide, which will not react with the other nucleobases under these conditions . The prind acp3U , 46 but 4 [4 treatment revealed that D, ac4C, and s4U are reduced under rather harsh reaction conditions [4 is a ureidopropanol riboside  has a saturated pyrimidine ring due to reduction of the double C=C bond between carbons 5 and 6. This considerably affects the stability of D both in the reduction by NaBH4 –86 and u4 . Initialnditions , while mriboside . FurtherAnother characteristic of D is its particular sensitivity to alkaline hydrolysis. Hydrolytic ring opening  of dihyd1 does not distinguish inosine residues present in RNA from guanosine residues and cleaves on their 3′ side. However, preliminary specific reaction with glyoxal allows RNase T1 to distinguish G and I residues [N1 and 2N of G residues and thus abolishes their recognition by RNase T1. The resulting covalent adduct is stabilized by boric acid. By contrast, inosine cannot react in the same way with glyoxal due to the absence of NH2-group at position 2 and the glyoxal-inosine adduct is unstable. Thus, RNase T1 cleavage of glyoxal-treated RNA proceeds only at inosine residues and not at G residues. The cleavage positions may be detected by primer extension analysis or other methods.The modified nucleotide inosine (I) is derived from adenosine (A) by enzymatic deamination catalyzed by specific adenosine deaminases , 90. RNaresidues  phenyl-3-boronic acid (APB) into polyacrylamide gels  in yeast tRNAnslation , 94.4 reduction, or hydrolysis of phosphodiester bonds under various conditions. Analysis of the cleavage position can be performed with 5′-32P-labeled RNA and separation of the cleaved fragments by electrophoresis on denaturing PAGE. However, these techniques require difficult and time-consuming purification to homogeneity of a sufficient amount (at least 0.5–1 μg) of a given RNA species and thus cannot be applied to low-abundance cellular RNAs.Specific chemicals like those described above may be used both for detection and the precise localization of modified residues in RNAs. The techniques developed for RNA sequencing using Maxam and Gilbert's approach also allow for detection of modified nucleotides, since many chemical reactions that are specific for modified nucleotides may be exploited to induce cleavage of RNA polynucleotide chain, often in combination with further chemical treatment. This is the case, for instance, for hydrazine cleavage, NaBHAn alternative approach for the detection of RNA modifications is based on the use of RT. A synthetic DNA oligonucleotide designed to target a specific sequence is annealed to cellular RNA without previous RNA purification. Primer elongation by reverse transcriptase allows both sequencing of RNA species by Sanger type reactions and, in addition, the detection of specific reagent-dependent stops, which are indicative of the presence and exact position of modified nucleotides. A limited number of natural RNA modifications altering Watson-Crick base pairing can be detected by this approach even in the absence of a preceding chemical treatment. For RT-silent modifications, a specific reagent is required to create a bulky chemical modification, which blocks base pairing and consequently progression of the reverse transcriptase, thus resulting in an RT stop. Alternatively, cleavages of the RNA chain generated by specific treatment may also be detected by primer extension. However, interpretation of RT profiles should be undertaken with caution, since the presence of stops or gaps in the RT profile is frequently hidden by natural pauses of the RT at the corresponding positions. O-methylated residues in RNA and uses primer extension by reverse transcriptase at low dNTP concentrations [O-methylation. The comparison of RT profile for natural (modified) RNA and unmodified RNA transcript may help to discriminate modification-dependent from other RT stops. It is noteworthy that pauses observed at 2′-O-methylated residues are sequence dependent and, depending on the sequence context, no pause may occur at some of the 2′-O-methylated residues. Therefore, the method does not allow an exhaustive identification of all 2′-O-methylated residues in RNA molecules. In some instances, RNA sequence changes resulting from RNA editing  can also be directly detected by comparison of RT profile for unmodified transcript and natural RNA species.For some RNA modifications, a simple primer extension under specific conditions may also be used for detection. Such method was developed for the precise mapping of 2′-trations , 56. It by in vitro transcription with the cDNA sequence obtained with the authentic RNA. Another recently developed method that is suitable for transcriptome-wide analysis of inosine is based on specific cyanoethylation of inosine [Other methods employing RT are based on particular base pairing properties of some modified nucleotides. An alternative approach for inosine detection consists in direct RNA sequencing with RT and comparison of the cDNA sequence obtained with the corresponding genomic sequence. Due to the absence of amino group at position 6, I base pairs with C residues instead of U residues, thus change the sequence of the cDNA synthesized by RT. Thus, one can detect unexpected A → G changes in the cDNA compared to the genomic sequence . To conf inosine .Despite extensive development of specific chemical reagents during several decades (starting in the 1970s), the spectrum of available chemicals capable of selective reaction with modified ribonucleotides remains rather limited. This is related to a limited difference of chemical reactivity between modified nucleotides and their unmodified counterparts. Furthermore, the low stability of RNA molecules during required reaction conditions presents a stringent limitation. Due to these limitations, development of specific reaction schemes was possible only for a small subset of the known modified residues in RNA. Future development in the field should fill these gaps and propose new chemical reagents for extensive analysis of RNA modification by RT and by MS techniques.


Until recently, RNA metabolism has been considered a purely academic topic that kept a small group of molecular biologists busy. Thanks to many emerging techniques—mainly, but not exclusively—systems approaches that employ high-throughput sequencing, such as UV cross-linking and immunoprecipitation (CLIP), ribosome profiling and RNAseq, we gained major new insights into the importance of RNA metabolism for brain function, as well as malfunction Darnell, . In thatThe last decade marked an expansion of genome-wide experimental and computational techniques that provide unprecedented insights into the mechanisms and physiological relevance of post-transcriptional regulation in the brain, and how it can go awry in disease Darnell, . The newA remarkable feature of neuronal and glial cells is their morphological and functional diversity. Such specialized functions are achieved through highly complex gene expression programs. The brain exhibits the highest levels of alternative splicing and RNA editing  gene may affect the dendritic transport of BDNF mRNA, and thereby cause deficits in memory. This remains an area of intense research, with a recent study suggesting an anterograde, rather than retrograde mode of BDNF action (Dieni et al., A recent study identified as many as 2550 transcripts that are transported to either axons or dendrites (Cajigas et al., mRNAs localized to dendrites have a key function in synaptic plasticity. In response to synaptic stimuli, local control of mRNA translation near synapses is required to facilitate long-lasting forms of synaptic plasticity, the cellular basis for learning, and memory formation (Kapeli and Yeo, Mutations in RBPs, toxic RNA repeats, or other defects in post-transcriptional regulation contribute to a variety of neurologic diseases, especially motor neuron diseases (Ramaswami et al., Taken together, we feel that the present collection of reviews on the mRNA life cycle in normal brain function and malfunction provides a timely update by leading researchers to reflect recent developments in key technologies, and summarizes the current understanding and future directions for the studies of mRNA metabolism in the brain.


Xenopus laevis oocytes. The possibility that expression of the downstream cistron in these constructs was due to alternative splicing or to cryptic promoter activity was ruled out. The HIV-1 variants exhibited significant 5′UTR nucleotide diversity with respect to the control sequence recovered from pNL4.3. Interestingly, translational activity from the 5′UTR of some of the HIV-1 variants was enhanced relative to that observed for the 5′UTR of pNL4.3. In an attempt to explain these findings we probed the secondary structure of the variant HIV-1 5′UTRs using enzymatic and chemical approaches. Yet subsequent structural analyses did not reveal significant variations when compared to the pNL4.3-5′UTR. Thus, the increased IRES-activity observed for some of the HIV-1 variants cannot be ascribed to a specific structural modification. A model to explain these findings is proposed.The 5′untranslated regions (UTR) of the full length mRNA of the HIV-1 proviral clones pNL4.3 and pLAI, harbor an internal ribosomal entry site (IRES). In this study we extend this finding by demonstrating that the mRNA 5′UTRs of natural variants of HIV-1 also exhibit IRES-activity. Cap-independent translational activity was demonstrated using bicistronic mRNAs in HeLa cells and in  Upon recruitment to the mRNA, the 40S ribosomal subunit scans downstream along the 5′ untranslated region (UTR) until the initiation codon is encountered. Alternatively, an RNA structure termed the internal ribosome entry site (IRES) can drive 40S ribosomal subunit recruitment and positioning on the mRNA either at or upstream of the start codon Initiation of protein synthesis in the eukaryotic cell is a complex process that leads to the assembly of the 80S ribosome at the start codon of the mRNA Retroviridae and the etiologic agent of AIDS, plays key roles during the viral life cycle gag IRES) The 5′ UTR of the full length or genomic RNA of the human immunodeficiency virus type-1 (HIV-1), prototype member of the lentivirus genus of the Since its first characterization Renilla luciferase gene (RLuc) and a downstream firefly luciferase gene (FLuc) [10]. All plasmids were sequenced . Variant HIV-1 5′UTR sequences were aligned against the 5′UTR of pNL4.3, used in this study as the reference IRES [10]. Thirteen dl VAR vectors arbitrarily numbered dl VAR 1 to dl VAR 13 were randomly selected to continue the study. The sequence differences between the VAR 5′ UTRs and pNL4.3 are highlighted in , Illkirch, France) following well established protocols [23]. After 24 h total proteins were recovered and the FLuc and RLuc activities were measured as described in Retroviridae and some retroelements , and strongly support the notion that the 5′UTRs of all natural variants of HIV-1 recovered from clinical samples harbor active IRES elements. Additionally, the observed variability in RTA and, by inference IRES activities is difficult to ascribe to anything other than to 5′UTR sequence variability between clones  was therefore evaluated using a bicistronic mRNA approach similar to that described by Brasey et al. (2003) and Gendron et al. (2011). To generate bicistronic VAR vectors , naturaln clones . InteresStudies to identify internal initiation in isolated viral or cellular RNA regions utilizing the transfection of bicistronic reporter plasmids as described above have seen strong criticism Xenopus laevis oocytes, an experimental system that has proven useful for the study of viral IRESes n vitro synthesized, capped and polyadenylated RNAs corresponding to the dl ΔEMCV, dl HIV-1 IRES, and the dl VAR vectors were microinjected into the cytoplasm of Xenopus laevis oocytes . Together, these data provide substantial evidence supporting the presence of IRESes within the VAR 5′UTRs examined. Furthermore, our observations are in agreement with previous studies Xenopus laevis oocytes.Next we evaluated if this bicistronic mRNA could indeed drive cap- and IRES dependent translation initiation. After several unsuccessful results using RNA transfection assays in cells, we decided to address this question in  oocytes . RLuc an oocytes  as stand oocytes . The RLu oocytes . As desc oocytes . FLuc fr oocytes .B, demongain see  the trangain see . FindingThe firefly luciferase reporter gene has recently been shown to exhibit a cryptic promoter activity that is detectable both in yeast and in mammalian cells via a cryptic promoter.Careful analysis of the data presented in 32P-labeled primer. For each run the reverse transcription (RT) pattern of the modified RNA was compared to the profile obtained with a non-modified RNA. Modifications were classified into two categories according to the relative intensity of the induced stop by comparison with the control. Modifications were classified as “weak” when inducing a 2- to 3-fold increase in intensity of the RT stop, or as “highly reactive” for higher intensities. Typical results using DMS covering the full HIV-1 5′leader are shown  complexes ′UTR of the laboratory adapted HIV-1 infectious recombinant proviral clone NL4.3 (pNL4.3) into the intercistronic region of a dual luciferase (dl) reporter construct Renilla luciferase gene (RLuc) upstream and the firefly luciferase gene (FLuc) downstream. To ensure that the two cistrons were independently translated, a defective encephalomyocarditis virus IRES (?EMCV), known to inhibit ribosome reinitiation and read-through ′UTR ′UTR was monitored using FLuc activity as the readout, while the RLuc reporter gene served as an upstream translational control. This approach demonstrated that, when in the context of a bicistronic mRNA the HIV-1 5′UTR of pNL4.3 was capable of driving efficient translation initiation ′UTR of the CXCR4 (X4)-tropic primary isolate HIV-LAI ′UTR of pNL4.3 or pLAI, raising the question as to the significance and validity of these findings in the context of naturally circulating viral variants. Here we provide evidence that HIV-1 5′UTR sequences recovered from clinically sourced RNA isolates do indeed harbor an IRES, suggesting that the ability of HIV-1 mRNA to drive cap-independent translation initiation is likely to be widespread if not ubiquitous and is not restricted to the laboratory adapted HIV-1 clone pNL4.3 or to the single primary HIV-1 isolate, pLAI.The HIV-1 IRES was identified by cloning the 5′UTR of viral RNA isolated from HIV-1 infected individuals (VAR 5′UTRs) was evaluated in the context of a bicistronic mRNA ′UTRs was demonstrated in HeLa cells and in Xenopus laevis oocytes  ′UTRs, which, as shown herein share RNA secondary structure similarity with the pNL4.3 5′UTR . However, responsiveness to hnRNPA1 over expression was not equivalent for all VAR 5′UTRs  from the serum of HIV-1 infected patients, normally discarded upon viral load determination, was randomly pooled and used in this study as the source of viral RNA. Pooled RNA samples were provided by the Laboratorio de Infectología, Facultad de Medicina, Pontificia Universidad Católica de Chile.′UTR of natural variants were recovered from the randomly pooled viral RNA extracts by RT-PCR using the SuperScript™III one step RT-PCR system with platinum® Taq DNA polymerase  using the primers HIV1-F (5′-CACGAATTCGGTCTCTCTG-3′) and HIV1-R (5′-CCATGGTCTCTCTCCTTC-3′). All new sequences identified in this study can be found in GenBank (JN642552 to JN642564). The dl VAR vectors were generated as previously described E. coli DNA Polymerase I Klenow fragment (Fermentas) to generate blunt ends, and ligated using T4 DNA ligase (Fermentas). All plasmids were confirmed by sequencing .The dl ΔEMCV and the dl HIV-1 IRES plasmids were previously described 2 atmosphere.HeLa cells, similar to those used in previous studies 5cell/well in 12-well plates. DNA transfection was performed at 60% confluency by the JetPei system  according to the manufacturer’s protocol. For the hnRNPA1 over expression experiments, the well characterized hnRNPA1 expression plasmid (125 ng) TM Assay System (Promega) according to the manufacturer’s instructions on a Sirius Single Tube Luminometer .Cells were seeded at 102HPO4·7H2O, 1.4 mM KH2PO4, pH 7.4) at 4°C and lysed in 100 µL of RLNa buffer (10 mM Tris–HCl (pH 8.0), 10 mM NaCl, 3 mM MgCl2, 1 mM DTT, 0.5% NP40 and 10 U/mL of RiboLock™ RNase Inhibitor (Fermentas) for 2 min on ice. Supernatants were mixed with 1 mL of TRIzol® Reagent (Invitrogen) and total RNA was extracted according to the manufacturer’s instructions. Total RNA was resuspended in 20 µL of nuclease-free water and treated using the DNA-free™ kit  according to the manufacturer’s protocol. The integrity of the RNA obtained was confirmed by electrophoresis on 1% agarose gels and quantified by spectrophotometry (NanoDrop Technology). DNA was extracted from cells using the EZNATM kit  according to the manufacturés protocol. DNA concentration was determined by spectrophotometry (NanoDrop Technology).Cells were washed with phosphate-buffered saline  and Pforluc (′-CATGACTTCGAAAGTTTATGATC-3′5), 100 ng of total DNA and the Go Taq®Green Master mix (Promega), according to the manufacturer’s protocol. The RT-PCR assay was carried out with SuperScript™ III One-Step RT-PCR System using Platinum® Taq DNA polymerase (Invitrogen) according to the manufacturer’s protocol, using 1 µg of RNA and the primers described above.The PCR assay was conducted using primers p2anti (′-AGGTGAAGTTCGTCGTCCAACATTATC-3′5) and RLucAS (′-GAAACTTCTTGGCACCTTCAACAATAGC-3′5) for RLuc amplification (193bp); or FLucS (′- ACTTCGAAATGTCCGTTCGG-3′5) and FLucAS (′- GCAACTCCGATAAATAACGCG-3′5) for FLuc amplification (135bp), using the Brilliant® II SYBR® Green QRT-PCR Master Mix KIT . The amplification reactions were conducted under the following conditions: 30 min at 45°C for RT step followed by 10 min at 94°C, continued by 40 cycles of 20s at 94°C, 20s at 60°C and 30s 72°C. The melting curve was performed between 60°C and 90°C to verify the reaction specificity. For each experiment, the amount of RNA-RLuc and RNA-FLuc for each bicistronic RNA was determined (pmoles) based on an individual standard curve constructed using in vitro transcribed RNA as a template. Values were corrected considering the amplification efficiency of each couple of primers. The RNA-FLuc/RNA-RLuc ratio was used to compare the amount of bicistronic RNA in each transfection assay.The RT-qPCR assay was conducted using the primers RLucS , 0.04 U RNase inhibitor (Applied Biosystems/Ambion), and incubated for 120 min at 37°C. To eliminated the DNA template, 10 µL of DNAse RQ1 (Promega) was added, and further incubated for 20 min at 37°C. The RNA was precipitated using 2.5 M LiCl (−20°C for 30 min), centrifuged at 16000×g (30 min at 4°C), washed with 70% ethanol and resuspended in 50 µL of nuclease-free water. RNA concentrations were determined spectrophotometrically (NanoDrop Technology) and RNA integrity was monitored by electrophoresis on denaturing agarose gels.Capped RNAs were synthesized using the mMESSAGE mMACHINE High Yield Capped RNA Transcription Kit  and polyadenylated using the Poly(A) Tailing kit (Applied Biosystems/Ambion) according to the manufacturer’s protocol. Uncapped RNA was synthesized by Xenopus laevis ovarian fragments by manual dissection as previously described in vitro transcribed capped and polyadenylated RNA was microinjected into each oocyte with glass micropipettes calibrated to deliver a final volume of 50 nl Oocytes were isolated from ′UTR in vitro transcribed RNAs (10 pMoles), which included the 5′ leader of HIV-1 (pNL4.3) and the first 58 nt of fluc open reading frame (recovered from the dl HIV-1 IRES and dl VAR plasmids using a primer T7HIVF ′-CCATATGTAATACGACTCACTATAGGTCTCTCTGGTTAGA-3′5 and Fluc30bp ′-CATCTTCCAGCGGATAGAATG-3′5), was probed using DiMethyl Sulfate , N-cyclohexyl-N-[N-methylmorpholino)-ethyl]-carbodiimid-4-toluolsulfonate  and RNAse V1 (Applied Biosystems/Ambion) as described previously 2 were added. Following a10 min incubation at 30°C, DMS , CMCT  or RNAse V1 (0.01 or 0.025 U) was added and the mixture was incubated for 5 min (10 min for CMCT). Mock controls, where the chemical was replaced by water, were also included. The modification reaction was stopped on ice by addition of 10 µg of total yeast tRNA, and precipitated on dry ice with ethanol and 5 M ammonium. RNA was then resuspended in 0.5 M ammonium acetate, ethanol precipitated and resuspended in 6 µL of nuclease-free water. Modifications were revealed by reverse transcription  using 32P-labelled primer  according to the manufacturer’s instructions (Promega). Reverse transcription products were resolved on an 8% denaturing urea-polyacrylamide gel, the resulting gel was scanned on a Typhoon Trio Variable Mode imager (Amersham Biosciences). The relative proportion of each product was determined by drawing profiles using Multi Gauge V3 software (Fujifilm).The secondary structure of the HIV-1 5


There was an error in affiliation 1 for authors Peihua Ni and Yang Lu. Affiliation 1 should be: Department of Pharmacy, Shanghai Jiaotong University, School of Medicine, Shanghai, China.


Mammalian transcriptomes mainly consist of non protein coding RNAs. These ncRNAs play various roles in all cells and are involved in multiple regulation pathways. More recently, ncRNAs have also been described as valuable diagnostic tools. While RNA-seq approaches progressively replace microarray-based technologies for high-throughput expression profiling, they are still not routinely used in diagnostic. Microarrays, on the other hand, are more widely used for diagnostic profiling, especially for very small ncRNA , employing locked nucleic acid (LNA) arrays. However, LNA microarrays are quite expensive for high-throughput studies targeting longer ncRNAs, while DNA arrays do not provide satisfying results for the analysis of small RNAs. Here, we describe a mixed DNA/LNA microarray platform, where directly labeled small and longer ncRNAs are hybridized on LNA probes or custom DNA probes, respectively, enabling sensitive and specific analysis of a complex RNA population on a unique array in one single experiment. The DNA/LNA system, requiring relatively low amounts of total RNA, which complies with diagnostic references, was successfully applied to the analysis of differential ncRNA expression in mouse embryonic stem cells and adult brain cells. The high-resolution analysis of 1% of the human genome by the ENCODE project has shown that up to 90% of the genome is being transcribed while only about 1.5% of these transcripts correspond to protein coding exons , 3, withThese novel exciting aspects of the cellular transcriptome content thus require novel methods for profiling ncRNAs expression in a high-throughput manner. Lately, the most widely used expression profiling technique has become high-throughput sequencing or RNA-seq , 6, withTm of 72°C and covers all miRNAs of miRBase (version 9.2).The miRCURY LNA miRNA array ready-to-spot probe set (reference 208010) was purchased from Exiqon (Denmark) as an LNA capture probe set for short ncRNAs detection. This set comprises 2,056 capture probes designed to have a uniform Tm, desalted and diluted in 3xSSC, 1.5 M Betaine buffer to a final concentration of 20 μM.The DNA probes were purchased from Microsynth (Switzerland). They were 5′-C6 amino-modified, designed so as to comply with a 72°C The LNA-based capture probe set for short ncRNAs as well as the self-designed DNA-based capture probe set for long ncRNAs was spotted on HiSens epoxy-coated glass slides (Nexterion) using the MicroGrid II Microarray Spotter . Every probe  was spotted twice on the slide in four replicates  to ensure quality assurance and reliability.Hybridizations have been performed using the Tecan HS400 hybridization station according to the Exiqon protocol for hybridization with the miRNA LNA platform. Hybridizations were performed at 56°C or 64°C.The ncRNA chip was scanned using the Axon instruments GenePix 4000B.μg–5 μg) and total mouse embryonic stem cell RNA (2 μg) were directly labeled employing the NCode Rapid miRNA Labeling System (Invitrogen), following the manufacturer's protocol with the following modifications: (i) prior to poly-A tailing, RNA was denatured at 90°C for 3 min, centrifuged, and cooled on ice for 2 min and (ii) the reaction buffer provided with the kit was replaced by a custom reaction buffer containing 50 mM Tris-HCl (pH 8.0), 250 mM NaCl, and 10 mM MgCl2. For differential expression, 2 biological replicates of total mouse brain RNA and total mouse embryonic stem cell RNA were used.Total mouse brain RNA was extracted from C57/Bl6 mice (4–8 weeks old) and total mouse embryonic stem cell RNA from E14 stem cells with TriReagent (Sigma-Aldrich) following the manufacturer's protocol. RNA was quantified employing a nanodrop spectrophotometer (Fischer Scientific). Total mouse brain RNA  according to the manufacturer's protocol. Five hundred nanograms of total RNA were poly-A tailed and reverse transcribed to cDNA using the microRNA 1st strand synthesis kit  following the manufacturer's protocol. The cDNA was used as template for the real-time PCR. The universal reverse primer provided with the kit was used together with the following forward primers: mmu-miR-125-5p 5′-TCCCTGAGACCCTAACTTGTGA-3′, mmu-miR-293 5′-AGTGCCGCAGAGTTTG-TAGTGT-3′, SNORD113 5′-GGGTGCTGTATGAGTCGTGTATTATGA-3′, 7SK 5′-CCATTGTAGGAGAACGTAGGGTAG-3′, SNOZ39 5′-TGATGAAGCAAATCAGTATGAATAAAATG-3′, SNORA18 5′-TGACTCACAGGACTGACTGTTAGGCCTG-3′, SNORD55 5′-CACCTGGGTATGCTGCACACTCC-3′, SNORA71 5′-CTGCCGGTGCCCTGGTCATTG-3′, U6 5′-CTCGCTTCGGCAGCACA-3′.t-test was applied to compare between two groups. Differences were considered significant when P < 0.05.Primers were purchased from Sigma-Aldrich. Real-time PCR was performed using Power SYBR Green PCR Master Mix . Reactions were performed for 40 cycles with annealing step temperature set at 60°C. All results from three technical replicates were normalized to U6 and expressed as ΔCt values. Relative expression ratios were calculated by the ΔΔCt method . Five inWe investigated whether a combined DNA and LNA platform, dedicated to the expression analysis of long ncRNAs as well as small ncRNAs, respectively, could be used for the expression profiling of a heterogeneous population of ncRNAs. To that end, we generated a custom microarray spotted with (i) DNA capture probes for tRNAs, 7SK RNA as well as C/D and H/ACA box snoRNAs (Supplementary Table  1) and (ii) the commercially available miRCURY LNA miRNA ready-to-spot probe set from Exiqon. To generate a mixed DNA/LNA microarray, all probes spotted had to exhibit the same melting temperature. We opted for a fixed hybridization temperature to avoid elevated background due to unspecific hybridization, as observed when using temperature gradients  see the. 7SK RNAμg total mouse brain RNA for our initial proof of concept experiments. To exclude dye bias effects [2+ cations were reported to stimulate unspecific activity of poly-A polymerase activity in vitro [2+ custom poly-A tailing buffer  while labeling of 1 μg of RNA resulted in satisfying results  or two (MM2) positions. At 64°C, the snoRNA SNOZ39 was only detected by the antisense and one nucleotide mismatch capture probes, while the signal with the two nucleotides mismatch probe SNOZ39_6-60MM2 was falling below threshold , indicatμg of total mouse brain RNA and 2 μg of total mouse embryonic stem cells RNA. Hybridizations were performed at 64°C. As expected, differences in ncRNA expression between mouse adult brain RNA and mouse embryonic stem cell RNA could be detected  of labeled total mouse brain RNA. Diagrams of average intensity values show filtered results in logarithmic scale. The y axis represents values of AlexaFluor3 dye measurement at 532 nm and the x axis represents values of AlexaFluor5 dye measurement at 635 nm. Red spots represent all signals from LNA and DNA probes spotted on the microarray slide. Detection of 7SK RNA from antisense probes (a) without any modifications to the RNA labeling protocol, or (b) with the modified labeling protocol (see results). (c) Detection of tRNAs with the modified labeling protocol (see results).Supplementary Figure 1: Self-self hybridization at 56°C of 5 Supplementary Table 1: Sequences of the ncRNA corresponding DNA probes spotted on the microarray. Nucleotide mismatches are shown in bold and positions with deletions are underlined. Hybridization position of the probe on the ncRNA is indicated with numbers at the end of the probes name. Abbreviations: MM1: one nucleotide mismatch; MM2: two nucleotides mismatches; D1: one nucleotide deletion; D2: two nucleotides deletions; RC: sense probe.Supplementary Table 2: Filtered results corresponding to Figure 6. Fold changes are indicated as log2 values. Only ncRNAs differentially expressed above 2 folds are mentioned.Click here for additional data file.


With increasing life expectancy the number of people affected by multimorbidity rises. Knowledge of factors associated with health-related quality of life in multimorbid people is scarce. We aimed to identify the factors that are associated with self-rated health (SRH) in aged multimorbid primary care patients.Cross-sectional study with 3,189 multimorbid primary care patients aged from 65 to 85 years recruited in 158 general practices in 8 study centers in Germany. Information about morbidity, risk factors, resources, functional status and socio-economic data were collected in face-to-face interviews. Factors associated with SRH were identified by multivariable regression analyses.Depression, somatization, pain, limitations of instrumental activities (iADL), age, distress and Body Mass Index (BMI) were inversely related with SRH. Higher levels of physical activity, income and self-efficacy expectation had a positive association with SRH. The only chronic diseases remaining in the final model were Parkinson’s disease and neuropathies. The final model accounted for 35% variance of SRH. Separate analyses for men and women detected some similarities; however, gender specific variation existed for several factors.In multimorbid patients symptoms and consequences of diseases such as pain and activity limitations, as well as depression, seem to be far stronger associated with SRH than the diseases themselves. High income and self-efficacy expectation are independently associated with better SRH and high BMI and age with low SRH.MultiCare Cohort study registration:ISRCTN89818205. Multimorbidity is an issue of increasing importance to the health care system. The prevalence of multimorbidity rises from 10 percent in the 0 to 19 year-olds up to 78 percent in people aged 80 years and older [A persons’ own subjective rating of their health status was found to be an important predictor of morbidity and mortality, as well as a useful indicator of health-related quality of life ,3.Factors associated with health-related quality of life have been studied in the general population as well as in patient-populations suffering from different chronic diseases. Several studies indicated a positive association between higher education and income with better self-rated health (SRH) -6. The rAlthough a lot is known about factors correlated with SRH in the general population, knowledge of corresponding factors in aged multimorbid patients is scarce. Important questions remain: Is SRH in multimorbid patients more strongly affected by the presence of single disease states or by the sequelae of illnesses ? What are demographic , lifestyle  and psychological factors  associated with SRH in this patient group? Is SRH determined by the same factors in men and women or are there gender-specific differences? Therefore, the aim of this study was twofold: First, to identify the factors that are independently related to SRH in a multimorbid primary care sample of elderly people; and second, to identify possible gender-related differences in these factors.Multimorbidity is usually defined as the presence of two or more illnesses at the same time. In the inspection of the diagnosis distribution it however became clear that two or more chronic illnesses were present in practically all our elderly patients. We thereupon defined multimorbidity as the presence of at least three chronic illnesses. In addition, in order to ensure a large number of patterns of multimorbidity, the very frequent illnesses with a prevalence of over 25%  were not considered for the inclusion in the sample. Nevertheless, these highly prevalent diagnoses are frequently combined with the relatively lower prevalent ones and are therefore still part of the sample. A detailed list of the 29 diseases used for inclusion of multimorbid patients can be found elsewhere . This liData analyzed in this study came from the baseline investigation of the German MultiCare-Study , conductPatients were recruited in 8 study centers across Germany . In each city about 20 general practitiosners (GPs) were recruited and asked to provide the study group with a list containing all of their patients between 65 and 85 years (date of birth 1.7.1923 to 30.6.1943), who had at least one consultation in the most recent quarter.In each surgery, approximately 50 patients of those who suffered from at least three different chronic diseases out of a reference list of 29 chronic conditions  and did Exclusion criteria were:● Residence in a nursing home● Severe illness probably lethal within three months according to the GP● Insufficient ability to speak and read German language● Insufficient ability to consent (e.g. due to dementia)● Insufficient ability to participate in interviews ● Patients with no regular consultations and therefore poorly known to the GP● Participation in other studiesPatients who met the inclusion criteria and were willing to participate were visited at home or in the GP practice and interviewed by a trained investigator. A set of standardized questionnaires was used to collect variables belonging to the area of socio-demography, lifestyle, psychological and illness-related factors. Table SRH was measured with the visual analogous scale (VAS) of the EuroQoL-5D (EQ-5D) . With onSocio-demographic variables included age, gender, marital status and living conditions. Educational level was categorized as low, intermediate or high according to the CASMIN  classification , a certiLifestyle variables included physical activity (IPAQ-7) , alcoholPsychological variables ascertained in the patient-interview included perceived social support (F-SOZU K-14) , patientPatients were asked for the presence of chronic diseases. The interviewer read 32 chronic diseases  and uses this value for imputation . If moreM) and standard deviations (SD) were calculated for continuous variables and frequencies, as well as percentages, for categorical variables. Group differences were tested for statistical significance either by χ2-test or t-test as appropriate. Linearity of the relationship between independent variables and SRH was controlled by visual inspection. Bivariate associations between risk factors and SRH were analyzed by Pearson’s correlation and differences by t-test.The data were analyzed using the Statistical Package for the Social Sciences . Means  and by the Ethics Committees of the participating study centers. Written consent was obtained from every participant after being completely informed about the study.A total of 50,786 patients in the database of the participating GPs fulfilled the age criterion and had at least one GP contact in the last quarter. Out of those 24,862 were randomly selected and checked for the presence of at least three chronic diseases and exclusion criteria. After exclusion of the patients without multimorbidity and those who met the exclusion criteria, 7,172 patients remained and were contacted. Out of those 3,317 agreed to participate (response rate 46.2%). In total, data of 3,189 patients were included in the final analyses. The difference of 128 cases between the 3,317 patients who agreed to participate and the 3,189 whose data it was possible to include in the statistical analysis is due to the fact that patients died before they could be interviewed or that exclusion criteria became obvious only after sample selection. A more detailed description of sampling and response rate, as well as a non-responder analysis can be found elsewhere .SD 18.4) compared to 61.6 (SD 18.0) in women  correlations with the dependent variable SRH , age (p = 0.001), psychological distress (p = 0.01), a self-reported diagnosis of Parkinson’s disease (p = 0.003) and neuropathies (p = 0.008) had a significant negative effect on SRH. Significantly positive relationships were found between SRH and physical activity, independency in instrumental activities, higher monthly income  and self-efficacy expectation (p = 0.002).Table p < 0.001). Physical activity (p < 0.001) had a positive effect on SRH. Low or intermediate education (p < 0.001) and a self-reported diagnosis of cardiac arrhythmia (p = 0.004) were associated with reduced SRH in men. In women pain intensity, high BMI, distress , age (p = 0.001) and chronic low back pain (p = 0.004) were significantly negatively associated with SRH. SRH was better among women with higher monthly income (p = 0.009).Factors associated with SRH when multiple regression analyses were conducted for men and women separately are presented in Table p < 0.001), in women restrictions in instrumental activities (p < 0.001) resulted in significantly lower SRH values.In both genders a negative association between SRH and restrictions in activities of daily living was found. Whereas in men lower SRH scores were associated with restrictions in basic activities of daily living (The present study aimed to identify socio-demographic, lifestyle, psychological and disease-related factors associated with SRH in a large sample of elderly multimorbid general practice patients. To the best of our knowledge there are no studies that investigated SRH in multimorbid samples of comparable size and age.In our sample the mean EQ-VAS score was slightly below the general population’s mean score ,31-34, bWhereas in the bivariate analyses nearly all variables showed significant correlations with SRH, the final regression model for the whole sample included 13 of the initially more than 50 variables. Lifestyle variables like current smoking and alcohol consumption ,37 that In our multimorbid sample the association between socioeconomic factors and SRH seems to be weaker than in other samples . Age andNot surprisingly, among the disease-related factors single diseases were less influential for SRH in our sample than sequelae of diseases like pain intensity, disability caused by pain and restrictions in instrumental activities. It appears as if general factors like pain, disability, depression, somatic complaints, restrictions in physical activity and independent living, which accumulate in multimorbid patients due to the presence of multiple chronic diseases, affect SRH more than single self-reported diseases. Based on the fact that every patient in our sample having at least three diagnoses, it is not surprising that most diseases do not contribute independently to the explained variance of SRH. It might be, that in a general population sample with a lower prevalence of diagnoses the result would be different.Gender-specific analyses indicated that there are no differences in the relationship between somatization, depression, pain associated disability, and low physical activity and SRH in men and women. It seems that in both genders consequences of and complaints due to multimorbidity explain most of the variance of SRH. Besides these most important factors, we found different variables to be associated with SRH specifically in men and women. As found in a previous study , educatiMost importantly, we found SRH to be predominantly associated with modifiable factors. This suggests that SRH could be improved through specific interventions at the level of primary care. Main focus should be on modifiable aspects and consequences of multimorbidity: appropriate interventions of pain treatment and reduction; thorough exploration of somatic symptoms, since they could reflect sequelae of multimorbidity as well as potential side and interaction effects of polypharmacy. In order to improve SRH, physical complaints should be relieved, be it by improving patients compliance to or by adjusting the medication. Besides the reduction of pain and somatic complaints, depression provides another important starting point for improvement of SRH. The high burden of physical complaints and symptoms could make it difficult to reliably detect and diagnose depression in elderly multimorbid patients. Screening for and when indicated, treatment of depression should be standard in multimorbid patients. In addition, patients’ health-related behavior should be the target of interventions in order to improve SRH. Possible interventions are: participation on special training programs for elderly that focus on weight reduction and simultaneously increase physical activity; rehabilitation interventions for those patients who have restrictions in functional abilities.A major strength of our study was the large number of multimorbid general practice patients assessed that were spread over eight study centers distributed throughout Germany. In contrast to other studies of comparable size, which are generally based on postal or telephone surveys, our study data was collected by face-to-face interviews. In our study a larger set of variables was collected and tested for associations than in comparable studies. To enhance accuracy of the diagnoses used for inclusion of multimorbid patients, GPs’ diagnoses were used for selection of participants. Nevertheless, GPs’ diagnoses are also not entirely valid . DespiteWe decided to measure the health related life quality by means of a global visual analog scale. To this it could be objected that such a simple scale were not capable of representing all facets of the complex construct: life quality. According to Idler and Benyamini , howeverHowever, the present study also had some limitations. Most of the information was obtained through self-report, which may reduce the validity of the information. For example, questions about alcohol consumption, smoking behavior and physical activity may have been answered in a socially acceptable manner. Conversely, it can be seen as an advantage of our study that information regarding BMI was directly measured by the physician and the ability to perform instrumental and basic activities was rated by the interviewer based on the patients’ narration. Although electronic health records are more reliable in detecting multimorbidity in younger patients, the prevalence of multimorbidity in elderly patients appears to be the same for electronic health records as for self-reports . PatientSRH is a pivotal indicator of quality of life. The identification of factors influencing health-related quality of life in elderly multimorbid patients gains in importance in our aging society.We found the strongest correlation between SRH and disease sequelae, but only few diseases and only those with a high burden of symptoms or limitations were independently associated with low SRH. In women SRH seemed to be more strongly associated with pain, whereas, in men threats of limitations of activity seemed to play a larger role.In conclusion, perception of health and health-related quality of life at an older age might be improved by treatment of disease sequelae such as pain and discomfort. To enhance quality of life in the elderly, particular attention might be paid to the diagnosis and treatment of depression.AUDIT-C: Alcohol use disorders identification test; BMI: Body mass index; CASMIN: Comparative analysis of social mobility in industrial Nations; COPD: Chronic obstructive pulmonary disease; EQ-5D: EuroQoL-5D; F-SOZU K-14: Fragebogen zur sozialen Unterstützung; GCPS: Graded chronic pain scale; GDS: Geriatric depression scale; GPs: General practitioners; iADL: Instrumental activities of daily living; IPAQ-7: International physical activities questionnaire; M: Mean; OECD: Organization for economic cooperation; PAOD: Peripheral arterial occlusive disease; SD: Standard deviation; SPSS: Statistical package for the social sciences; SRH: Self-rated health; SWE: Self-efficacy expectation; VAS: Visual analogous scale; 4DBL: Four dimensional symptom questionnaire.The authors declare that they have no competing interests.AN and HB performed the statistical analyses and interpretation of the data, and drafted the manuscript. IS, BW, MS and HvB conceived and designed the study. GS and BW prepared the data for analysis. AD, AF, JG, HHK, SRH, WM and SW participated in study design and implementation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2296/15/1/prepub


Kaposi's Sarcoma-Associated Herpesvirus (KSHV) expresses a highly abundant, 1.1 kb nuclear noncoding RNA of unknown function. We observe that this polyadenylated nuclear (PAN) RNA avidly binds host poly(A)-binding protein C1 (PABPC1), which normally functions in the cytoplasm to bind the poly(A) tails of mRNAs, regulating mRNA stability and translation efficiency. During the lytic phase of KSHV infection, PABPC1 is re-localized to the nucleus as a consequence of expression of the viral shutoff exonuclease (SOX) protein; SOX also mediates the host shutoff effect in which host mRNAs are downregulated while viral mRNAs are selectively expressed. We show that whereas PAN RNA is not required for the host shutoff effect or for PABPC1 re-localization, SOX strongly upregulates the levels of PAN RNA in transient transfection experiments. This upregulation is destroyed by the same SOX mutation that ablates the host shutoff effect and PABPC1 nuclear re-localization or by removal of the poly(A) tail of PAN. In cells induced into the KSHV lytic phase, depletion of PAN RNA using RNase H-targeting antisense oligonucleotides reveals that it is necessary for the production of late viral proteins from mRNAs that are themselves polyadenylated. Our results add to the repertoire of functions ascribed to long noncoding RNAs and suggest a mechanism of action for nuclear noncoding RNAs in gamma herpesvirus infection.During the lytic phase of infection, the gamma herpesvirus  Kaposi's Sarcoma-Associated Herpesvirus (KSHV), a noncoding viral RNA is synthesized that resembles an mRNA in that it is transcribed by RNA polymerase II, is methyl-G capped at the 5′ end, and is polyadenylated at the 3′ end; yet this RNA is never exported to the cytoplasm for translation. Rather, it builds up in the nucleus to exceedingly high levels. We present evidence that the function of this abundant, polyadenylated nuclear (PAN) RNA is to bind poly(A) binding protein, which normally binds poly(A) tails of mRNAs in the cytoplasm but is re-localized into the nucleus during lytic KSHV infection. The interaction between PAN RNA and re-localized poly(A) binding protein is important for formation of new virus, in particular for the synthesis of proteins made late in infection. Our study provides new insight into the function of this noncoding RNA during KSHV infection and expands recent discoveries regarding re-localization of poly(A) binding protein during many viral infections.Almost all eukaryotic messenger RNAs (mRNAs) have a string of 150–200 adenylates at the 3′ end. This poly(A) tail has been implicated as important for regulating mRNA translation, stability and export. During the lytic phase of infection of  Kaposi's Sarcoma-Associated Herpesvirus (KSHV) is the causative agent of several human cancers and immunoproliferative disorders, including Kaposi's Sarcoma, Multicentric Castleman's Disease and Primary Effusion Lymphoma polyadenylated nuclear (PAN) RNA, also known as T1.1 or nut-1, begins to be synthesized at unusually high levels expression and nuclear retention element (ENE), serves to stabilize the RNA in the nucleus Upon KSHV entry into the lytic phase, an intronless viral noncoding (nc)RNA called poly(A)-binding protein C1 (PABPC1). PABPC1 normally functions in the cytoplasm where it binds the poly(A) tails of mRNAs, regulating their stability by either antagonizing or enhancing the activity of cytoplasmic deadenylases shutoff exonuclease (SOX) protein, as transient transfection of SOX into uninfected cells causes nuclear accumulation of PABPC1 in the absence of any other viral gene product To address how PAN RNA contributes to lytic infection of KSHV, we began by investigating protein components of the PAN RNP and identified Here, we demonstrate that PAN RNA interacts with PABPC1 after it has re-localized to the nucleus during the lytic phase of KSHV infection. In transient transfection experiments, SOX strongly upregulates the levels of PAN RNA by a mechanism that is dependent on the ability of SOX to mediate the host shutoff effect, on the re-localization of PABPC1 into the nucleus and on the existence of a poly(A) tail on PAN RNA. In infected cultured cells activated into lytic phase, PAN RNA expression is coincident with the host shutoff effect, and correlates with PABPC1 re-localization from the cytoplasm into the nucleus. Yet, knockdown of PAN RNA using RNase H-targeting oligonucleotides shows that it is not required for the shutoff effect or the nuclear re-localization of PABPC1. Instead, the amount of virus released into the supernatant of cultured cells is severely reduced upon knockdown. This striking effect on viral titer is explained by our observation that knockdown of PAN RNA adversely affects the expression of a subset of viral genes during the late stage of lytic infection.BCBL1 TReX-vector and TReX-RTA cells  were maintained in RPMI supplemented with pen/strep, L-glutamine and 20% tetracycline-compatible FBS (Clonetech). iSLK.219, 293 DC-SIGN  and 293T cells were maintained in DMEM supplemented with pen/strep, L-glutamine and 10% FBS. 293 tet-on cells were maintained in DMEM supplemented with pen/strep, L-glutamine and 10% tetracycline-compatible FBS. Anti-orf6/SSB was a gift from G. Hayward (Johns Hopkins University), anti-LANA1 and anti-orf4 were gifts from Y. Chang (University of Pittsburg), anti-K8.1 antibody was from Advanced Biochemicals Incorporated, anti-orf65 antibody was a gift from G. Miller , anti-myc antibody was from Santa Cruz Biotechnology. Electroporation was conducted in 0.4 cm electroporation cuvettes at 975 µF/220 mV for BCBL1 cells, and 975 µF/210 mV for iSLK.219 cells. 293T cells were transfected with MirusTransIT reagent according to the manufacturer's protocol. For some oligonucleotide knockdown experiments in BCBL1 TReX-RTA cells, cultures were first synchronized into S phase by serum starvation for 24 hours, followed by the re-addition of serum and incubation for 16 hours 2, 1 mM DTT, 10% glycerol, 1 mM ATP, protease inhibitor cocktail  and RNase Inhibitor (Roche), and syringe lysed by 10 passages through a 20G needle, 15 passages through a 25G7/8 needle and 20 passages through a 27G needle. Lysate was clarified by spinning at 14,000 x g for 10 minutes at 4°C, before nutating with 500 µL bed volume DEAE-sepharose resin that had been pre-equilibriated with lysis buffer. After 4 hours at 4°C, the resin was pelleted at 3000 rpm for 1 minute at 4°C, washed twice with lysis buffer, and subjected to successive batch elutions with increasing concentrations of KCl in lysis buffer. Maximal yield of the PAN RNP was observed in the 0.3 M KCl fraction. 1 mM MgCl2, 1.5 mM ATP, 5 mM creatine phosphate and 2–5 pmol/µL of biotinylated 2′-O-methylated anti-PAN oligonucleotides, complementary to nucleotides 70 to 89 and 993 to 1012 of PAN RNA, was added to 35 µL (175 µg) of the 0.3 M KCl fraction in a 100 µL total volume. 150 µL bed volume of streptavidin beads was pre-blocked at 4°C for 15 minutes in 100 µg/mL E. coli tRNA, 100 µg/mL glycogen and 1 mg/mL BSA, washed three times in 20 mM Tris pH 7.6, 50 mM NaCl and 0.01% NP-40. Extract was nutated with streptavidin beads at 4°C for 1 hour, before pelleting, removing the supernatant, washing beads twice with lysis buffer and eluting bound proteins by treatment with micrococcal nuclease  supplemented with 2 mM CaCl2 at 37°C for 30 minutes. Proteins were fractionated by 10% SDS PAGE; the PAN RNP-specific bands were excised and identified by mass spectrometry (Columbia University). Further details on all peptides identified in each band can be found at links provided in KSHV-infected BCBL1 TReX-vector or TReX-RTA cultures were grown to a density of 0.8 million cells/mL, and then treated with 2 ug/mL doxycycline for 24 hours. 100 million cells were pelleted, washed with PBS, resuspended in 500 uL 50 mM Tris-HCl, pH 7, 100 mM KCl, 10 mM MgClACAAATGCCACCTCACTTTGTCGC; SB85: CGCTGCTTTCCTTTCACATT; SB88: GTGAAGCGGCAGCCAAGGTGACTGG), which were labeled with digoxigenin-dUTP using the DIG oligonucleotide Tailing Kit (Roche), were hybridized with the samples in 50% formamide, 10% dextran sulfate, 2X SSC, 0.1% RNase-free BSA, 500 µg/mL salmon sperm DNA, 125 µg/mL E. coli tRNA and 1 mM vanadyl ribonucleoside complexes and detected using FITC-conjugated anti-DIG antibody . PABPC1 was visualized with anti-PABPC1 mouse monoclonal or rabbit polyclonal antibodies , and anti-mouse or anti-rabbit Alexa Fluor 594 antibody (Invitrogen). FLAG-PABPC1-NRS, a gift from B. Glaunsinger (UC Berkeley), was visualized with rabbit anti-FLAG antibody (Sigma) and anti-rabbit Alexa Fluor 594 (Invitrogen) BCBL1 TReX-vector or TReX-RTA cells were fixed onto glass slides pre-treated with poly-L-lysine, fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton-X. Anti-PAN RNA oligonucleotides  and 20 units/mL DNase One (New England Biolabs) for 1 hour at 37°C to degrade extracellular RNA and any viral DNA that had not been properly packaged into viral capsid protein and released into the supernatant. The supernatant was then spun at 15,000 rpm using a Beckman TLA 100.2 rotor for 2 hours at 4°C, and the resulting pellet was resuspended in 600 µL of lysis buffer  and incubated at room temperature for 10 minutes to inactivate the DNase. Following proteinase K treatment , 3 ng of a control plasmid DNA was added to each sample as a normalization control for loss of DNA during subsequent steps of the purification process. After extraction with phenol:chloroform:isoamyl alcohol (25∶24∶1), 600 µL of the aqueous phase was precipitated with isopropanol, sodium acetate and GlycoBlue (Ambion), and viral DNA levels were quantified using qRT-PCR. The viral DNA signal was normalized to signal from the control plasmid DNA .For measuring virus production from iSLK.219 cells, cells were induced with doxycycline for 2-3 days, and supernatant was collected and passed through a 0.45 micron filter. 50 µL of supernatant was added to 300 µL of DMEM +10% FBS and placed on 293 DC-SIGN cells that had been grown in 12-well plates. Cells were then spun at 2000 rpm for 1 hour at room temperature in a Beckman Coulter J6-MI centrifuge fitted with a JS-4.2A rotor BCBL1 TReX-RTA cells were electroporated and induced as above for 3 days. 5 million live cells were washed with PBS, resuspended in 250 mM Tris pH 8.5, 125 mM EDTA, 1 mg/mL protease K and 1% SDS, and incubated at 60°C for 2 hours. 1/5th volume of 5 M potassium acetate pH 5.2 was added, lysate was incubated on ice for 20 minutes, and clarified by centrifugation at 12,000 rpm for 15 minutes at 4°C. Supernatant was transferred to a fresh tube, and incubated with 20 µg/mL RNase A at 37°C for 15 minutes. Then, 2.5-fold volume of ice-cold 100% ethanol was added, and DNA was precipitated overnight at −20°C. Pellets were resuspended in 100 µL water, and viral DNA levels were measured by qRT-PCR as above, normalizing to human DNA using GAPDH-specific primers.To determine what proteins bind to PAN RNA in the nucleus of lytically infected cells, we isolated the PAN RNP from a KSHV-infected cell line containing a doxycycline-inducible version of RTA (BCBL1 TReX-RTA), the viral transcription factor that is necessary and sufficient to promote entry into the lytic phase of viral infection Since PAN RNA is exclusively nuclear, proteins that interact with PAN RNA must reside in the nucleus. While PABPC1 is essentially a cytoplasmic protein, nuclear re-localization of PABPC1 during KSHV lytic phase had been reported in the literature Additionally, we noted that unique peptides in the mass spectrometry data also identified PABPC4 as a protein that co-purifies with PAN RNA . RecentlSince SOX induces both host shutoff and the nuclear accumulation of PABPC1 The effect of SOX on PAN levels was also seen by immunofluorescence . When a The SOX protein is a dual-function protein, acting both as the mediator of the host shutoff effect and as an alkaline exonuclease that is important for processing the viral DNA To address whether nuclear localized PABPC1 alone can upregulate levels of PAN RNA, in the absence of SOX, we co-transfected genes for PAN RNA and a mutant PABPC1-NRS that is retained in the nucleus A previous study showed that although SOX and its murine herpesvirus-68 homolog reside in both the nucleus and cytoplasm, it is the cytoplasmic fraction of these proteins that is responsible for the host shutoff effect and for PABPC1 re-localization If increased levels of PAN RNA in the nuclei of lytically reactivated cells are due to PABPC1 re-localization, as observed in transiently transfected cells , then PATo ask whether lytic induction results in the accumulation of other abundant, polyadenylated noncoding RNAs coincident with PABPC1 re-localization, we measured the levels of host MALAT1 and NEAT1 RNAs by northern blot and qRT-PCR. MALAT1 RNA exists either as a long polyadenylated transcript that localizes to nuclear speckles, or a shorter transcript that localizes to the cytoplasm O-methylated and phosphorothioate-substituted antisense oligonucleotides that target endogenous RNase H to cleave PAN RNA , anti-GFP, anti-K7 or anti-PAN RNA oligonucleotides revealed that knockdown of PAN RNA affects late rather than early KSHV proteins . SeveralAlthough non-specific toxic effects were experienced upon electroporation followed by lytic induction, particularly in the BCBL1 TReX-RTA cells see , similarLikewise, several lines of evidence indicate that the decreased levels of late protein expression observed upon knockdown of PAN RNA are not simply due to a non-specific decrease in RTA expression. Although some of the 9 immunoblot experiments revealed a decrease in RTA expression upon transfection with the K7 and PAN RNA oligonucleotides , in otheThe results of PAN RNA knockdown in iSLK.219 cells were also assessed . KnockdoSince knockdown of PAN RNA adversely affects expression of important late viral genes, we predicted that its knockdown would affect overall viral yield. To assess the role of PAN RNA in the production of new virus, we harvested virus from the supernatant of induced BCBL1 TReX-RTA cells 8 days post-induction. As seen in The effect of PAN RNA knockdown on production of infectious virus from iSLK.219 cells was also assayed by harvesting the supernatant from induced iSLK.219 cells and infecting target 293 cells that stably express the DC-SIGN receptor. Infection was assessed by visualization  and by FSince viral DNA replication is required for the expression of herpesvirus late proteins 6 transcripts per nucleus) corresponds well to the abundance of PABPC1 (estimated at 7×106 copies in the average HeLa cell), given that the length of PAN RNA's poly(A) tail is estimated to be similar to that of the average host mRNA  and that each PAN RNA thus likely binds 8–10 PABPC1 molecules The data presented here indicate that the highly abundant PAN RNA binds to the normally cytoplasmic poly(A) binding protein PABPC1 once it has been re-localized to the nucleus of KSHV-infected cells . This isSince PAN RNA expression is concurrent with the host shutoff effect in the context of viral lytic infection , PAN RNAUltimately, knockdown of PAN RNA adversely affects release of new infectious virus into the supernatant from BCBL1 TReX-RTA cells , as woulKSHV targets host gene expression at several levels during the lytic phase. First, host mRNA levels are specifically downregulated by the SOX protein, a phenomenon that is itself related to nuclear re-localization of PABPC1, a highly abundant translation factor that displays nanomolar affinity for the polyadenylate tails of mRNAs Our data support a model in which the highly abundant PAN RNA contributes importantly to the viral manipulation of gene expression. Aspects of viral infection that downregulate host gene expression, such as host shutoff and PABPC1 re-localization, do not require PAN RNA, but instead affect the expression level of PAN RNA itself both in transiently transfected cells and in bona fide infected cells. PAN RNA then impacts the expression of at least a subset of viral genes; possible molecular mechanisms for this regulation are presented below.Poly(A) binding protein is a central player in cellular gene expression and is targeted by a number of viruses, usually as a means to achieve shutdown of host gene expression. Some, such as picornaviruses and caliciviruses, have evolved specific proteases that cleave PABPC1 cleavage and polyadenylation stimulation factor (CPSF) within the assembled 3′-end processing machinery PABPN1, a distinct nuclear poly(A) binding protein Given the striking abundance of PAN RNA and the role that it plays in KSHV gene expression within the context of PABPC1 re-localization, it might be expected that homologs of this RNA would be found in related herpesviruses in which re-localization of PABPC1 occurs. Viruses whose mRNAs lack poly(A) tails, such as rotavirus and Bunyamwera virus, would be exceptions. Indeed, the EBV homolog of the SOX protein, BGLF5, has been shown to mediate host shutoff in cells infected with EBV and to drive PABPC1 re-localization Interestingly, the best-characterized viral noncoding RNAs, the VA RNAs of adenovirus, which facilitate viral translation by suppressing protein kinase R and competitively inhibiting miRNA export and processing By what molecular mechanism might PAN RNA enhance the expression of late viral genes, and could this role be related to its ability to bind re-localized PABPC1 in the nucleus of KSHV-infected cells? It is first critical to understand why PABPC1 re-localization might be harmful to the cell's normal functioning. PABPC1 is proposed to have three major functions: 1) to synergistically enhance mRNA translation by interaction with eIF4G, 2) to regulate mRNA fate by inhibiting deadenylation of the poly(A) tail or promoting interaction with deadenylating factors, and 3) to facilitate nuclear export of mRNA d ∼7 nM) could displace PABPN1 (Kd ∼555 nM) from polyadenylated mRNAs and interfere with PABPN1's role in export How might nuclear re-localized PABPC1 contribute to host mRNA degradation? Under normal conditions, PABPN1 is required for the synthesis of and co-transciptionally binds to the newly-extended poly(A) tails of mRNAs. It has been suggested that PABPN1 is further required for proper export of mRNA via its interaction with nuclear export factors If a highly abundant, inert, polyadenylated RNA were expressed in the nucleus, then the re-localized PABPC1 would be stoichiometrically bound, or nearly so. Thus, PAN RNA could serve as a “buffer” against changes in nuclear PABPC1 concentration . The facA challenge for the model  is explaFigure S1Induction of PAN RNA in BCBL1 TReX-RTA cells and purification of the PAN RNP from cell lysate. A. Northern blot of total RNA harvested from BCBL1 TReX-RTA cells treated with doxycycline for the indicated number of hours. B. Northern blot of fractions collected during purification of the PAN RNP by anion exchange and affinity chromatography steps from lysate prepared at 24 hours post-induction.(EPS)Click here for additional data file.Figure S2Proteins identified by mass spectrometry, including PABPC1 (top band), hnRNPC1 and hnRNPC2 (middle and bottom bands), as co-purifying with PAN RNA. A. List of proteins identified and the number of unique peptides identified for each protein in each band. B. Silver-stained gel showing better resolution of the top band, containing PABPC1, from a closely migrating non-specific band, and a zoomed-in image of the silver-stained gel in (EPS)Click here for additional data file.Figure S3Confocal images of PABPC1 (red) and rRNA (green) in latent or lytic BCBL1 TReX-vector and TReX-RTA RTA cells, respectively. Both cell lines were treated with 1 µg/mL doxycycline for 24 hours before fixation and staining with anti-PABPC1 and Y10B anti-rRNA antibodies. Red and green channels merge into yellow in the cytoplasm of latently infected cells, showing that both PABPC1 and rRNA are predominantly cytoplasmic during latency. However, while PABPC1 (red) re-localizes into the nucleus during lytic phase, rRNA (green) remains cytoplasmic.(EPS)Click here for additional data file.Figure S4Manual scoring of images taken with a laser scanning confocal microscope indicates that PAN RNA expression and PABPC1 re-localization are highly correlated. Left panels show wide field images of PABPC1  and PAN RNA . Right panels show these same images, except that saturated pixels are identified in white and background pixels are in gray. Saturated and background signals aided scoring since cytoplasmic PABPC1 stained very intensely compared to re-localized PABPC1 and since the anti-PAN RNA oligonucleotide probes described in (TIF)Click here for additional data file.Figure S5Replacing the poly(A) signal of PAN RNA with a U7 snRNP-dependent 3′-end formation signal (PAN RNA Δ-poly(A) tail) (TIF)Click here for additional data file.Figure S6qRT-PCR analysis of GAPDH mRNA in total RNA extracted from BCBL1 TReX-RTA cells that were electroporated with the indicated modified oligonucleotides and induced with doxycycline for the indicated times.(EPS)Click here for additional data file.Figure S7Immunoblots probed with anti-RTA, anti-vIL-6 and anti-K8.1 antibodies, from 9 independent knockdown experiments. Blots were developed either with a G:BOX (Syngene)  or by x-ray film . For the former, densitometry analysis was performed with GeneTools software (Syngene), ensuring use of non-saturated signal. Furthermore, linearity of signal was confirmed by titration of lysate. For data collected with x-ray film, a titration of exposure time was performed for each blot to avoid analysis of x-ray film that had been overexposed. X-ray films were then scanned and densitometry was performed using Image J software (NIH). Average RTA, vIL-6 and K8.1 signal from cells treated with mock, anti-GFP, anti-K7 and anti-PAN RNA oligonucleotides are provided in (EPS)Click here for additional data file.Figure S8Manual scoring of RTA and K8.1 expression in BCBL1 TReX-RTA cells electroporated with no oligonucleotide (mock), anti-GFP, anti-K7 or anti-PAN oligonucleotides. A. Representative images from those used to score RTA and K8.1 expression in latently infected (top) and lytically infected (bottom) cells. First, DAPI was used to identify healthy cells correctly positioned on the z-axis (panels marked DAPI). Then, RTA-myc expression was scored by overlaying the Alexa Fluor 594 signal (panels marked DAPI+RTA). Finally, K8.1 expression was scored by overlaying the Alexa Fluor 488 signal (panels marked DAPI+RTA+K8.1). B. Quantification of 4 independent experiments . 10 randomly chosen fields, each containing about 60–70 cells, were scored per treatment for each experiment. Standard error of the mean is shown, and Student's t-test values are as follows: anti-GFP versus anti-K7 = 0.987, anti-GFP versus anti-PAN = 0.023, anti-K7 versus anti-PAN = 0.045. For one of these 4 experiments, a second person independently scored K8.1 and RTA expression, confirming a significant decline in K8.1 expression comparing anti-K7 and anti-PAN treated cells (Student's t-test = 0.009). The difference between K8.1 expression in anti-GFP versus anti-PAN treated cells was less significant, as individually scored by this person (Student's t-test = 0.105).(EPS)Click here for additional data file.


Small non-coding RNAs constitute an important class of gene expression regulators that control different biological processes in most eukaryotes. In plants, several small RNA (sRNA) silencing pathways have evolved to produce a wide range of small RNAs with specialized functions. Evidence for the diverse mode of action of the small RNA pathways has been highlighted during plant–microbe interactions. Host sRNAs and small RNA silencing pathways have been recognized as essential components of plant immunity. One way plants respond and defend against pathogen infections is through the small RNA silencing immune system. To deal with plant defense responses, pathogens have evolved sophisticated mechanisms to avoid and counterattack this defense strategy. The relevance of the small RNA-mediated plant defense responses during viral infections has been well-established. Recent evidence points out its importance also during plant–bacteria interactions. Herein, this review discusses recent findings, similarities and differences about the small RNA-mediated arms race between plants and these two groups of microbes, including the small RNA silencing pathway components that contribute to plant immune responses, the pathogen-responsive endogenous sRNAs and the pathogen-delivered effector proteins. Immune responses against pathogens are present in most multicellular organisms. Plants and animals have evolved complex and effective innate immune systems to protect themselves from invading microorganisms . Plants trans-acting small interfering RNAs (ta-siRNAs), natural antisense transcript-derived small interfering RNAs (nat-siRNAs), repeat-associated small interfering RNAs (ra-siRNAs) or heterochromatic small interfering RNAs (hc-siRNAs), and long small interfering RNAs  of Table 1).Several studies have shown that plant small RNAs are directly involved in bacterial disease responses. The first sRNAs identified to participate in plant immunity during bacterial infections were miRNAs. Plants tested with pathogenic bacteria showed several changes in miRNA accumulation, particularly in auxin signaling related miRNAs . Virus-rArabidopsis was reported to restrict P. syringae invasion; however, no mechanism was known that could be involved in triggering this resistance. Analyzing gene expression profilings of seedlings challenged with flg22, TIR1, AFB2, AFB3), targets of miR393, was repressed upon treatment with flg22. However, one of the auxin receptors AFB1 was not affected possibly due to a slightly different miR393-binding site. This result suggested a role for miR393 in regulating defense responses during P. syringae infection. Further experiments confirmed this role for miR393a since its overexpression enhanced plant bacteria resistance and as consequence reduced virulent P. syringae pv. tomato (Pst) DC3000 in planta growth  by transferring bacterial DNA to the plant genome. Both, disarmed and oncogenic strains induced miR393 in an early stage of infection. Interestingly, the flg22 of Agrobacterium tumefaciens that is completely inactive to the receptor kinase FLAGELLIN INSENSITIVE2 (FLS2) as a ligand, maintained unaltered miR393 expression levels  and a cytochrome c oxidase subunit V (COX5), is reduced only in plants challenged with avirulent strains such as Pst DC3000 avrRpm1 and Pst DC3000 avrRpt2 . RNAi-mediated gene silencing of DMT2, and a different DNA methyltransferase (DMT1), results in reduced tumor formation during Agrobacterium infection  stresses  favors reduction of DCL4 and DCL3 two important DCLs in charge of producing vsiRNAs  and the Tobacco rattle virus (TRV) presented reduced accumulation of miR482. In accordance, two miR482 NBS-LRR mRNA targets were induced in infected plants, especially in plants infected with TCV. A secondary siRNA, product of one of these phased resistance loci that targets a defense-related mRNA and forms a sRNA regulatory cascade, was also suppressed during viral and bacterial infections. Taking into account these observations, they proposed that miRNA-regulated R genes might participate in an uncommon non-race-specific immunity mechanism when this small RNA regulation is blocked by pathogen-encoded suppressors of RNA silencing to release the defense resistance targets. Although further experiments are necessary to extensively validate this hypothesis, previous observations suggest this could be a possible defense mechanism. For example, Tobacco mosaic virus (TMV) in Nicotiana benthamiana. Additionally, it was previously reported that overexpression of miR482 miRNA resulted in hypernodulated soybeans, that miR482 is induced after Bradyrhizobium japonicum inoculation, and that miR1507 accumulates upon rhizobia infection in the roots of a supernodulated mutant  revealed an enhanced accumulation of miR158 and miR1885  containing proteins in Arabidopsis. However, further studies are still needed to determine the precise role of these miRNAs in antiviral immunity besides the identity of their targets because it is well-known that the viral suppressor of TuMV HC-Pro increased miRNA accumulation and inhibits miRNA cleavage function  to regulate gene expression. Now, several studies have confirmed that the opposite strand (star or passenger strand) of a defined miRNA could also be functional in plants and animals. In relation with small RNA-mediated plant defense responses, the star strand miR393* has also been shown to play a role in plant immunity . In Arab protein .N in tobacco. The immune receptor N was the first virus-related resistance gene identified. It confers resistance to the TMV. Expression of these two miRNAs in N. Benthamiana was found to reduce N-mediated resistance to the TMV. Interestingly, the 22-nt miRNA nta-miR6019 triggers production of secondary siRNAs from the N gene similar to the miR482 miRNA family. In this study, eight more miRNA families from tobacco, tomato, and potato that may target R genes were identified, including the miR482 family  and PPRL (pentatricopeptide repeat-like gene). It targets the 3′ UTR region of PPRL. In plants infected with Pst (avrRpt2), nat-siRNAATGB2 strongly and specifically accumulates. Surprisingly, this siRNA has a role in disease resistance against pathogenic bacteria by silencing the PPRL gene which negatively regulates the coiled-coil NBS-LRR type R protein RPS2. Induction of this siRNA also depends on the disease resistance gene RPS2 and on the NDR1 gene that is required for RPS2-specified resistance. Plants overexpressing a resistant version of PPRL (without UTR) against nat-siRNAATGB2 that were inoculated with Pst avrRpto showed delayed HR, reduced level of cell death and enhanced pathogen growth. All these results suggest that nat-siRNAATGB2 plays a positive role in disease resistance through regulation of PPRL.Previous to the discovery of nat-siRNAATGB2, siRNAs were known to be involved in antiviral defense mechanisms in plants and animals. Even so, there was a lack of information regarding siRNA-mediated regulation of antibacterial defenses in plants. In this context, finding of nat-siRNAATGB2 revealed, for the first time, the importance of siRNAs and, particularly nat-siRNAs, in controlling antibacterial immunity in plants . This siP. syringae), avrRpt2) infection. The most functionally characterized lsiRNA is AtlsiRNA-1. This siRNA is generated from the overlapping region of the SRRLK (putative leucine-rich repeat receptor-like protein kinase) and atRAP (an expressed protein that contains a putative RNA-binding domain) natural antisense transcripts. AtlsiRNA-1 is complementary to the 3′ UTR of the antisense gene AtRAP and therefore regulates its expression. In mutant atrap plants less growth of both virulent and avirulent Pst was observed, suggesting a negative regulatory role for this gene in plant resistance responses. Based on these results, AtlsiRNA-1 may promote resistance against Pst avrRpt2 infection because of the particular regulation of its target. Almost all the discovered AtlsiRNAs still have to be confirmed to play a role in plant defense responses. Nonetheless, taking into account the cases exposed here of siRNAs related to plant immunity during bacterial infections, together with the growing knowledge generated around small RNAs, for example Pol IV-dependent siRNAs (p4-siRNAs), more siRNAs could also be involved in regulating bacteria stress responses.In an effort to identify other small RNAs specifically induced in a pathogen interaction (Table 2). Consequently, the sRNA silencing pathway components may be altered during bacterial infection to precisely affect production of the sRNAs that contribute to plant resistance. Besides, many components of the small RNA silencing pathways involved in PTGS form the antiviral RNA silencing defense mechanism. Functional studies have assigned a concise or a redundant function in antiviral immunity to various elements of the sRNA silencing pathways. The importance of some of these elements in regulating antiviral plant defenses is reflected in the attempt of many viral suppressors to disturb their activity. Coincidentally, some of these small RNA biogenesis factors have a dual role in plant defense responses against both bacterial and viral infections.Previously described sRNAs that play important roles in bacterial and viral infections are produced by diverse small RNA silencing pathways. Some small RNA biogenesis factors are directly involved in plant immunity against pathogens  strain NPS3121, which does not infect Arabidopsis, the non-pathogenic P. fluorescens Pf-5 and the E. coli W3110 strains showed enhanced growth  and to the non-host pathogen P. syringae pv. tabaci  that carries a viral suppressor that probably targets AGO2. In N. benthamiana, AGO2 plays an important antiviral role against the Tomato bushy stunt virus  and the Cabbage leaf curl virus (CaLCuV) was observed  was shown to interact with DRB4 to accomplish its replication. Furthermore, plants expressing P6 induce similar symptoms as the drb4 mutant . DRB4 was reported to be induced upon infection with this virus and it was observed that this protein controls viral coat protein accumulation. Besides, DRB4 was found to interact in vitro with a RNA translational enhancer of the TYMV, suggesting a role for DRB4 in repressing viral RNAs at a translational level  infection, suggesting the presence of a viral suppressor with this activity in ORMV (Zucchini yellow mosaic virus (ZYMV) HC-Pro suppressor inhibits the activity of HEN1 in vitro  gene was found to disturb PTGS. SDE3 encodes a RNA helicase-like protein. Infection of sde3 plants with CMV increased accumulation of viral RNA and severity of symptoms (SDE5) gene, a putative homolog of a human mRNA export factor, is involved in antiviral immunity. Mutants of sde5 were hyper-susceptible to the CMV but not to the TuMV  infections was not altered in sgs3 plants . Silenced RDR1 maize plants were more susceptible to the infection of SCMV and accumulated more viral RNA  strand RNA viruses because of the primary role DCL4 has in antiviral defense. In absence of DCL4, the DCL2-dependent 22-nt class of vsiRNAs is the most abundant. The amount of 22-nt vsiRNAs when an active DCL4 is present constitutes only a small portion of the total vsiRNAs  could be involved  and TYMV. The drb4 plants infected with these two viruses showed reduced accumulation of 21-nt vsiRNAs  is the consequence of the chlorophyll biosynthetic gene (CHL1 mRNA) downregulation mediated by Y-Sat-derived siRNAs. The CHL1 mRNA from N. tabacum was found to possess a 22-nt sequence site complementary to the Y-Sat. Interestingly, it was also reported that other two Nicotiana species that do not exhibit yellowing symptoms when infected with the CMV Y-Sat are due to a single nucleotide polymorphism presented in the CHL1 mRNA sequence  and the Grapevine rupestris stem pitting-associated virus  oncogene and the agropine synthase (ags) gene were detected after 3 days of post-infiltration with A. tumefaciens in N. benthamiana leaves. Interestingly, like in several cases of viral RNA silencing, sRNAs were predominantly 21-nt long and originated from sense and antisense strands, suggesting an important role for DCL4 and RDR6 in the biogenesis of this bacteria-derived sRNAs.In bacteria, the widely studied pathogenic Figure 1).Plant–microbe interactions are sophisticated and dynamic, involving the continuous improvement of complex defense and counter-defense strategies from both sides. Several microbes introduce effector proteins into plant cells in order to suppress PTI. The contribution of sRNA-mediated silencing in PTI and ETI suggested the existence of bacterial suppressors of RNA silencing (BSRs) and VSRs. Suppressors of RNA silencing may impact small RNA silencing pathway proteins, long double-stranded RNAs, small RNAs, DNA methylation, or sRNA-derived genes to modify the biogenesis, maturation, or function of endogenous and microbe-derived small RNAs. VSRs constitute a diverse group that is widely distributed among viruses. In contrast, only few BSRs have been identified; however, VSRs and BSRs share common strategies like AGO1 disturbance , NS3 (Influenza A virus), 2b (Tomato aspermy virus and CMV), P14 (Pothos latent virus), and P38 (TCV) bind size-independent dsRNAs (Beet yellows virus), P15 (Peanut clump virus), γB (Barley stripe mosaic virus), HC-Pro (Tobacco etch virus), P122 (TMV), NS3 (Rice hoja blanca virus), Pns10 (Rice dwarf virus), and the tospoviral NSs proteins bind mostly size-dependent double-stranded sRNAs having 2-nt 3′ overhangs (Cucumber vein yellowing virus (CVYV) interacts with similar affinity to double-stranded sRNAs with a phosphoryl group or a free OH at their 5′ ends and to duplexes with 2-nt 3′ overhangs or blunt-ends (African cassava mosaic virus (ACMV) binds only single-stranded miRNAs and siRNAs (Sweet potato mild mottle virus (SPMMV)] interact with the AGO1 protein using GW/WG motifs commonly employed by some endogenous RNA silencing components to assemble functional RISCs. Point mutations in the GW residues of P38 resulted in resistant plants against the TCV infection. Interestingly, the AGO1-binding activity of the P1 protein of SPMMV allows this suppressor to inhibit mature assembled AGO1-containing RISCs and their de novo assembly  E3 ubiquitin ligase complex. For this reason, it was thought that P0 promoted AGO1 ubiquitination and degradation via the ubiquitin proteasome system (UPS); however, a recent report showed that P0-induced degradation of AGO1 occurs through the autophagy pathway  acts as a silencing suppressor using a particular approach that involves its endonuclease activity. This RNase was shown to cleave small RNAs of 21, 22, and 24-nt long into unfunctional small RNAs of 14 base pair size  and the L2 protein of BCTV suppress RNA silencing by inhibiting the adenosine kinase (ADK) activity, which plays relevant roles in adenosine salvage and methyl cycle maintenance. Inactivation of ADK resulted in suppression of RNA silencing as occurred with the incorporation of the two geminivirus proteins. These two proteins can reverse TGS of a GFP transgene introduced in N. benthamiana. In addition, the AL2 and L2 proteins were found to cause ectopic expression of an endogenous loci silenced by methylation and a global reduction in cytosine methylation (Beet severe curly top virus (BSCTV). The C2 protein interacts with the S-adenosyl-methionine decarboxylase 1 (SAMDC1) attenuating its proteasome-dependent degradation. The SAMDC1 protein participates in the polyamine biosynthesis, but is also important for SAM/dcSAM balance and transmethylation (Tomato yellow leaf curl China virus (TYLCCNB) encodes a protein called βC1 that acts as a suppressor of methylation-mediated TGS. The βC1 protein interacts with the S-adenosylhomocysteine hydrolase (SAHH) enzyme that is involved in the methyl cycle and therefore plays a role in TGS. The expression of the βC1 protein decreases cytosine methylation of the viral and host genomes. Also, this protein was shown to reverse TGS applied to a transgene and an endogenous locus (Specific viral suppressors that employ different and particular strategies to avoid RNA silencing have been identified as well. For example, the V2 protein of TYLCV is a RNA silencing suppressor that interacts directly with SGS3 and binds dsRNA. Disruption of V2–SGS3 interaction with a point mutation version of V2 stops RNA silencing suppression. These results suggested that V2 affects the interaction of SGS3–RDR6 to avoid small RNA amplification . Notablyair size . The viroduction . Other Voduction . Furtherhylation . Anotherhylation . The betus locus . As prevus locus .The small RNA-mediated plant defense responses have emerged as relevant components of the innate immune system. Increasing evidence has highlighted the warfare that takes place between plants and microbes around the RNA silencing system. In this review, the recent findings, similarities and differences related to the RNA-mediated arms race between plants and two important group of microbes such as bacteria and viruses were discussed. In general, evident biological differences between these two groups of microbes are reflected in big differences regarding small RNA-mediated antiviral and antibacterial immunity; however, there are also specific similarities in plants defense responses through RNA silencing against bacteria and viruses. Likewise, similar strategies have been identified related to the microbes counter defense responses against RNA silencing. Bacteria-responsive miRNAs with potential roles in regulating bacterial immunity have been reported for several plant–bacteria strains interactions, flg22 treatment and even during symbiotic nitrogen fixing bacteria inoculation and nodule development . Most ofThe 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.


Controlling gene expression via small interfering RNA (siRNA) has opened the doors to a plethora of therapeutic possibilities, with many currently in the pipelines of drug development for various ocular diseases. Despite the potential of siRNA technologies, barriers to intracellular delivery significantly limit their clinical efficacy. However, recent progress in the field of drug delivery strongly suggests that targeted manipulation of gene expression via siRNA delivered through nanocarriers can have an enormous impact on improving therapeutic outcomes for ophthalmic applications. Particularly, synthetic nanocarriers have demonstrated their suitability as a customizable multifunctional platform for the targeted intracellular delivery of siRNA and other hydrophilic and hydrophobic drugs in ocular applications. We predict that synthetic nanocarriers will simultaneously increase drug bioavailability, while reducing side effects and the need for repeated intraocular injections. This review will discuss the recent advances in ocular siRNA delivery via non-viral nanocarriers and the potential and limitations of various strategies for the development of a ‘universal’ siRNA delivery system for clinical applications. Pharmaceutical treatment of retinal degenerative diseases affecting the posterior segment of the eye is made challenging by restrictive blood ocular barriers such as the blood aqueous barrier (BAB) and the blood retinal barrier (BRB), which separate the eye from systemic circulation . AdditioIt is estimated that following instillation, only 5% of topically applied drugs enter the anterior chamber of the eye, either through trans-corneal permeation Figure , arrow 1.-cholesterol) have also been used to deliver siRNA successfully and may present opportunities to combine desired features to create novel lipid-based nanocarriers -N,N,N-trimethylammonium chloride (DOTMA) and N,N-dioleyl-N,N-dimethylammonium chloride (DODAC)) and forms nanoscale complexes. Liposomes are probably the most commonly used artificial gene delivery vector since their ability to transport the preproinsulin gene to the liver was demonstrated nearly 30 years ago . Liu et  in vitro. After i in vitro. Lipid carriers .3) Protein nanocarrierset al. have developed a novel cell-penetrating peptide (CPP) for ocular delivery of small and large molecules, including siRNA, fluorescent probes, plasmid DNA and quantum dots to RPE, photoreceptor and ganglion cells in vitro and in vivo-cholesterol; DODAC: N,N-dioleyl-N,N-dimethylammonium chloride; DOPE: 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine; DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane; DOTMA: N-[1-propyl]-N,N,N-trimethylammonium chloride; dsRNAs: Double stranded RNAs; EVA: Ethylene vinyl acetate; FAc: Fluocinolone acetonide; FDA: US Food and Drug Administration; 2’-F-RNA: 2’-Fluoro-RNA; HD-Ad: Helper-dependent adenovirus; miRNA: microRNA; MMP-2: Matrix metalloproteinase-2; ODNs: Oligodeoxynucleotides; PACT: dsRNA-binding protein; PAMAM: Poly(amidoamine); PB: Boranophosphonate; PEG: Polyethylene glycol; PEI: Polyethylenimine; PKR: Protein kinase R; PO4: Phosphodiester; PPI: Poly(propylene imine); PS: Phosphorothioate linkage; PVA: Polyvinyl alcohol; RISC: RNA-induced silencing complex; RNAi: RNA interference; RPE: Retinal pigment epithelium; scAAV: Self-complementary AAV; siRNA: Small interfering RNA; TaT: HIV transactivator of transcription; TLR: Toll-like Receptors; TRBP: HIV-1 TAR RNA-binding protein; VEGFA: Vascular endothelial growth factor-A; VEGFR1: Vascular endothelial growth factor receptor-1.No competing interests to declare.AT, SF, AZ, KK and BM contributed towards writing and editing the manuscript. GH and HS critically evaluated the manuscript for publication. All authors read and approved the final manuscript.No information to share.


Science 2009, 326, 1279–1283.Commentary on Tawar, R.G.; Duquerroy, S.; Vonrhein, C.; Varela, P.F.; Damier-Piolle, L.; Castagné, N.; MacLellan, K.; Bedouelle, H.; Bricogne, G.; Bhella, D.; Eléouët, J.-F.; Rey, F.A. Crystal structure of a nucleocapsid-like nucleoprotein-RNA complex of respiratory syncytial virus.  Paramyxoviridae; order: Mononegavirales). The viral RNA of this group of viruses is always bound to the viral nucleoprotein (N) and this N-RNA structure is the template for viral transcription and replication by the viral RNA-dependent RNA polymerase complex consisting of L  and P . In a recent article in Science, Tawar and coworkers published the crystal structure of the RSV nucleoprotein in complex with cellular RNA [Mononegavirales order form helical structures and, because these are very flexible, they are not simple objects for structural biology studies. Fortunately, recombinant nature has helped a bit; when the nucleoproteins of vesicular stomatitis virus (VSV), rabies virus and RSV were expressed in bacteria or insect cells, they bind to short cellular RNAs (possibly tRNA molecules) and form short N-RNA complexes that close up into rings through protein-protein contacts [RSV is a non-segmented, negative strand RNA virus (family: ular RNA . All thecontacts –3. When Mononegavirales order but influenza virus is a segmented negative strand RNA virus. The nucleoproteins of rabies virus, RSV and BDV have a conserved helical C-terminal domain (top domain in the structures in Paramyxoviridae, the nucleoprotein has an even longer and natively disordered C-terminal tail, called NTAIL [TAIL forms an α-helical molecular recognition element that consists of a dynamic and interchanging population of overlapping helices [TAIL [The rabies virus and RSV nucleoproteins have extended N-terminal strands (in blue in ed NTAIL . A seque helices . The vires [TAIL –12. Theres [TAIL ,14. The es [TAIL ,16. TherApart from opening up the possibility of developing new drugs against RSV, the structure of the nucleocapsid-like RSV complex also clearly shows what is conserved in the nucleoproteins of the negative strand RNA viruses and what is not. Because the conservation of structure and RNA binding is now so clearly shown, it becomes even more obvious how different the influenza virus NP is. Finally, all those who have been working on the structure and function of Sendai and measles virus transcription and replication know that the nucleoproteins of these viruses bind six nucleotides –19 and t