Bacillus subtilis DEAD box helicase YxiN illustrate the versatility of RNA recognition motifs as RNA-binding platforms. Comparison of chemical shift perturbation patterns elicited by different RNAs, and the effect of sequence changes in the RNA on binding and unwinding show that the RBD binds a single-stranded RNA region at the core and simultaneously contacts double-stranded RNA through its C-terminal tail. The helicase core then unwinds an adjacent RNA duplex. Overall, the mode of RNA binding by Hera is consistent with a possible function as a general RNA chaperone.DEAD box helicases catalyze the ATP-dependent destabilization of RNA duplexes. Whereas duplex separation is mediated by the helicase core shared by all members of the family, flanking domains often contribute to binding of the RNA substrate. The Thermus thermophilus DEAD-box helicase Hera (for “heat-resistant RNA-binding ATPase”) contains a C-terminal RNA-binding domain (RBD). We have analyzed RNA binding to the Hera RBD by a combination of mutational analyses, nuclear magnetic resonance and X-ray crystallography, and identify residues on helix α1 and the C-terminus as the main determinants for high-affinity RNA binding. A crystal structure of the RBD in complex with a single-stranded RNA resolves the RNA–protein interactions in the RBD core region around helix α1. Differences in RNA binding to the Hera RBD and to the structurally similar RBD of the  DEAD box helicases unwind RNA duplexes in an adenosine triphosphate (ATP)-dependent reaction . MembThermus thermophilus (in vivo role of Hera is currently unclear.The ‘heat-resistant RNA-dependent ATPase’ Hera is a DEAD box protein from mophilus . Hera comophilus A 15–18)Thermus tmophilus , even atmophilus , Linden mophilus . Both RNmophilus  to a commophilus . Hera unmophilus . HoweverThe Hera RBD forms a modified RNA recognition motif (RRM) . The firBacillus subtilis DEAD box helicase YxiN  and X-ray crystallography. Fluorescence anisotropy titrations and NMR chemical shift perturbation (CSP) experiments identify residues on α-helix α1 and the C-terminal tail as the main determinants for high-affinity RNA binding to the Hera RBD. Despite its important role in RNA binding, the C-terminal tail does not adopt a regular secondary structure in the presence of RNA, although its flexibility is reduced. A crystal structure of the RBD in complex with a tetranucleotide RNA resolves the RNA-protein interactions in the central region of the binding site, and illustrates major differences to RNA binding by the RBD of the ase YxiN . CompariEscherichia coli Rosetta(DE3) in autoinducing medium (2+-nitrilotriacetate (NTA) sepharose and an S75 size-exclusion column, as described , Hera_208-419 (Hera_RecA_C_DD) and Hera 370-510 , suppleUGGGC UGUUC GCCCAUU-3′ (32mer), 5′-CGAGGUCCCAAGGGU UGGGC UGUU GCCCAUU-3′ (32mer_loop4), 5′-CGAGGUCCCAAGGGU UGGGC UGUUUC GCCCAUU-3′ (32mer_loop6), 5′-CGAGGUCCCAAGGGU UGGGC UUUAC GCCCAUU-3′ (32mer_loopmut), 5′-CGAGGUCCCAAGGGU UGGC UGUUC GCCAUU-3′ (32mer_stem4), 5′-CGAGGUCCCAAGGGU UAGGGC UGUUC GCCCUAUU-3′ (32mer_stem6), 5′-CGAGGUCCCAAGGGU UGGGA UGUUC UCCUAUU-3 (32mer_stemmut), 5′-UUGGGACCU-3′ (9mer), 5′-GGGUUGGGCUGUUCGCCCAUU-3′ (21mer), 5′-CACUUGGGCUGUUCGCCCAUU-3′ (21mer_mod), 5′-GGGUUGGGCUAUUCGCCCAUU-3′ (21mer_modloop), 5′-GUUGGGCUGUUCGCCCAUU-3′ (19mer), 5′-GGGCC-3 (5mer) and 5′-GGGC-3′ (4mer). The regions forming the stem of hairpin 92 are underlined. Sequence variations are indicated in italics. The four nucleotides of the 19mer that were observed in the crystal structure are highlighted in bold.RNA oligonucleotides were from Purimex or Dharmacon. The sequences were: 5′-GCAGGUCCCAAGGGU 15N-labeled 21mer was generated by in vitro transcription from a linearized plasmid using T7 polymerase and 15N-labeled GTP and UTP (Silantes GmbH). Linearization was performed by cleavage at an XbaI-restriction site inserted after the region coding for the 21mer, resulting in four additional nucleotides (CUAG) at the 3′-end. The transcript was purified by denaturing polyacrylamide gel electrophoresis (PAGE), heated to 95°C for 5 min and folded by 5-fold dilution into ice-cold H2O, as described previously , 5′-labeled with fluorescein, in 50 mM Tris/HCl, pH 7.5, 150 mM NaCl with Hera, Hera_core or GST-Hera_RBD at 25°C, as described . Data were analyzed using the solution of the quadratic equation that describes a 1:1 complex formation wherFor stoichiometic titrations, 5 µM of 5′-fluorescein-labeled 32mer were titrated with Hera and GST-Hera_RBD until saturation was achieved.d values by electrophoretic shift assays, 6 µM of 5′-fluorescein-labeled substrate RNA (32mer or 21mer) were mixed with increasing concentrations of GST-Hera_RBD in 50 mM Tris/HCl, pH 7.5, 150 mM NaCl and 12% (v/v) glycerol, incubated at 25°C for 30 min and subjected to electrophoresis on a 15% (32mer) or 18% (21mer) polyacrylamide gel. Gel electrophoresis was performed at 140 V for 30 min in 1 × TBE buffer at 4°C, and the fraction of single- and double-stranded RNA was quantified by densitometry.To determine K2 in the presence of 5 mM ATP at 25°C, and stopped and analyzed as described (RNA unwinding reactions with subsequent separation of 32/9mer and released (3′-fluorescently labeled) 9mer by native PAGE were performed with 10 µM Hera and 5 µM 32/9mer in 50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2 at 25°C with 0.5 µM RNA substrate, 5 µM Hera, Hera_core or Hera_RBD, 2 mM ATP and 5 µM unlabeled 9mer RNA as a trap to prevent re-annealing of labeled RNA after strand separation. For unwinding assays with Hera and Hera_core, protein and ATP were added sequentially, whereas Hera_RBD was added simultaneously with ATP. The donor (Cy3) was excited at 554 nm (1 nm bandwidth), and unwinding was followed as a decrease in acceptor (Cy5) emission at 666 nm (2 nm bandwidth). Unwinding experiments where substrate and product were separated by native PAGE and with direct detection of unwinding by FRET determined similar unwinding rates of 12 × 10−3 s−1 and 14 × 10−3 s−1, confirming that the decrease in FRET and in acceptor fluorescence is indeed due to strand separation .RNA unwinding was followed in real time using a fluorescence resonance energy transfer (FRET)-based assay. The 32/9mer substrate was generated by incubating a 2-fold molar excess of a 3′-Cy3-labeled 9mer with the 5′-Cy5-labeled 32mer in 50 mM HEPES/NaOH, pH 7.5, at 25°C for 1 h. Unwinding reactions were performed in 50 mM Tris/HCl pH 7.5, 150 mM NaCl and 5 mM MgClFar-UV circular dichroism (CD) spectra of 10 µM Hera, 10 µM Hera_RBD and 10 µM RNA in 50 mM potassium phosphate buffer, pH 7.5, were measured at 25°C from 200 to 260 nm in 1 nm steps with an integration time of 5 s/nm in a 0.1 mm quartz cuvette in an Aviv 62A DS CD spectropolarimeter, accumulated 5-fold and corrected for buffer contributions.2O at 37°C and Hera_RBD concentrations of 55 or 100 µM (titration experiments), or 300 µM (triple resonance experiments). HNCO, HNCACB and HNCA spectra of Hera_RBD and the RBD/21mer complex (RNA:protein 1.33:1) were recorded at 37°C using standard pulse sequences  Dequences . NMR datequences .1H (ΔH) and 15N (ΔN) chemical shifts according to CSP values were calculated from the difference in d values of RNA/RBD complexes were determined from the concentration dependence of the CSP using max instead of ΔFmax, and an offset r0 of zero). CSPs of at least 10 amino acids located along the entire protein sequence were analyzed independently.K2. Crystals were obtained by 1:1 (v/v) mixing of the complex with reservoir solution containing 1.06 M Na-malonate, pH 6.0, 0.1 M Tris/HCl, pH 7.5, 0.13 M K/Na-phosphate and incubation for 3 months during which time the RNA decomposed. The Hera_RBD_R444A/R449A double mutant was crystallized by diluting a 2.1 mM protein solution in 50 mM Tris/HCl, pH 7.5, 500 mM NaCl with water to a concentration of 462 µM and 1:1 (v/v) mixing with un-buffered reservoir solution containing 26% PEG8000 and 0.1 M Zn(OAc)2. Hera_RBD_K463A was crystallized by mixing a 750 µM protein solution in 25 mM Tris/HCl, pH 7.5, 250 mM NaCl 1:1 (v/v) with 3.2 M ammonium sulfate and 0.1 M Bicine/HCl pH 9.0. Crystals (except those of the double mutant) were cryoprotected with paraffin oil and embedded in a vitreous matrix by hyperquenching (Thermus thermophilus Hera_RBD (PDB-ID 1I31) was used as the search model for molecular replacement with PHASER (www.pymol.org).Crystallization was performed at 20°C in the sitting drop vapor diffusion setup. A stoichiometric Hera_RBD/RNA complex at 0.45 mM concentration was formed by mixing 6.1 µl of 1.47 mM Hera_RBD in 50 mM Tris/HCl, pH 7.5, 500 mM NaCl with 9 µl of 1 mM 19mer RNA, 4.8 µl water and 0.1 µl of 2 M MgCluenching . Data weuenching  and scaluenching  (K463A mh PHASER . Models h PHASER  and refih PHASER . The trid = 0.13 ± 0.07 µM (Hera), Kd = 0.45 ± 0.036 µM (GST-Hera_RBD) and Kd = 1.0 ± 0.18 µM (Hera_core). These values are in agreement with RBD as the primary RNA-binding site of Hera, with minor contributions from the helicase core in the absence of ATP. To test for possible contributions of the DD to RNA binding, we also performed titrations with Hera_370-510  and with Hera_208-419 . Whereas the Hera_370-510/RNA complex showed a similar Kd as observed for the RBD only, the Kd for the Hera_208-419/RNA complex was increased ∼70-fold. Thus, the DD or the RecA_C domains do not contribute to RNA binding to Hera. Overall, our results demonstrate that Hera_core has a low affinity for (single-stranded) RNA, and the RBD confers high-affinity RNA binding to Hera in the absence of ATP. Stoichiometric titrations  are consistent with a 1:1 stoichiometry of 32mer RNA per Hera protomer, or per Hera_RBD.Hera consists of a helicase core formed by two RecA domains , a DD and a C-terminal RBD ; Figure . We haved values of complexes with the 32mer RNA were determined for all mutants in fluorescence anisotropy titrations  with no or only a moderate (<5-fold) effect on RNA binding , (ii) with an intermediate (5- to 10-fold) effect on RNA binding (R444A and Y472A) and (iii) mutants that showed a >25-fold increase in the Kd values . On the RBD surface, the residues involved in RNA-binding are clustered in two regions, one involving α-helix α1 and β-strand β2, and one formed by the C-terminus . The unassigned resonances correspond to the seven carbonyl carbon atoms of amino acids preceding a proline, and one Cβ-signal (P481). From the assigned HN-, NH-, Cα-, Cβ- and CO chemical shifts, the chemical shift index was calculated with PECAN . The N-terminal sub-domain exhibits a moderate propensity for β-sheet formation, helix α2 present in canonical RRMs is absent and the chemical shift index values of the C-terminal tail following β4 are in agreement with random coil conformation. Thus, the structural features that set the Hera RBD apart from canonical RRM folds are also evident in solution.NMR CSP experiments allow mapping of regions involved in RNA binding through changes in amide proton chemical shifts on RNA binding. A Hera RBD A shows wth PECAN   experiments provide information on protein dynamics, with HetNOE-values of >0.5, indicating conformational rigidity. For Hera_RBD, high HetNOE values are indicative of a rigid peptide backbone from aa 425 to 499 , despite an intermediate to fast exchange on the NMR timescale. In titration experiments, large changes in chemical shifts were observed for a number of resonances, saturating at a ∼1.5:1 ratio of RNA to protein. From the concentration dependence of chemical shifts, a Kd of ∼6 µM  can be estimated. Kd values from electrophoretic mobility shift assay and isothermal titration calorimetry experiments  are somewhat higher (20–40 µM), possibly because of the higher ionic strength. Interestingly, the resonance for K463 disappears in the RBD/RNA complex, possibly owing to exchange effects. Mutation of K463 abolishes RNA binding (see previously). From HSQC spectra of the RBD and the RBD/21mer complex, in combination with an HNCA experiment for the RBD/21mer complex, all backbone amide signals (except K463) for the RBD bound to RNA were assigned. Large changes in chemical shift were identified for residues in the N-terminal sub-domain (aa 425-435), in α-helix α1 (aa 443-454), in β-strand β2 (aa 460-467) and in the C-terminal tail (aa 498-504) . In contrast, the RBD complex with a shortened 21mer that comprises the stem-loop region but lacks the largest part of the 5′-single stranded region yielded high-quality spectra (498-504) B. The di the RBD B, region the RBD A.d), suggesting that α-helix α1 mainly contacts the RNA through the positively charged side-chains of R444 and R449.The changes in chemical shifts, as well as the mutational analysis, indicate a crucial role of residues in helix α1 for RNA binding. We therefore probed the contributions of S446, A452 and Q457, all located within this helix, to RNA binding in anisotropy titrations of the RBD carrying the S446C, A452C, K455A and Q457C mutations B. Only td values were 1.0 ± 0.17 µM and 1.1 ± 0.13 µM  led to a 25-fold decrease in 32mer affinity, suggesting an important role of this region in RNA binding to the Hera RBD. In the crystal structure of the RBD , residue2 of the RBD and physically block the RNA-binding site . Sizeable movements of the C-terminal region would also be in agreement with the observed large changes in chemical shifts for residues 498-504 on RNA binding (see previously). In the crystal structures, the proline peptide bonds at P501 and P507 are in the trans conformation, although 1H,15N-HSQC-spectra showed two signals with different intensities for some amino acids in the C-terminal tail, suggesting that both cis- and trans-conformations can be populated . Altogether it can be concluded that the C-terminus of Hera_RBD exhibits considerable conformational plasticity.To visualize the structure of the C-terminal tail of the Hera_RBD, we crystallized several constructs and mutants of Hera_RBD. Two of the mutants, Hera_RBD_K463A and R444A/R449A, yielded structures with their entire C-terminal sequences visible in the electron density maps up to aa 510. In all structures where the C-terminal part is ordered, residues 493-498 fold back onto the outermost β-strand βSupplementary Figure S4B). However, the spectrum of the complex was virtually identical to the sum of the individual spectra . Similarly, the chemical shift indices of amino acids in the C-terminal tail are similar for Hera_RBD and the RBD/21mer complex . Altogether, these findings imply a possible direct role of the C-terminal tail in RNA binding, albeit without the formation of a regular secondary structure in the RBD/RNA complex. The HetNOE values of the C-terminal amino acids in the RNA/complex do not reach the values typical of rigid conformations, indicating that the interaction between the C-terminus of the RBD and the RNA is dynamic in nature and most likely based on unspecific electrostatic interactions.To investigate whether the C-terminal tail adopts a defined structure on RNA binding, we measured far-UV CD spectra of Hera_RBD, the 32mer and a RBD/32mer complex  in complex with a tetranucleotide were obtained and the structure of the complex was determined to 2.3 Å resolution. The tetranucleotide must have been generated Supplementary Figure S4A). In the RBD/RNA complex structure, the C-terminal tail is visible up to aa 499, whereas the remainder appears to be disordered. Residues 492-499 pack closely to the RBD without engaging in contacts with the bound RNA  of the GGGPyr sequence is base-specifically bound by the N-terminal subdomain of Hera_RBD, a small addendum of 12 residues (A424-W435) unique to Hera . The Kd value was 11 µM. For the Hera_RBD_ΔC-tail/32mer RNA complex, a similar Kd of 13 µM was determined , suggesting a similar binding mode of both RNAs to this part of Hera_RBD. However, in contrast to the 21mer, binding of the 5mer RNA to the RBD does not induce changes in the chemical shifts of amide protons from residues in the C-terminal tail . Overall, the crystal structure thus captures an RBD/RNA complex that reflects Hera’s natural preference for RNA substrates. The N-terminal sub-domain creates a specific guanine binding pocket, and sets Hera apart from RBDs of other helicases (see Discussion).To further confirm that the binding mode of the short oligonucleotide in the crystal structure reflects binding of larger RNAs to Hera_RBD, CSP experiments were performed with a 5mer RNA (5′-GGGCC-3), similar to the RNA in the crystal, that includes the 3′-phosphate of Cyt4. For residues in the RBD core (aa 424-499), changes in chemical shifts of amide protons on RBD/5mer complex formation are similar to the ones observed for the RBD/21mer complex, both in magnitude and pattern . In the RBD/21mer complex, the resonance at 11.6 ppm shows a slight change in chemical shift, and additional signals are present between 10.0 and 11.5 ppm . In the 1D-HSQC-spectrum of 15N-labeled RBD in complex with unlabeled 21mer, and in the 1D- and 2D HSQC-spectra of the reverse complex of 15N-labeled 21mer and unlabeled RBD, two of these resonances (at 10.2 and 11.1 ppm) show no spectral correlation to nitrogen atoms in either the RBD or the RNA, implying that they correspond to hydroxyl groups. NOEs between these resonances and resonances of aromatic protons within the RBD suggest that they belong to tyrosine, and experiments with the free RBD confirm that these signals are already present in the absence of the RNA . Notably, one of the additional signals in the complex (at 10.5 ppm) can be assigned to a guanine imino proton that becomes protected from proton exchange with the solvent only on complex formation, pointing towards a direct interaction of this guanine with the RBD . The 21mer contains four unpaired guanines, three at the 5′-end and one in the apical loop. To identify which of these guanines is contacted by Hera_RBD, we performed NMR CSP experiments with a 19mer RNA, lacking two of the terminal guanines, a modified 21mer (21mer_mod) where the three guanines at the 5′-end have been substituted by CAC and a modified 21mer (21mer_modloop) where the guanine in the apical loop has been replaced by an adenine , in agreement with the absence of one recognition element, the GGGU sequence. Interestingly, two Kd values are observed for the 21mer_mod/Hera_RBD complex: for resonances of the RBD core residues (aa 424-493), a Kd,1 value of 40–70 µM can be inferred, whereas resonances from residues from the C-terminal tail yield a higher Kd,2 value of 150–300 µM. These two different Kd values indicate the 21mer_mod RNA can either interact with the C-terminal tail or contact the RBD core region. Possibly, the G in the loop can bind to the pocket formed by the N-terminal sub-domain in the absence of the preferred GGGPyr sequence.From the dependence of amide proton chemical shifts on the RNA concentration, a KSupplementary Figure S7). As shown previously (Overall, our data are thus in-line with the conclusion that the single-stranded GGGU sequence of the 32mer and the 21mer binds to the core of the Hera RBD. This GGGU sequence is immediately adjacent to the double-stranded region of the 32/9mer unwinding substrate. We therefore tested whether binding of the 32/9mer to the RBD already destabilizes the duplex in FRET unwinding assays with Hera, Hera_core and Hera_RBD , where aWe have shown here that the Hera RBD binds RNA independent of the helicase core, and is the major RNA-binding platform for Hera. We have mapped the regions important for RNA binding to the RBD in a mutational and NMR study to the N-terminal sub-domain, the α-helix α1 and to the C-terminal tail of the RBD. The RBD core region binds single-stranded GGGPyr stretches, as revealed by the crystal structure of the RBD/RNA complex. NMR CSP experiments with different RNA substrates are consistent with the RBD core binding to a single-stranded GGGU region in the RNA substrate, and the C-terminal tail interacting with the stem of an adjacent hairpin. A neighboring duplex can then be unwound by the Hera helicase core in the presence of ATP.B. subtilis DEAD box protein YxiN (With the 32mer derived from 23 S rRNA, Hera binds to the same RNA as the ein YxiN . Howeverein YxiN ,24, wherein YxiN  (Figure ein YxiN B. The diA closer comparison of the two RNA complex structures, however, reveals different sequence specificities A. The onThe differences in interactions with Gua1 and Pyr4 in Hera and YxiN mark diversion points in the RNA traces of the Hera_RBD and YxiN_RBD complexes A. The rein vivo function of Hera remains ill-defined. A function of Hera as a general RNA chaperone would predict that it acts on a variety of RNA substrates. Future mechanistic studies will therefore have to focus on the action of Hera on the yet-to-be-identified natural RNA substrates.Unwinding of the 32/9mer RNA by Hera is not caused by binding of the RBD to the region adjacent to the duplex, but requires the helicase core. In contrast to YxiN ,40, HeraPDB-IDs 4I69, 4I68 and 4I67.Supplementary Data are available at NAR Online: Supplementary Table 1 and Supplementary Figures 1–7.


Oxytricha trifallax, the macronuclear DNA is highly processed to yield gene-sized nanochromosomes with telomeres at each end. Here we report that soon after mating of Oxytricha trifallax, abundant 27 nt small RNAs are produced that are not present prior to mating. We performed next generation sequencing of Oxytricha small RNAs from vegetative and mating cells. Using sequence comparisons between macronuclear and micronuclear versions of genes, we found that the 27 nt RNA class derives from the parental macronucleus, not the developing macronucleus. These small RNAs are produced equally from both strands of macronuclear nanochromosomes, but in a highly non-uniform distribution along the length of the nanochromosome, and with a particular depletion in the 30 nt telomere-proximal positions. This production of small RNAs from the parental macronucleus during macronuclear development stands in contrast to the mechanism of epigenetic control in the distantly related ciliate Tetrahymena. In that species, 28–29 nt scanRNAs are produced from the micronucleus and these micronuclear-derived RNAs serve as epigenetic controllers of macronuclear development. Unlike the Tetrahymena scanRNAs, the Oxytricha macronuclear-derived 27 mers are not modified by 2′O-methylation at their 3′ ends. We propose models for the role of these “27macRNAs” in macronuclear development.Ciliated protozoans possess two types of nuclei; a transcriptionally silent micronucleus, which serves as the germ line nucleus, and a transcriptionally active macronucleus, which serves as the somatic nucleus. The macronucleus is derived from a new diploid micronucleus after mating, with epigenetic information contributed by the parental macronucleus serving to guide the formation of the new macronucleus. In the stichotrichous ciliate  DNA elimination also occurs within macronuclear-destined regions resulting in splicing out of internally eliminated sequences (IESs) and rejoining of macronuclear-destined sequences (MDSs)  while the micronucleus, which is not transcribed, serves as the genetic repository (the germ line nucleus). During sexual reproduction, the parental micronuclei undergo meiosis and haploid micronuclei are exchanged between conjugating (mating) cells to form a new diploid micronucleus. This new diploid micronucleus divides by mitosis, and one of its daughter nuclei develops into the new macronucleus. This new developing macronucleus is referred to as the anlage. While the new macronucleus develops from the anlage, the parental macronucleus is destroyed. The process of macronuclear development from a diploid micronucleus requires extensive DNA amplification and polytenization, followed by elimination of micronucleus-specific sequences. This elimination results in fragmentation to smaller chromosomes, to which telomeres are added Stylonichia, Euplotes and Oxytricha species), take the processing of micronuclear sequences into macronuclear chromosomes to an extreme. Using re-association kinetics, it was measured that the macronuclear genome possesses only 5% of the sequence complexity of the micronuclear genome Oxytricha genes are not capable of being expressed without extensive DNA processing that occurs during macronuclear development. This includes the removal of internally eliminated sequences (IESs) followed by splicing together of the surrounding macronuclear destined sequences (MDSs). These IESs total over 100,000 in number Among the ciliates, the stichotrichous ciliates  by labeling an equivalent amount of total RNA from the Chlorogonium elongatum food source (data not shown). Equivalent amounts of total Chlorogonium RNA, labeled and run in parallel lanes, did not yield detectable bands at these sizes. We also did a mock mating with the ALXC9 strain, starving it and moving it into Pringsheim buffer for 24 hours, to test whether these 27 nt RNAs are induced by starvation as opposed to mating. This treatment, equivalent to the mating procedure but done with only one strain that does not self mate, did not lead to production of 27 nt small RNAs , indicating that it too lacks modifications of the terminal ribose.Having demonstrated that Oxytricha trifallax macronucleus Oxytricha trifallax, an extensive but incomplete assembly of macronuclear sequence data called WGS2.1.1 is available In order to better understand the small RNA species observed in 32P-labeling results indicate are present in all samples, both vegetative and mating . We used the micronuclear and macronuclear sequences as targets for mapping and counted the number of sequence hits to each gene of either nuclear origin. Micronuclear and macronuclear gene sequences compared in this study were trimmed so that sequence length differences for each pair are only due to the presence of IESs and pointer sequence duplication in the micronuclear version. Oxytricha trifallax genome consisting of two chromosomes, chr1 and chr2, which we call “oxytri1”. chr1 corresponds to the concatenated complete nanochromosomes and chr2 corresponds to the concatenated partially-assembled nanochromosomes that were used in our Bowtie macronuclear alignments  and the later mating libraries (55–72hrs - mat55_10 and mat72_11). DESeq determined an unadjusted p-value for the difference in relative expression of each of the 27 mers between these two sets of libraries. 32P labeling of total RNA during a developmental time course  the joining of MDSs and importantly the unscrambling of the micronuclear genome Stylonichia has been performed Another potential function of 27macRNAs is to direct chromatin modifications in the developing macronucleus. A thorough analysis of chromatin modifications present in the developing macronucleus in the stichotrichous ciliate Alternative models for 27macRNA function can be proposed in which this class of RNAs do an important job during macronuclear development but do not provide epigenetic information. They may function to change the chromatin state of the parental macronucleus in which they are made, helping to down-regulate gene expression from the parental macronucleus. They may act as siRNAs, targeting mRNAs from the parental macronucleus for destruction. They may function to promote DNA elimination during destruction of the parental macronucleus. Further studies to identify the protein binding partners of the 27macRNAs and to determine the subcellular localization of these RNAs during macronuclear development will be required to shed light on a functional role for the 27macRNAs.Oxytricha trifallax .Note: Wenwen Fang and colleagues  have shared with us that they have also observed production of 27nt-long mating-specific RNAs of macronuclear origin in Oxytricha trifallax strains JRB310 and JRB510 Chlorogonium elongatum (UTEX Collection Strain B203) as a food source.Klebsiella pneumoniae culture per 300 ml of mating cells as food. Cells began to show strong levels of aggregation by 5 hours and mating pairs were first visible after 8 hours. In order to refresh the JRB310 and JRB510 stocks to provide for a more robust mating, exconjugants with anlage were individually isolated from a JRB310 x JRB510 mating and these clonal isolates were grown as cultures. After six weeks of vegetative growth, we tested the mating compatibility of each of 12 clonal cultures by pairwise mixing. Two of the new strains, ALXC2 and ALXC9 showed strong mating ability with each other, and their progeny had high survival rates. ALXC2 cultures have a tendency to self mate if starved, and surprisingly these self-progeny are also viable. No ALXC9 selfing is evident. When equal numbers of ALXC2 and ALXC9 are mixed together, a highly efficient mating occurs, with ∼70% of cells possessing anlage 48 hours after mixing.Mating competent strains were grown separately until they had nearly exhausted their food. Cells were filtered through cotton to remove algal debris and were then concentrated on 10 µM Nitex filters. Cells were washed off of the filters into inorganic salts media and the individual cultures were counted. Cells of the different mating strains were mixed together in equal numbers, concentrated again on Nitex filters and washed on the filters into Pringsheim salts buffer. Timing of mating begins at this mixing step. Cells were put into Pyrex dishes at a concentration of 1200–2000 cells/ml in Pringsheim solution with 1 ml of stationary Oxytricha trifallax cultures were washed through cotton and then concentrated on 10 µM Nitex filters. Cells were put into microcentrifuge tubes and gently pelleted at 1000 x g for 2 minutes. Supernatants were removed except for the 50 µl above the cell pellet. 200 µl of mirVana Lysis/Binding Buffer from the mirVana miRNA Isolation Kit (Ambion) was added to each tube. Total RNA was purified according to the kit's protocols for total RNA purification (not the miRNA purification protocol). Typical total RNA yields from this protocol starting with 300 ml of Oxytricha trifallax culture were 100 µg.2O. 32P labeling was done using T4 polynucleotide kinase (NEB) and gamma-32P-ATP in 10 µl reaction mixtures. An equal volume of formamide dyes were added to the reaction mixtures and then heated to 95°C. Labeled RNA samples were separated on 40 cm long 15% acrylamide (19∶1) urea gels in TBE buffer. 32P labeled Decade marker (Ambion) was used for a 10 nt ladder size reference. Gel images were recorded using a Typhoon PhosphorImager  and ImageQuant software.3 µg of total RNA were treated with 30 units of calf intestine alkaline phosphatase (New England Biolabs) in a 100 µl reaction mixture using the manufacturer's protocol. The reaction mixture was acid phenol:CHCl3 extracted, Na-Acetate added to 0.3 M and ethanol precipitated with 4 volumes of ethanol and 1 µl glycoblue (Ambion) as carrier. Precipitate pellets were washed with 70% ethanol, dried and then resuspended in 10 µl of deionized H32P labeled total RNA was prepared and reaction mixtures were raised to 95°C for 2 minutes after the kinase reaction in order to inactivate the T4 polynucleotide kinase enzyme. In addition, 250 pmoles of a 21 nt synthesized RNA with the C. elegans lin-4 microRNA sequence and 250 pmoles of a 23 nt synthesized RNA with the C. elegans mir-90 sequence were 32P labeled at their 5′ ends in 10 µl reaction mixtures to serve as positive controls for beta elimination, as these synthetic RNAs have a 2′ and 3′ OH group on their 3′ terminal ribose. After labeling and heat inactivation, 1.5 µg of unlabeled total Oxytricha RNA was added to each tube so that each control RNA was treated in the beta elimination assay in the same complex RNA background as the labeled total RNA. Beta elimination reaction conditions were essentially those described by Horwich et al. 32P kinase reaction mixture were added to 100 µl of 25 mM Na-meta-periodate in 60 mM Borax/60 mM Boric acid buffer pH 8.6. Tubes were incubated at room temperature for 30 minutes. Then, 13 µl of 1 M NaOH was added to each tube in order to raise the pH from 8.6 to 9.5. Reaction mixtures were then incubated for 90 minutes at 45°C to allow the beta elimination reaction to occur. RNA was recovered after acid phenol:CHCl3 extraction and ethanol precipitation, and dried pellets were raised in 10 µl of formamide loading dye. Products were separated on 15% polyacrylamide urea sequencing gels and visualized by autoradiography.The beta elimination assay was used to test for modifications at the 3′ end of RNAs. Small RNAs had linkers ligated to them and bar-coded cDNAs were prepared using the TruSeq Small RNA Sample Prep Kit (Illumina) following the manufacturer's instructions. Library preparation began with 1 µg of total RNA from mating cells in which small RNAs are abundant, or 2 µg of total RNA from vegetative cells in which small RNAs  are rare. Individual libraries were analyzed on a BioAnalyzer (Agilent) for the presence of linkered cDNA at the appropriate size (135–165 bp) and 11 bar-coded libraries were pooled into one sample by mixing 2.0 ng of the 135–165 bp peak from each sample as determined by the BioAnalyzer. The 135–165 bp peak of the pooled cDNAs was purified from the mixed sample using the Pippin Prep DNA Size Selection System (Sage Science), and confirmed by BioAnalyzer.Sequencing of the pooled libraries was performed in one lane of the Illumina HiSeq2000 Sequencer at the UCSC Genome Technology Center. 100 bp paired-end reads of the libraries were obtained. After indexing and trimming of linker sequences, those reads of at least 16 nt in length that had 100% identity in the two directions  were further analyzed.Oxytricha trifallax non-coding RNA sequences from the list of non-coding RNAs published by Jung et al. 5 to filter out any RNAs derived from telomeres. This non-coding RNA filter was used as a first pass filter of the libraries prior to alignment to the macronuclear genome.We extracted Oxytricha trifallax is currently incomplete, an extensive assembly of whole genome shotgun sequencing contigs, WGS2.1.1, was released in the supplemental material of Jung et al. Oxytricha trifallax mitochondrial genome sequence In order to be able to understand where the 27 nt small RNAs originate, an assembly of macronuclear sequences was needed. While the macronuclear genome sequence of Bowtie In order to determine whether the 27 nt small RNAs were macronuclear or micronuclear in origin, we compared 26–28 nt sequence reads that passed through the non-coding RNA filter for the ability to align to a micronuclear version of a gene as compared to its macronuclear counterpart. To do this, we used the micronuclear and macronuclear versions as Bowtie filters for the sequence reads, and identified and counted the number of sequence reads that aligned to each set in the pair. This allowed us to create a Venn diagram  of smallRaw sequence files for all 11 Illumina sequencing libraries, and files listing the sequences of 26–28 nt RNAs that survived the non-coding RNA filter from each of the 7 mating libraries, have been deposited in GEO - Accession Number GSE37390.


DICER is an RNase III family endoribonuclease that processes precursor microRNAs (pre-miRNAs) and long double-stranded RNAs, generating microRNA (miRNA) duplexes and short interfering RNA duplexes with 20~23 nucleotides (nts) in length. The typical form of pre-miRNA processed by the Drosha protein is a hairpin RNA with 2-nt 3' overhangs. On the other hand, production of mature miRNA from an endogenous hairpin RNA with 5' overhangs has also been reported, although the mechanism for this process is unknown.in vitro and generates an intermediate duplex with a 29 nt-5' strand and a 23 nt-3' strand, which was eventually cleaved into a canonical miRNA duplex via a two-step cleavage. The previously identified endogenous pre-miRNA with 5' overhangs, pre-mmu-mir-1982 RNA, is also determined to be a substrate of rDICER through the same two-step cleavage.In this study, we show that human recombinant DICER protein (rDICER) processes a hairpin RNA with 5' overhangs The two-step cleavage of a hairpin RNA with 5' overhangs shows that DICER releases double-stranded RNAs after the first cleavage and binds them again in the inverse direction for a second cleavage. These findings have implications for how DICER may be able to interact with or process differing precursor structures. DICER plays a key role in RNA interference pathways through the biogenesis of microRNA (miRNA) and small interfering RNA (siRNA) -3. Most Giardia intestinalis showed that the hydrophobic pocket of the PAZ domain was responsible for the binding of the 3' dinucleotide overhangs of the substrate and the connector helix between the PAZ domain and RNase III domain functioned as a molecular ruler measuring the distance from the 3' end of pre-miRNA to the cleavage site . N-terminally FLAG-tagged human DICER1 protein was purified from 293T cells transfected with the plasmid pCA-FLAG-DICER1. This vector contained the full-length human DICER1 protein FLAG-tagged at the amino terminus in a pCA-FLAG-DEST vector  ATP (6000Ci/mmol) at 37°C for 4 h.The probe sequences in this study were as follows: probe-1 (5'-TCAACATCAGTCTGATAAGCTA-3'), probe-2 (5'-ACAGCCCATCGACTGGTGTTG-3'), probe-3 (5'-CCATGAGATTCAACAG-3'), probe-4 (5'-CCTCCCCAGGACCCTCCCAA-3') and probe-5 (5'-CTGTGGGAGAACATAGGGTGAGA-3'). The probes were 5'-end labelled using T4 polynucleotide kinase (TaKaRa Bio) with  ATP (3000Ci/mmol) at 37°C for 4 h. The 5'-end labeled transcript was PAGE-purified as described above and the RNA pellet was resuspended in water at a final concentration of approximately 0.5 pmol/μl. One microliter of this was used for the processing reaction by rDICER. These processed samples were run on 7.5 M urea-denaturing 20% polyacrylamide gels in 1x TBE buffer with RNA molecular marker or the products of alkaline hydrolysis of the same RNA molecule. The alkaline hydrolysis ladder was generated by incubating the labelled RNA in alkaline hydrolysis buffer (Ambion) at 100°C for 10 min. The signals were detected by autoradiography and quantified using ImageJ software . The signal intensities were calculated as the mean of pixel value of selected area.For the 5'-end labelling, RNA (45 pmol) was dephosphorylated with CIP at 37°C for 60 min. The reaction was inactivated by phenol-chloroform extraction and precipitated by sodium acetate-ethanol at -20°C. The pellet was resuspended in an appropriate volume of water. The dephosphorylated transcript was 5' end-labelled using T4 polynucleotide kinase (TaKaRa Bio) with [γ-YA conceived the study, designed and performed experiments and drafted the manuscript. YM and AM participated in the experimental design and performed experiments. RK and JC participated in the experimental design and purified recombinant FLAG-DICER fusion proteins. AMB, HS and KM participated in the design of the study and revised the manuscript. YH designed the research project, provided funding, supervised the study and critically reviewed the manuscript. All authors read and approved the final manuscript.Supplementary informationClick here for file


The TAR hairpin is present at both the 5′ and 3′ end of the HIV-1 RNA genome. The 5′ element binds the viral Tat protein and is essential for Tat-mediated activation of transcription. We recently observed that complete TAR deletion is allowed in the context of an HIV-1 variant that does not depend on this Tat-TAR axis for transcription. Mutations that open the 5′ stem-loop structure did however affect the leader RNA conformation and resulted in a severe replication defect. In this study, we set out to analyze which step of the HIV-1 replication cycle is affected by this conformational change of the leader RNA.We demonstrate that opening the 5′ TAR structure through a deletion in either side of the stem region caused aberrant dimerization and reduced packaging of the unspliced viral RNA genome. In contrast, truncation of the TAR hairpin through deletions in both sides of the stem did not affect RNA dimer formation and packaging.These results demonstrate that, although the TAR hairpin is not essential for RNA dimerization and packaging, mutations in TAR can significantly affect these processes through misfolding of the relevant RNA signals. Human immunodeficiency virus type-1 (HIV-1) is a retrovirus with an RNA genome of approximately 9 kb that contains nine open reading frames and untranslated regions at the 5′ and 3′ end. The highly conserved leader RNA at the 5′ end contains several important regulatory RNA motifs that are involved in both early and late replication steps . The firIn vitro studies demonstrated that the HIV-1 leader RNA cannot only fold the branched multiple hairpin (BMH) conformation, but also an alternative conformation in which DIS sequences interact with the polyA region [in vivo, i.e. in the context of the replicating virus, were hampered because mutations in this element cause a severe transcription and replication defect [in vitro, resulting in uncontrolled dimerization of leader RNA transcripts due to induced exposure of the DIS hairpin [A region -22. ThisA region ,23,24. MA region . This U5A region . Indeed,A region -28, packA region ,21,29,30A region -34. Prevn defect ,29,35. W hairpin . In thisin vitro produced leader RNA transcripts demonstrated that opening of the 5′ TAR hairpin resulted in stabilization of the polyA hairpin, which forced the leader RNA in the BMH conformation [We previously designed an HIV-1 variant in which the Tat-TAR transcription mechanism was inactivated through mutation and functionally replaced by the doxycycline (dox)-inducible Tet-On gene regulation system  and cultured with dox for two days. Because we previously observed that opening of the TAR hairpin at the 3′ end of the viral genome reduced the polyadenylation efficiency of the viral transcripts [We first examined the effect of the TAR mutations on the production of HIV-1 RNA and its packaging into virions. C33A cervix carcinoma cells were transfected with the HIV-rtTA molecular clones that contain either the wild-type or modified TARnscripts , all molnscripts .5+3, B5+3 and AB5+3) was similar to that observed with the wild-type HIV-rtTA construct  and purified the virions from the culture supernatant by ultracentrifugation. RNA isolated from cells and virions was first used for cDNA/PCR analysis with primers that specifically detect unspliced and spliced transcripts . Whereas the level of the single spliced and double spliced RNA in the wild-type and AB5 virions was too low to detect by Northern blot analysis, the 2 kb double spliced RNAs were apparent for the A5 and B5 mutants  and B5 mutated RNAs  by co-transfecting cells with both HIV-rtTA constructs and cDNA/PCR analysis of the unspliced and spliced RNAs present in the cells and virions . As controls, cells were transfected with only the wild-type HIV-rtTA or the B5 variant (TARm and B5 lanes). Analysis of the intracellular RNA from the TARm/B5 transfected cells revealed a similar production of unspliced and spliced transcripts from the TARm and B5 constructs  and AB5 RNAs (with a truncated but stable hairpin structure) by co-transfecting cells with both HIV-rtTA mutants  we thus observed that opening of the 5′ TAR structure element does not only reduce the packaging efficiency of the unspliced RNA but also that of the spliced viral RNAs, which argues against a direct positive effect of TAR opening on packaging of spliced RNAs. It thus seems likely that the increased level of spliced RNAs observed in the A5 and B5 mutated virions  caused increased dimerization of leader RNA transcripts, whereas the AB5 transcripts showed the wild-type pattern of regulated dimerization [m) and the A5, B5 and AB5 variants. Analysis of these cellular samples under non-denaturing conditions revealed that the wild-type HIV-rtTA transcripts formed large intracellular complexes, which resulted in a discrete peak of RNAs with very low gel mobility (indicated with 'complex'). This HIV-rtTA sample did not show discrete RNA peaks corresponding to monomeric and dimeric 9-kb RNAs, but a smear of bands was observed at the expected positions  reduces the formation of complexes, increases the level of monomers and leaves unchanged the relative level of dimers. In contrast, 5’ TAR truncation (AB5) did not result in such a shift from large RNA complexes to monomers. As shown in Figure 5 and B5 virus particles, whereas the AB5 virions contained a wild-type level of this unspliced RNA. Analysis of these virion RNA samples under non-denaturing conditions revealed that the wild-type HIV-rtTA and AB5 virions contained predominantly a discrete RNA species that migrates in the gel as genomic RNA dimers, whereas the RNA from the A5 and B5 virions migrated more diffusely and did not show any discrete peak  does not influence the production and splicing of viral RNA, which is in agreement with previous observations [5) exhibited the wild-type RNA pattern in cells and normal packaging and dimerization in virions. The observed changes in RNA configuration, dimerization and packaging thus correlate with the virus replication phenotype.This study is based on the surprising finding that the HIV-rtTA variant, which does not need the 5′ TAR RNA element for Tat-mediated activation of transcription, is severely replication impaired by a deletion in the left or right side of the TAR stem that opens the hairpin structure (A and B mutants), but not by the combined deletion that truncates this stem-loop element (AB). We demonstrate that introduction of the A and B deletions in the 5′ TAR element (Arvations . Howevercis-acting RNA elements and the trans-acting nucleocapsid (NC) domain of the Gag protein [The unspliced HIV-1 transcript is used as mRNA for the synthesis of the Gag and Pol proteins and as genomic RNA that is encapsidated and present as dimer in the viral particle. During this packaging process, the genomic RNA must be selected from a multitude of cellular and spliced viral RNAs. This selection involves  protein . The maj protein . In addi protein ,28,39,42 protein ,37,39. Wm and AB5 virions, these RNAs were largely present as complexes in the corresponding cells. The A5 and B5 mutations resulted in less intracellular RNA complexes and more monomeric RNA. Whereas the intracellular level of RNA dimers was seemingly unaffected by the A5 and B5 mutations, the RNA dimer level in the corresponding virions was found to be reduced. Although it has been observed that dimerization and packaging can be dissociated [m and AB5, respectively) are more efficiently packaged in virus particles than dimeric RNA molecules with an opened 5’ TAR structure (A5 and B5). On the other hand, the direct correlation that we observe between the intracellular complex level and the RNA dimer content of the corresponding virions  may suggest that the RNA complexes are the actual targets for packaging, which are later transformed into dimers. However, we have not verified the configuration of the RNA molecules in newly released virions, and it has been shown that RNA monomers (or RNAs that appear as monomers when assessed by gel electrophoresis) can also be efficiently packaged and subsequently mature into dimers [We not only observed genomic RNA monomers and dimers when analyzing the RNA content of cells and virions, but also larger RNA complexes Figure . The natsociated -46, a cosociated ,47-53. Io dimers ,46.in vitro RNA structure probing experiments indicated that opening of the TAR structure resulted in an interaction between unpaired TAR nucleotides and downstream U5 sequences, which caused extension of the adjacent polyA hairpin [et al.[5 and B5 mutations do indeed reduce packaging of the unspliced RNA. Moreover, we previously measured aberrant dimerization of TAR-mutated leader RNA molecules in in vitro assays. The A and B leader RNAs were found to dimerize more efficiently than the wild-type and AB molecules, which may be due to the increased exposure of the DIS signal in these short RNAs [in vivo analysis confirms that the A5 and B5 mutations affect the dimerization process, but in this case a more complex pattern is observed. The mutations did not seemingly affect the intracellular RNA dimer level, but reduced complex formation and increased the monomeric RNA level in cells. Moreover, these TAR opening mutations reduced the RNA dimer content of the virions. These results may seem contradictory, but we note that there is good evidence that in vitro RNA dimerization only partially mimics the in vivo dimerization process. For instance, the DIS drives in vitro RNA dimerization via a loop-loop kissing interaction, but a 4-nt deletion in the loop-exposed palindrome surprisingly does not prevent HIV-1 replication and does not affect the stability of the in vivo dimer [Previous  hairpin . Stabili hairpin ,17,54. A hairpin . Alterna hairpin -25, exte hairpin . Ooms etn [et al. previousort RNAs . The curvo dimer . This anvo dimer ,56 indicet al. very recently demonstrated that TAR deletion did not significantly affect RNA dimerization, NC binding and RNA packaging [We previously showed that HIV-rtTA and a similarly designed dox-controlled SIV-rtTA variant replicate efficiently upon complete deletion of the TAR hairpin ,57. Thesackaging . Howeverackaging . Our comackaging ,30,59,60HIV-1 does not require TAR for dimerization and packaging of its RNA genome, but mutations in TAR can affect these processes in an indirect way. We demonstrate that opening of the TAR hairpin structure alters the leader RNA conformation, which reduces packaging and dimerization of the unspliced viral RNAs.2. Construction of the infectious HIV-rtTA molecular clone and variants with a deletion in the 5′, the 3′ or both TAR elements was described previously [C33A cervix carcinoma cells (ATCC HTB31)  were groeviously ,62. In aeviously .2 wells and transfected with 5 μg HIV-rtTA plasmid by calcium phosphate precipitation as previously described [et al.[C33A cells were cultured to 60% confluency in 3 ml complete medium in 10-cmescribed . Cells wescribed . RNA wasd [et al.. The cel2 plates to 60% confluency, transfected with 40 μg HIV-rtTA and cultured with 1 μg/ml dox. The culture supernatant was harvested 2 days after transfection and cells were removed by low-speed centrifugation (10 min at 1500 rpm). The supernatant was filtered through a 0.45-μm filter and virion particles were pelleted by centrifugation at 32,000 rpm  for 90 min at 4°C in a Beckman SW32 Ti rotor. The virions were resuspended in 0.6 ml RLT buffer. The RNA was isolated as described above and resuspended in 50 μl water.For the isolation of RNA from purified virions, C33A cells were cultured in 20 ml complete medium in 75-cmFor denaturing Northern blot analysis, 5 μg cell-derived RNA in 10 μl water or 10 μl virion-derived RNA were mixed with 10 μl denaturing sample buffer , heated at 65°C for 10 min and subsequently electrophoresed on a 1% agarose gel in MOPS buffer  with 7% formaldehyde at 100 V for 4 h. For non-denaturing Northern blot analysis, 10 μl RNA was mixed with 5 μl sample buffer (30% glycerol and 0.25% bromophenol blue), incubated for 10 min at 37°C (or 55°C when indicated) and electrophoresed on a 0.9% agarose gel in 1 x Tris-Borate-EDTA buffer at 100 V for 4 h. The RNA was subsequently denatured by soaking the gel in 3 volumes of 10% formaldehyde at 65°C for 30 min.32P-labeled probe corresponding to the nearly complete HIV-rtTA genome was generated by random-primed labeling  of the 3.9 and 5.5 kb SalI fragments derived from the HIV-rtTA plasmid. Prehybridization and hybridization was done in ULTRAhyb buffer (Ambion) at 55°C for 1 and 16 hours, respectively. The membrane was then washed two times at room temperature for 5 min with low-stringency buffer  and two times for 10 min at 50°C in high stringency buffer . Images were obtained using the PhosphorImager (Amersham Biosciences) and data analysis was performed with the ImageQuant software package.The RNA was transferred onto a positively charged nylon membrane (Boehringer Mannheim) with 20 x SSC by means of capillary force for 16 h. The RNA was linked to the membrane using a UV crosslinker (Stratagene). A 25 and random hexamers primers. The cDNA product was used as template in a PCR assay with primers 1  and 2  to detect unspliced RNA, with primers 3  and 4  to detect the single spliced RNA, and with primers 3 and 5  to detect the double spliced RNA. The cDNA was denatured at 94°C for 5 min and PCR-amplified in 30 cycles of 1 minutes 95°C, 1 minutes 55°C, 2 minutes 72°C and a final extension time of 7 minutes at 72°C. The PCR products were visualized on a 1% agarose gel stained with ethidium bromide. In the co-transfection experiment, the unspliced transcript was detected with primers 1wt/B  or 1A/AB  plus 2+348 , and the splice products were detected with primers 1wt/B or 1A/AB plus 4 (single spliced) or 5 (double spliced). The PCR products were digested with NarI before gel analysis.RNA was reverse transcribed with ThermoScript reverse transcriptase at 50°C (Invitrogen) using the supplied oligo(dT)The authors declare that they have no competing interests.ATD designed research, analyzed data and wrote manuscript. MMV designed and performed experiments and wrote manuscript. AH performed experiments and edited manuscript. BB designed research and wrote manuscript. All authors read and approved the final manuscript.


Cucumber mosaic virus(CMV) Y satellite RNA (Y-sat) is a non-coding subviral RNA and modifies thetypical symptom induced by CMV in specific hosts; Y-sat causes a bright yellowmosaic on its natural host Nicotiana tabacum. The Y-sat-inducedyellow mosaic failed to develop in the infected Arabidopsis andtomato plants suggesting a very specific interaction between Y-sat and its host.In this study, we revealed that Y-sat produces specific short interfering RNAs(siRNAs), which interfere with a host gene, thus inducing the specific symptom.We found that the mRNA of tobacco magnesium protoporphyrin chelatase subunit I hada 22-nt sequence that was complementary to the Y-sat sequence, including fourG-U pairs, and that the Y-sat-derived siRNAs in the virus-infected plantdownregulate the mRNA of ChlI by targeting the complementarysequence. ChlI mRNA was also downregulated in the transgeniclines that express Y-sat inverted repeats. Strikingly, modifying the Y-satsequence in order to restore the 22-nt complementarity toArabidopsis and tomato ChlI mRNA resultedin yellowing symptoms in Y-sat-infected Arabidopsis and tomato,respectively. In 5′-RACE experiments, the ChlI transcriptwas cleaved at the expected middle position of the 22-nt complementary sequence.In GFP sensor experiments using agroinfiltration, we further demonstrated thatY-sat specifically targeted the sensor mRNA containing the 22-nt complementarysequence of ChlI. Our findings provide direct evidence that theidentified siRNAs derived from viral satellite RNA directly modulate the viraldisease symptom by RNA silencing-based regulation of a host gene.Symptoms on virus-infected plants are often very specific to the given virus. Themolecular mechanisms involved in viral symptom induction have been extensivelystudied, but are still poorly understood.  Cucumber mosaic virus (CMV) Y satellite RNA (Y-sat) is aninteresting subviral RNA because it changes the green mosaic induced by CMV intoa bright yellow mosaic in Nicotiana tabacum. The molecularbasis underlying the induction of symptoms by viruses is not well understood,and this Y-sat-mediated modification of symptoms has been a long-standingmystery. In this study, we discovered the molecular mechanism involved in theY-sat-induced yellowing. First, we showed that transgenic N.benthamiana plants that expressed the inverted-repeatsequence of Y-sat also developed a yellow phenotype, similar to theY-sat-infected plants. Then, we found that tobacco magnesium protoporphyrinchelatase subunit I gene  was downregulated in the transgenic plants and in theY-sat-infected plants. We then identified a 22-nt long sequence that iscomplementary to the Y-sat including four G-U pairs in the ChlImRNA. Finally, we demonstrated that a short interfering RNA (siRNA) derived fromY-sat specifically targeted and downregulated the ChlI mRNA,thus impairing the chlorophyll biosynthesis pathway. This discovery of themolecular basis of the symptom modification induced by Y-sat is the firstdemonstration that a subviral RNA can induce disease symptoms by regulating hostgene expression through the RNA silencing machinery. Plants infected with viruses often display various symptoms, which can be veryspecific to given viruses. Despite past efforts, the molecular bases underlyingvirus-induced diseases symptoms are still poorly understood. Subviral non-coding RNAmolecules such as satellite RNAs (satRNAs) or defective interfering (DI) RNAs areoften associated with plant viruses and can modify the symptoms induced by helperviruses Cucumber mosaic virus (CMV) are dependent on helperviruses for their replication and encapsidation and often attenuate the diseasesymptoms induced by CMV. Specifically, Y-satellite RNA (Y-sat) modifies the symptomsand exacerbates the pathogenicity of CMV in specific hosts; Y-sat induces a brightyellowing of leaves of Nicotiana tabacum  andother related species ,which is yellower than a typical chlorosis, whereas it induces systemic necrosis ontomato SatRNAs of RNA silencing is a conserved, sequence-specific gene regulation system, which has anessential role in development and maintenance of genome integrity. RNA silencingrelies on short RNA (sRNA) molecules (21–24 nt), which are the key mediatorsof RNA silencing-related pathways in almost all eukaryotic organisms Subviral RNAs such as satRNA and DI RNA of tombusvirus have been also used tounderstand the roles of RNA silencing in viral replication and in symptomdevelopment. The DI RNA-induced RNA silencing response is known to control the levelof helper virus, facilitating the long-term co-existence of the host and the viralpathogen ChlI have the yellow phenotype ChlI gene of tobacco or cotton was targeted by virus-inducedgene silencing (VIGS) Arabidopsis mutantdefective for ChlI also had pale-green to yellow leaves ChlI is downregulated by Y-sat in thevirus-infected plants.Magnesium (Mg)-chelatase is the key enzyme in chlorophyll biosynthesis, and threesubunits  of the tobacco magnesium protoporphyrin chelatase arerequired for the proper function of the enzyme N.benthamiana plants develop a yellow phenotype when expressingthe inverted-repeat sequence of Y-sat, similar to the symptoms of the Y-sat-infectedplants. Moreover, we provided evidence that Y-sat targets the ChlIgene using the host RNA silencing machinery in such a way that Y-sat-derived siRNAsefficiently downregulate ChlI mRNA through RNA silencing-mediatedcleavage. Our findings strongly suggest that this yellow phenotype is the result ofa disorder in chlorophyll synthesis caused by the downregulation of theChlI gene.In this study, we show that transgenic N. benthamiana plants that express the Y-satsequence, expecting the yellow phenotype to be induced without CMV as a helpervirus. We have used this strategy to avoid any effect of virus replication onhost gene expression, because virus infection itself has been shown to regulatethe expression of numerous genes N.benthamiana plants, we observed that the transgenicN. benthamiana lines (16c:YsatIR) had ayellow phenotype (N. benthamiana that expressed dsRNA of GUS(16c:GUSIR), demonstrating that the expression of dsRNA of an unrelated sequencein the same Y-sat IR transformation cassette does not cause a yellow symptom  .Among tana 16c) , furtherana 16c) . More inana 16c) , indicatChlI) gene (accession AF014053). Because ChlI is a componentof the primary enzyme that catalyzes the first step in chlorophyll synthesis viathe tetrapyrrole biosynthesis pathway ChlI gene of N.benthamiana. We then found that both theChlI genes from N.tabacum and N.benthamiana had the 22-nt sequence complementary to theY-sat sequence (ChlI gene and the Y-sat sequence as the yellow region (YR)and satellite yellow region (SYR), respectively  between the yellow-inducing domain of Y-satsequence .Hereaftectively . We thenh plants andconftoplasts , confirmChlI gene using VIGScan induce similar yellow symptoms in the absence of Y-sat. The 150-bp ofChlI (817 to 966) was inserted into the two CMV vectors,CMV-A1 and CMV-H1; CMV-A1 lacks the C-terminal one-third of the intact 2bprotein N. benthamiana plants infected with either of the viralvectors had systemic yellow symptoms similar to those induced by the replicatingY-sat in the presence of the helper virus (ChlI mRNA was downregulated in both CMV-A1:ChlI150- andCMV-H1:ChlI150-infected N. benthamiana plants compared tocontrol plants infected with one of the empty vectors (ChlI sequence replicated and accumulated to a similarlevel in the systemic leaves at 14 days post-inoculation (dpi) (We next examined whether silencing of the er virus . Althoug vectors . Using eon (dpi) .ChlI genes of pepper, tomato and Arabidopsisthaliana were obtained from the gene database, and the 22-ntcomplementary sequences of the ChlI genes and Y-sat werealigned  genes   genes . We nextctively) . When tothamiana . Quantited plant . There wthamiana , confirmChlI gene seemed to have a specificinteraction through their sequence complementarity, we then examined thepossible involvement of RNA silencing in the Y-sat-mediated yellow phenotype.First, we tested whether the Y-sat-derived siRNAs can be hybridized and detectedby ChlI mRNA probe. As shown in ChlI sense RNAprobe. On the other hand, we failed to detect antisense siRNAs from Y-sat byNorthern blots using the ChlI antisense RNA probe. This resultseems reasonable because the YR and SYR sequences do not share complementarityin the antisense orientation , which agrees with the expected cleavage site(s) driven by the21-nt and 22-nt siRNAs (N. benthamiana leaves that had bright yellowsymptoms (GFP mRNA (To clarify whether the t siRNAs . To verit siRNAs . The consymptoms . GFPaccGFP mRNA . The accGFP mRNA . These rRCC1 gene in Arabidopsisinfected with Cauliflower mosaic virus (CaMV) was downregulated byvirus-derived siRNAs, but contrary to expectations, the decrease in gene expressiondid not affect either viral accumulation or symptoms. It is, in fact, quitedifficult to clarify the relationship between such small RNAs and viralpathogenicity although the idea that host gene silencing against a particular genemight contribute to the specific expression of symptoms is very attractive.Plant RNA silencing has often been implicated as a molecular mechanism for symptominduction caused by viruses or viral subviral agents. Viral suppressors of RNAsilencing (VSRs) are able to compromise the endogenous RNA silencing pathways ChlI gene, resulting in the inhibition of chlorophyllbiosynthesis. Here we provide several lines of evidence that Y-sat-induced brightyellow mosaics are the outcome of specific interference between the pathogen-derivedsiRNAs and a host gene. First, the 22-nt long region of Y-sat (SYR) producesspecific siRNAs that were complementary, including four G-U pairs, to the 22-nt longregion of tobacco ChlI mRNA (YR). Second, the ChlImRNA could detect Y-sat-derived siRNAs in Northern blots. Third, 5′-RACEexperiments revealed that the ChlI mRNA was cleaved exactly in theexpected middle of the YR. Fourth, the levels of the ChlItranscript significantly decreased in both Y-sat-infected plants and the transgenicplants expressing Y-sat dsRNA. Fifth, the Y-sat mutants that had the modified SYR toeither Arabidopsis ChlI1 mRNA or tomato ChlI mRNAwere able to induce yellow symptoms in these host plants. In contrast, thesemodified Y-sat lost the ability to induce yellow symptoms on tobacco. Sixth, the GFPsensor construct carrying the YR sequence was specifically targeted inY-sat-infected plants. Considering all these results, we propose a model thatexplains that the Y-sat-mediated yellow symptom results from the cleavage of hostChlI mRNA by RNA silencing machinery  of satRNAs, their evolutionary strategy andbiological significance have long been intriguing topics. Since the originalisolation of Y-sat in Japan more than 30 years ago Nicotiana benthamiana, Capsicum annuum,Solanum lycopersicum and Arabidopsisthaliana were used as host plants for the analysis.Nicotiana benthamiana line 16c having a single copy of theGFP transgene N. benthamiana lines expressing the inverted repeat(IR) of Y-sat were generated by transforming N. benthamiana 16cwith the binary vector pIG121-Hm carrying the IR of Y-sat under the CaMV 35Spromoter. In the sense and antisense orientations, the 317-bp (53 to 369) Y-satsequence (GenBank accession D00542) was inserted in the pJM007 vector Transgenic ChlI gene, we used two CMV-based vectors,CMV-A1 and CMV-H1. CMV-A1 and CMV-H1 are derived from RNA2 of CMV-Y, and CMV-A1lacks the C-terminal one-third of the intact 2b protein as a consequence ofintroducing a multiple cloning site ChlI gene (817 to966) was inserted into the CMV vectors to create CMV-A1:ChlI150 andCMV-H1:ChlI150, respectively. To avoid severe mosaic symptom induction by CMV-Y,we used a pseudorecombinant virus that contains RNA components derived from RNA1and RNA3 of CMV strain L together with RNA2 of the vector. Each plasmidcontaining a full-length cDNA clone of RNA1 to RNA3 was transcribed in vitroafter linearization with a restriction enzyme N. benthamiana weredusted with carborundum and rub-inoculated with the RNA transcripts. Forinoculation of tomato plants, leaves of young plants were rub-inoculated withthe sap from virus-infected tissues of N.benthamiana. Successful systemic infection with the viruscontaining the full insert sequence was confirmed by RT-PCRs. Viral accumulationwas examined by conventional ELISA CMV strain Y (CMV-Y) was used as a helper virus for satellite RNA. To induce genesilencing to the N. benthamiana ChlI cloneincluding the entire ORF was amplified by RT-PCR using the primer pair designedfrom the tobacco ChlI sequence . Quantitative real-time RT-PCR was performed essentially asdescribed before N. benthamiana ChlI gene were as follows:5′-CTTATTGGTTCGGGTAATCCTG-3′ for forward primerand 5′-GCTGAGTCGATTTGGTTCTG-3′ for reverse primer.The N. benthamiana actin gene was amplified using5′-GCGGGAAATTGTTAGGGATGT-3′ for forward primerand 5′-CCATCAGGCAGCTCGTAGCT-3′ for reverse primerand used for data normalization. Northern blot hybridization was performedessentially as previously described ChlI gene was generated by PCR with the PCR DIG ProbeSynthesis Kit (Roche Diagnostics) to amplify the 371 bp (634 to 1004) of3′-terminal regions of the ChlI gene using the primerpair ChlI-634F (5′-GAGCCTGGTCTTCTTGCTAAAGC-3′) and ChlI-1004R(5′-GCTGAGTCGATTTGGTTCTG-3′). In the Northernblots of the small RNAs corresponding to the 22-nt complementary sequence region, the SYRs were detected by using32P-labeled locked nucleic acid (LNA) oligonucleotide probesdescribed previously Total RNAs were extracted by either a conventional phenol/chloroform method ChlI mRNA cleavage sites were analyzed by modifiedRNA-ligase mediated 5′-RACE + mRNA was ligated to the GeneRacer RNA Oligo adaptorusing the GeneRacer Kit (Invitrogen). Ligated RNAs were reverse transcribedusing the gene-specific reverse primer for the ChlI gene,ChlI-1004R (5′-GCTGAGTCGATTTGGTTCTG-3′). The 5′end ofthe cDNA was then amplified by PCR using the GeneRacer 5′ primer and thegene-specific reverse primer used for the reverse transcription for the firstPCR. The GeneRacer 5′ nested primer was also used for the subsequentnested PCR. The amplified product from the nested PCR was excised from1.2% agarose gel and cloned into pGEM-T Easy (Promega) forsequencing.The N.benthamiana as described before ChlI and CAB gene were measuredby quantitative real-time RT-PCR . Primers for quantitative real-timeRT-PCR for the CAB gene were 5′-CGGCCGATCCAGAAACTTT-3′ for forward primerand 5′-GCCCATCTGCAGTGAATAACC-3′ for reverseprimer.Protoplasts were prepared from leaves of N.benthamiana plants. Small RNAs were isolated essentially asdescribed Total RNA was extracted from CMV and Y-sat-infected BamHI andSacI sites in the pBE2113 vector. The Ti-plasmid constructwas then introduced into Agrobacterium tumefaciens KYRT1strain, which was supplied by Dr. G. B. Collins .Agrobacterium infiltration was carried out essentially as described The GFP-YR sensor gene was inserted between the Total proteins were extracted from the sample tissues by grinding in Laemmlibuffer, separated by SDS-PAGE, and /transferred onto a PVDF membrane . Anti-GFP antibodies were purchased from Roche and used at a1∶1000 dilution. For immunostaining, an alkaline phosphatase-conjugatedgoat anti-rabbit antibody was added to the blots at a 1∶3000 dilutionfollowed by colorimetric development with BCIP and NBT.Figure S1Nicotianabenthamiana 16c  and N.benthamiana 16c:YsatIR . Redcircles in the gel of 16c indicate the spots that decreased in 16c:YsatIRcompared to 16c. Blue circles in the gel of 16c:YsatIR indicate the spotsthat increased in 16c:YsatIR compared to 16c. Among these spots, we selectedfive spots (M1-M5) that had markedly changed between 16c and 16c:YsatIR forLC-MSMS analysis. The analyzed proteins were identified as follows: M1,ribulose bisphosphate carboxylase large chain (RuBisCo large subunit); M2,ribulose bisphosphate carboxylase activase; M3, glyceraldehyde-3-phosphatedehydrogenase A ; M4, ribulose bisphosphate carboxylase small chain 1 ; M5, ribulose bisphosphate carboxylase small chain 1. This proteome analysis revealed thatchloroplast-related proteins were significantly altered in 16c:YsatIR, andthat the mobility of the RuBisCo small subunit had shifted in atwo-dimensional gel, suggesting that RuBisCo small subunit in 16c:YsatIR wasmodified at the posttranslational level.Two-dimensional electrophoresis of extracted proteins from (TIF)Click here for additional data file.Figure S2ChlI gene probe inNorthern blots. RNAs were prepared from 16c:YsatIR, 16c and CMV-infectedN. benthamiana with or without Y-sat.Left panel, detection of sense small RNAs of Y-sat by the hybridization withthe ChlI sense RNA (mRNA) probe. Right panel, detection ofantisense small RNAs of Y-sat by the hybridization with theChlI antisense RNA probe. For the RNA probe, theamplified ChlI fragments  werecloned downstream of the T7 promoter in the pGEM-T easy vector (Promega).The sense and antisense RNA probes specific to the ChlIwere prepared using DIG RNA Labeling Mix (Roche). Arrows indicate small RNAsof Y-sat. The 22-nt sequence complementarity between theChlI and Y-sat is shown below each panel. A continuous22-nt complementary sequence including G-U pairs is formed between theChlI sense RNA and Y-sat sense RNA, but there are fourmismatches in the region between the ChlI antisense RNA andY-sat antisense RNA.Detection of Y-sat small RNAs by the (TIF)Click here for additional data file.Figure S3Arabidopsis. RNAs were prepared fromArabidopsis leaves infected with CMV or CMV+Y-satmut-Ara1. For the RNA probe, the amplified ChlI1 fragments were cloned downstream of the T7 promoter in thepGEM-T easy vector (Promega). The sense RNA probe specific to theArabidopsis ChlI1 was prepared using DIG RNA LabelingMix (Roche). Note that Arabidopsis ChlI1 sense RNA probedetected small RNAs from Y-sat mut-Ara1 (shown by an arrow) in the lane forY-sat mut-Ara1-infected leaves.Northern blots of Y-sat mut-Ara1 small RNAs in Y-sat mut-Ara1-infected(TIF)Click here for additional data file.Figure S4Confirmation of the relative abundance of Y-sat small RNAs by Northern blothybridization. The small RNAs derived from the hot spots that were observedin the Y-sat small RNA profiles  were det(TIF)Click here for additional data file.Figure S5ChlI small RNAs inN. benthamiana plants infected withCMV-Y and Y-sat. Location and frequency of the ChlI-derivedsmall RNAs (21- to 24-nt) were mapped to the ChlI sequencein either sense (above the x-axis) or antisense (below thex-axis) orientation. Data from 21-, 22-, 24-nt smallRNA are color-coded in green (21 nt), red (22 nt), and yellow (24 nt). Tablein graph gives the number of small RNA reads and percentage of each size.Note that the small RNAs are mostly generated from the 3′ regiondownstream of the cleavage site indicated by an arrow.Deep-sequencing analysis of the (TIF)Click here for additional data file.Table S1Genes downregulated in 16c:YsatIR 40% less than in 16c plants inmicroarray analysis. Among the 134 genes, 31 genes were chloroplast-relatedgenes.(DOC)Click here for additional data file.Text S1Supplementary materials and methods for the DNA microarray  and two-(DOC)Click here for additional data file.


Triptorelin lactate was successfully synthesized by porcine pancreatic lipase (PPL) in organic solvents. The effects of acyl donor, substrate ratio, organic solvent, temperature, and water activity were investigated. Under the optimum conditions, a yield of 30% for its ester could be achieved in the reaction for about 48 h. Peptides play important roles in the pharmaceutical and food fields –5. It iset al. [C-terminal amino acid residue in peptides with various other groups . The advantages of enzymatic peptide modification are freedom from racemization, minimal activation and side-chain protection, mild reaction conditions, high regio- and stereoselectivity. Furthermore, the reactions can be carried out in a mixture of organic solvent and water, devoid of the problem of low solubility of protected peptides in the organic solvents employed in chemical synthesis.The use of enzymes for the modification of peptides has been investigated in the past decades. For example, Sih and co-workers –10 inveset al.  investigLipases are the most commonly used enzymes in industrial processes. They can catalyze the acylation of peptides in organic solvents. Acylation of the peptide might affect its functions, including its stability, DNA binding, protein-protein interaction, and peptide-receptor recognition. However, there still remains relatively little research so far on the applications of lipase in the modification of peptides. Herein, we report a facile lipase-catalyzed acylation of a serine-containing peptide (triptorelin) in organic media , wherebyThe catalytic reaction of lipase depended mainly on the type and origin of the enzyme . Six comThe rate of an enzymatic catalytic reaction also depends on the concentrations of the substrate . In thisP (log P [n-octanol and water) were selected to investigate the effect of the reaction media. Log P is the most frequently used parameter to denote the polarity or hydrophobicity of a solvent. As shown in Various organic solvents with different log P . As can be observed in aw = 0.51. Further decrease or increase in the initial aw value resulted in an obvious decrease in enzyme activity.One of the most important factors, which may affect the enzyme behavior in nonaqueous media is the water activity . In the aw in organic solvents, the decrease in enzyme activity can be attributed to the excessively flexible conformation of lipase. The water in the reaction mixture may have acted as a competing nucleophile for the acyl-enzyme, thus suppressing the expected acyl transfer and causing an unfavorable equilibrium position in reversed hydrolysis. Hence, a high aw had a negative effect on the thermodynamic balance, shifting the equilibrium towards hydrolysis [At low water activity, the conformation of PPL was excessively rigid, which might have disturbed the “induced-fit” process of PPL, and the enzyme activity was decreased . At highdrolysis .It could be observed  that thePseudomonas sp. (PSL), Pseudomonas fluorescens lipase  and Candida cylindracea A.Y. lipase (AYL) was purchased from Amano Pharmaceutical Co., Ltd. (Japan). Novozym 435 was purchased from Novo . Candida rugosa lipase (CRL) was purchased from Sigma . porcine pancreatic lipase (PPL) was purchased from Shanghai Dongfeng Biochemical Reagent Co., Ltd. (China). Octreotide acetate (purity > 99%) was purchased from Shanghai TASH Biotechnology (China). Triptorelin was kindly donated by Wang Yingwu, Jilin University, China. l-lactic acid was purchased from Sigma-Aldrich Chemical Co. and was of analytical grade. Other regents were purchased from Shanghai Chemical Reagent Company (China). All the reagents and solvents were used without further purification.Lipase from aw) were prepared by adding a certain amount of water. The resulting samples were pre-equilibrated at the desired temperature for 24 h in a sealed vial before being subjected to aw measurement. The water activity (aw) was measured by Hygrolab Humidity Detector  before performing the reaction [The used organic solvents were previously dried in a vacuum of 1 mmHg for 12 h. Then all the reaction mixtures with specific water activity (reaction .aw = 0.51) and lipase (5.0 mg) while stirring at 40 °C. To determine the concentrations of the produced ester, the organic samples were withdrawn from the reaction mixture and analyzed by high performance liquid chromatography (HPLC). The synthetic activity of enzyme (μmol/h/g) was defined as the amount (in micromoles) of triptorelin ester produced per hour per gram of enzyme. Enzyme activity was determined while the conversion was controlled in the range of the 15%–30%. All experiments were repeated three times and the errors did not exceed 5%. All graphs were based on the average values.The reaction was performed by using triptorelin , lactic acid , benzene (1.0 mL), water activity , which consisted of a binary pump, an autosampler, an oven, and a Kromasil-C18 column . Chromatography was performed at 25 °C with a 20 min linear gradient elution from 10% to 50% acetonitrile in water containing 1% TFA at a flow rate of 1.5 mL/min. Octreotide acetate (purity > 99%) was used as internal standard. The products were detected at 280 nm. Baseline separation of triptorelin and its lactate  was obtained. Conversions were determined based on the peptide ester increase.y and b series of fragment ions of triptorelin lactate were obtained by in-source collision-induced dissociation (CID) and the results were shown in m/z of triptorelin lactate was 1383.6 (M + H)+ and the structure of triptorelin lactate was confirmed from the results of CID.We used an Agilent 1100-LC system (Agilent Technologies) coupled to a 6510-ESI TOF MS (Agilent Technologies). The molecular weight and amino acid sequence of triptorelin lactate were determined by electrospray ionization mass spectrometry (ESI-MS). The aw = 0.51, while lactic acid was used as acyl donor (the optimum molar ratio of lactic acid/triptorelin was 100:1). However, the activity was not satisfactory even under optimum conditions. Some techniques  are currently in the process of being studied to further improve the properties of the enzyme and will be reported on in due course.In conclusion, we describe here an effective method for the acylation of triptorelin by esterification in nonaqueous media, and PPL showed a higher catalytic performance. Under optimum conditions, the triptorelin lactate was successfully synthesized by catalyzation by PPL in benzene at 40 °C and


Foamy viruses (FVs) differ from all other genera of retroviruses (orthoretroviruses) in many aspects of viral replication. In this review, we discuss FV assembly, with special emphasis on Pol incorporation. FV assembly takes place intracellularly, near the pericentriolar region, at a site similar to that used by betaretroviruses. The regions of Gag, Pol and genomic RNA required for viral assembly are described. In contrast to orthoretroviral Pol, which is synthesized as a Gag-Pol fusion protein and packaged through Gag-Gag interactions, FV Pol is synthesized from a spliced mRNA lacking all Gag sequences. Thus, encapsidation of FV Pol requires a different mechanism. We detail how WT Pol lacking Gag sequences is incorporated into virus particles. In addition, a mutant in which Pol is expressed as an orthoretroviral-like Gag-Pol fusion protein is discussed. We also discuss temporal regulation of the protease, reverse transcriptase and integrase activities of WT FV Pol.  Foamy viruses (FVs) are ancient and highly successful retroviruses. The integration of retroviral genomes creates endogenous retroviral elements that have been left as a fossil record in host species. Although rare, cases of endogenous foamy viruses have been reported in the genomes of sloths  and the Spumaretroviridae. FVs reverse transcribe their encapsidated RNA genome during assembly and/or budding, leading to the production of DNA containing virions. FVs package RNA; but, infectious virions contain double-stranded DNA (dsDNA), and in this way, FVs resemble hepadnaviruses, such as hepatitis B virus (HBV), whose genomic organization differs from that of retroviruses. Thus, FVs bridge the gap between retroviruses and hepadnaviruses . The env RNA, and there is an undefined mechanism to exclude spliced RNA from virions (reviewed in .,31.cis-aackaging ,33. In Fnstructs ,35. In tSeveral mechanisms have been proposed for FV Pol encapsidation. One mechanism is that Pol directly binds to the genomic RNA concurrently with RNA packaging via Gag binding ,31. ThusAs Pol incorporation requires binding to the PES in genomic RNA, the number of Pol molecules per virion would be limited. Each of the two copies of FV genomic RNA would accommodate one or at most a few Pol dimers. Surprisingly, quantification of Pol molecules within purified FV particles found that an approximate ratio of Gag to Pol is 16:1, which is a higher level of Pol than that predicted by the RNA-only model . AlternaIn orthoretroviruses, the Pol precursor (PrPol) is incorporated into virions as a Gag-Pol fusion protein and cleaved by protease into three components, protease (PR), reverse transcriptase/RNase H (RT) and integrase (IN). FV PrPol is cleaved only once between RT and IN, yielding two mature proteins, PR-RT and IN. Only FV PrPol, not the individual cleavage products, is incorporated into virus particles ,39. A Poin vitro only at NaCl concentrations of 2–3 M [in vivo. Orthoretroviral Gag is cleaved into at least four proteins, whereas FV PR only cleaves Gag once, releasing a 3 kD peptide (p3) from the C-terminus. As a result, FV particles never mature. Infectious FV resembles the immature orthoretroviral virions in the assembly stage prior to Gag cleavage. The single cleavages in Gag and Pol are absolutely required for FV infectivity and replication ,42,43. Lof 2–3 M ,48. Howeof 2–3 M ,50. In aof 2–3 M . Given tVarious mechanisms have been proposed for the activation of FV PR. One proposed mechanism for PFV Pol dimerization is that a dimerization domain within IN is required . As retrpol region of genomic RNA stimulates PR activity of PR-RT proteins [in vivo, a Gag-PR-RT fusion protein lacking IN was created and expressed [Another mechanism has been proposed for FV PR activation. It was shown that a specific protease-activating RNA motif (PARM) located within the PES in the proteins . The disproteins , which cxpressed . In thisxpressed , supportin vitro studies have revealed two major differences in polymerase activity relative to some orthoretroviral RTs. First, FV RT is more processive and more active on a variety of templates [RTs have two enzymatic activities, polymerase and RNase H, which cleaves the RNA strand of an RNA-DNA duplex. These two enzymatic activities are both necessary and sufficient for RT to convert the single-stranded viral RNA genome into dsDNA (reviewed in ). While emplates . Second,emplates ,59,60. Femplates  and as pemplates ,61. Much effort has been devoted to determining the precise mechanism by which reverse transcription occurs, and models have been derived for both orthoretroviruses (reviewed in ) and hepOther viral or cellular factors may be involved with RT activity during viral assembly. A recent study using a FV four-vector system found that Gag processing is required for initiation of reverse transcription . It is nIntegrase functions early in the viral lifecycle to integrate reverse-transcribed dsDNA into the cell genome, and this is also true for FVs. FV IN, like orthoretroviral INs, contains an N-terminal zinc finger domain, a critical aspartic acid in the active site and a DNA binding domain. Studies of purified FV IN demonstrated both endonuclease and integrase activities ,70. FV IDespite much effort to determine the crystal structure of retroviral IN proteins, to date, PFV IN is the only such protein for which high-resolution structures have been obtained. The recent studies of PFV IN in complex with viral DNA have provided structural insights into retroviral IN enzymes and specifically PFV IN structures ,54,75. AIn orthoretroviruses, such as HIV-1, the PR domain in the Gag-Pol fusion protein can form only a weak transient dimer, resulting in low PR activity, until it is assembled into virions ,80,81. ADuring normal FV infection, PR and RT are not cleaved from each other. This is the only known example of a protein that has both protease and nucleic acid polymerizing activity. Like orthoretroviruses, FV PR requires dimerization for activation. As described above, FV PR-RT exists predominantly as a monomer in solution and forms only transient dimers ,51. FV RFV Pol is not synthesized as a Gag-Pol protein, as in orthoretroviruses. Rather, it is expressed from a spliced mRNA. Thus, there are unique mechanisms for regulation of Pol expression and packaging. Only Pol precursor protein, PR-RT-IN, is packaged. Protease cleavage of PrPol results in PR-RT and IN proteins, but neither protein by itself is packaged. Pol packaging requires RNA motifs in genomic RNA, called PES (Pol encapsidation sequences). Pol packaging mediated through PES requires both the Pol IN domain, as well as a GR box in the Gag protein. It is likely that a Gag-Pol complex is packaged through binding to the PES, but it remains to be determined how this complex is formed. The FV Pol precursor dimerizes through the IN domain. There may also be a role for an RNA sequence, PARM. All retroviral proteases must dimerize to create an active site. Dimerization of FV PrPol is required for PR activation. FV RT is active as a monomer. This poses a problem for FV, since PR and RT are expressed as a fusion protein. A model is presented to explain this paradox. PrPol has protease activity, which results in cleavage of IN from the precursor. The resultant PR-RT is now monomeric and has RT activity. FV reverse transcription occurs during virus assembly and/or budding, resulting in dsDNA-containing infectious virions. The timing of FV reverse transcription is unique among retroviruses and is independent of the mode of Pol expression. The timing of RT is not the result of the absence of Gag in the precursor. Instead, RT timing appears to be intrinsic to the Pol protein.