The flavivirus nonstructural protein 3 (NS3) bears multiple enzymatic activities and represents an attractive target for antiviral intervention. NS3 contains the viral serine protease at the N-terminus and ATPase, RTPase, and helicase activities at the C-terminus. These activities are essential for viral replication; however, the biological role of RNA remodeling by NS3 helicase during the viral life cycle is still unclear. Secondary and tertiary RNA structures present in the viral genome are crucial for viral replication. Here, we used the NS3 protein from dengue virus to investigate functions of NS3 associated to changes in RNA structures. Using different NS3 variants, we characterized a domain spanning residues 171 to 618 that displays ATPase and RNA unwinding activities similar to those observed for the full-length protein. Interestingly, we found that, besides the RNA unwinding activity, dengue virus NS3 greatly accelerates annealing of complementary RNA strands with viral or non-viral sequences. This new activity was found to be ATP-independent. It was determined that a mutated NS3 lacking ATPase activity retained full-RNA annealing activity. Using an ATP regeneration system and different ATP concentrations, we observed that NS3 establishes an ATP-dependent steady state between RNA unwinding and annealing, allowing modulation of the two opposing activities of this enzyme through ATP concentration. In addition, we observed that NS3 enhanced RNA-RNA interactions between molecules representing the ends of the viral genome that are known to be necessary for viral RNA synthesis. We propose that, according to the ATP availability, NS3 could function regulating the folding or unfolding of viral RNA structures. Proteins with RNA helicase activity have been identified in the genome of plus stranded RNA viruses Flavivirus genus in the Flaviviridae family, together with other important human pathogens such as Yellow Fever virus (YFV), West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV) Flavivirus genus DENV is a member of the NS3 is a multifunctional protein essential for flavivirus replication Hepacivirus genus in the Flaviviridae family, has been extensively studied The helicase activity of hepatitis C virus (HCV) NS3, member of the In this study, we investigated enzymatic properties of the DENV NS3 protein in regard to its ability to modulate the formation of RNA structures. Using in vitro assays to evaluate RNA unwinding and annealing reactions, we identified a new RNA strand annealing activity of NS3. In contrast to the requirements for the helicase activity, we demonstrated that the RNA annealing activity does not require ATP. The viral NS3 establishes an ATP-dependent steady state between RNA unwinding and annealing, allowing modulation of the two opposing activities of this enzyme through ATP concentration. Our findings provide novel information for functional roles of the DENV NS3 in regulating viral RNA structures.The flavivirus NS3 protein contains two defined domains with distinct enzymatic activities kcat) of 2.2±0.1 s−1 and 2.8±0.1 s−1; and Michaelis Menten constants (KM) of 31±3 µM and 17±2 µM, respectively  showed hyperbolic kinetics as a function of substrate concentration, with catalytic constants  of 4.57±0.05 nM and 4.54±0.11 nM, and apparent RNA unwinding rate constants of 0.16±0.01 min−1 and 0.31±0.02 min−1, for NS3-FL and NS3-hel, respectively  disappears. A steady state, indicating equal velocity between forward (RNA unwinding) and reverse  reactions, is observed. However, it was uncertain whether the steady state reflected the equilibrium between the helicase-mediated RNA unwinding and the spontaneous RNA annealing, or if the latter reaction was also accelerated by NS3.−1  are able to utilize diverse NTPs Using RNA molecules with complementary regions involved in local interactions, and employing conditions in which spontaneous hybridization was unfavourable, high strand annealing activity of the DENV NS3 protein was observed . AccelerRNA annealing is not considered a general biochemical characteristic of RNA helicases. However, a growing number of proteins with RNA remodelling activities are being reported, including enzymes that accelerate RNA strand annealing It is still unclear how viral RNA helicases participate in viral replication. Moreover, the physiological significance of combined RNA unwinding and RNA annealing activities in a viral protein has not been investigated. We have recently provided a model in which the DENV genome is a dynamic RNA molecule and at least two mutually exclusive conformations are crucial for infectivity Although we found that the protease domain does not modulate the helicase activity of DENV NS3 in vitro, it is likely that in the infected cell the two enzymatic activities (or other functions of NS3) are necessary in the same polypeptide. Viral polyprotein processing and RNA replication occur in distinct compartments known as convoluted membranes and vesicle packets, respectively  and reverse primer AVG119 (which incorporates the nucleotide sequence encoding the GGGGSGGGG linker sequence at the 3′ terminus). The DNA fragment corresponding to amino acid residues 1 to 618 of NS3 protein was amplified by PCR using the forward primer AVG120 (which incorporates the nucleotide sequence encoding the GGGGSGGGG linker sequence at the 5′ terminus) and the reverse primer AVG16 (which incorporates a stop codon and a HindIII restriction site at the 3′ terminus). From these two PCR products, an overlapping PCR was carried out using primers AVG492 and AVG16 and then cloned into a pET-28a vector (Novagen) between BamHI and HindIII restriction sites to generate plasmid pET-CF47-NS3-FL. This plasmid encodes for a polypeptide consisting of an N-terminal hexahistidine tag (derived from the vector) followed by the central hydrophylic region of NS2B linked by a GGGGSGGGG sequence to the full-length NS3 protein. Recombinant protein preparations from this recombinant plasmid resulted in NS3 degradation due to the presence of the active two-component NS2B-NS3 protease domain. To avoid this problem, the active site of the protease was mutated . For this mutation, plasmid pET-CF47-NS3-FL was used as template and primers AVG118, AVG541, AVG492 and AVG16 were used to generate plasmid pET-CF47-NS3-FL-H51A by overlapping PCR. His51 to Ala replacement greatly decreased auto-proteolysis.Constructs encoding NS3 proteins were derived from the cDNA of an infectious clone of DENV2 strain 16681 (GenBank accession number U87411) NdeI restriction site at the 5′ terminus) and reverse primer AVG16. The PCR product was digested with NdeI and BamHI and cloned into a modified pET-28a plasmid using T4 DNA ligase to generate plasmid pET-NS3-hel. This plasmid encodes for a polypeptide consisting of an N-terminal hexahistidine tag followed by the polypeptide corresponding to the helicase domain of NS3 protein (residues 171 to 618). In order to generate an ATPase-defective variant of NS3-hel, Asp284 and Glu285 residues were replaced for alanine by site-directed mutagenesis. Plasmid pET-NS3-hel-D284E285-AA was generated by overlapping PCR using plasmid pET-NS3-hel as template and primers AVG943, AVG1263, AVG1262 and AVG16.The DNA fragment encoding for DENV2 NS3 helicase domain (residues 171 to 618) was obtained by PCR from plasmid pET-CF47-NS3-FL using forward primer AVG943 .E. coli BL21 (DE3) Rosetta pLac cells were transformed with expression plasmids. Transformed cells were grown at 37°C in LB medium containing kanamycin (50 mg/L) and chloramphenicol (34 mg/L) to an optical density at 600 nm of 0.6–0.8. Then, 0.5 mM IPTG was added in order to induce protein expression. After 4 hours at 28°C, cells were harvested by centrifugation and stored at −80°C. Cells were suspended in 3 ml of buffer A  supplemented with 2 mM DTT, 2 mM MgCl2, 2 mg/ml DNAse I, 10 units protease inhibitor cocktail (Roche), and then lysed by a French press  and clarified by centrifugation . The cell extract supernatant was applied to a HisTrap HP column  previously equilibrated in buffer A and eluted in a gradient with buffer B  using an AKTA FPLC . Fractions containing the protein of interest were pooled, dialyzed exhaustively against buffer D  at 4°C using a 14 kDa cut-off dialysis tube (Spectrum Laboratories Inc.).The sample was then concentrated to 10 mg/ml using an Amicon Ultra centrifugal filter unit (Millipore Corporation). Particulate material was removed by centrifugation at 16,000×g for 30 minutes at 4°C. The supernatant fraction was stored at −70°C.Competent 32P-γ-ATP (Perkin Elmer) and T4 polynucleotide kinase (New England Biolabs).Synthetic RNA oligomers (up to 30-mer) were purchased from Integrated DNA technologies Inc. Longer RNAs  were obtained by in vitro transcription using T7 RNA polymerase (Ambion) and treated with RNase-free DNase I to remove DNA templates. These RNAs were purified using illustra MicroSpin G-50 Columns  to remove free nucleotides, and then quantified spectrophotometrically. Their integrities were verified by electrophoresis on agarose gels. All numbers given below in parentheses refer to nucleotide positions of a dengue virus type 2 strain 16681 infectious cDNA clone (GenBank accession number U87411). The sequences corresponding to 5′DV-159 (nucleotides 1 to 159) and 3′SL (nucleotides 10617 to 10723) were amplified by PCR using primers AVG689 and AVG130, and AVG11 and AVG5, respectively, from a DENV2 cDNA clone CUGACUGCGAUCCGACUGUCCUGCAUGAUGCAGGACAGUAACAGGUUCU-3′5′-GGAUCGCAG) was designed to fold in two hairpins including a sequence that is complementary to the Cy5-RNA30 probe (underlined in the sequence).RNA58 . The reaction was initiated by adding ATP (including an equimolar concentration of MgCl2) in 150 µl buffer N and carried out in a spectrophotometer microcuvette. The initial velocities of ATP hydrolysis were calculated as previously described kcat and KM constants. RNA-stimulated ATPase activity was determined as described above, including different concentrations of a synthetic RNA (20-mer) and 2 mM ATP-MgCl2.ATP hydrolysis was determined by using EnzCheck Phosphate Assay Kit . Purified recombinant NS3 variants at a concentration of 40 nM were preincubated for 10 minutes at 25°C in 150 µl buffer N  annealed to a shorter RNA strand of 15 nucleotides (5′-CUGUCCUGCAUGAUG-3′). When the 15-nucleotide long molecule was radio-labeled, a polynucleotide kinase (New England Biolabs Inc) and 32P-gamma-ATP (Perkin-Elmer Inc) were used. The 5′Cy5 labeled 30-mer RNA was purchased from IDT.In order to generate substrates for helicase assays, two synthetic oligonucleotides  were annealed by heating at 90°C and cooled slowly to room temperature. The duplex RNA substrate contained an RNA strand of 30 nucleotides in length . Unwinding reactions were initiated by adding ATP in the indicated concentrations. Simultaneously the RNA trap (100 nM of unlabeled oligonucleotide) was added in order to prevent re-annealing of the probe. Reactions were terminated at different incubation times by adding 2 µl of buffer S  in order to inactivate the enzyme and to dissociate NS3 from the RNA probe, preventing formation of RNP complexes. Reaction mixtures were resolved on non-denaturing 9% polyacrylamide gels in TBE buffer  supplemented with 5% glycerol using Mini-PROTEAN electrophoresis system . The fluorescent signal from Cy5-labeled probes was detected by scanning the gels with a Storm 865 device . The radioisotopic probes were detected by using autoradiographic plates (Kodak Inc.). The signal was quantified with ImageJ software (NIH). The ATP regeneration system included creatine phosphokinase (32 µg/ml) and creatine phosphate (16 mM) .RNA unwinding reactions were performed at 30 or 37°C in a final volume of 20 µl. RNA substrate (5 nM) and NS3 (250 nM) were preincubated for 10 minutes in buffer R  and the complementary RNA strand (20 nM or at the indicated concentration). Reaction mixtures were incubated with NS3 (250 nM) and Mg-ATP at different concentration as indicated in each case. Annealing reactions were initiated by mixing aliquots of the two RNA strands in buffer R. Reactions were terminated by cooling the sample on ice to detain the annealing process. Simultaneously, 2 µl of buffer S were added to inactivate the enzyme and to prevent formation of RNP complexes. Single-stranded and duplex RNAs were separated and detected as described above for the helicase activity assay.kunwobs and A, respectively) for unwinding reactions were determined by fitting time courses to a monoexponential function of time:1+ssRNA2 = dsRNA); and the initial velocities were obtained as, vi = (d [dsRNA]/dt)t = 0 from the slopes of the fitting curves at their zero time.The amount of ssRNA and dsRNA were determined from the intensity of the respective bands in the gels measured by ImageJ 1.45 software . Observed rate constants and amplitudes (Equations and numerical solution of the kinetic models were adjusted to the experimental data by non-linear regression analysis using free software COPASI. This program provides not only the best fitting values of the parameters but also their standard errors


H replacement occurs through RAG-mediated recombination between the cryptic recombination signal sequence (cRSS) near the 3′ end of a rearranged VH gene and the 23-bp RSS from an upstream unrearranged VH gene. Due to the location of the cRSS, VH replacement leaves a short stretch of nucleotides from the previously rearranged VH gene at the newly formed V-D junction, which can be used as a marker to identify VH replacement products. To determine the contribution of VH replacement products to mouse antibody repertoire, we developed a Java-based VH Replacement Footprint Analyzer (VHRFA) program and analyzed 17,179 mouse IgH gene sequences from the NCBI database to identify VH replacement products. The overall frequency of VH replacement products in these IgH genes is 5.29% based on the identification of pentameric VH replacement footprints at their V-D junctions. The identified VH replacement products are distributed similarly in IgH genes using most families of VH genes, although different families of VH genes are used differentially. The frequencies of VH replacement products are significantly elevated in IgH genes derived from several strains of autoimmune prone mice and in IgH genes encoding autoantibodies. Moreover, the identified VH replacement footprints in IgH genes from autoimmune prone mice or IgH genes encoding autoantibodies preferentially encode positively charged amino acids. These results revealed a significant contribution of VH replacement products to the diversification of antibody repertoire and potentially, to the generation of autoantibodies in mice.V H, DH (for heavy chain only), and JH gene segments catalyzed by the recombination activating gene products (RAG1 and RAG2) H, DH, and JH gene segments is directed by the recombination signal sequences (RSSs) The variable region exons of the immunoglobulin (Ig) genes are generated through sequential rearrangement of previously separated VH to JH recombination on one allele of the IgH loci in early progenitor (pro) B cells followed by recombining a VH gene segment to the DJH joint in late pro B cells L segment and a JL segment in the small precursor (pre-) B cells Rearrangement of Ig heavy (IgH) chain genes starts with a DL gene to a downstream JL gene LJL joint is deleted during the editing process Igκ alleles can initiate de novo rearrangements at the Igλ locus H replacement process H replacement occurs through RAG-mediated recombination between a cryptic RSS embedded at the 3′ end of the rearranged VH gene with the 23 bp RSS from a upstream VH gene H replacement was originally observed in murine pre-B cell leukemia cells, which generated functional IgH genes from non-functional IgH rearrangements H replacement in editing IgH genes encoding anti-DNA antibodies was demonstrated in a series of studies using engineered mouse models carrying knocked-in IgH V(D)J rearrangements encoding anti-DNA antibodies H replacement was employed to diversity the antibody repertoire in mouse carrying knocked-in IgH genes encoding anti-NP antibodies H replacement during B cell development in normal mouse and contribution of VH replacement products to the mouse antibody repertoire were lacking for a long time Editing of rearranged IgL genes can occur through RAG-mediated secondary recombination between any upstream VH germline gene, VH replacement renews almost the entire VH coding region but leaves a short stretch of nucleotides from the previously rearranged VH gene at the newly formed V-D junction H replacement footprints to trace the occurrence of VH replacement and to identify potential VH replacement products through analyzing IgH gene sequences H replacement products contribute to about 5% of the primary B cell repertoire in human H replacement footprints H replacement products were also found in IgH genes obtained from the wild type B6 mice Due to the location of the cRSS at the 3′ end of VH replacement products to the diversification of mouse IgH repertoire, we developed a Java based VH replacement footprint analyzer (VHRFA) program and analyzed 17,179 mouse IgH gene sequences from the National Center for Biotechnology Information (NCBI) database to identify VH replacement products. These results revealed a significant contribution of VH replacement products to the murine IgH repertoire and the enrichment of VH replacement products in several strains of autoimmune prone mice.To explore the contribution of VH replacement products, we developed a Java based VH Replacement Footprint Analyzer (VHRFA) program. Using the VHRFA program, we analyzed 17,179 mouse IgH gene sequences from the NCBI databases to identify VH replacement products. First, the potential VH, DH, and JH germline gene usage were assigned using the IMGT/V-QUEST program by sending batches of sequences using the VHRFA program  functional IgH genes using the IGHV5/VH7183 family of VH genes. The other families of VH genes, including IGHV4/X-24, IGHV11/CP3, IGHV12/CH27, IGHV13/3609N, and IGHV15/VH15A, are used at much lower frequencies  regions of these IgH genes. VH replacement can only introduce VH replacement footprint in the N1 region. As an internal control, we searched for similar VH replacement footprint motifs in the DH-JH junction (N2) regions of these IgH genes, which are likely generated by random nucleotide addition. The frequencies of 3, 4, and 5-mer VH replacement footprint motifs in the N1 regions are significantly higher than those in the N2 regions  regions. As internal controls, we also searched for the similar motifs in the DH-JH junction (N2) regions. The frequencies of identified VH replacement footprints with 3, 4, 5, 6, or 7 nucleotides in the N1 regions are significantly higher than those in the N2 regions J recombination, they are similarly targeted for secondary recombination during VH replacement. As an internal negative control, we analyzed the N1 regions of IgH genes using the DH proximal VH5-2/7183.2 gene. Among the 56 functional IgH genes using the VH5-2/7183.2 gene, there is no pentameric VH replacement footprints in the N1 regions. Such result provides supporting evidence that the presence of pentameric footprints in the N1 regions of mouse IgH genes is contributed by VH replacement.As we showed earlier, different Vequences . The fre similar . These rH replacement in mouse, we analyzed the distribution of VH replacement products in IgH genes correlating with different keywords in the NCBI database. Based on the identification of 5-mer VH replacement footprints within the N1 regions, the frequencies of VH replacement products in IgH genes derived from C57BL/6 and BALB/c strains of mice are 3.17% and 5%, respectively , the frequency of VH replacement products is 15.38%. In IgH genes from the Sle1/Sle3 mice, the frequency of VH replacement products is 30%. These frequencies are significantly higher than that in the BALB/c or C57BL/6 mice   . The eleHRFA program, we also analyzed the frequencies of VH replacement products based on the 4- or 3-mer of VH replacement footprints in IgH genes derived these diseased subcategories. Extending the assignment of VH replacement products with considering the 4- and 3-mer VH replacement footprints clearly increases the frequencies of VH replacement products in IgH genes from all subcategories. With considering the 4-mer VH replacement footprints, the frequencies of VH replacement products in IgH genes derived from NZB/NZW, MRL/lpr, SLE1, SLE1/SLE3 and IgH genes encoding anti-DNA and ANA antibodies are significantly higher than that in the BALB/c mice   (H replacement products are enriched in IgH genes derived from different strains of autoimmune prone mice and in IgH genes encoding autoantibodies.Using the Vre test) ; with core test) . Taken tH replacement products in human IgH genes showed that the VH replacement footprints preferentially encode charged amino acids into the IgH CDR3 regions H replacement products from mouse IgH genes showed that 64% of the amino acids encoded by the identified VH replacement footprints contribute charged amino acids, including K, R, D, E, N, and Q. Such frequency is significantly higher than the overall frequency of charged amino acids in the N1 regions (p<0.0001) (H replacement footprints are significantly higher than those encoded by the N1 regions of non-VH replacement products (p<0.0001)   . Moreove<0.0001) . The pre<0.0001) . In non-products . Such reH replacement was considered as a receptor editing process to change non-functional IgH rearrangements or IgH genes encoding autoantibodies H replacement footprints preferentially encoded charged amino acids, especially R and K residues, is contrast to the original goal of VH replacement to eliminate autoreactive IgH genes. Because charged residues within the IgH CDR3 might contribute to autoreactivity. Interestingly, when we analyzed the amino acids encoded by the identified 3-mer VH replacement footprints, the usages of charged residues, including R, K, and E, are significantly reduced; meantime, the usages of several neutral residues, including H, L, and Y, are significantly increased  (H replacement.During V t test) . These rH replacement footprints is likely predetermined by the 3′ end sequences of VH germline genes. Based on the 3′ end sequences of VH germline genes, VH replacement footprints can contribute almost equal numbers of positively or negatively charged residues . The other 80% of IgH gene sequences have no identifiable VH replacement footprints H replacement footprints are deleted during VH replacement recombination. Thus, even if using the minimal length of VH replacement footprints with 4 or 3 nucleotides, we may still under-estimate the actual frequency of VH replacement products in the murine IgH repertoire. Theoretically, 66.7% of the IgH rearrangements generated during V(D)J recombination will be out of reading frame and cannot produce functional IgH proteins; about 44% of the pro B cells undergoing V(D)J recombination should carry non-functional rearrangements on both IgH alleles. If VH replacement can efficiently rescue these pro B cells, at least 44% of the expressed IgH genes should be generated by VH replacement.It has been shown previously that in mice carrying two non-functional alleles of IgH genes, VH replacement footprints may have false positive calls. Theoretically, there are no VH replacement footprints in the N2 regions. In some of the IgH sequences, we identified similar 3, 4, or 5 mer VH replacement footprint motifs in the N2 regions, although the frequencies of such motifs in the N2 regions are significantly lower than those in the N1 regions. The presence of such VH replacement footprint motifs in the N2 regions could be due to random nucleotide addition during V(D)J recombination. In this regard, a low frequency of identified footprints might be false positive.We should also point out that this sequence analysis based approach in identification of VH replacement footprints to assign VH replacement products, the frequencies of VH replacement products in IgH genes derived from BALB/C or C57BL/6 mice are about 5% or 3.2%, respectively, which may represent the basal level of VH replacement product in these two strains of mice. Interestingly, the frequencies of VH replacement products are significantly elevated in IgH genes derived from different strains of autoimmune prone mice, including MRL/Lpr and Sle1/Sle3 mice. It has been well demonstrated that these mice spontaneously produce anti-DNA or anti-ANA antibodies and develop lupus like symptom H replacement products are significantly elevated in IgH genes encoding anti-DNA antibodies or ANA autoantibodies derived from mice with lupus glomerular nephritis. These results suggested a potential contribution of VH replacement products to the generation of autoantibodies. When we consider the 4- or 3-mer VH replacement footprints to assign VH replacement products, the frequencies of VH replacement products are elevated in all the sub-categories of IgH genes. Nevertheless, the frequencies of VH replacement products in IgH genes derived from different strains of autoimmune prone mice and IgH genes encoding anti-DNA and ANA antibodies are significantly higher than that in the BALB/c mice.If we use the 5-mer VH replacement will leave a short stretch of VH replacement footprints to elongate the IgH CDR3 region H replacement footprints preferentially encode charged amino acids in the newly formed CDR3 regions. Such features are commonly found in VH replacement products identified from human and mouse IgH genes H replacement products are significantly elevated in IgH genes encoding anti-DNA and ANA autoantibodies in mouse. Theoretically, the VH replacement footprints can encode either positively or negatively charged residues. Analysis of the amino acids encoded by the identified VH replacement products from different strains of autoimmune prone mice and IgH genes encoding autoantibodies showed that the frequencies of positively charged residues encoded by VH replacement footprints are significantly elevated; while the frequencies of negatively charged residues encoded by VH replacement footprints are significantly reduced. Previous studies have shown that positively charged residue like Arg within the IgH CDR3 is critical for DNA binding H replacement products from autoimmune prone mice have been positively selected. Such notion is also supported by the accumulated mutations in these identified VH replacement products.Due to the location of the cRSS, VH replacement was originally recognized as a receptor editing process to change either non-functional IgH genes or IgH genes encoding autoreactive antibodies H replacement products in IgH genes from different strains of autoimmune prone mice and in IgH genes encoding autoantibodies are surprising findings from this study. Currently, it is not clear why VH replacement products are accumulated in autoimmune prone mice. Like any recombination process, VH replacement is a random process that can generate non-functional IgH genes or IgH genes encoding autoreactive antibodies. Previous studies have shown that VH replacement products generated through replacing the knocked-in anti-DNA IgH genes can produce high affinity anti-DNA antibodies during chronic graft-versus-host (cGVH) response H replacement recombination, the newly generated IgH genes should be subjected to strict negative selection again to eliminate B cells expressing autoreactive BCRs. The observed accumulation of VH replacement products in autoimmune prone mice could be due to the defective negative selection processes in these mice. In autoimmune prone mice, the newly generated VH replacement products encoding autoreactive antibodies cannot be efficiently eliminated, but are rather positively selected and contribute to the generation of autoantibodies. To this extend, the different strains of autoimmune prone mice will be excellent experimental models to dissect how the VH replacement products are selected and enriched during early B cell development.VH replacement footprints also uncovered an interesting finding that short VH replacement footprints, especially the 3-mer footprints, encode less charged residues. These results suggested that if the VH replacement footprints were trimmed down to 3-mer during either primary or secondary recombination, they will be less likely to contribute charged amino acids into the IgH CDR3 regions. Given the fact that 33.55% of IgH genes contain 3-mer VH replacement footprints at their N1 regions, it is reasonable to conclude that the majority of these VH replacement products successfully edited the IgH genes without introducing of extra charged residues into the newly formed CDR3 regions. The observed accumulation of VH replacement products based on the identification of 5-mer footprints in the N1 regions in IgH genes derived from autoimmune prone mice may represent the failed VH replacement attempts either due to defects in negative selection or defects in trimming down the VH replacement footprints during primary or secondary recombination. Such findings raised several interesting questions that require further studies.Our analyses of the amino acid residues encoded by the identified VH replacement products to the diversification of mouse IgH repertoire. Identification of enriched VH replacement products in IgH genes derived from different strains of autoimmune prone mice and IgH genes encoding autoantibodies indicated that abnormal regulation of VH replacement may contribute to the generation of autoreactive antibodies.In conclusion, analysis of large number of mouse IgH gene sequences from the NCBI database provides a comprehensive view of the IgH repertoire of the available mouse IgH genes in the NCBI database and reveals a significant contribution of Vhttp://www.ncbi.nlm.nih.gov/projects/igblast/) on May 07, 2011, which were used to download GenBank records of the sequences from NCBI. There were total 17,179 mouse IgH gene sequences retrieved at that time. The IDs of these IgH genes and their VH, DH, and JH gene assignments are included in H, DH, JH genes, clonally redundant sequences were stripped out based on their identical CDR3 regions. The resulting 11,308 unique sequences were further analyzed. Clonally related sequences with mutations within their CDR3 regions still remain. The 17179 mouse IgH sequences were derived from 861 published studies , identification of VH replacement footprints with different lengths, analysis of amino acids encoded by the identified VH replacement footprints, calculation of the amino acid usage encoded by the identified VH replacement footprints, and correlation of the identified VH replacement products with different keywords and publications associated with the sequences in the NCBI database.We developed a Java-based Vhttp://www.imgt.org/IMGT_vquest/share/textes/) for assign potential germline VH, DH, JH genes, allowing 1 mutation at the 3′ end of VH genes and at the 5′ end of JH genes. All the IgH gene sequences were analyzed in batches containing 50 sequences each batch and the results were downloaded to a local computer as Excel files. These processes were conducted using the VHRFA program.Mouse IgH sequences in the GenBank format were converted to FASTA format and submitted to IMGT/V-QUEST  or DH-JH junction  regions of the IgH genes to identify matched footprint motifs. For each IgH gene, the VHRFA program started by searching the longest footprint motifs (11 mer) from the 5′ to 3′ of the DNA sequences and then goes to search footprints with one nucleotide shorter. The identified footprints were listed if it does not overlap with any previously identified footprint within this region. For examples, the end results of footprint analyses of with specified 5 mer included all the footprints with 5, 6, 7, 8, 9, 10, and 11 mer from the VH replacement footprint library. The end result was exported as a CVS file that contains the gene ID, functionality, VH, DH, JH gene assignment, VH replacement footprint in N1 (N1 signatures) or N2 (N2 signatures), together with other information from the original Excel file provided by the IMGT V-QUEST program. The identified footprints were shown in parenthesis within the N1 or N2 region sequences.All the rest steps were conducted on a local computer by the Vdatabase . Basicaln length . The VHRH replacement footprints within the N1 regions, the VHRFA program further analyzed the amino acids encoded by the VH replacement footprints and the usages of different amino acid. Each result was exported as an individual Excel file.After identification of the VHRFA program can also analyze the original GenBank file to correlate the keywords and publication information with each IgH gene sequence. Basically, the VHRFA program parses the source GenBank file for keywords in the KEYWORDS and FEATURES sections of each entry sequence and output the keyword list in correlation with the sequence IDs, VDJ assignments, N1 footprints, and N2 footprints. Through this analysis, we can determine the distribution of VH replacement products in different diseases.The VHRFA program only calculated the mutation rate of IgH VH genes with >80% similarities to the assigned germline VH genes.For mutation analysis, the VChi-square test with Yates’ correction or non paired student t test. Significant difference was determined if the p value <0.05.Statistical significance was determined by using either the two tailed Table S1Analyses of mouse IgH genes and identification of VH replacement products.(XLSX)Click here for additional data file.Table S2Number of sequences from each publication.(XLSX)Click here for additional data file.Table S3Mouse VH genes containing the TACTGTG cRSS.(DOCX)Click here for additional data file.Table S4H replacement footprint motifs with different length.Potential mouse V(DOCX)Click here for additional data file.Table S5H replacement footprint motifs in mouse IgH sequences.Identification of 4-mer V(DOCX)Click here for additional data file.Table S6H replacement products in IgH genes correlating with different keywords.Identification of V(DOCX)Click here for additional data file.


Box C/D snoRNAs guide the site-specific 2’-O-ribosemethylation of nucleotides in rRNAs and small nuclear RNAs (snRNAs). A number ofbox C/D snoRNAs and their fragments have recently been reported to regulatepost-transcriptional modifications and the alternative splicing of pre-mRNA.Artificial analogues of U24 snoRNAs directed to nucleotides in 28S and 18SrRNAs, as well as pre-mRNAs and mature mRNAs of human heat shock cognate protein(hsc70), were designed and synthesized in this study. It was found that afterthe transfection of MCF-7 human cells with artificial box C/D RNAs in complexwith lipofectamine, snoRNA analogues penetrated into cells and accumulated inthe cytoplasm and nucleus. It was demonstrated that the transfection of culturedhuman cells with artificial box C/D snoRNA targeted to pre-mRNAs induce partialsplicing impairments. It was found that transfection with artificial snoRNAsdirected to 18S and 28S rRNA nucleotides, significant for ribosome functioning,induce a decrease in MCF-7 cell viability The class of small nucleolar RNAs (snoRNAs) consists of two major families: box C/DRNAs and box H/ACA RNAs. These RNAs act as a recognizing and targeting element inRNA–ribonucleoprotein complexes and participate in the modification ofnucleotides in eukaryotic ribosomal RNAs. The RNAs belonging to the box C/D RNAfamily guide the 2’-O-methylation of rRNA nucleotides. Box C/D RNAs containconserved structural elements, CUGA (box D) and RUGAUGA (box C), near the 5’and 3’ termini, respectively. The box C/D RNAs possess a guide sequence region(a sequence complementary to the region of an RNA target). Certain RNAs contain twoguide sequences and two box C/D pairs  .Cavaille J. et al. [. et al. demonstr. et al. .de  novo 2’-O-methylation [2C R [One of the key approaches to the study of the properties of box C/D RNAs is thedesign of DNA constructs that are expressed in a cell yielding either shortnon-natural snoRNAs or pre-mRNA fragments, the processing of which results in theformation of snoRNAs targeted to the pre-specified rRNA nucleotides . This aphylation , 4. The on [2C R , 6. ThusThe effect of synthetic analogues of natural box C/D RNAs on pre-mRNA-target splicingand processing of 18S and 28S rRNA in human cells was investigated in this study.The analogues of human U24 box C/D RNA directed to pre-mRNA of human heat shockcognate protein hsc70 and human rRNA were designed. The transfection of the MCF-7cell line (human breast adenocarcinoma cells) with synthetic analogues was shown toresult in a partial disruption of splicing (the elimination of an exon from thepre-mRNA-target). It was found that the transfection of MCF-7 cells with syntheticanalogues of box C/D RNAs directed to rRNAs induces a decrease in cellviability.Artificial box C/D RNAs synthesisin  vitro transcription of PCR-amplified DNA templates with T7 RNApolymerase .Synthetic analogues of box C/D RNAs were obtained via the Transfection of MCF-7 cells with synthetic RNAs. Isolation of total cellularRNAо С. Synthetic analogues of box C/DRNAs were pre-incubated with the lipofectamine reagent according to the manufacturer’s protocol and were subsequently added to theculture medium. After the MCF-7 cells had been incubated for 18 h, total RNA wasisolated using the Trizol reagent  according to themanufacturer’s protocol.MCF-7 cells  were cultured in an IMDMmedium with 10 mM L-glutamine and 40 µg/ml of gentamicin in the presence of 10%fetal bovine serum at 37 Isolation of cytoplasmic and nuclear fractions of MCF-7 cell lysateg . The isolation of RNA from the supernatant (the cytoplasmicfraction of MCF-7) and nucleolar precipitate suspended in buffer A was carried outusing the Trizol reagent. RNA concentrations in the samples were determinedspectrophotometrically (λ = 260 nm), taking into account the extinctioncoefficient for RNA (ε 260  = 25 l/mol cm). Upon completion of incubation, the MCF-7 cells were ice-cooled. The medium wascollected. The cells were washed twice with phosphate buffered saline (PBS) andlysed on ice for 10 min . The lysate was suspended and coated onto a 10% sucrosesolution in buffer A, followed by centrifugation for 20 min at 600 Analysis of pre-mRNA HSPA8 splicing variants by RT-PCRHSPA8  and cDNAamplification was performed in the reaction mixture for RT-PCR “Real BestMaster Mix RT”  using the primers hsp2.1(5’-ACTGAACGGTTGATCGGTGA-3’) and hsp8.2(5’-AGATGAGCACGTTTCTTTCT-3’). The products were analyzed in a 4%polyacrylamide gel. The quantity of amplification products in the gel wasascertained using the Gel-Pro Analyzer 3.1 software. Sanger sequencing was performedusing fluorescently labelled terminators of DNA-polymerase in the BigDye 3.1mixture, followed by the separation of DNA on an ABI3100 analyzer .The reverse transcription of pre-mRNA in the Fluorescently labelled box C/D RNA synthesis and analysis of the accumulationof fluorescently labelled RNA in human cellsin  vitro transcription by T7 RNA polymerase using Flu-12-UTP . The RNA transcript was isolated viaion-pair RP-HPLC on a Milichrome A-02 liquid chromatograph using theProntoSIL-120-5-C18 sorbent and a 2.0 × 7.5 mm column. The accumulation offluorescently labelled RNA in MCF-7 cells was analyzed via fluorescence microscopy. For this purpose, 3 × 10 4 MCF-7 cells were seeded onto a slide plate of the Culture Slide chamber , then they were transfected with fluorescently labelled RNAafter 24 h, followed by incubation for 18 h. The medium was removed post-incubation.The cells were washed twice with PBS; the specimens were embedded into a drop of theDAPI/Antifade dye  and covered with a cover slip. Thespecimens were analyzed on an Axioskop 2 Plus microscope .Fluorescently labelled RNA was obtained via Analysis of 2’-O-methylation of G1702 of the 18S rRNA2 CO3 solution (pH 9.0) for 18 min at 90 о С. Thehydrolysis products were recovered by ethanol precipitation. Reverse transcriptionwas carried out using primer 18.1702 (5’-GCCGATCCGAGGGCCTCACT-3’),complementary to the region 1731–1750 of the 18S rRNA, using M-MLV reversetranscriptase . The rRNA region was sequenced viareverse transcription in the presence of ddNTP, according to [2’-O-methyl groups of rRNA were detected via partial alkaline hydrolysis aspreviously described in . The totAnalysis of MCF-7 cell viability via the MTT assay4  cellsper well). After 24 h, the RNA–lipofectamine complex was added to the culturemixture until concentrations of 3.0, 10.0, and 70.0 nM were obtained. MCF-7 cellswere incubated with RNA for 3 days; the MTT solution in PBS was subsequently addedto the medium until a final concentration of 0.5 mg/ml. The mixture was incubated at37°С for 90 min. After the medium was removed, MTT-formazan crystals weredissolved in 100 µl of isopropanol. The absorbance of the solution was determined on an Apollo 8 LB 912 multichannelspectrophotometer . The data were presented as adecrease in viability (100% – MTT index) against the control .In order to analyze the effect of the analogues of box C/D RNAs on the viability ofMCF-7 cells, the cells were cultured in a 96-well plate   [ ).The effect of the synthetic analogues of box C/D RNAs on RNA processing in humancells was studied using the designed analogues of human natural U24 box C/D RNA.Human U24 RNA contains CUGA and AUGAUGU (GUGAUGA) sequences (for D and C (C’)boxes, respectively) and two guide sequences directing the 2’-O-methylation ofC2338 and C2352 in 28S rRNA (  .  1A)  . The resHSPA8 gene encoding the human heat shock cognateprotein (hsc70). Suppressing the expression of hsp-70-related proteins, includingthe hsc70 protein, results in the death of cultured cancer cells. The HSPA8 gene is considered to be a promising target for gene-targeted cancertherapy [Table 1 .One of the guide sequences (the D-box-dependent one) was directed to pre-mRNAnucleotides of the therapy . NucleotHSPA8  gene that both variants of alternative splicing of thispre-mRNA (the major and minor ones) can be identified in the control MCF-7 cells . It was demonstrated viathe sequencing of these forms that they differ by either the presence or absence ofthe second exon. The content of the minor form, i.e., the form without the secondexon, increases in the cells transfected with synthetic box C/D RNAs directed to thenucleotides that are key ones for pre-mRNA splicing . Thus, it was found that the analoguesof box C/D RNAs directed to the selected pre-mRNA nucleotides of the HSPA8 gene have an impact on the pre-mRNA target splicing and result in theexcision of the second exon.It was ascertained via the transfection of MCF-7 cells with synthetic analogues ofbox C/D RNA and analysis of the variants of the alternative splicing of pre-mRNA ofthe Fig. 3 that, 3   h after the passive transfection of the cellswith PM.8 RNA in the absence of lipofectamine, RNA could be detected neither in thenuclear nor in the cytoplasmic fraction of MCF-7 cells .  After the cells were transfected with theanalogue of box C/D RNA in the presence of lipofectamine, PM.8 RNA was found both inthe cytoplasmic and nuclear fractions even 26 h post-transfection .  All these facts leadone to the conclusion that synthetic box C/D RNAs in the presence of lipofectaminecan efficiently penetrate into the cell cytoplasm and nucleus, where they canparticipate in the processing of the pre-mRNA-target. The transfection efficiency, distribution, and stability of the synthetic analoguesof box C/D RNAs were assessed via RT-PCR of nuclear and cytoplasmic RNA of the MCF-7cells transfected with PM.8 RNA. It is clear from the data shown in Fig . 4 ).The RT-PCR data are in close correlation with the results of the analysis of thedistribution of FAM-labelled PM.8 RNA in MCF-7 cells via fluorescent microscopy.Thus, after the cells are incubated in a medium with FAM-labelled PM.8 box C/D RNAin complex with lipofectamine, RNA is captured, internalized, and then distributedboth over the cytoplasm and the cell nucleus  . These dFig .  5 ). Thepresence of the 2’-O-methylated nucleotide within rRNA results in a decreasein the yield of the cDNA product , the length of which is determined by the positionoccupied by a nucleotide that is downstream adjacent to the 2’-O-methylatednucleotide [Fig. 5 that the yield of the cDNA transcript corresponding to the2’-O-methylated G1702 of 18S rRNA does not decrease as a result of thetransfection of human cells with any analogue of box C/D RNAs.The induction of 2’-O-methylation of the target of the second guide sequence(G1702 of the 18S rRNA) was analyzed in order to assess the ability of an analogueof box C/D RNA to direct rRNA modification. Since G1702 is one of the keynucleotides of the decoding site of human ribosomes, it was selected to be thetarget . No addi The targets selected are the key nucleotides of the ribosomal functionalsites: U1827 of the 18S rRNA can be found in the ribosomal decoding centre; G4499,U4500, and U4502 of the 28S rRNA, in the peptidyl transferase centre [Table2 ).The effect of synthetic analogues of box C/D RNAs on the post-transcriptionalmodification of rRNAs in human cells was analyzed using the designed and obtainedanalogues of U24 box C/D RNA, whose first guide sequence was directed to thenucleotides of human 18S and 28S rRNA. The nucleotide sequences of the analogues ofbox C/D RNAs are listed in e centre , 4. TheFig.6 that the greatest reduction in viability (>  35%) was induced by box C/D RNAs directed to U1827 of the 18SrRNA, G4499, U4500, and U4502 of the 28S rRNA. Meanwhile, the decrease in cellviability and proliferation caused by the analogues of U24 RNA, whose first guidesequence was directed to pre-mRNA of the hsc70 protein, was less than 22% .A comparative analysis of the effect of the analogues of box C/D RNAs on MCF-7 cellviability was performed. The effect of the analogues of box C/D RNA was assessedafter the cells were incubated in a medium with initial RNA concentrations of 3.0,10.0, and 70.0 nM. It is clear from Table 2 ) in order to analyze theinduction of de novo  2’-O-methylation of target nucleotides;2’-O-methylated rRNA nucleotides were detected by the partial alkalinehydrolysis method. It was ascertained that the contribution of rRNA forms containingthe 2’-O-methylated target nucleotide in the total RNA pool of the transfectedMCF-7 cells was lower than the sensitivity threshold of the detection technique.MCF-7 cells were transfected with the analogues of box C/D RNA  andD/D’ (CUGA) boxes, and the guide sequence ensure the ability of these RNAs toparticipate in the formation of a catalytic complex with proteins of themethyltransferase complex and direct 2’-O-methylation of a pre-specifiednucleotide , 2, 15. rRNAs and snRNAs are the major targets of box C/D RNAs in eukaryotic cells.Meanwhile, snoRNAs participating in pre-mRNA processing have been detected . It was HSPA8  gene encoding heat shock cognate protein (hsc70) weredesigned and obtained in the present study . It was ascertained that the transfection of humancells with synthetic analogues of box C/D RNA directed to the splice donor andsplice acceptor sites of the second intron, adenosine the splicing branch point, thefirst and the second nucleotides of the first intron of pre-mRNA of theHSPA8 ( Table1 ) results in an increase in the amount of the splicing productof pre-mRNA without the second exon . Two key mechanisms can be proposed for the influence ofthe analogues of box C/D RNA on pre-mRNA target processing: 2’-O-methylationof the target nucleotide and complementary interaction between the antisense regionof the box C/D RNA with the target RNA. Both pathways may theoretically result inthe inhibition of certain splicing steps and, consequently, in a change in variantsof alternative splicing of the pre-mRNA target.The analogues of U24 small nucleolar box C/D RNA directed to pre-mRNA nucleotides ofthe The methods for detection of 2’-O-methylated nucleotides, which are currentlyin widespread application, do not allow to reveal these modifications in mRNA andpre-mRNA with an adequate level of efficiency. Therefore, it cannot be unambiguouslystated whether the methylation of the nucleotide target actually occurs, and whetheror not the effect on splicing is caused by 2’-O-methylation of the pre-mRNAtarget. The key points of pre-mRNA splicing are sensitive to the modifications ofthe 2’-OH groups of ribose residues to different extents . The resFigs .  3, 4 ). It is clear fromFig .  3  that the analogue of box C/D RNA wasreliably detected by RT-PCR 26 h following the single transfection in the presenceof lipofectamine in the nuclear and cytoplasmic RNA fractions of MCF-7 cells. Theresulting data attest to the fact that the artificial box C/D RNAs are potentiallyavailable for interaction with RNA targets located inside the nucleus. Theaccumulation of synthetic RNA in cells was confirmed via fluorescence microscopy . Furthermore, it was ascertainedthat the synthetic analogues of box C/D RNA in the presence of lipofectamine can beefficiently conserved and detected via RT-PCR in human cells 72 h after the singletransfection (the data are not illustrated). Meanwhile, in the case of transfectionin the absence of lipofectamine, synthetic RNAs cannot be detected in human cellsvia RT-PCR as early as 3 h after having been introduced into the culture medium .In order to participate in splicing, artificial RNA has to interact with the pre-mRNAtarget inside the nucleus. It was demonstrated that the analogues of box C/D RNA inthe presence of lipofectamine are capable of efficient penetration into human cells( Table 1 ). It wasshown earlier that the transfection of human cells with synthetic analogues of boxC/D RNAs directed to rRNA nucleotides ( Table 2 ) induces the termination of reverse transcriptionon the target nucleotides [Fig . 5 ). Neither of the de novo 2’-O-methylation of rRNA nucleotides – the targets of the analoguesspecified in Table 2 (aftercell transfection with the corresponding RNAs) – was revealed. Meanwhile, thepositions of a number of known 2’-O-methylated nucleotides in human rRNA wassuccessfully determined using this method.For all the analogues of U24 snoRNAs obtained, one of the two guide sequences wasdesigned so as to direct the 2’-O-methyaltion of G1702 in human 18S rRNA . Itis . et al. , 4, 25 , results in a decreasein their viability by 36–48%; the initial concentration of synthetic RNA inthe medium being 70.0 nM .Meanwhile, synthetic analogues simultaneously directed to the pre-mRNA of heat shockcognate protein hsc70 and on G1702 in 18S rRNA ( Table 1 ) reduced the cell’s viability byonly 20–25% within an initial concentration range of 3.0–70.0 nM .The effect of the analogues of box C/D RNA on the viability of human MCF-7 cells wasassessed via the MTT assay. The data obtained made it possible to arrive at theconclusion that cell transfection with synthetic analogues, whose first guidesequence is directed to the nucleotides of 18S and 28S rRNA .The rates of proliferation and monolayer formation decreased considerably, and cellmorphology changed after the MCF-7 cells were transfected with synthetic analoguesof box C/D RNA directed to U1827 in 18S rRNA, G4499, U4500, and U4502 in 28S rRNA.The rate of the decrease in viability varied for different analogues and depended onthe target nucleotide and the initial RNA concentration in the culture medium (Table2 ). It was shown earlier by Liu B. et al.[The difference in the influence of synthetic analogues of box C/D RNA on MCF-7 cellviability attests to the fact that these RNAs are involved in the regulation ofvital processes in human cells during the transfection. The fact that thisdifference is a result of changes in the structure of the guide sequence allows oneto assume that post-transcriptional processing of pre-rRNA is the major processmodulated by the analogues of box C/D RNA. It should be noted that rRNA nucleotidescomprising the ribosomal active sites (the decoding and peptidyl transferase ones)were selected as targets  with synthetic analogues of box C/D RNA directed to pre-mRNAnucleotides of heat shock cognate protein hsc70 results in the disruption ofsplicing of the pre-mRNA target. The transfection of MCF-7 cells with analogues ofbox C/D RNA directed to the nucleotides in 18S and 28S rRNA that play the key rolein ribosome functioning induces a decrease in cell viability. The data obtainedattest to the high potential of designing constructs for the regulation of humangene expression and translation based on the snoRNA structure.


Pyrococcus furiosus and a reconstituted transcription system, we demonstrate that the TL is essential for initial RNA synthesis until a complete DNA–RNA hybrid is formed. The archaeal TL is further important for transcription fidelity during nucleotide incorporation, but not for RNA cleavage during proofreading. A conserved glutamine residue in the TL binds the 2’-OH group of the nucleoside triphosphate (NTP) to discriminate NTPs from dNTPs. The TL also prevents aberrant transcription termination at non-terminator sites.The trigger loop (TL) forms a conserved element in the RNA polymerase active centre that functions in the elongation phase of transcription. Here, we show that the TL also functions in transcription initiation and termination. Using recombinant variants of RNA polymerase from  RNA polymerases (RNAPs) carry out transcription in all living organisms. One RNAP is present in bacteria and archaea, whereas eukaryotes possess three to five specialized nuclear RNAPs I-V. RNAP structures suggested mechanisms of transcription that can now be analysed biochemically. Analysis of eukaryotic RNAP II mutants is however restricted to viable yeast strains, but archaeal RNAP, which is closely related to RNAP II ,2, is av2+ ions to catalyse RNA chain growth by phosphodiester bond formation.Archaeal RNAP and eukaryotic RNAP II use the same core promoter elements, the TATA box and TFIIB recognition element (BRE), and interact with homologous general transcription initiation factors (GTIFs), TATA-binding protein (TBP) and transcription factor B (TF(II)B), which govern promoter DNA recognition and opening ,10–12. APyrococcus furiosus (Pfu) RNAP, the TL is part of the subunit A'', which together with subunit A' corresponds to RNAP II subunit Rpb1. Five distinct TL conformations have been observed: open, closed, wedged, trapped and locked (The trigger loop (TL) is a conserved mobile element of the RNAP active centre . In Pyrod locked . Nucleosd locked ,18,21–23d locked . When RNd locked , until id locked . Finallyd locked ,30.P. furiosus A'' L83, Escherichia coli β' M932) and H1085   ,19,23. Tβ' H936) ,31. The β' H936) . The TL β' H936)  and by tβ' H936) ,31.Saccharomyces cerevisiae Rpb1 N479, P. furiosus A' N456, Thermus thermophilus β' N737) was suggested to recognize the 2'-OH group of the NTP  contacts the NTP 2'-OH group  and [α-32P]UTP at 0.15 MBq (110 tBq/mmol) for 15 min at 70°C. Transcription initiation was carried out with the template gdh-C9, where the first cytidine occurs at position +10, 40 μM priming RNA (Sigma), [α-32P] complement NTP at 0.22 MBq (110 tBq/mmol). RNA was extracted with phenol-chloroform and analysed on an 8% (run off) or 28% (trinucleotides and short transcripts) polyacrylamide gel containing 7 M urea.Standard transcription template gdh-C20  was geneescribed . Run-offescribed . Brieflygdh-C20 promoter in reactions containing 90 nM TFE, 50 nM RpoE', 100 nM TBP and 70 nM TFB.Thymidine residues in open complexes were detected by treatment with potassium permanganate as previously established . Reconst32P] ATP. For EC assembly, RNAP was incubated with a hybrid of the DNA template strand annealed to the RNA in Elongation Buffer  for 5 min, then with the 3′end-biotinylated non-template DNA strand for 5 min and then with 2.5 g Heparine for 5 min at 20°C. Beads were subsequently washed three times with EB pre-heated at 50°C. Beads were resuspended in EB. For 1 nt RNA extension assays, 100 µM of NTP  (NEB Biolabs) were added, and the mixture was incubated at 50°C. For the elongation and termination assays, 30 µM of NTPs mix were added. The mixture was incubated at 70°C for elongation assays and at 70, 80 or 90°C for termination assays. For TFS-induced cleavage assays, 150 nM TFS were added, and the mixture was incubated at 70°C. Reactions were stopped by transferring the sample into cold loading buffer containing formamid, and samples were heated to 95°C and analysed on a 28% polyacrylamid gel containing 7 M urea. For elongation assays and termination assays, the samples were analysed on a 15% polyacrylamid gel containing 7 M urea. The radioactively 5′-labelled RNA products were visualized with a FLA-5000 scanner (Fujifilm). Gel bands were quantified using Aida Image Analyzer (Raytest).Bead-based elongation complexes (ECs) containing complete complementary scaffolds were assembled and immobilized on Dynabeads M-280 Streptavidin (Invitrogen) essentially as described ,60. The 3CO2K (pH 9), 10 mM Mg(CH3COO)2, 0.1 mg BSA] and incubated at 70°C. The reactions were stopped by transferring the sample to ice. Beads were washed one time with CB and resuspended with CB with loading buffer containing formamid. Samples were heated to 95°C and analysed on a 28% polyacrylamid gel containing 7 M urea. The radioactively 5′-labelled RNA products were visualized with a FLA-5000 scanner (Fujifilm). Gel bands were quantified using Aida Image Analyzer (Raytest).For intrinsic RNA cleavage assays, ECs were assembled in Cleavage Buffer /(KM + [NTP]), where kpol is the maximum NTP incorporation rate of the enzyme, [NTP] is the substrate concentration and KM is the Michaelis constant. Kinetic data  were fitted to a single exponential equation using non-linear regression in Sigmaplot (Systat Software Inc.). Discrimination ratios of complementary ATP (cATP) over c2′dATP and cATP over non-complementary UTP (ncUTP) were calculated by using the equation, / and /, respectively.The nucleotide (mis)incorporation rates obtained for various substrates and concentrations were fitted to the Michaelis–Menten equation, Pfu RNAP variants with TL mutations (Sc Rpb1 A1087-K1093), where the mobile tip of the TL is deleted. We also prepared a TL deletion mutant, ΔTL , where the size of the deletion was designed based on the structural information to prevent destabilization of the enzyme. In yeast, equivalent mutations were lethal  promoter in the presence of GTIFs  that was complementary to the +1 and +2 positions in the template DNA strand . By addition of the radioactive nucleotide [α-32P]-UTP, we tested whether mutant RNAPs could elongate the GpU RNA by 1 nt. The L83A variant synthesized the trinucleotide product but was less active than the WT enzyme . The TL deletion mutants and H87A were not active.To investigate whether the initiation phase of transcription requires the TL, we carried out an abortive transcription assay, using closed and pre-opened versions of the Supplementary Figure S1)  or around the putative site of opening (−10). Transcription activity was also not increased by addition of TFE, which stimulates promoter opening and abortive transcription (gure S1) . Permanggure S1) C. Thus, We next analysed the capacity of WT RNAP and TL variants to extend a short RNA 3–9 nt in length by 1 nt in the presence or absence of GTIFs A. These Supplementary Figure S2). Therefore, the two successive RNA synthesis steps from RNA6 to RNA8 are necessary to maintain transcriptional activity, and they could not be obtained through a pre-synthesized RNA that binds the initiation complex. In contrast, extension activity is observed with RNA8 and RNA9 and was independent of GTIFs (The WT enzyme efficiently added 1 nt to short RNAs (3–6 nt) in these scaffolds, whereby the activity was dependent on GTIFs, and the synthesis of a 5 nt RNA was most effective D. On scaof GTIFs D. Exceptin vitro assembly of nucleic acids originally described by Kireeva et al.  and incubation with 2'dNTP at 50°C. The results of this test series are summarized in To explore the role of the archaeal TL during transcription elongation in more detail, we used an a et al. . Elongata et al. A. On thikpol of 13 s-1, similar to other RNAPs tested under in vitro conditions . Among all mutants tested, the ΔTL variant had the strongest effect on the incorporation rate, resulting in an ∼30-fold decrease, whereas the substrate affinity was decreased ∼3-fold. Thus, the archaeal TL is important, but not essential for NTP substrate binding and catalysis during elongation.Under our conditions, the WT enzyme added cATP at a nditions ,31. The kpol by ∼10-fold and reducing substrate affinity by ∼2-fold . Although the reaction rates with ncUTP were similar with mutant and WT enzymes, the KM values were lower for all TL mutants. Accordingly, the discrimination for ncUTP was relatively high at ∼240 000-fold for the WT RNAP, whereas the ability to recognize the correct NTP was reduced by ∼25-, 40- and 90-fold for the H87A, L83A and ΔTL mutant RNAPs, respectively  and N479 (Pfu A' N456) with the 2'-OH group of the ribose moiety (Pfu A'' Q80) with the 2’-OH group was observed recently (Pfu A'' Y88) could improve the 2'dNTP discrimination . These results show that a novel contact of a conserved glutamine residue in the TL with the NTP 2'-OH group may contribute to selection of NTP over 2'dNTP substrates.These results prompted us to investigate the effect of analogous substitutions at homologues residues in the archaeal RNAP. In agreement with results from the eukaryotic system, mutation A'' Y88S resulted in a phenotype that improved discrimination of cATP over 2'dATP . AlthougTo decipher the role of the archaeal TL during intrinsic RNA hydrolysis, we used the mismatched scaffold templates MEC(C) and MEC(G) in which the terminal nucleotide at the 3′-end (CMP or GMP) of RNA15 is non-complementary to the corresponding template DNA base, whereas the upstream residues of template and RNA strands in the 9-bp hybrid region were complementary A. Such cSupplementary Figures S6 and S7). This agrees with one study in the bacterial system . In contrast, with MEC(G), the first and major cleavage product is RNA13, whereas RNA14 was a minor cleavage product arising later during the course of the experiment. Only WT RNAP was able to produce the RNA14 cleavage product on the MEC(G) template, whereas cleavage with the H87A, ΔTLtip and ΔTL RNAPs yielded RNA13 and smaller cleavage products only . These results indicate that H87 and other residues in the TLtip region may influence the translocation state of the EC.On MEC(C), the first cleavage product produced was RNA14, for both WT and mutant RNAPs   . To test' H1242) E. UnlikeSupplementary Figure S8). Moreover, TFS efficiently stimulated RNA hydrolysis in the absence of a 3′-mismatch (scaffold template EC(U), Supplementary Figure S8) , indicating that the TL affects the translocation state on matched templates also in the presence of TFS. The slight decrease in RNA cleavage activity noted with the ΔTL and ΔTLtip mutants  was also observed in the bacterial system and can be explained by a minor disarrangement of the active site caused by the deletion (The RNA cleavage activity in the presence of TFS occurred with WT and mutant RNAPs faster than without TFS (gure S8) ,60. Howedeletion . Our resSupplementary Figure S9). This effect was not reduced by increased NTP concentration .To investigate whether the TL plays a role during transcription termination, we compared the termination ability of WT and ΔTL RNAPs. Elongation assays were performed as described earlier in the text, but at increasing temperatures  on template EC(U) and ECTerm, which contains a hepta-dT (T1 to T7) transcription termination sequence A. WT RNASupplementary Figure S9). We conclude that the ΔTL RNAP mutant exhibits a temperature-sensitive processivity defect and is prone to termination at T-rich sequences. These results show that the TL is required for suppressing termination at non-terminator sites.Previously, the non-template DNA strand (NT) and RNAP subunits E' and F  were shown to be required for full processivity . We therAlthough previous TL analysis concentrated on transcription elongation, we report here functions of the TL during transcription initiation and termination . The arcOur results also provided insights into TL function during the discrimination of NTPs from 2'dNTPs. Mutagenesis of residues that were proposed to interact with the NTP 2’-OH group showed that A' R423 and N456 moderately affected 2'dNTP discrimination, whereas residue A'' Q80 was more important, consistent with its proposed role to couple 2'-OH recognition with TL closure ,32, A'' Transcription fidelity also relies on proofreading, a post-incorporation mechanism that involves cleavage of a dinucleotide from the RNA 3′-end containing the misincorporated nucleotide. In our assays, dinucleotide cleavage was preferred over mononucleotide cleavage, consistent with a catalytic role of the RNA 3′-end . The TL Finally, our results revealed a function of the TL in transcription termination. We show that the sensitivity of the archaeal RNAP to poly-T sequences is increased on TL truncation, showing that the TL prevents aberrant termination at non-terminator sites, to ensure processive RNA synthesis. In the absence of the TL or on its mutation, RNAP apparently is more prone to pausing and termination at T-rich sites.Supplementary Data are available at NAR Online: Supplementary Figures 1–9 and Supplementary Methods.The Deutsche Forschungsgemeinschaft . Funding for open access charge: DFG [SFB 960].Conflict of interest statement. None declared.


RNA Seq provides unparalleled levels of information about the transcriptome including precise expression levels over a wide dynamic range. It is essential to understand how technical variation impacts the quality and interpretability of results, how potential errors could be introduced by the protocol, how the source of RNA affects transcript detection, and how all of these variations can impact the conclusions drawn. Multiple human RNA samples were used to assess RNA fragmentation, RNA fractionation, cDNA synthesis, and single versus multiple tag counting. Though protocols employing polyA RNA selection generate the highest number of non-ribosomal reads and the most precise measurements for coding transcripts, such protocols were found to detect only a fraction of the non-ribosomal RNA in human cells. PolyA RNA excludes thousands of annotated and even more unannotated transcripts, resulting in an incomplete view of the transcriptome. Ribosomal-depleted RNA provides a more cost-effective method for generating complete transcriptome coverage. Expression measurements using single tag counting provided advantages for assessing gene expression and for detecting short RNAs relative to multi-read protocols. Detection of short RNAs was also hampered by RNA fragmentation. Thus, this work will help researchers choose from among a range of options when analyzing gene expression, each with its own advantages and disadvantages. Interest in quantifying levels of gene expression has been high ever since the first methods were developed to assess RNA differences across cell types and growth conditions. The methods and technologies have progressively improved to allow better sensitivity and inclusion of an ever greater number of transcripts in each assay. In the recent past, various forms of expression arrays have been the stalwart of gene expression analysis, but this technology is now rivaled by a more accurate, sensitive and versatile technology, RNA Seq  except for one experiment in which selected hexamers (avoiding sequences that prime rRNA) were used Because ribosomal RNAs (rRNA) and mitochondrial RNAs (mitoRNA) are so common but generally of less interest than other transcripts, they were removed prior to analysis. To compare expression levels across samples that have differing numbers of reads, it is necessary to normalize the total number of reads to a constant value. This is accomplished by multiplying the filtered read counts from all non-rRNA/mitoRNA reads by a constant factor to generate a total of 1,000,000 reads per sample. Expression is thus given as Reads Per Million (RPM). Elsewhere, this is frequently further adjusted to Reads per thousand (K) nucleotides Per Million reads (RKPM). As discussed later, this additional normalization introduces a variety of problems so, except for spike-in data, all comparisons will be made using RPM rather than RPKM.With total RNA, >80% of aligned reads correspond to rRNA, reducing the number of reads arising from transcripts of higher interest. Because the precision with which any RNA Seq measurement can be made is directly dependent on the number of times each transcript is counted, eliminating rRNA from sequencing allows a much higher number of counts to be obtained from all other RNAs. However, selective removal of any class of RNA introduces the potential risk of inadvertently altering the concentration of other RNAs To assess the impact of RNA selection on RNA Seq results, total RNA from human liver, human brain, and K562 cells, derived from human leukemia cells For K562 cells, three channels of total RNA, two channels each for two replicate samples of RiboMinus RNA prepared in parallel, and duplicate single channels of two replicate samples of polyA RNA were compared. The UCSC reference transcriptome included 28,808 non-ribosomal, non-mitochondrial annotated transcripts, each representing a collapsed set of transcript annotations. Transcript counts for all channels used in this analysis can be found in the GEO database under accession GSE28123 and sequence reads are available at the Sequence Read Archive under accession number SRP006040. The 69 individual samples available are listed in When different samples of K562 polyA RNA were compared to each other, it did not matter whether the duplicate runs or replicate samples were compared as the correlations were >0.99 in all pair-wise comparisons . One paiTo determine the generality of the patterns observed with the K562 samples, the same analysis was repeated with liver RNA . With thThe polyA samples discussed thus far were prepared using a single selection with oligo-dT magnetic beads. Another common method involves one or two selections using oligo-dT cellulose. According to the product manufacturers, the oligo-dT on the beads we used was 25 nt and the oligo-dT on the cellulose was a mix ranging from 12–25 nt. polyA RNA selected once with oligo-dT cellulose shows a similar amount of rRNA (33%) to polyA RNA selected once with beads. A second round of oligo-dT cellulose selection yielded a sample with only 3.6% rRNA. As shown in The non-rRNA reads in the flow through mirror what is selected but there are also interesting differences. Most of the reads that are underrepresented in the second selection relative to the flow through are either short non-coding RNAs or very long mRNAs. The former may have shorter than average polyA tails and hence less likely to stably bind the matrix. Many are detected in DGE experiments, suggesting some level of polyadenylation. The very long RNAs are probably overrepresented in the flow through because they are more likely to suffer degradation during processing. Conversely, the transcripts that tend to be underrepresented in the flow through include short, ribosomal protein encoding mRNAs that are probably less susceptible to degradation. Transcripts that show the greatest variation among the different polyA selection strategies are those that change most dramatically upon examination of total RNA versus any polyA RNA.Another indirect method for elimination of rRNA reads has been described in vitro transcription and combined in known absolute proportions Certain samples were also spiked with a set of seven RNAs produced by Fragmentation of RNA and/or cDNA has been used previously In addition to RNA Seq methods in which reads are captured from throughout the transcript length, it is also possible to use tag-based approaches in which reads are obtained only from the 5′ or 3′ end of each transcript. This should lead to improved counting though at the expense of characterizing splice and other sequence variants. Single-molecule Digital Gene Expression (DGE) has been described using yeast polyA RNA Because DGE should produce one read rather than many reads per RNA molecule, the agreement between the two methods is not perfect . DGE alsA noticeable impact of the double polyA selection is a loss in complexity of the sample with many genes missing. It is pronounced even at the level of annotated transcripts and has been shown to be even more extreme among unannotated transcripts While examination of static gene expression levels is instructive, it is generally of more interest to study changes in gene expression. However, care must be taken in such analyses. For example, it has been previously noted Transcripts were binned by length in groups of 400 and the fraction of genes that were differentially expressed plotted versus mean transcript length of the bin. As observed previously with other sequencing platforms and expected based on increased sampling, a higher proportion of long transcripts are observed to be differentially expressed . Howeverc-Myc and the upregulation of THBS1 and CTSD, all of which change by more than 1000 RPM and greater than 20×.Comparison of a tissue with a cell line provides a rich source of differentially expressed transcripts but may not adequately reflect the issues faced with systems of greater biological interest. For a more relevant model, RNA was prepared from HL60 cells growing in standard growth media or growing in the presence of t-retinoic acid (t-Ret) to induce differentiation SLBP is also downregulated 2.9× with t-Ret treatment in total RNA and 1.9× each in polyA or DGE samples. Thus, these expression changes, which are similar to other conditions in which DNA replication in cells ceases The importance of looking beyond just polyA RNA is highlighted by our observations of differential expression with many non-coding and histone RNAs in total RNA. Histone mRNAs are known to lack a polyA tail and their 3′ ends are processed by a variety of proteins including Stem Loop Binding Protein Because of the limitations imposed by incomplete annotation of the transcriptome and oversampling of longer transcripts, we have explored other methods for analyzing expression. By examining bins of uniform size across the whole genome, it is possible to eliminate both the effect of transcript length and the issue of collapsing a complex set of overlapping and poorly characterized transcripts into a master list. As an example, we compared K562 and liver RNA samples . The genMeasurement of RNA expression levels has been critical for understanding many pathways and biological systems. Up until recently, it has only been possible to examine a small number of transcripts over a wide range of expression levels using qPCR or a large number of transcripts over a narrower range of expression levels using microarrays. With the advent of RNA Seq and DGE technology, it is now possible to measure both a wider dynamic range of expression and in an unbiased manner at a genome-wide scale, a combination unachievable with any other technology. With the exquisite sensitivity and precision now made possible by next generation sequencing technologies, choice of sequencing platform, sample preparation methods, and the nature of the source RNA are increasingly important in setting the overall precision and reproducibility of the measurements allowed by this technique. Thus, these choices can significantly influence the results obtained.Measurement of expression levels for transcripts having a high number of reads is inherently more precise than measuring transcripts with fewer reads. However, when using random hexamer priming for generating cDNA, the number of reads arising from a given transcript depends not only on the number of such transcripts in a cell but also on its length because longer RNAs have more opportunities for priming (and hence more reads). Thus, the absolute number of reads is frequently further adjusted to Reads per thousand (K) nucleotides Per Million reads (RKPM). Unfortunately, this adjustment eliminates the simple relation between read count and precision. Furthermore, any length normalization is imperfect because so many annotated loci have multiple transcripts of different lengths. Length normalization is intended to fix the differences in the number of hexamer priming sites, but this is not an ideal surrogate as RNA secondary structure and GC content also play major roles in priming efficiency Furthermore, attempting to correct RNA Seq data for length increases the correlations among all pairs of datasets, not just those that should be dependent on length. As discussed in One method for avoiding overcounting of long transcripts is the use of a tagging approach in which only a single read arises from each transcript. This eliminates many of the counting issues introduced by multiple tag approaches but reduces the evenness of coverage. The DGE protocol generates a single read predominantly from the 5′ end of the transcript via selective priming from the 3′ end RNA and cDNA fragmentation are frequently used to improve evenness of coverage across the length of transcripts so splice junctions and SNPs can be more efficiently detected. However, this improved coverage comes at the expense of reduced coverage of short RNA species. Short RNA species are produced from many locations Total RNA and rRNA-depleted RNA have significantly more reads mapping to unannotated regions of the genome than does polyA RNA, suggesting they should be used as the source of RNA in transcriptome profiling if a comprehensive survey is desired. While rRNA-depleted samples still contain a significant number of rRNA reads, the fraction corresponding to non-rRNA, non-mitochondrial reads increases by several fold and allows sufficient precision to make differential expression experiments practical for most transcripts. Furthermore, we have observed that the use of a larger number of probes (30 versus 8) or other commercial kits such as RiboZero for rRNA removal significantly improves depletion (data not shown) thus enhancing the number of informative reads.Annotated sequences provide genomic bounds to which one can either map the sequence reads directly or within which one can sum all the mapped reads to generate transcript counts. Unannotated transcripts do not offer this opportunity. Computational methods have been developed to reconstruct transcript structures based on overlapping reads There are also technical limitations that force particular choices on experimenters. Most next-gen sequencing platforms require amplification just prior to sequencing and, in many cases, also amplify cDNA earlier in the protocol. These steps can introduce bias based on GC-content and length. One recent protocol eliminated cDNA amplification While it would be desirable to ignore the costs of any experimental plan and make technical choices solely based on optimizing the experimental outcome, the reality is that the cost of sequencing and analysis means that a cost-blind approach is rarely possible. Thus, each technical choice must be a trade-off between cost and quality of results. For example, for a full view of RNA expression, sequencing polyA RNA results in the loss of a tremendous number of transcripts, but, use of total RNA can yield 5–25-fold fewer non-ribosomal, non-mitochondrial reads There is no perfect set of technical choices for transcriptome analysis but, armed with the knowledge of the impact that various choices have on the outcome, experimenters can choose the best set of parameters to match their scientific needs and resources and realize the potential limitations in their analysis. RNA Seq, DGE and related methods provide valuable tools for biologists that allow detailed characterization of both the level of expression of known transcripts with unparalleled precision and the identification of novel transcripts. However, these techniques are subject to artifacts and biases, no matter what the sequencing platform or method of RNA processing. Less sensitive or comprehensive technologies have clear deficits with respect to what can be seen and not seen. The large amount of data generated by sequencing-based technologies can lull the experimenter into thinking one has a complete and accurate picture of gene expression. This is clearly not the case as technical choices affect the results. These choices and their associated caveats should be made knowingly so that conclusions can be drawn in as biologically-relevant a manner as possible while understanding the limitations of those conclusions.Total RNA was obtained from commercial sources from the following cell lines and tissues: K562 (Ambion), normal liver and normal brain (Clontech), and HL60 . The HL60 RNA was extracted from cells grown under 2 different conditions : 1. untreated, 2. treated with retinoic acid (RA) to induce differentiation (0.1%DMSO+1 µM RA for 5 days).Before further fractionation, total RNA was treated with DNase I as follows: 50 µg of total RNA was mixed with 10 µl of 10× DNase I buffer (Roche), 2 µl of RNaseOut (Invitrogen) and 8 µl of recombinant DNase I  and incubated for 45 minutes at 37°C. The RNA was then purified using the RNeasy MinElute kit (Invitrogen).The DNase I-treated total RNA was either unfractionated  or fractionated using one of the following methods: 1. Depleted of ribosomal RNA (rRNA) using the RiboMinus kit (Invitrogen) 2. polyA fraction was selected using a magnetic bead-based purification kit  or, 3. polyA fraction was selected using the oligo-dT cellulose method (Micro Poly(A)Purist Kit, Ambion). Some RNA samples were fragmented by heating at 95 C for 10 min prior to cDNA synthesis.2 were added. The samples were returned to the thermocycler and allowed to incubate at 15°C for 20 min. Then, 2.5 µl of RNaseOut and 2.5 µl of SuperScript III reverse transcriptase were added and the samples were incubated at 25°C for 10 min, 42°C for 40 min, 55°C for 50 min and 70°C for 10 min.100–400 ng of DNase I -treated RNA, except where noted, was mixed with the following reagents from the SuperScript III kit (Invitrogen). First 10 µl of 50 ng/µl Random Hexamers and 2 µl of 10 mM dNTPs were added in the total volume of 25 µl. When employing selected primers, the same conditions were used. The mixture was then placed in a thermocycler and heat denatured at 65°C for 5 min followed by rapid cooling on ice. Next, 5 µl of 10× cDNA synthesis buffer, 5 µl of 0.1 M DTT and 10 µl of 25 mM MgClAfter reverse transcription, RNA was removed by adding 1 µl of RNaseH (Invitrogen) and 1 µl of RNase If (New England BioLabs) to each sample and incubating at 37°C for 30 min. The cDNA was then purified by two rounds of purification over Performa columns (EdgeBio) and quantified using a NanoDrop spectrophotometer.2, 5 µl of 10× terminal transferase (TdT) buffer (New England BioLabs), 5 µl of 50 µM dATP and 1.5 µl of TdT  was then added and the samples were incubated at 42°C for 1 hr, and at 70°C for 10 min.Next, a 3′ poly-A tail was added to the cDNA samples. cDNA (100 ng) was mixed with a control oligo to monitor tail length and water in a total volume of 33.5 µl. The mixture was denatured at 95°C for 5 min followed by rapid cooling on ice. 5 µl of 2.5 mM CoClThe 3′ ends of the polyA-tailed cDNA were then blocked with biotin-ddATP. The sample was denatured at 95°C for 5 min followed by rapid cooling on ice. 0.3 µl of 1 mM biotin-ddATP (Perkin Elmer) and 1.5 µl of TdT were added followed by incubation at 37°C for 45 min and 70°C for 10 min.The control oligo was removed by digestion with the USER enzyme (New England BioLabs). 1 µl of the USER enzyme (1 U) was added to the sample and incubate at 37°C for 30 min.The sample was then purified using AMPure beads (Agencourt) by bringing the volume up to 60 µl with water and adding 72 µl of the AMPure beads followed by incubation at room temperature for 30 min with agitation. The beads were then captured on a magnetic stand and washed twice with 70% ethanol. The beads were then allowed to air dry for 5–7 min, resuspended in 20 µl of water and left open for 30 min on the magnet. The eluate was collected, the beads were resuspended again in 20 µl of water and left for 5 min on the magnet. The eluate was collected again and combined with the first eluate.Typically, the samples were hybridized to the HeliScope flow cell at a loading concentration of 100–350 pM.Digital Gene Expression was performed as described Figure S1Varying methods of polyA selection. polyA selection of liver RNA was carried out as described in (TIF)Click here for additional data file.Figure S2Artifactual correlations induced by length corrections. Four different liver RNA samples were compared pair-wise for correlations with varying methods of correcting for transcript length.(TIF)Click here for additional data file.Figure S3Selected versus random hexamer priming in HL60 total RNA. cDNA was synthesized from HL60 Total RNA using either random hexamers or hexamers selected to avoid cDNA synthesis from ribosomal RNA. Expression levels for transcripts with more than 5 RPM in either sample are shown on a log-log plot.(TIF)Click here for additional data file.Figure S4Fragmented versus unfragmented liver polyA. polyA RNA was prepared from liver. Prior to cDNA synthesis, RNA was fragmented by heating to 95° in MgCl2. RNA was then prepared identically and sequenced. RPM for fragmented and unfragmented RNA is shown on a log-log plot.(TIF)Click here for additional data file.Figure S5Ratio of expression for unfragmented/fragmented liver polyA RNA. Liver RNA was fragmented after polyA selection as described in (TIF)Click here for additional data file.Figure S6Spike-in expression. Spike-in RNAs were provided by Dr. Brian Willi in Dr. Barbara Wold's laboratory. These were added to a liver RNA sample and prepared as described above. Because the absolute number of molecules in the sample was known, the counts were adjusted for the known lengths and then plotted versus the known spike-in concentration. The number of transcripts per nanoliter of spike is shown on the horizontal axis and RPKM on the vertical axis. The single point below the diagonal is AGP which is only 325 nt long and represented by 3 reads and thus the least precise measurement among the spiked RNAs, reinforcing the issues with converting RPM to RPKM. Although two transcripts have lower RPKM than AGP, they are, in fact represented by more reads  because they are much longer (9786 and 1451 nt).(TIF)Click here for additional data file.Table S1Sequencing data summary. The sequence data channels used in the analyses and various figures and tables are listed with the machine used for sequencing, run date, and channel number. The figures in which each channel are included on the X or Y axis are noted as such or, if included in some other fashion, by “Yes”.(XLS)Click here for additional data file.Text S1Additional details on how sequence reads were aligned and counted, how normalization and correlations of data were carried out, and how spikes and expression differences were analyzed is provided.(DOC)Click here for additional data file.


Cas9/CRISPR has been reported to efficiently induce targeted gene disruption and homologous recombination in both prokaryotic and eukaryotic cells. Thus, we developed a Guide RNA Sequence Design Platform for the Cas9/CRISPR silencing system for model organisms. The platform is easy to use for gRNA design with input query sequences. It finds potential targets by PAM and ranks them according to factors including uniqueness, SNP, RNA secondary structure, and AT content. The platform allows users to upload and share their experimental results. In addition, most guide RNA sequences from published papers have been put into our database. Gene engineering technology has always been a hot topic in life science research. With the development of gene modification technology, certain genes can be knocked out or knocked down to a lower level. The appearance of zinc finger nuclease (ZFN) and tale nuclease (TALEN) has greatly accelerated progress in this field, but their efficiency is often unpredictable and it is difficult to target selected genes –8.Recently, Cas9/CRISPR has been reported to successfully induce targeted gene disruption and homologous recombination in both prokaryotic and eukaryotic cells with higher efficiency compared with ZFN and TALEN . AdditioHowever, there is no available tool for the guide RNA design of Cas9/CRISPR silencing system. Although Mali et al. have reported the construction of unique whole human genome guide RNA library, covering more than 40% human exons [Existed library also did not take into consideration related influencing factors, such as SNP, deletion or insertion on the genome, and potential RNA secondary structure. According to our current understanding of the gRNA maturing process, the secondary structure of gRNA is crucial for Cas9-gRNA complex . The 20 http://cas9.cbi.pku.edu.cn/), with DNA variants information integrated. This tool helps researchers design their candidate guide RNA sequences more easily and provides assistance for users to choose better candidates based on preliminary results.Thus, we developed an online platform for the guide RNA design of the Cas9/CRISPR silencing system (Both guide RNA sequences and their corresponding efficiency were manually collected from the literature and stored in our database. For designing guide RNA, we used a Java framework mainly containing 5 steps, and connecting to Tomcat web server. 20NGG sequence pattern principle, where NGG represents PAM sequence, by utilizing Java regular expression matching. In the second step, the program would put all the candidate sequences to a fasta file and run bowtie 0.12.9 to check if they could be mapped on selected model organism's genome uniquely [In the first step, the program would find any candidate sequences based on the Nuniquely . The paruniquely –18, if tuniquely . In the Multiple gene sequences are allowed for batch gRNA design and the streamline of this platform is shown in Recently, Jiang et al. report that only the first six base pairs near PAM are of great importance for recognition efficiency in bacteria . It is uPVALB, the first target sequence is 50% more efficient than the rest two, since the first has 0 SNP while the rest have 3 or 2 SNPs. The first target sequence has fewer base pairs involved in RNA secondary structure loops, allowing it to bind more with target genome, while the rest two both have 9 base pairs in loops. For the given gene AAVS1, the first target is more than twofold efficient than the other, since the other one has an off-target site in genomes. For the given gene VEGFA, the first one is about half efficient with the rest two, since it has 1 SNP while the rest have none.We conducted a validation by using those reported results in our platform on factors, such as uniqueness, SNP, and base in loops (AT content is crucial factor as those previously mentioned, since evidence is not clear. Thus, we list it here as a consideration for users.Our platform is an easy-to-use software to identify potential efficient gRNA sites within given sequences for model organisms, avoiding off-target effects and SNPs. This platform also allows users to search existing guide RNA/protospacer sequences and share their results. We have manually extracted most reported gRNA/protospacer sequences into our database for reference and will expand it with newly published work.