Picornaviruses constitute a large group of viruses comprising medically and economicallyimportant pathogens such as poliovirus, coxsackievirus, rhinovirus, enterovirus 71 andfoot-and-mouth disease virus. A unique characteristic of these viruses is the use of aviral peptide (VPg) as primer for viral RNA synthesis. As a consequence, all newly formedviral RNA molecules possess a covalently linked VPg peptide. It is known that VPg isenzymatically released from the incoming viral RNA by a host protein, called TDP2, but itis still unclear whether the release of VPg is necessary to initiate RNA translation. Tostudy the possible requirement of VPg release for RNA translation, we developed a novelmethod to modify the genomic viral RNA with VPg linked via a ‘non-cleavable’bond. We coupled an azide-modified VPg peptide to an RNA primer harboring a cyclooctyne[bicyclo[6.1.0]nonyne (BCN)] by a copper-free ‘click’ reaction, leading to aVPg-triazole-RNA construct that was ‘non-cleavable’ by TDP2. We successfullyligated the VPg-RNA complex to the viral genomic RNA, directed by base pairing. We showthat the lack of VPg unlinkase does not influence RNA translation or replication. Thus,the release of the VPg from the incoming viral RNA is not a prerequisite for RNAtranslation or replication. Picornaviruses constitute a large group of small non-enveloped RNA viruses including manyimportant human and animal pathogens like poliovirus (PV), coxsackievirus, rhinovirus,enterovirus 71 and foot-and-mouth disease virus. These viruses possess a single-stranded RNAgenome of positive polarity ranging from 7.0 to 8.5 kb in length. The genome consists of asingle open reading frame flanked by two highly structured untranslated regions (UTRs). The5′ UTR contains the internal ribosomal entry site essential for viral RNA translation.Additiopro proteinase , at the 5′ terminus of the genomic RNA . This peoteinase , thetyroteinase ,14. The oteinase . As a cooteinase .Soon after the discovery that picornavirus virion RNA contained covalently linked VPg,viral RNA molecules lacking VPg were detected in infected cells . Thus, Vin vitrotranscribed RNA, consequently lacking VPg, is translated and replicated efficiently. BecauseVPg is released from the viral RNA on introduction in the cytoplasm, it has been suggestedthat the presence of the VPg may hamper the formation of the translation initiation complex gene under control of theviral internal ribosomal entry site  was useUnmodified RNA primers, containing a 5′-terminal hydroxyl (OH) group, as well asRNA primers possessing a 5′biotin, amine or Cy5 modification, were purchased fromSigma. The RNA primer modified with bicyclo[6.1.0]nonyne (BCN) ,27 wassThe 250-nt RNA fragment used for RNA ligations was produced by run-off RNA transcriptionusing the T7 RiboMAX kit (Promega) for 2 h at 37°C. As template, a 250-bp PCR productwas used (Fw: 5′-TAATACGACTCACTATAGG-3′ and Rv:5′-GTAGTTGGCCGGATAACGAACG-3′) spanning the 5′-end of theRLuc-CVB3-CLΔ1-6 + 8 genome containing a T7 promoter sequence. Fortranscription of genomic RLuc-CVB3 wild-type (wt) RNA, RLuc-CVB3-Δ1-6 + 8 RNAand RLuc-CVB3-Δ1-6 + 5 RNA, the infectious clone was linearized with BamHI, andrun-off RNA transcripts were made using the T7 RiboMAX kit (Promega) for 2 h at 37°C.RNA transcripts were purified and concentrated using LiCl precipitation (Ambion) followedby polyphosphatase treatment (Epicentre) for 2 h at 37°C. Finally, the RNA waspurified once more using LiCl precipitation (Ambion) and used for RNA ligations.For RNA primer ligation, 200 pmol of the 12-nt RNA primers(5′-UUCCACCGCUAA-3′) or 9-nt RNA primers (5′-UUAAAACAG-3′) wasincubated with 20-pmol genomic RNA in the presence of 20 U of RNA ligase 2 (NEB) for 4 hat 37°C . Excess RNA primer was washed away using a GenEluteMammalian total RNA miniprep column (Sigma). To assess RNA primer ligation efficiency, a250-nt RNA fragment was released from 1.25 pmol of genomic RNA ligation product using DNAprimer-directed RNase H digestion (10-µl volume). RNA was incubated with 12.5 pmolof DNA primer (5′-GTAGTTGGCCGATAACGAACG-3′) and 5 U of RNase H (Fermentas) for20 min at 50°C. The released RNA fragment was analyzed on an 8-M urea, 8%polyacrylamide gel electrophoresis (PAGE) gel and stained with Stains-All (Sigma).Renilla luciferase assay system according to manufacturer’sinstructions (Promega).To determine RLuc translation levels, 100 ng of genomic RLuc-CVB3 run-off transcript RNA(wt) or RNA ligation product were transfected (in triplicate) into 100 000 HeLa cellsusing Lipofectamine2000 (Invitrogen) in the presence of 2.5 mM GuHCl. Cells were lysed inpassive lysis buffer (Promega) 8 h post-transfection, and RLuc values were measured usingthe Renilla luciferase assay system (Promega). Theincrease of RLuc values in the absence of replication inhibitor GuHCl illustrates RNAreplication.To measure RNA replication, 1 ng of genomic RLuc-CVB3 run-off transcript RNA (wt) or RNAligation product were transfected (in triplicate) into 100 000 HeLa cells usingLipofectamine2000 (Invitrogen) in the presence or absence of 2.5 mM GuHCl. Cells werelysed in passive lysis buffer (Promega) 8 h post-transfection, and RLuc values weremeasured using the To determine RNA stability, 100 ng of genomic RLuc-CVB3 run-off transcript RNA (wt) orRNA ligation product were transfected (in triplicate) into 100 000 HeLa cells usingLipofectamine2000 (Invitrogen) in the presence of 2.5 mM GuHCl. At 1, 4 and 8 hpost-transfection, total RNA was isolated using the GenElute mammalian total RNA miniprepkit (Sigma-Aldrich). Isolated RNA was treated with DNAse I (Invitrogen) before reversetranscription. cDNA synthesis was performed with the TaqMan reverse transcription reagentskit (Applied Biosystems) using random hexamer primers. Quantitative analysis of viral RNAlevels was performed using the LightCycler 480 (Roche).To couple the VPg peptide to the BCN-containing RNA primer, a strain-promotedalkyne–azide cycloaddition (SPAAC) was used. Azide-modified CVB3 VPgand poliovirus VPg were synthesized by ALMAC , and a 100-foldexcess of VPg (225 nmol) was incubated with 2.25 nmol of BCN group containing RNA primer(IBA) in a total volume of 50 µl of phosphate-buffered saline for 1 h at roomtemperature. Coupling efficiency of the peptides to BCN was determined by 8-M urea,20% PAGE gel analyses and stained with Stains-All (Sigma). VPg-containing RNAprimers were used for RNA ligation reactions.Equimolar amounts  of VPg peptides and VPg-containing RNA ligationproducts were spotted on an Immobilon-P membrane (Millipore). Membranes were washed oncewith washing buffer (PBS + 0.1% Tween20) and incubated 1 h in blocking buffer. Membranes weresuccessively incubated for 1 h with a rabbit polyclonal antibody (1:500) raised againstthe PV VPg  followed2, 1 mM dithiothreitol, 5% v/v glycerol) in the presence orabsence of 1 pmol of GST-TDP2 enzyme. Unlinkase reactions were performed for 30 min at30°C, and treated RNA was purified over a GenElute Mammalian total RNA miniprep column(Sigma). Purified RNA was used for dot blot analysis, and VPg was detected using anantibody raised against the PV VPg (as described earlier). Signal intensity was quantifiedusing the supplied Image Studio Software of LI-COR.The VPg unlinkase reaction was essentially performed as described before ,30 with in vitrotranslation. RRL mixtures (in triplicate) were pre-incubated at 30°C for 5 min, andRNA  was added. Reactions were incubated for 30 min at30°C, translation was stopped by adding a 10-fold excess of H2O, and theRLuc values were measured using the Renilla luciferase assay system(Promega).Rabbit reticulocyte lysate (RRL) from Promega was used for 2O stopped the translationreaction, and the RLuc values were measured using the Renilla luciferaseassay system (Promega).Translation in HeLa S10 extract was performed as previously described . RNA (0.Renilla luciferase upstream of the capsid region(RLuc-CVB3)  . Initialuc-CVB3) A for basuc-CVB3) A. This Ruc-CVB3) A. FigureTo validate this base pairing-directed RNA primer ligation procedure, an unmodified 12-ntRNA primer was ligated to a 250-nt RNA fragment harboring the CLΔ1-6 +8-modified CL structure. For this RNA ligation reaction, two different enzymes weretested, and ligation efficiency was visualized by separating the RNA ligation products bypolyacrylamide gel electrophoresis in the presence of urea (urea-PAGE). As shown in To modify the 5′ terminus of the full-length RLuc-CVB3 genomic RNA, several 12-ntRNA primers containing different 5′ termini were ligated to the genomic RLuc-CVB3-CLΔ1-6 + 8 RNA. Because the RLuc-CVB3genomic RNA is too large (>7500 nt) to allow differentiation of a 12-nt modification ona urea-PAGE gel, a 250-nt RNA fragment was released from the genomic RNA using DNAprimer-directed RNase H digestion. Analysis of these 250-nt fragments showed efficientligation of all different RNA primers to the genomic RLuc-CVB3 genomic RNA C. In conTo determine whether the different RNA modifications affect translation, anRLuc-translation assay was performed. To ensure that we only measured translation of theincoming RNA, the RNA ligation products were transfected into HeLa cells in the presenceof the replication inhibitor guanidine hydrochloride ( + GuHCl) to prevent RNAreplication. Modification of the CL structure (CLΔ1-6 + 8) hampered RLuctranslation significantly D.RestorTo investigate whether modifications at the 5′ terminus of the genomic RNAinfluence replication, RNA ligation products were transfected in HeLa cells but this timealso in the absence of GuHCl (−GuHCl). This allows translation and also replicationof the RNA, which can be measured by an increase in RLuc values. When wt RLuc-CVB3 RNA wastransfected in HeLa cells without any drug addition, increased RLuc values were measuredsee wt, F. UnfortTo allow the analysis of 5′ modifications of genomic RLuc-CVB3 RNA on bothtranslation as well as replication, a new infectious clone was generated. This new cloneholds the same deletion of 6 nt in the 5′ strand of stem A but now possesses only a5-nt insertion in the 3′ strand of stem A  again showed a slight, butsignificant, effect on RLuc translation C. As obs again shBCN is a reactive cyclic alkyne that can be used to link azide-containing molecules via aSPAAC, also called copper-free ‘click’ reaction A 26,34,,34,35. TUsing the genomic RNA possessing VPg via a ‘non-cleavable' bond, we were able tostudy the prerequisite of VPg unlinkase for translation of the incoming RNA and successiveRNA replication. RNA ligation products were subjected to the RLuc translation assay inHeLa cells. As observed before, ligation of the BCN-modified RNA primer to theCLΔ1-6 + 5 genomic RNA resulted in similar translation levels as RNA lacking a5′ modification (OH) and the wt RNA A. ImportTo test whether the inability to release VPg interfered with RNA replication, an RNAreplication assay was performed in the presence and absence of GuHCl. Also, in thisreplication assay, the ‘non-cleavable’ VPg bond did not negatively influencethe RNA replication levels D. TheseOver 35 years ago, the discovery was made that genomic RNA of picornaviruses possessed acovalently linked VPg peptide. Soon after this discovery, it was recognized that VPg isreleased from the incoming genomic RNA by a cellular enzyme. The importance of VPg unlinkasefor efficient translation and/or replication has remained unknown ever since. It has beentechnically difficult to study the possible importance of VPg unlinkase because modificationof the 5′ terminus of large RNA molecules, like the picornavirus genomic RNA, hasproven to be challenging. Here we present a novel method to covalently derivatize the5′ terminus of large RNA molecules with a diverse set of modifications. We exploitedthe highly folded CL structure in the 5′ UTR to allow base pairing-directed ligationof modified RNA primers. Combining this RNA ligation method with the new ‘click’chemistry principle, we were able to modify the large genomic RNA with VPg via a‘non-cleavable’ bond. Using these modified genomic RNA molecules in a series oftranslation and replication assays, we show that the inability to release VPg from theincoming viral RNA does not affect translation and replication efficiency.For the ‘click’ reaction, we used a novel ring-strained alkyne, the BCN group. The maiin vitro unlinkase assay. Funding for open access charge: the Netherlands Organization forScientific Research (NWO).Conflict of interest statement. None declared.


RNA interference (RNAi) is a set of intracellular pathways in eukaryotes that controls both exogenous and endogenous gene expression. The power of RNAi to knock down (silence) any gene of interest by the introduction of synthetic small-interfering (si)RNAs has afforded powerful insight into biological function through reverse genetic approaches and has borne a new field of gene therapeutics. A number of questions are outstanding concerning the potency of siRNAs, necessitating an understanding of how short double-stranded RNAs are processed by the cell. Recent work suggests unmodified siRNAs are protected in the intracellular environment, although the mechanism of protection still remains unclear. We have developed a set of doubly-fluorophore labeled RNAs  to probe in real-time the stability of siRNAs and related molecules by fluorescence resonance energy transfer (FRET). We find that these RNA probes are substrates for relevant cellular degradative processes, including the RNase H1 mediated degradation of an DNA/RNA hybrid and Dicer-mediated cleavage of a 24-nucleotide (per strand) double-stranded RNA. In addition, we find that 21- and 24-nucleotide double-stranded RNAs are relatively protected in human cytosolic cell extract, but less so in blood serum, whereas an 18-nucleotide double-stranded RNA is less protected in both fluids. These results suggest that RNAi effector RNAs are specifically protected in the cellular environment and may provide an explanation for recent results showing that unmodified siRNAs in cells persist intact for extended periods of time. C. elegans [1], is a set of conserved post-transcriptional gene silencing pathways in eukaryotes. Following the first demonstration that RNAi is functional in human cells and receptive to using synthetic small interfering (si)RNA effector molecules [2], significant progress has been made in harnessing the RNAi pathway for functional genomics and for therapies targeting previously “undruggable” genetic targets RNA interference (RNAi), first described in the nematode The inherent sensitivity of RNA molecules both inside and outside the cell towards degradation by ribonucleases motivated an immediate focus on development of chemically modified siRNAs that can withstand nucleolytic extracellular environments and reach the cellular milieu intact [3]. Investigations into the determinants of siRNA stability have focused on extracellular stability, intracellular stability, and potency of silencing. First, to mimic the extracellular environment several groups have used blood serum and purified nucleases to determine the sensitivity of siRNAs to degradation. Chemical modifications were found to impart a considerable degree of nuclease resistance to siRNAs while retaining the ability to induce silencing through the RNAi pathway The intracellular milieu, which is the functional environment of the siRNA, is the second environment to which the siRNA is exposed. By monitoring the expression of a target gene, several groups were able to assess the potency and duration of siRNA effects, including a number of studies that focused on determining whether chemically modified siRNAs are more potent than unmodified siRNAs . A recent study has shown, however, that enhanced intracellular nucleolytic stability is not necessarily correlated with increased duration of the silencing effect [8]. In fact, the authors found that silencing in non-dividing cells persisted for up to one month from a single dose of an unmodified siRNA, suggesting that siRNAs may be quite stable inside the cell.Several groups have addressed questions of intracellular siRNA stability and localization by introducing fluorophore modified siRNAs into live cells and using various microscopy techniques [20]. siRNAs are actively exported from the nucleus [21], except in cases where the RNA target is located in the nucleus [22]. Fluorescence fluctuation spectroscopy has been utilized in a separate study to assess the integrity of labeled intracellular RNAs, revealing that doubly-labeled RNA suitable for fluorescence resonance energy transfer (FRET) measurement between the fluorophores is relatively unstable in single-stranded form compared to the corresponding siRNA duplex [23]. Intracellular FRET imaging of dsRNAs has also been employed to show that intact siRNA duplexes accumulate in cellular foci identified as P-bodies . We have used FRET labeled single-stranded RNAs to show that secondary structure in general attenuates degradation in human cell extracts [26].The precise origin of the apparent intracellular protection of siRNAs is unclear. Long duration of silencing in non-dividing cells may be indicative of the stability of the single-stranded guide strand incorporated into RISC. Prior to RISC maturation, it can be assumed that some stability is derived from the decreased nuclease sensitivity of double-stranded RNA compared to single-stranded RNA . Previous intracellular fluorescence studies [21–25] have focused, however, only on canonical 21–22 nucleotide  siRNAs, while the observation that the RNAi pathway can utilize 21- to 27-nt double-stranded RNAs [27] suggests that intracellular double-stranded RNAs are discriminated based on length.To better evaluate the potential of siRNAs in gene therapeutics, a real-time characterization of their degradation kinetics under extracellular and intracellular conditions is necessary. Such an assay should include rapid and precise assessment of RNA stability as a function of bodily fluid, base paring partner, and RNA size and should eventually be amenable to high-throughput screening for optimizing siRNA drugs. To this end, we here have developed a generalizable series of RNAs modified for FRET measurements, where two uridines in positions 7 and 16 of the 21-nt guide strand were replaced with amino-modified deoxythymidines to introduce two fluorophores that undergo distance dependent FRET Figure 1.pp-luc luciferase gene . We fluorophore labeled the strand that dominantly acts as guide for the RNAi machinery and is antisense to the luciferase mRNA . We first sought to compare the degradation of these dsRNAs with those of the corresponding, relatively unstructured single-stranded RNA  at 37°C.We here have developed a set of fluorescein (FL) and tetramethylrhodamine (TMR) modified RNAs  of either fetal bovine serum (FBS) or HeLa S100 cytosolic cell extract, equivalent to 11.1 mg/ml total serum and 2.49 mg/ml total cellular protein, respectively. The blood serum contains a significant fraction of RNase A-like enzymes [29], whereas the cell extract contains the protein components of the RNAi pathway and was previously tested as RNAi-active [12].Figure S1). In cell extract, the fast phase(s) decreased significantly . Consistent with our qualitative observations by gel electrophoresis, the fastest observed rate constants are found for the decay of ssRNA in serum  and particularly the DNA/RNA hybrid  are less efficiently degraded.Applying our real-time FRET degradation assay to the three fluorophore labeled RNA variants allowed us to determine their decay rate constants over a range of total protein concentrations from added serum or cell extract . We find that the secondary structured dsRNA is most strongly protected in cell extract, leading to only a slow decay . In both cell extract and blood serum, the formation of dsRNA thus protects ssRNA against nucleolytic degradation.The relative decay kinetics in HeLa cell extract . These observations indicate that human RNAse H1 is (one of) the enzyme(s) responsible for degrading a DNA/RNA hybrid in HeLa cell extract.One of the most prominent features of our decay data is the fast degradation of the DNA/RNA hybrid in HeLa S100 cytosolic cell extract, contrasting with its strong protection in blood serum. We hypothesized that the efficient degradation of the DNA/RNA hybrid in cell extract results from the activity of human RNase H enzymes. RNase H activity degrades the RNA in a DNA/RNA hybrid and is important in nucleic acid processing in the cell, including in DNA replication. Two DNA aptamers have previously been Interaction with Dicer is one of the earliest and most crucial steps in RNAi, wherein larger dsRNAs are cleaved into 21-nt fragments that Dicer then loads into the RISC complex [31–34]. To test whether our 24-nt dsRNA is an authentic substrate for Dicer, we employed both FRET assays and gel based analysis. First, we collected steady-state FRET time traces for our 18-, 21-, and 24-nt dsRNAs upon addition of purified human Dicer . This trend correlates with the increasing distance of the fluorophores from the ends as well as the higher thermodynamic stability of the longer duplexes . In contrast to the serum result, the 21- and 24-nt dsRNAs exhibit the same low vmax values, signifying that they are degraded at about the same rate in cell extract . The same trend was observed using the 5′ 32P-radiolabeled dsRNAs . These results suggest that the size dependence of the protection of dsRNA from decay in cell extract is distinct from that in serum. It should be noted that we also observed that the deoxythymidine and fluorophore modifications offered a general, modest protection to the RNAs, as may be expected (data not shown).Next, we employed our steady-state FRET assay to the 18-, 21-, and 24-nt dsRNAs and extracted the rate constants from the time traces that were identified in duplexes Figure 132P-labeled RNAs incorporated into Complex D is less than that of 32P-labeled 21-nt dsRNA devoid of any modification, consistent with the slow processing of 24-nt dsRNA by Dicer , comprising the proteins Dicer, Ago2, and TRBP . Since cellular delivery of 21-nt siRNAs induces RNAi, albeit not as effectively as does delivery of Dicer substrates [27], both types of dsRNAs must be interacting with the components of the RNAi machinery, in contrast to shorter dsRNAs. The similar protection of 21- and 24-nt dsRNAs in our assays relative to the corresponding 18-nt dsRNA Figure 5in vitro FRET assays established here open up the possibility for future high-throughput screening of the nucleolytic stability of additional chemically modified RNAi effector molecules.In summary, our results are consistent with established steps in the RNAi pathway where dsRNAs of ∼21 nucleotides per strand are bound by the RLC or other RNAi components and remain largely hidden from nucleolytic decay either as the base-paired siRNA duplex or as the single guide strand processed into active RISC. The real-time CGGTCGCTCCGTGTGGCTTGGGTTGGGTGTGGCAGTGAC; V-2, GCTGGTCTCTGCGGGTTGTTGCGCCGCGGCACCCTTGGCA; S1-DNA, CGTACGCGGAATACTTTGAAA.All RNAs were synthesized by the HHMI Biopolymer/Keck Foundation Biotechnology Resource Laboratory at the Yale University School of Medicine, deprotected, TMR labeled (as necessary), and purified as previously described [39–41]. More specifically, the guide strand was labeled during synthesis with Fluorescein dT and Amino-Modifier C6 dT in the positions indicated in 2 (chosen to mimic physiologic conditions). After the 10-min cooling period at room temperature, RNA solutions were incubated in a circulating water bath at 37°C for 5 min before the samples were added to a fluorometer cuvette, also controlled at 37°C. After the initiation of collection of donor (F520) and acceptor fluorescence emission signal (F585) as described previously [41], a baseline measurement was recorded for approximately 50–100 seconds before a fraction of either HeLa S100 cytoplasmic cell extract , fetal bovine serum (Gibco), or purified Dicer  was added and the resulting signal evolution recorded. The FRET ratio Q (F585/(F585+F520)) was plotted over time t and fit where appropriate with either the single- or double-exponential form of the following expression to extract the observed rate constants kobs :A final concentration of 50 nM FL and TMR doubly-labeled RNAs was annealed either alone (single-stranded RNA) or with 100 nM  of an appropriate DNA or RNA complement by heating to 70°C for 2 min and cooling to room temperature over 10 min in a standard buffer of 50 mM Tris-HOAc, pH 7.4, 80 mM KCl, 20 mM NaCl, and 1 mM MgClThese assays were carried out over a range of 0–30% (v/v) HeLa cell extract and serum volume additions, equivalent to 0–2.5 mg/ml total HeLa protein (concentration measured by Bradford assay) and 0–11.1 mg/ml total serum protein (concentration provided by Gibco). The observed rate constants were plotted as a function of total protein content and fit with the following hyperbolic binding equation that does not assume cooperativity:maxv is the maximal rate constant at saturation, and 1/2protein is the half-titration point.In this equation, Fluorometer assays to monitor RNase H1 inhibition by DNA aptamers VI-2 and V-2 [30] were conducted identically to the fluorometer assays described above with the following exceptions. The HeLa cell extract used in these assays was pre-incubated with a defined concentration of the pre-annealed DNA aptamer or the negative control DNA S1 for 30 min at 37°C before mixing with the DNA/RNA hybrid in the fluorometer. In addition, the DNA/RNA hybrid was prepared in the presence of the respective aptamer or control DNA at a concentration equal to that in the HeLa cell extract. Again, the FRET Ratio Q for each trace was extracted and fit with a single-exponential expression as above, and the observed rate constants were plotted as a function of aptamer concentration and fit with the following hyperbolic binding equation below where no cooperativity was assumed:minv is the minimal rate constant reached at saturation and 1/2aptamer is the half-titration point.Here, 2. The T1 and OH lanes in the gels refer to reference ladders using RNase T1 and alkali (OH−) digestion, respectively, prepared as described [12] except that no carrier tRNA was added to the RNase T1 digest. For all reactions, 10 pmol/lane of labeled RNA was mixed with loading buffer for a final concentration of 1×TBE, 0.025% (w/v) bromophenol blue, and 40% (v/v) formamide, and the products were separated on a denaturing, 8 M urea, 20% (w/v) polyacrylamide gel. Fluorescence detection of the RNA was accomplished using a FluorImager SI fluorescence scanner with ImageQuant software (Molecular Dynamics) as previously described .Denaturing gel electrophoresis was paired with FRET detection of the RNA to gain qualitative insight into the processing of the labeled RNAs by Dicer, cell extract, and serum. The doubly-labeled RNA strand was prepared at 500 nM final concentration in standard buffer, the complementary RNA or DNA as necessary added to 1 µM concentration and annealed as described above. After cooling, Dicer, cell extract, or serum was added as indicated and the samples were incubated at 37°C until the reactions were stopped by mixing with a final concentration of 10% (v/v) Contrad 70 at pH 9.3 (Decon Labs) as previously described [26]. For the samples of the FRET gel in 32P labeled with T4 polynucleotide kinase (PNK) and γ-32P-ATP, mixed with loading buffer to give a final concentration of 1×TBE, 0.025% (w/v) bromophenol blue, and 40% (v/v) formamide, and gel purified on a denaturing, 8 M urea, 20% (w/v) polyacrylamide gel. The radiolabeled full-length RNA was excised from the gel, diffusion eluted into 1 mM EDTA overnight, and ethanol precipitated. The dried RNA was dissolved in water.The 21-nt non-phosphorylated, doubly-fluorophore labeled RNA was 5′-32P labeled RNA  was mixed with 50 nM non-radiolabeled, but 5′ phosphorylated, doubly-fluorophore labeled RNA and 100 nM of the complementary 21-nt RNA strand, and annealed as described above in standard buffer. Degradation was initiated by the addition of a final total protein concentration of 2.49 mg/ml cell extract or 11.1 mg/ml serum at 37°C. For the analysis in , the assays were carried out over a range of 0–30% (v/v) HeLa cell extract. Time points were taken after 2, 5, 10, 20, 40, and 80 min for the cell extract sample and 5, 10, 20, 40, 80, 120, and 180 min for the serum sample. For the analysis in 5′-t and fitted with a simple linear regression line to estimate the observed rate constant kobs:For the radioactive dsRNA degradation data in These assays were carried out over a range of 0–30% (v/v) added HeLa cell extract, equivalent to 0–2.5 mg/ml total HeLa protein (concentration measured by Bradford assay). The observed rate constants were plotted as a function of total protein content and fitted as above with hyperbolic binding equation 2.32P labeled RNA in standard buffer at 4°C. After 2 h incubation the reaction was stopped by mixing with non-denaturing loading buffer to a final concentration of 1×TBE, 0.025% (w/v) bromophenol blue and 0.025% (w/v) xylene cyanol and the RNA-associated complexes were separated by non-denaturing 4% (w/v) polyacrylamide gel electrophoresis at 4oC, quantified and normalized to the sum of all bands in a lane using a Typhoon 9410 Variable Mode Imager with Image Quant software , essentially as described [35]. Three independent analyses gave similar results.The gel electrophoretic mobility shift assays of RISC-related RNA:protein complexes were initiated by the addition of a final total protein concentration of 2.49 mg/ml cell extract to the annealed 5′-Figure S1FRET ratio time trace upon incubating doubly fluorophore labeled 21-nt dsRNA with either serum (red) or 2 ng/ml of purified RNase A (black). The real-time FRET traces for each of these conditions reveal that both serum and the purified nuclease manifest a significant initial increase in FRET ratio.(DOC)Click here for additional data file.


Caenorhabditis elegans unc-13 gene is required for SV exocytosis, and encodes long and short isoforms (UNC-13L and S). Fast release was mediated by UNC-13L, whereas slow release required both UNC-13 proteins and was inhibited by Tomosyn. The spatial location of each protein correlated with its effect. Proteins adjacent to the dense projection mediated fast release, while those controlling slow release were more distal or diffuse. Two UNC-13L domains accelerated release. C2A, which binds RIM , anchored UNC-13 at active zones and shortened the latency of release. A calmodulin binding site accelerated release but had little effect on UNC-13’s spatial localization. These results suggest that UNC-13L, UNC-13S, and Tomosyn form a molecular code that dictates the timing of neurotransmitter release.Synaptic transmission consists of fast and slow components of neurotransmitter release. Here we show that these components are mediated by distinct exocytic proteins. The DOI:http://dx.doi.org/10.7554/eLife.00967.001 Neurons communicate with one another at junctions called synapses. When an electrical signal known as an action potential travels along a neuron and arrives at a synapse, the neuron releases a package of transmitter chemicals into the synapse. These chemicals then diffuse across the gap and bind to receptors on a second neuron, conveying the signal to the target neuron.The strength of a synapse depends in part on the number of packages, or vesicles, of transmitter chemicals that are available for release. Most synapses contain multiple populations of vesicles: some that are released within a few milliseconds of the arrival of an action potential, and others that are released more slowly. The vesicles that are released rapidly are found close to sites at which calcium ions enter the neuron, whereas the others are located further from these sites. However, little is known about the molecular basis of the differences between fast and slow vesicle release.C. elegans, a nematode worm that is often used in neuroscience because it has a simple nervous system, consisting of just 302 neurons, and a well-characterized genome. Hu et al. showed that the release of synaptic vesicles at the neuromuscular junction between neurons and muscles in C. elegans also has slow and fast components. A long form of UNC-13, which is also found in mammals, promotes fast release of transmitter vesicles. Slow release is mediated by an independent pathway that involves both long and short UNC-13 proteins, as well as a protein called Tomosyn. As in mammals, long UNC-13 is localized to the sites at which calcium ions enter neurons, whereas short UNC-13 is more widely distributed throughout neurons.Now Hu et al. have studied the proteins involved in these two processes in The work of Hu et al. provides a molecular explanation for how the timing of transmitter release is determined. Because the UNC-13 and Tomosyn proteins are evolutionarily conserved, this mechanism is likely to be present in other animals too.DOI:http://dx.doi.org/10.7554/eLife.00967.002 Caenorhabditis elegans expresses two. It is not known if each Munc13 protein has distinct functions. Do they mediate different forms of plasticity? Do they promote exocytosis of different subpopulations of SVs?The amount of neurotransmitter released at a synapse, and consequently synaptic strength, is often limited by the number of synapse vesicles (SVs) available for release. To become competent to undergo calcium-evoked fusion, SVs must first physically attach to the plasma membrane (termed docking), and then must undergo a second process that has been termed priming. Docking and priming are both promoted by UNC-13/Munc13 proteins, whereas Tomosyn inhibits both processes . MammalsAt most synapses, neurotransmitter release comprises multiple populations of SVs that fuse with distinct kinetics and distinct release probabilities . These kAlthough a great deal is known about how synchronous and delayed release are regulated by calcium and by activity , relativC. elegans cholinergic neuromuscular junction (NMJ) as a model to address these questions. Transmission at this synapse is mediated by graded release of neurotransmitter, whereby release varies with the strength of depolarization . Slow release is mediated by two UNC-13 isoforms (UNC-13L and S) and is inhibited by a third priming protein, the syntaxin-binding protein Tomosyn. These results suggest that UNC-13L, UNC-13S, and Tomosyn form a protein code that dictates the timing of neurotransmitter release.unc-13 gene contains two promoters. The upstream promoter drives expression of long isoforms, while a downstream promoter (which lies in the intron between exons 13 and 14) drives expression of short isoforms . UNC-13L and UNC-13S share a common domain  that contains the C1, C2B, C2C, and MUN domains (C. elegans UNC-13L (mCherry-tagged) and UNC-10 RIM (GFP-tagged) proteins were colocalized at presynaptic terminals  mutants. The s69 allele corresponds to a 5 base pair deletion in an exon shared by UNC-13S and UNC-13L (exon 21), which shifts the reading frame thereby inactivating both isoforms (unc-13(s69) mutants  mutants  mutants, indicating that both isoforms efficiently promote SV priming. The sucrose-evoked charge observed in strains containing UNC-13S transgenes was significantly larger than those observed in wild-type controls, consistent with our prior studies  quantal content defect .To assess changes in the kinetics of ACh release, we recorded stimulus-evoked EPSCs . To estiunc-13(s69) produced evoked responses with kinetics similar to wild-type controls  between calcium entry and exocytosis is determined by how rapidly calcium reaches primed SVs and binds to their calcium sensors. Tighter coupling is expected when SVs are physically closer to calcium channels or when they have higher intrinsic calcium sensitivity. Thus, differences in evoked EPSCs kinetics could be mediated by differential coupling of primed SVs to the sites of calcium entry. To test this idea, we analyzed the effects of the calcium chelator EGTA on evoked responses. Because EGTA binds calcium slowly, it inhibits SV fusions that occur later in time, that is the SVs that are more loosely coupled with calcium entry .C. elegans NMJ can be distinguished by their calcium coupling, we analyzed the effect of EGTA on evoked EPSCs , evoked EPSC amplitudes were very similar to those observed in 1 mM CaCl2 with EGTA  of unc-13 (e51 and e1091 mutations are not included in the unc-13S mRNA (encoded by exons 14–31)  can function independently to promote fast and slow release. Because transgenes are typically expressed at higher levels than endogenous genes, we wanted to determine if endogenously expressed UNC-13 isoforms also function independently. To address this question, we analyzed f unc-13 . These mf unc-13 . The e51s 14–31) . Thus, eunc-13 [L−S+] mutants should be similar to that observed in UNC-13S-rescued animals. Contrary to this idea, the evoked EPSC  mutants, consistent with prior studies  [L−S+] (22% WT) but was slightly larger than in unc-13(s69) mutants    (McEwen  p<0.05) . Thus, iunc-13 [L−S+]-evoked EPSC defect was fully rescued by a transgene expressing UNC-13L (unc-13 [L−S+] mutants was indistinguishable from that in wild-type controls and was significantly larger than in UNC-13L-rescued unc-13(s69) mutants (unc-13(e1091) [L−S+] mutants were indistinguishable from wild-type controls (unc-13(e1091) [L−S+] mutants  mutants reconstituted only fast release. These results suggest that endogenous UNC-13S requires UNC-13L to perform its function.The  UNC-13L , indicat mutants . The actcontrols . EGTA tr mutants . Thus, runc-13 [L−S+] mutant phenotypes could result from a failure to express UNC-13S in unc-13 [L−S+] mutants. To test this idea, we measured the abundance of UNC-13S mRNA by quantitative PCR. We found that UNC-13S mRNA levels were modestly increased in unc-13 [L−S+] mutants compared to wild-type controls . Consequently, decreased expression of the UNC-13S promoter is unlikely to explain the synaptic transmission defects observed in mutants lacking UNC-13L.The discrepancy between the UNC-13S rescue and unc-13 [L−S+] synaptic defect is that UNC-13S function is actively inhibited by an endogenous regulatory mechanism. In this scenario, overexpressing UNC-13S could titrate out the inhibitor (through direct binding or indirectly via a shared binding partner), thereby restoring synaptic function. Several studies suggest that the Tomosyn protein could provide this inhibitory function. In mutants lacking Tomosyn, neurotransmitter release is increased  double mutants (tom-1 unc-13 [L−S+] double mutants was significantly larger than in unc-13 [L−S+] single mutants (tom-1 unc-13 [L−S+] double mutants was accompanied by a corresponding increase in the sucrose-evoked charge (tom-1 unc-13 [L−S+] double mutants was significantly higher than in unc-13 [L−S+] single mutants (tom-1 unc-13 [L−S+] double mutants was significantly greater than in tom-1 unc-13(s69) double mutants. The only genetic difference between these strains is that tom-1 unc-13 [L−S+] double mutants retain endogenous UNC-13S, while tom-1 unc-13(s69) double mutants lack UNC-13S. Consequently, these results suggest that Tomosyn inhibits the priming activity of endogenously expressed UNC-13S.To determine if TOM-1 inhibits UNC-13S function, we analyzed  mutants . As prev mutants  and the  mutants  were sig priming . Evoked  mutants , implyin mutants . Increasd charge , implyin mutants . Thus, etom-1 unc-13(s69) double mutants than in unc-13(s69) single mutants  [L−S+] and unc-10 RIM mutants. Because both mutations dramatically reduce release, we analyzed double mutants lacking TOM-1/Tomosyn to increase the sensitivity of our experiments. The activation kinetics of evoked EPSCs in tom-1 unc-13(e1091) and in tom-1; unc-10 double mutants were significantly slower than those in tom-1 single mutants (tom-1 unc-13(e1091) and in tom-1; unc-10 double mutants were also significantly less EGTA-resistant than those in tom-1 single mutants  . UNC-10/Our analysis of ΔC2A suggests that in addition to the C2A domain, other sequences in the UNC-13L NTD contribute to the faster release kinetics. The NTDs of Munc13 proteins have amphipathic helixes that bind calmodulin . CalmoduTo test its functional importance, we constructed a chimeric UNC-13 protein containing the predicted calmodulin-binding site (residues 556–610) fused to the R domain (CaMR) . An mCheC. elegans NMJs is a composite of fast and slow ACh release, and we describe a molecular mechanism that dictates release kinetics. Fast release comprises fusion of SVs that are tightly coupled to calcium entry, and is mediated by UNC-13L. Slow release consists of SV fusions that are loosely coupled to calcium entry is promoted by UNC-13S and L acting in concert, and is inhibited to TOM-1 Tomosyn. UNC-13L accelerates release by two mechanisms: increasing the spatial proximity of primed SVs to calcium channels and enhancing the calcium sensitivity of release by binding to calmodulin. These experiments define a molecular code that dictates the kinetics of neurotransmitter release during evoked responses. Below we discuss the significance of these findings.Here we show that synaptic transmission at the tom-1 mutants was eliminated by EGTA treatment, indicating that the extra release was mediated by fusion of SVs that are loosely coupled to calcium entry. In transgenic animals, TOM-1 inhibited UNC-13S-mediated slow release far more potently than UNC-13L-mediated fast release. The apparent selectivity for inhibiting slow release could result from the distal localization of the TOM-1 protein, as immuno-EM studies indicate that TOM-1 staining peaks ∼300 nm from the dense projection  are localized near dense projections . Slow reunc-13(e1091) [L−S+] mutants (<100 nm from dense projections) and in unc-10 RIM mutants (<50 nm from dense projections), whereas docking of distal SVs was unaffected in both mutants (unc-13(e1091) [L−S+] and unc-10 RIM mutations made evoked release slower and more loosely coupled to calcium entry, both indicating that SVs docked proximal to the dense projection are required for fast release. By contrast, inactivating TOM-1/Tomosyn increased docking of distal SVs , and sensitivity to EGTA inhibition all support a model whereby fast ACh release is mediated by the subpopulation of SVs that are docked adjacent to dense projections (<100 nm), while slow release is mediated by fusion of distal SVs (100–350 nm from dense projections).ca = 200 µm2/s). Thus, it seems likely that exocytosis of UNC-13S primed SVs is intrinsically slower than that of UNC-13L primed vesicles.Are differences in the spatial location of primed vesicles sufficient to explain the kinetics of fast and slow release? Several results argue against this idea. The rise time of fast and slow release differ by ∼2 ms. If this difference was caused solely by a diffusional delay in calcium binding, slow SVs would have to be 3 μm farther from calcium channels than fast SVs  also contribute to its physical location because the ΔC2A protein was strongly colocalized with UNC-10/RIM. The calmodulin-binding site in UNC-13L’s NTD also accelerated release. The analogous calmodulin-binding site in mouse Munc13-1 and Munc13-2 is required for augmentation, a form of short-term plasticity . Based ounc-13 [L−S+] mutants, implying that UNC-13L did not alter expression of the UNC-13S promoter. UNC-13L could directly bind to UNC-13S thereby altering its activity or stability. UNC-13L could prevent inhibition of UNC-13S by Tomosyn. Finally, UNC-13L could increase the release probability of UNC-13S primed SVs. Further experiments will be required to determine how UNC-13L regulates slow release.In addition to promoting fast release, UNC-13L also acts in conjunction with UNC-13S to promote slow release. Mutations inactivating UNC-13L dramatically reduced fast and slow release, and both forms of release were re-instated by restoring UNC-13L expression. Thus, UNC-13L promotes but is not required for slow release. The UNC-13L requirement for slow release was reduced (but not eliminated) when the total number of primed SVs was increased (by inactivating Tomosyn or by overexpressing UNC-13S). UNC-13L could regulate slow release by a variety of mechanisms. UNC-13S mRNA levels were not decreased in Our results do not exclude the idea that other potential mechanisms also contribute to differences in UNC-13L and UNC-13S release kinetics. For example, UNC-13 and TOM-1 could regulate the kinetics or duration of calcium entry or could alter the spatial distribution of open calcium channels. Further experiments will be required to test these ideas.Our results suggest that fast and slow release are mediated by exocytosis of distinct subpopulations of SVs, and that distinct priming molecules mediate fast and slow SV fusions. In this manner, fast and slow release could be independently adjusted by modulatory pathways. It is intriguing that the fast and slow release components appear to function in parallel. We were able to construct mutants in which evoked release is mediated purely by either the fast or slow mechanism. Thus, both forms of release can occur in isolation. These results imply that fast SVs are not derived from maturation of slow SVs, and vice versa. Our results are more compatible with the idea that fast and slow release comprise independent exocytosis mechanisms that operate in parallel. Fast and slow release share a requirement for UNC-13L, which provides a potential mechanism for coordinately regulating changes in these two release pathways.Adjusting the contributions of fast and slow release will alter the duration of postsynaptic responses. This could have several important consequences for circuit function. Prolonged EPSCs will result in increased integration of synaptic inputs, which could alter the rate of postsynaptic spiking. Shifting the kinetics of postsynaptic responses could also alter spike timing-dependent plasticity. In sensory circuits, prolonged postsynaptic responses could alter the temporal integration of sensory inputs, and may alter the kinetics of sensory evoked responses. All these changes could have profound effects on circuit development, cognition, and behavior. Consistent with this idea, we recently showed that mutations linked to Autism alter the kinetics of neurotransmitter release . For theIn summary, we propose that the kinetics of neurotransmitter release are dictated by a protein code consisting of fast  and slow  priming factors, which mediate fusion of proximal and distal SVs, respectively. All these molecules are conserved across phylogeny, and orthologous proteins are found at both NMJs and central synapses. Thus, we propose that the basic mechanisms governing the kinetics of neurotransmitter release are ancient, and will apply to many synapses. Consistent with this idea, a recent study showed that Munc13 proteins that lack C2A domains (bMunc13-2 and Munc13-3) are located more distally and mediate slow release at the Calyx of Held .Escherichia coli was utilized. The following strains were utilized in this study:Wild type, N2 bristolunc-13(s69)KP6901 unc-13(e1091)KP6902 unc-13(e51)KP6903 tom-1(nu468)KP3707 tom-1(nu468) unc-13(s69)KP3810 tom-1(nu468) unc-13(e1091)KP6904 tom-1(nu468) unc-13(e51)KP6905 tom-1(nu468);unc-10(md1117)KP7162 acr-16(ok789)KP5982 unc-29(x29)KP5984 snb-1::UNC-13L];unc-13(s69)KP6893 nuEx1515 [Psnb-1::UNC-13L];unc-13(e1091)KP6894 nuEx1516 [Psnb-1::UNC-13L];tom-1(nu468) unc-13(s69)KP6895 nuEx1517 [PnuIs46 [Punc-13::UNC-13S::GFP];tom-1(nu468) unc-13(s69)KP6896 nuIs486 [Punc-129::UNC-13L::mCherry]KP6897 acr-2::UNC-10::mCherry]KP6898 nuEx1518 [PnuIs165 [Punc-129::UNC-10::GFP]KP3928 nuIs46 [Punc-13::UNC-13S::GFP];unc-13(s69)KP6899 snb-1::UNC-13SL];unc-13(s69)KP7151 nuEx1590 [Psnb-1::UNC-13ΔC2A];unc-13(s69)KP7152 nuEx1591 [Psnb-1::UNC-13R];unc-13(s69)KP7153 nuEx1592 [Psnb-1::UNC-13C2AR];unc-13(s69)KP7154 nuEx1593 [Psnb-1::UNC-13CaMR];unc-13(s69)KP7155 nuEx1594 [Psnb-1::UNC-13caMR(F574R)];unc-13(s69)KP7156 nuEx1595 [Punc-129::UNC-13ΔC2A::mCherry]KP7157 nuEx1596 [Punc-129::UNC-13C2AR::mCherry]KP7158 nuEx1597 [Punc-129::UNC-13CaMR::mCherry]KP7159 nuEx1598 [Punc-129::UNC-13S::mCherry]KP7160 nuEx1599 [PnuIs490 [Punc-129::UNC-13R::mCherry]KP7161 Strain maintenance and genetic manipulation were performed as described . Animalsmyo-2::NLS-GFP (KP#1106) or Pmyo-2::NLS-mCherry (KP#1480) as coinjection markers. Integrated transgenes were obtained by UV irradiation of strains carrying extrachromosomal arrays. All integrated transgenes were out-crossed at least six times. UNC-13L and UNC-13S expression constructs contained full-length cDNAs, with the exceptions noted below. For UNC-13L, we utilized a full-length ZK524.2e cDNA , whose expression as an SL1 spliced mRNA we confirmed by qPCR. The ZK524.2e cDNA corresponds to the LR mRNA previously described (nuIs46) (unc-13 promoter (UNC-13S rescue) or the snb-1 promoter . For imaging experiments, UNC-13  and UNC-10  (unc-129 promoter.Transgenic strains were isolated by microinjection of various plasmids using either Pescribed . For UNCescribed . We coul(nuIs46)  for elec tagged)  transgenWorm tracking and analysis were performed as previously described  with minAll quantitative imaging was done on a Zeiss Axioskop, using an Olympus PlanAPO 100 × 1.4 NA objective and a CoolSNAP HQ CCD camera . Worms were immobilized with 30 mg/ml BDM (Sigma). Image stacks were captured and maximum intensity projections were obtained using Metamorph 7.1 software . GFP fluorescence was normalized to the absolute mean fluorescence of 0.5-mm FluoSphere beads . For dorsal cord imaging, young adult worms, in which the dorsal cords were oriented toward the objective, were imaged in the region midway between the posterior gonad bend at the tail. Line scans of dorsal cord fluorescence were analyzed in Igor Pro (WaveMetrics) using custom-written software .C. elegans as previously described . Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and Erik Jorgensen (peer reviewer).The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.C. elegans neuromuscular junction, which displays two clearly distinct kinetic components of release and exploit the relative ease of genetic manipulations in this system to provide important new information regarding two sources of heterogeneity: localization of vesicles relative to Ca-sources and intrinsic “willingness” of vesicles to fuse. They relate these aspects to the only two isoforms of the UNC-13 protein, which are present in C. elegans, suggesting roles of the C2A domain of UNC-13L in proper localization of vesicles relative to the dense projection and the calmodulin-binding domain of the same isoform in accelerating release. Furthermore, they describe an inhibitory/competitive role of Tomosyn, acting on UNC-13S mediated release. The data are of highest relevance for the understanding of synaptic function.Hu et al. provide exciting new data on a few longstanding questions in synaptic research regarding heterogeneity of release-probability of synaptic vesicles. They use the eLife. No additional experiments are necessary prior to publication. However, several issues should be addressed in the text prior to resubmission and publication. In general the manuscript could be improved by re-organization. This would take considerable work and is not necessary for publication, but it is a suggestion that the authors might wish to consider. Two textual revisions are considered essential for publication, highlighted as #1 and #2 under “Essential revisions”.All of the reviewers consider this work high profile and appropriate for publication in Essential revisions:++ sensitivity of the diffusely distributed CamMR would result in prolonged, EGTA-sensitive release. A decreased Ca++ sensitivity, on the other hand, would produce short-lived EGTA-insensitive release, since release would be restricted to the peak-Ca, both temporally and spatially, since release probability is low and a high-power function of local [Ca++]. It should also be noted that the UNC-13L rescue has very low quantal content (1) The reviewers strongly suggest a more cautious interpretation of the CaM-domain data. In essence, does the CaM-domain increase or decrease sensitivity? A high Ca content , as does2) The absence of detailed cartoons diagramming the constructs makes a careful reading of the manuscript difficult and, at times, confusing. It was not clear what the constructs looked like and how much of the surrounding region was included or whether there was a flexible linker. Given that supplements are now commonplace, it seems that DNA constructs can be described precisely, so that future researchers will be able to either repeat or interpret the experiments. 1) The reviewers strongly suggest a more cautious interpretation of the CaM-domain data. In essence, does the CaM-domain increase or decrease sensitivity?We agree that it was unwise to speculate about how the CaM binding site alters the overall calcium sensitivity of release. As suggested, we deleted that speculation from our Discussion, which now reads: “Our results suggest that this calmodulin binding site also accelerates release. Thus, the faster kinetics of UNC-13L mediated release results from both tighter spatial coupling to calcium channels, and faster intrinsic kinetics of exocytosis.”2) The absence of detailed cartoons diagramming the constructs makes a careful reading of the manuscript difficult and, at times, confusing. It was not clear what the constructs looked like and how much of the surrounding region was included or whether there was a flexible linker. Given that supplements are now commonplace, it seems that DNA constructs can be described precisely, so that future researchers will be able to either repeat or interpret the experiments.As requested, we now provide a new figure,


Accumulating evidence demonstrates that non-coding RNAs (ncRNAs) are indispensable components of many organisms and play important roles in cellular events, regulation, and development.Trichophyton rubrum by constructing and sequencing a cDNA library from conidia and mycelia. We identified 352 ncRNAs and their corresponding genomic loci. These ncRNA candidates included 198 entirely novel ncRNAs and 154 known ncRNAs classified as snRNAs, snoRNAs and other known ncRNAs. Further bioinformatic analysis detected 96 snoRNAs, including 56 snoRNAs that had been annotated in other organisms and 40 novel snoRNAs. All snoRNAs belonged to two major classes—C/D box snoRNAs and H/ACA snoRNAs—and their potential target sites in rRNAs and snRNAs were predicted. To analyse the evolutionary conservation of the ncRNAs in T. rubrum, we aligned all 352 ncRNAs to the genomes of six dermatophytes and to the NCBI non-redundant nucleotide database (NT). The results showed that most of the identified snRNAs were conserved in dermatophytes. Of the 352 ncRNAs, 102 also had genomic loci in other dermatophytes, and 27 were dermatophyte-specific.Here, we analysed the small non-coding RNA (ncRNA) transcriptome of T. rubrum. These results also provide important information to complement the current annotation of the T. rubrum genome, which primarily comprises protein-coding genes.Our systematic analysis may provide important clues to the function and evolution of ncRNAs in  Numerous studies have demonstrated that non-coding RNAs (ncRNAs) are widely expressed in both prokaryotes and eukaryotes -4. FurthNcRNAs include highly abundant and functionally important RNAs, such as transfer RNA (tRNA) and ribosomal RNA (rRNA), as well as other small, stable RNAs, such as small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), RNase P and mitochondrial RNA processing (MRP) RNA, signal recognition particle (SRP) RNA, and telomerase RNA. These RNAs have been characterised and are involved in splicing, ribosome biogenesis, translation, and chromosome replication ,11. ReceSchizosaccharomyces pombe. Functional analyses of 20 Box H/ACA snoRNAs indicated that the snoRNAs evolved in coordination with rRNAs to preserve post-transcriptional modification sites among distant eukaryotes . We collected gel bands containing RNAs of 70–500 bp, excluding the 5.8S rRNA band. RNAs were passively eluted and then ethanol-precipitated. RNA size and concentration were quantified with the Agilent 2100 Bioanalyser and the Agilent RNA 6000 Pico Kit according to the manufacturer’s protocols. The fractionated RNA was dephosphorylated with FastAP (Fermentas) and ligated to the 3′-adaptor oligonucleotide  by T4 RNA ligase (Promega). Subsequently, the RNA was reverse transcribed using oligo 3RT (CTGGGTACCGTGGTCAAA) and converted into double-stranded cDNA with a SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen). The ds-cDNA was purified using the MinElute Reaction Cleanup Kit (Qiagen) according to the manufacturer’s protocol.et al.[For 454/Roche sequencing, approximately 5 μg of the size-fractionated cDNA sample (70–500 bp) was blunted. The pieces were then ligated with short adaptors prior to amplification and sequencing. The sequencing run was performed using the method of Margulies et al..T. rubrum and six related dermatophytes  were downloaded from the Broad Institute web site (http://www.broadinstitute.org/annotation/genome/dermatophyte_comparative/MultiDownloads.html).After 454 sequencing, the 5′ and 3′ adaptors were removed from the reads. Genome data for The high-quality reads were mapped to the genome using BLAST (version 2.2.22) . Then, reads that were 80% mapped to the genome were clustered according to their genomic position and assembled into contigs according to the genomic sequence at the corresponding loci. The ORFs in the contigs were predicted using getorf in the EMBOSS program (version 6.3.1). Contigs with less than 80% ORF were aligned to TrED EST sequences and the NCBI non-redundant protein sequence database (NR) ,39. The T. rubrum snRNAs are compared with the homologs in other fungi using the multiple sequence alignment software ClustalW2. The secondary structures of aligned sequences are predicted by RNAalifold [T. rubrum were downloaded from GenBank under the accession number JX431933.Aalifold . The putAalifold ,18. The T. rubrum and all snRNAs identified in our data [To predict the two classes of snoRNAs and their putative targets in our data, we used the Snoscan and SnoGPS programs, defining the potential target sequences as the 5.8S, 18S, and 25S rRNAs of our data ,18.32P)-ATP and hybridised at 45°C for 16 hr. After stringency washes, the blots were exposed to phosphor storage screens, which were then scanned with a Typhoon 9200 imager .For the northern blot analysis, 10 μg of total RNA was separated by electrophoresis on an 8% polyacrylamide gel containing 7 M urea and then electrotransferred onto a nylon membrane  using a semi-dry blotting apparatus (BioRad). A total of 24–30 mer DNA oligonucleotides antisense to snRNAs and 15 randomly selected ncRNA candidates were end-labelled with (γT. rubrum were submitted to GenBank under the following accession numbers: KC352999 – KC353350.The 352 ncRNAs sequences of The authors declare that they have no competing interests.TL, TX and XX performed experiments. XR, TX and JY analyzed and interpreted data. JD and LS carried out the ncRNA cDNA sequencing. TL and XR wrote the paper. RC and QJ proposed the research goal, supervised the whole studies and provided a critical review of the manuscript. All authors read and approved the final manuscript.T. rubrum.Detailed information on ncRNAs identified in Click here for fileSecondary structure predictions of aligned snRNAs.Click here for filePotential base-paring between H/ACA box snoRNAs and rRNAs predicted by snoGPS.Click here for fileConversed sncRNAs in all seven dermatophytes.Click here for file


Next-generation sequencing of RNA (RNA-Seq) is a powerful tool that can be applied to a wide range of biological questions. RNA-Seq provides insight at multiple levels into the transcription of the genome. It yields sequence, splicing and expression-level information, allowing the identification of novel transcripts and sequence alterations. We have been developing and comparing methods for samples that present a challenge: that is, those with low quantity and/or quality RNA.0t hybridization with a duplex-specific nuclease for light normalization and NuGEN’s Ovation RNA-Seq kit. We demonstrated that these three methods successfully reduce the fraction of rRNA to less than 13%, even when starting from degraded RNA. We compared the performance between these methods and with ‘gold standard’ RNA-Seq data , using quantitative criteria that evaluate effectiveness for genome annotation, transcript discovery and expression profiling. The application of these methods to samples that contain degraded RNA and/or very low input amounts of RNA will also be presented.RNA-Seq methods that start from total RNA and do not require the oligo(dT) purification of mRNA will be valuable for such challenging samples. Such methods use alternative approaches to reduce the fraction of sequencing reads derived from rRNA. We will present results from multiple approaches, including the use of not-so-random (NSR) primers for cDNA synthesis, low-C


RanBPM (Ran-binding protein in the microtubule-organizing centre) was originally reported as a centrosome-associated protein in human cells. However, RanBPM protein containing highly conserved SPRY, LisH, CTLH and CRA domains is currently considered as a scaffolding protein with multiple cellular functions. A plant homologue of RanBPM has not yet been characterized.Arabidopsis thaliana. AtRanBPM protein has highly conserved SPRY, LisH, CTLH and CRA domains. Cell fractionation showed that endogenous AtRanBPM or expressed GFP-AtRanBPM are mainly cytoplasmic proteins with only a minor portion detectable in microsomal fractions. AtRanBPM was identified predominantly in the form of soluble cytoplasmic complexes ~230 – 500 kDa in size. Immunopurification of AtRanBPM followed by mass spectrometric analysis identified proteins containing LisH and CRA domains; LisH, CRA, RING-U-box domains and a transducin/WD40 repeats in a complex with AtRanBPM. Homologues of identified proteins are known to be components of the C-terminal to the LisH motif (CTLH) complexes in humans and budding yeast. Microscopic analysis of GFP-AtRanBPM in vivo and immunofluorescence localization of endogenous AtRanBPM protein in cultured cells and seedlings of Arabidopsis showed mainly cytoplasmic and nuclear localization. Absence of colocalization with γ-tubulin was consistent with the biochemical data and suggests another than a centrosomal role of the AtRanBPM protein.Based on sequence similarity, we identified a homologue of the human RanBPM in Arabidopsis RanBPM protein physically interacts with LisH-CTLH domain-containing proteins. The newly identified high molecular weight cytoplasmic protein complexes of AtRanBPM showed homology with CTLH types of complexes described in mammals and budding yeast. Although the exact functions of the CTLH complexes in scaffolding of protein degradation, in protein interactions and in signalling from the periphery to the cell centre are not yet fully understood, structural conservation of the complexes across eukaryotes suggests their important biological role.We showed that as yet uncharacterized  Arabidopsis genome contains three genes encoding AtRan  was cloned into Gateway-compatible plant binary vectors for N-terminal GFP and C-terminal GFP fusion. r et al. . Transfo799 was cArabidopsis extracts, the antibody recognized a band of 52 kDa, the predicted molecular mass (MW) of AtRanBPM, and a band corresponding to the MW of GFP-AtRanBPM   while the truncated version of human RanBPM is present in centrosomal and ectopic microtubule nucleation sites , AtRanBPRanBPM belong to genes with unknown functions. We found that the product of AtRanBPM is predominantly a cytoplasmic protein that is a part of protein complexes with a molecular mass of approximately 230 – 500 kDa. A large protein complex of RanBPM was described by Nishitani et al.  was obtained by PCR amplification using P fusion  were useArabidopsis suspension cultures of Landsberg erecta ecotype stably expressing GFP-AtRanBPM or transiently expressing AtRanBPM-GFP were prepared according to the protocol of Mathur et al.  for V. vinifera, [Swiss-Prot:B9MWC1] for P. trichocarpa, [Swiss-Prot:C5XUT1] for S. bicolor, [Swiss-Prot:Q6ZI83] for O. sativa, [Swiss-Prot:B6UAR9] for Z. mays, [Swiss-prot: Q6VN20] for human RanBP10, [Swiss-prot: A3KMV8] for RanBP10 from Bos taurus, [Swiss-prot: B5LX41] for RanBP10 from Felis catus, [Swiss-prot: Q6VN19] for RanBP10 from Mus musculus, [Swiss-prot: Q1LUS8] for RanBP10 from Danio rerio, [Swiss-prot: Q9PTY5] for RanBP9 from Xenopus laevis, [Swiss-prot: Q96S59] for human RanBP9, [Swiss-prot: P69566] for RanBP9 from Mus musculus, [Swiss-prot: Q4Z8K6] for RanBP9/10 from Drosophila melanogaster and [Swiss-prot: P53076] for Gid1/Vid30 homologue from Saccharomyces cerevisiae. Distance bars are given bottom left and bootstrap values are indicated at the nodes. software . Branch Click here for fileImmunopurification of GFP-AtRanBPM protein. Immunopurification of GFP-AtRanBPM from extracts of GFP-AtRanBPM expressing cell cultures (IP GFP-RanBPM). GFP immunopurification from extracts of wild type Ler Arabidopsis cells (IP WT) was used as a negative control. A- Proteins were silver stained after separation on SDS-PAGE. Bands corresponding to MW similar of the proteins copurified with GFP-AtRanBPM (IP GFP-RanBPM) were not present in the negative control (IP WT). B- Signal for AtRanBPM was absent in the negative control (IP WT) after detection with anti-AtRanBPM antibody on Western blots. C- Proteins identified by MALDI-MS in negative control (IP WT in A) were background contamination. Click here for fileIdentities and similarities between proteins copurifying with AtRanBPM and human CTLH complex members. Identities and similarities between Arabidopsis and human proteins were analysed in WU-BLAST. Click here for fileAdditional proteins copurified with AtRanBPM. The proteins were identified by LC-MALDI-MS/MS and the identity of the matched peptides was confirmed by high-resolution MALDI-FTMS with mass accuracy below 1 ppm. Click here for fileGFP-AtRanBPM inArabidopsisroot cells. AtRanBPM-GFP signal is dynamic and moving with the cytoplasmic stream. Click here for fileCellular localization of C-terminal GFP and N-terminal GFP AtRanBPM fusion proteins. Cells of Arabidopsis expressing C-terminal GFP AtRanBPM (AtRanBPM-GFP) showed weak cytoplasmic and nuclear signal and accumulation of perinuclear GFP signal similarly as observed for N-terminal GFP AtRanBPM (GFP-AtRanBPM). Click here for file


RNA-Seq is a recently developed sequencing technology, that through the analysis of cDNA allows for unique insights into the transcriptome of a cell. The data generated by RNA-Seq provides information on gene expression, alternative splicing events and the presence of non-coding RNAs. It has been realised non-coding RNAs are more then just artefacts of erroneous transcription and play vital regulatory roles at the genomic, transcriptional and translational level. Transcription of the DNA sense strand produces antisense transcripts. This is known as antisense transcription and often results in the production of non-coding RNAs that are complementary to their associated sense transcripts. Antisense tran-scription has been identified in bacteria, fungi, protozoa, plants, invertebrates and mammals. It seems that antisense tran-scriptional ‘hot spots’ are located around nucleosome-free regions such as those associated with promoters, indicating that it is likely that antisense transcripts carry out important regulatory functions. This underlines the importance of identifying the presence and understanding the function of these antisense non-coding RNAs. The information concerning strand ori-gin is often lost during conventional RNA-Seq; capturing this information would substantially increase the worth of any RNA-Seq experiment. By manipulating the input cDNA during the template preparation stage it is possible to retain this vital information. This forms the basis of strand-specific RNA-Seq. With an ability to unlock immense portions of new in-formation surrounding the transcriptome, this cutting edge technology may hold the key to developing a greater under-standing of the transcriptome. Drosophila melanogaster has approximately half this amount, while the salamander has twenty times this number of genes [As sequencing techniques become more sophisticated and our understanding of molecular biology increases, it has become apparent that the pathway from gene to protein is an intricate and multifaceted process. Organism complexity highlights this; there is no correlation between genome size or number of genes and the complexity of an organism . Intuitiof genes -4. One tof genes . Up 98% of genes -8. It isOne component of the transcriptome of particular interest is transcription from the DNA sense (or plus) strand. Transcription from this strand produces transcripts known as antisense transcripts or antisense RNAs (asRNAs) . CurrentRNA-Seq is a next-generation sequencing technique that allows for an in depth look into the transcriptome . It is p2. 2.1. DNA exists as a double stranded molecule; one strand is known as the sense (or plus) strand the other is known as the antisense (or minus) strand. The antisense strand contains all of the pertinent information for the formation of proteins; it was originally thought that this strand was the only strand that underwent transcription. Transcription from the sense strand is less common and it will produce what is knows as antisense transcripts or asRNAs. It was previously thought that this form of transcription was an aberration of the norm, however antisense transcription has been identified at higher then expected levels in prokaryotes and eukaryotes; including humans and other mammals , 17-21. 2.2. There exist a variety of pathways in which antisense transcripts can act as regulatory elements. It is possible to divide these pathways into three broad categories; transcription modulation, hybridization of sense-antisense RNA partners and chromatin modification. As the field of non-coding RNA is still relatively young and the field of asRNA comparatively unexplored, it is likely that more mechanisms of regulation by these elements will be elucidated in the future.2.2.1. Saccharomyces cerevisiae and bioinformatic studies have suggested that it does occur in mice and humans [The act of antisense transcription, rather than asRNA molecule itself can modulate gene expression levels. During transcription RNA polymerase binds to the promoter region of the gene and proceeds along the strand. If transcription occurs on the DNA sense strand and antisense strand simultaneously it can result in the RNA polymerases colliding. This is known as the transcriptional collision model . While id humans , 26. Thed humans . As expe2.2.2. Antisense transcripts are complementary to their sense partners; this means that there is always a chance of hybridization, and the formation of a RNA duplex. Duplex formation can exert regulatory functions in diverse ways. Factors that impact on the regulatory implications of the RNA duplex include length of hybridization, location along the RNA transcript of hybridization and whether the duplex forms in the nucleus or the cytoplasm. The formation of RNA duplexes is probably the main way in which antisense transcripts exert regulatory functions.THRA) has splicing patterns that can be altered by the presence of its antisense transcript partner [Antisense transcripts have the potential to mask many regulatory components of sense RNA transcripts. Alternative splicing is a process by which exons and introns of primary transcripts can be included, excluded or skipped to form unique mRNAs. Splicing is controlled by the presence of exonic splicing enhancers/silencers and intronic enhancer/silencers, the ratios of these elements impact on the splicing pattern . These e partner . It can  partner . The numSPI1 or PU.1) its non-coding antisense transcript partner stalls translation between the initiation and elongation steps [BACE1) has been associated with Alzheimer’s disease (AD) [APP), leading to a build up amyloid-β peptides. It has been shown that the antisense transcript of BACE1 is up-regulated in AD brains [BACE1 antisense transcript and BACE1 will form a duplex and this increases the stability of the transcript through the formation of secondary and tertiary structures. Formation of these secondary and tertiary structures protects the transcript from degradation while still allowing it to function as a cleaving enzyme. Furthermore, as amyloid-β peptides levels are increased the levels of BACE1 antisense transcripts are also elevated, creating a positive feedback loop [RNA duplex formation in the cytoplasm may alter the ability of a transcript to be translated. It is possible that the duplex formation blocks the ability of the transcript to associate with the ribosome hence altering the efficiency of the translation machinery. In the case of the gene spleen focus forming virus proviral integration oncogene spi1 (on steps . Furtherase (AD) . It playD brains . It has ack loop .2.2.3. Genomic DNA in eukaryotic cells must be assembled appropriately to fit within the nucleus of the cell. Generally, genomic DNA in its packaged state exists as 146bp segments tightly encompassing a histone protein octamer and these units are known as nucleosomes . Each nuIt is thought that long non-coding RNAs are involved in the regulation of chromatin, possessing the ability to remodel the nucleosome complex , 36. HisXIST) [XIST coats one of the X-chromosomes, the coated X will go on to become the inactive X [XIST known as TSIX, is a 40 kb RNA that is located 15 kb downstream of XIST [TSIX is suspected to play a role in X-inactivation, by keeping XIST from coating both X-chromosomes. It is suspected that TSIX silences XIST through the modification of the chromatin structure by recruiting protein complexes that are involved in heterochromatinization [XIST is repressed by TSIX it follows that the X-chromosome will remain active. Strand specific FISH probes have demonstrated that TSIX expression is associated with the active X, while no TSIX is found in cells that have entered the X-inactivation pathway [Perhaps the best example of an antisense transcript being involved in chromatin remodeling is X-inactivation . X-inactXIST) . The higactive X . The asR of XIST . TSIX isnization . If XIST pathway .3. RNA-Seq is a recently developed next-generation sequencing technology, that through the analysis of cDNA allows for unique insights into the transcriptome of a cell. The data generated by RNA-Seq provides information on gene expression, alternative splicing events, locations of transcription factor binding sites and the presence of non-coding RNAs. It has been realised non-coding RNAs are more than just artefacts of erroneous transcription and play vital regulatory roles at the genomic, transcriptional and translational level. It is thought that these additional levels of regulation are the greatest contributors to the complexity seen in higher eukaryotic organisms such as humans , 42. Thiet al., 2012 [http://galaxyproject.org/) have been developed to make these programs and various other more complex tools user friendly and accessible to all wet-lab research scientists even those with little or no bioinformatics training [The data generated by RNA-Seq can then be analyzed using free, open source bioinformatics software tools such as TopHat and Cufflinks . TopHat,l., 2012 . Open, wtraining -48.RNA-Seq has several advantages over other gene expression quantification platforms such as exon arrays and reverse transcription quantitative real-time PCR. RNA-Seq is a high throughput method that requires low levels of initial input RNA, it has a high resolution in terms of determination of transcripts structure and their quantification, low levels of background noise and produces fewer false positive results . Further4. http://www.illumina.com/products/truseq_rna_sample_prep_kit_v2.ilmn). This method makes use of a bead capture procedure, where the beads will selectively bind rRNA molecules, the RNA bound beads are then removed using a magnet. This leaves the diverse RNA species representing an intact transcriptome.Despite differences in sequencing reaction chemistries and base calling all of the RNA-Seq platforms share commonalities in terms of major steps in template preparation . The conAfter selection of the appropriate RNA fraction is completed, the molecules must be fragmented into smaller pieces, to a size between 200-500bp depending on the sequencing platform being used . This frhttp://www.illumina.com/products/tru seq_rna_sample_prep_kit_v2.ilmn).For the direct fragmenting of double stranded (ds) cDNA, first the RNA must be reverse transcribed. This can be done using either random hexamers or oligo(dT) primers. Again oligo(dT) primers will fail to pick up any RNAs that are not polyadenylated. The ds cDNA is then fragmented and primer adapters are ligated . Alterna5. The standard method of RNA-Seq library generation fails to preserve the information pertaining to which DNA strand was the original template during transcription and subsequent synthesis of the mRNA transcript. Since antisense transcripts are likely to have regulatory roles that are distinctly different from their protein coding complement, this loss of strand information results in an incomplete understanding of the transcriptome , 17, 53.1 and Figure 1.Methods for the construction of strand-specific RNA-Seq libraries can be split into two categories (i) the use of known orientation strand-specific adapters, and (ii) the chemical modification of strands . The mai5.1. Adapter methods for strand-specific RNA-Seq are conceptually difficult and require more consideration and planning when compared to standard RNA-Seq. There has been an array of different protocols outlined. These methods utilize the known sequences at the 5’ or 3’ end and the relative orientations of the RNA transcript to derive strand information.1A) [In the strand-specific 3’-end RNA-Seq method anchored oligo(dT) primers are first used to select for mRNA, which results in production of double-stranded cDNA molecules . Adapter1A) . This pr1A) . It is a1B) [Another strand-specific RNA-Seq protocol makes use of single-stranded (ss) cDNA and Illumina adapters  . The sta1B) . These s1B) . 1C). [Flowcell reverse transcription sequencing (FRT-Seq), is another category of RNA-Seq that maintains strand information. Of all the methods discussed it diverges the greatest amount from the standard RNA-Seq protocol, but it can be loosely classified as an adapter method . , 59. FRTet al. 2010 [http://www.illumina.com/products/truseq_small_rna_sample_prep_kit.ilmn), and the Life Technologies  SOLiD® Total RNA-Seq Kit that preserves strand specificity through the addition of adapters in a directional manner (http://products.invitrogen.com/ivgn/product/ 44453 74).There are other adapter methods for strand-specific RNA-Seq. These include direct strand-specific sequencing (DSSS) developed by Vivancos, al. 2010 , which i5.2. Another method of carrying out strand-specific RNA-Seq involves the marking the RNA strand through chemical modification, either on the RNA itself or during second strand cDNA synthesis . These m1D). Through the addition of bisulfite mix, all cytidine residues of the RNA strand are changed to uridine residues [The first method of chemical modification involves marking the original RNA template through the use of bisulfite treatment  [The second method of chemical modification, known as dUTP second strand, involves a modified second-strand synthesis step. During this step dUTPs are incorporated into reverse transcription reaction, this results in ds cDNA where the original strand has deoxythymidine residues while the complementary strand contains deoxyuridine residues. Through the use of Uracil-DNA-Glycosylase (UDG) these residues are degraded, which leaves the first strand intact  -66.et al., 2012 [® Stranded Total RNA Sample Prep Kit  uses a similar method to maintain strand information. For the Illumina protocol second-strand synthesis also involves the incorporation of dUTPs, however instead of using UDG to degrade the residues a special polymerase is used during amplification that will not incorporate these nucleotides. In a comprehensive comparison of various strand-specific RNA-Seq protocols using RNA extracted from Saccharomyces cerevisiae the dUTP second strand method was identified as the leading protocol [When sequencing proceeds the polarity information from the original RNA molecules is maintained. A recent protocol by Zhang l., 2012  further protocol  and it iprotocol -66.6. Antisense transcription and the expression of antisense transcripts add another layer of complexity to the transcriptome and gene regulation. The modes of regulation by antisense transcripts are diverse and, as a greater amount of research is directed to this area, it is expected that more modes of regulation will be revealed. Strand-specific RNA-Seq holds the key to fully understand the antisense transcriptome. When using RNA-Seq to annotate the transcriptome of higher eukaryotic organisms the following might be suggested. Ribosomal RNA depletion should be used for the selection of the RNA fraction to be sequenced, random hexamers for the generation of double-stranded cDNA libraries and strand-specific RNA-Seq for sequencing. By following these guidelines the entirety of the transcriptome can be annotated including the vital non-processed RNAs, such as ncRNAs and the strand of origin information will be retained. These recommendations might help to avoid generation of a limited snapshot of the transcriptome consisting almost entirely of protein coding mRNAs and processed RNAs, which does not fully demonstrate the complexity of the transcriptome.


RNA polymerase (pol) III transcription is responsible for the transcription of small, untranslated RNAs involved in fundamental metabolic processes such mRNA processing (U6 snRNA) and translation (tRNAs). RNA pol III transcription contributes to the regulation of the biosynthetic capacity of a cell and a direct link exists between cancer cell proliferation and deregulation of RNA pol III transcription. Accurate transcription by RNA pol III requires TFIIIB, a known target of regulation by oncogenes and tumor suppressors. There have been significant advances in our understanding of how TFIIIB-mediated transcription is deregulated in a variety of cancers. Recently, BRF2, a component of TFIIIB required for gene external RNA pol III transcription, was identified as an oncogene in squamous cell carcinomas of the lung through integrative genomic analysis. In this review, we focus on recent advances demonstrating how BRF2-TFIIIB mediated transcription is regulated by tumor suppressors and oncogenes. Additionally, we present novel data further confirming the role of BRF2 as an oncogene, extracted from the Oncomine database, a cancer microarray database containing datasets derived from patient samples, providing evidence that BRF2 has the potential to be used as a biomarker for patients at risk for metastasis. This data further supports the idea that BRF2 may serve as a potential therapeutic target in a variety of cancers. Cancer is a major health problem afflicting millions of Americans annually and despite tremendous research and treatment advances, is still the leading cause of death amongst men and women younger than age 85 years . A dominLike all eukaryotic polymerases, RNA pol III cannot recognize its target promoters directly and accurate initiation requires TFIIIB -4. In hiB-related factor 2) shares structural features with TFIIB and BRF1  is a ubiquitous and conserved protein kinase with growth-promoting and oncogenic properties. CK2 is abnormally active in a variety of human cancers . COPA was previously described and utilized to identify oncogenic chromosomal aberrations such as the TMPRSS2:ETS fusion gene in prostate cancer by Tomlins et al . Based oLastly, we investigated if there was a correlation between BRF2 overexpression and clinical outcome. Using breast carcinoma studies, we performed a meta-analysis across 10 different breast carcinoma datasets studying recurrence, metastasis and death, Figure Recently, RNA pol III transcription has been the focus of a Phase I and pharmacokinetic study. Hammond-Thelin et al. studied the effects of a novel nucleoside analog inhibitor (TAS-106) of RNA pol I, II and III, in patients with advanced solid malignancies . PreviouBy elucidating the mechanism(s) by which RNA pol III transcription is both regulated and deregulated, it will be possible to further understand the mechanism(s) by which aberrant activity of the general transcription machinery contributes to cancer development. Deregulation of RNA pol III transcription in cancers coupled with the observation that TFIIIB, specifically BRF2-TFIIIB, is commonly a target of deregulation in a variety of cancers demonstrates that RNA pol III transcription is indeed a key player in tumorigenesis and could serve as a novel target in the development of pharmacological agents.The authors declare that they have no competing interests.SC reviewed the literature, wrote and drafted the manuscript; LS corrected and finalized the manuscript. All authors read and approved the final version.


Different models have been proposed for the HIV-1 RNA monomer and dimer structures, often by studying mutants that disrupt the palindromic nature of the dimerisation initiation site (DIS) to stabilise the monomer. We have developed a new technique known as “ingel SHAPE ”. Individual RNA structures within a mixed structural population are isolated by electrophoresis under native conditions, and secondary structural probing takes place within the gel. High-throughput analysis generates a secondary structural map of each RNA, making it possible to examine the structures of different conformers under native conditions, without the need to stabilise each structure by mutagenesis or by using non-physiological buffers. We have validated the technique using TAR RNA, and show it to be accurate and highly reproducible. We have then used it to show that under native conditions the unspliced HIV-1 RNA monomer and dimer have different acylation sensitivities across the packaging signal region, but that the DIS is unreactive in both monomer and dimer. These data imply that a structural switch takes place; further examination showed that our results fit most closely to a model where, in the monomer, the dimerisation initiation site interacts with the U5 region, and that the native dimer structure corresponds closely with previously proposed structures of the monomeric RNA. This new technique allows us to visualise the nucleotides involved in the RNA structural switch, and those that remain static.


There are errors in the "Gene chip hybridization" paragraph in Materials and Methods. The correct text is: "Total RNA was isolated using TRIzol Reagent  followed by a cleanup step with qiagen RNeasy mini columns  and quality-controlled by 2100 Bioanalyzer . The expression profile of rat tissues and cells was analysed using rat 230A-arrays from Affymetrix  according to the Genechip expression analysis technical manual 701025 Rev.2. Briefly, 5 μg RNA was used to synthesize double-stranded cDNA with the Superscript Choice system  using an oligo(dT) primer containing a T7 RNA polymerase promoter  and cDNA was in vitro transcribed to synthesize biotin-labeled antisense cRNA . After fragmentation at 94°C for 35 min in 40 mM Tris, 30 mM magnesium acetate, 10 mM potassium acetate, labeled cRNA was hybridized for 16 h on Affymetrix  expression arrays. Arrays were washed and stained with phycoerythrin-streptavidin (SAPE) in Affymetrix Fluidics Station 400 and scanned by Affymetrix 3000 GeneScanner."