Here we characterized a weak mutant allele of DBR1 in Arabidopsis, dbr1-2, and showed that a global increase in lariat RNAs was unexpectedly accompanied by a genome-wide reduction in miRNA accumulation. The dbr1-2 mutation had no effects on expression of miRNA biogenesis genes or primary miRNAs (pri-miRNAs), but the association of pri-miRNAs with the DCL1/HYL1 dicing complex was impaired. Lariat RNAs were associated with the DCL1/HYL1 dicing complex in vivo and competitively inhibited the binding of HYL1 with pri-miRNA. Consistent with the impacts of lariat RNAs on miRNA biogenesis, over-expression of lariat RNAs reduced miRNA accumulation. Lariat RNAs localized in nuclear bodies, and partially co-localize with HYL1, and both DCL1 and HYL1 were mis-localized in dbr1-2. Together with our findings that nearly four hundred lariat RNAs exist in wild type plants and that these lariat RNAs also associate with the DCL1/HYL1 dicing complex in vivo, we thus propose that lariat RNAs, as decoys, inhibit miRNA processing, suggesting a hitherto unknown layer of regulation in miRNA biogenesis.Lariat RNAs formed as by-products of splicing are quickly degraded by the RNA debranching enzyme 1 (DBR1), leading to their turnover. Null  DBR1 causes embryo lethality in both animals and plants. In animals, some debranched lariat RNAs could be further processed into mirtron miRNAs, a class of nonconventional miRNAs that bypass the microprocessor for their biogenesis. However, no mirtron has been functionally validated in plants, and how the accumulation of lariat RNA in dbr1 results in embryo lethality remains unclear. Here, we show that DBR1 is necessary for the regulation of genome-wide miRNA biogenesis in plants. By investigating the correlation between lariat RNA accumulation and miRNA processing, we showed that the DBR1-mediated lariat RNA debranching process provides a safeguard role for the binding of the dicing complex with miRNA precursors. As both the DBR1-mediated lariat RNA debranching process and miRNA biogenesis are common features in higher eukaryotes, the finding that lariat RNAs sequester the dicing complex in plants may have a broad implications for the non-coding RNA field.It is known that lariat RNAs formed during pre-mRNA splicing are debranched by DBR1 (RNA debranching enzyme 1). Loss of function of  In higher eukaryotes, splicing of mRNA precursors (pre-mRNA), a critical step for gene expression, comprises two catalytic steps . In the Saccharomyces cerevisiae) in a study aimed at identifying host cellular factors involved in transposition of the Ty1 retrotransposon , a , a dcl1-arginine . When DB rescued . We idenenotypes . Therefo dbr1-2) .dbr1-2 mutant resembles mutants that are defective in miRNA accumulation, we examined miRNA accumulation in dbr1-2 by northern blot analysis. Compared to those in Col-0, the levels of tested miRNAs were reduced in dbr1-2  to determine transcript levels of genes that have been shown to act in miRNA biogenesis [dbr1-2 were comparable to those in Col-0 . Pol II is responsible for MIR transcription in animals and plants [MIR genes by chromatin immunoprecipitation (ChIP), using an antibody against the second largest subunit of Pol II (NRPB2). Compared to the no antibody control  or the MIR390b promoter (MIR390b::GUS). The transgenic lines MIR172a::GUS and MIR390b::GUS were separately crossed with dbr1-2 and progeny plants homozygous for both transgene and dbr1-2 were analyzed. Both GUS activity  experiments. We performed RIP assays, using DCL1 antibody, with Col-0 and dbr1-2 inflorescences, and using HYL1 antibody with Col-0, dbr1-2, and hyl1-2 seedlings. Pri-miRNAs were detected by qRT-PCR from immunoprecipitated RNAs. The amounts of the pri-miRNAs bound by both DCL1  from Col-0 and dbr1-2 by RNA sequencing analysis. We found that most reads were predominantly mapped to the whole region of MIR genes. However because the boundaries of most pri-miRNAs are not clear, it was not feasible to calculate the abundance of pri-miRNAs, and thus we only counted reads uniquely mapped to pre-miRNA regions. We found that pre-miRNAs were overall enriched in both DCL1- and HYL1-immunoprecipitated samples in Col-0 (dbr1-2 or Col-0 libraries), in both DCL1- and HYL1-immunoprecipitated samples, were reduced in dbr1-2 -depleted RNAs, with or without RNase R-treatments , several tested lariat RNAs naturally present in Col-0, such as lariat24a, lariat28, lariat32, and lariat40, were also bound by both DCL1 and HYL1  with Col-0 and dbr1-2, to compare the binding capacity of recombinant HYL1 -RNA) to perform the competition assays. Increasing amounts (0.3 to 1.2 μg) of cold R(+)-RNA were added to the binding reaction mixture containing HYL1 and labeled pri-miR167b. As shown in pri-miR167b complex was decreased proportionally to the amount of cold circular RNA added from dbr1-2. Unexpectedly, the addition of cold circular RNA from Col-0 in the binding reaction also produced moderate competition effects  lines of lariat RNA and assessed miRNA accumulation. Although how some lariat RNAs escape debranching in wild type plants remains unknown, we hypothesized that over-expressing the corresponding genomic DNA should lead to increased levels of intron-derived lariat RNAs. Here, we selected lariat41, which is detectable in wild type plants , to inveArabidopsis plants expressing DBR1-GFP showed that DBR1 was distributed in both the nucleus and cytoplasm  and dbr1-2 were obtained. In Col-0, more than 85% of 1168 root cells harbored three or fewer dicing bodies in the nucleus and less than 10% of root cells harbored more than three dicing bodies  is wild type. Seeds of dbr1-3 (SALK_047099), hyl1-2 (SALK_064863), and MIR390b::GUS (CS66477) were obtained from the Arabidopsis Biological Resources Center . 35S::DCL1-YFP seeds were generated in our laboratory. pHYL1::YFP-HYL1 seeds were obtained from Yuda Fang’s lab. Wild-type and dbr1 fission yeast strains were purchased from Bioneer (http://pombe.bioneer.co.kr). Anti-DCL1 , anti-HYL1 , anti-SE , anti-Hsc70 , anti-GFP , and anti-FLAG  antibodies were purchased. Anti-DBR1, anti-NRPB2, and anti-AGO1 antibodies were generated by our laboratory.DBR1 was amplified from Col-0 genomic DNA with the primer pair DBR1F1/R1, cloned into pENTR-D/TOPO, and then transferred into the plant expression destination vector pMDC107 to construct DBR1-GFP, into pMDC163 (GUS was replaced by mRFP) to construct DBR1-RFP, into pEarleyGate302 to construct DBR1-Flag, into pEarleyGate101 (HA-GFP was replaced by mRFP) to construct 35S::DBR1-RFP, and into pB7YWG2 to construct 35S::DBR1-YFP. To construct 35S::DBR1-Flag, the DBR1 CDS was obtained using the primer pair DBR1F2/R2 and cloned into the plant expression vector pCambia1306. To construct 35S::DBR1-2-YFP, the CDS sequences of DBR1 was amplified from dbr1-2 with primer pairs DBR1F2/R2 and cloned into pCambia2302. To construct DBR1-GST, the CDS sequences of DBR1 was amplified with primer pairs DBR1F3/R2 and cloned into pGEX2TK. To construct DCL1-YFP, HYL1-RFP, and HYL1-Flag, the CDS sequences of DCL1 and HYL1, respectively, were obtained using the primer pairs DCL1F1/R1 and HYL1F1/R1, cloned into pENTR-D/TOPO, and then transferred into the plant expression destination vectors pEarleyGate101, pEarleyGate101 (YFP-HA was replaced by mRFP), and pEarleyGate202, respectively. To construct SE-Flag, the CDS sequence of SE was obtained using the primer pair SEF1/R1, and cloned into the plant expression vector pCambia1306. To construct lariat41-OE, the full length genomic region of At5g37720 was amplified using primer pair lariat41F2/R2 and ligated to pENTR1A, and transferred into pB7YWG2. To construct local41-OE, the full length coding region of At5g37720 was amplified from Col-0 cDNA using primer pair lariat41F2/R2 and ligated to pENTR1A, and transferred into pB7YWG2. To construct lariat42-OE, the intron sequences of At5g43100 were amplified using primer pair lariat42F2/R2 and inserted between nYFP and cYFP, and transferred into pB7WG2. To transfer lariat24a and lariat41 into the MS2 system, we first constructed lariat24a-pENTR-D/TOPO and lariat41-pENTR1A by introducing full length genomic fragments of At5g63530 and At5g37720 into pENTR-D/TOPO and pENTR1A using the primer pairs gL24F/R and gL41F/R, respectively, and then the 6XMS2 loops were amplified with primers 6XMS2Loops_F1/R1 and 6XMS2Loops_F2/R2 from 35S::GW-6XMS2, then ligated into lariat24a-pENTR-D/TOPO by XmnI and lariat41-pENTR1A by SacI, respectively, then transferred into the plant expression vector pB7WG2. To construct the mRNA control of At5g63530 into the MS2 system , the plasmid of lariat24a-pENTR-D/TOPO were introduced into 35S::GW-6XMS2 by LR reaction. To construct pri-miR163 into the MS2 system, we first constructed pri-miR163-pENTR-D/TOPO by introducing full length genomic fragments of MIR163 into pENTR-D/TOPO with the primer pair miR163-MS2-F/R, and then transferred into the plant expression vector 35S::GW-6XMS2. Primer sequences are listed in For construction of the DBR1-GFP, DBR1-RFP, DBR1-Flag, 35S::DBR1-RFP, and 35S::DBR1-YFP plasmids containing the endogenous promoter, A. thaliana reference genome using SOAP2 [http://www.mirbase.org/, v19) were found using BLASTN. Then, custom programs were used to calculate the normalized abundance  of mature miRNAs. miRNAs with at least 100 RPTM in either Col-0 or dbr1-2 were used to calculate log2 fold changes of their RPTM values. Deep sequencing datasets were deposited in the SRA database of National Center for Biotechnology Information with accession No. SRP062035.Illumina sequencing of small RNA libraries from inflorescences was performed by Genergy , and small RNA libraries were constructed according to Illumina's instructions using a TruSeq Small RNA Library Preparation kit. The small RNA sequencing profiles were analyzed as described . Brieflyng SOAP2 ; mature Total RNA was extracted using Trizol reagent from inflorescences. 5 μg small RNAs enriched by PEG8000 were separated by denaturing 15% (w/v) PAGE and transferred to a nylon membrane. 5’_biotin_labeled-oligo nucleotide sequences complementary to miRNA were synthesized as probes. Hybridization was performed using hybridization buffer (Ambion), and signals were detected using chemiluminescent nucleic acid detection module. U6 was used as a loading control. The probes are listed in pri-miR167b was synthesized by in vitro transcription. Using an RNA EMSA Kit (Thermo Scientific), electrophoresis mobility shift assay experiments were performed in a 20 μl reaction system containing binding buffer, 5% (v/v) glycerol, 2 μg tRNA, 2 nM of biotin-labeled pri-miR67b transcripts, and purified MBP-HYL1_D1D2 (only two DsRBD domains). For the competition assay, total RNA treated by RNase R was added to the reaction at different concentrations. The reaction was incubated for 30 min, electrophoresed on a 6.5% (w/v) native PAGE gel, and transferred to a nylon membrane, then detected using a chemiluminescent nucleic acid detection module.R-EMSA was performed according to  with moddbr1-2 was immunoprecipitated with anti-DCL1 and anti-HYL1 antibodies. For the DBR1 RIP assay, chromatin from inflorescences of Col-0 was immunoprecipitated with anti-DBR1 antibodies. RNA recovered from the immunoprecipitates was used for cDNA synthesis with Oligo dT. The primer sets used for PCR are listed in the Chromatin from inflorescences of Col-0 and dbr1-2, respectively. Immunoprecipitated DNA was quantified by qPCR relative to total input DNA. The results shown were consistent in three biological replicates. The primer sets used for the PCR are listed in ChIP was performed similar to RIP, the difference is that DNA not RNA is recovered. Pol II occupancy at miRNA loci was determined by ChIP using anti-RPB2 antibody and 2-week-old seedlings from Col-0 and dbr1-2 backgrounds was fixed in 90% acetone for 2–3 h and then stained for 12 h in 50 mM sodium phosphate buffer, pH 7.0, containing 0.2% Triton X-100, 5 mM potassium ferrocyanide, 5mM potassium ferricyanide, and 1 mM X-Gluc, then washed in 70% ethanol three times. Images were taken with a Leica DFC295 stereoscope.Three week-old seedlings from transgenic plants in Col-0 and dbr1-2 was first treated with a Ribo Zero kit (Epicenter) to obtain a ribosomal RNA-depleted RNA (ribo- RNA). Then ribo- RNA was incubated with or without RNase R (Epicentre) and then subjected to phenol:chloroform purification. Purified RNAs were further used for library preparation with Illumina TruSeq Stranded Total RNA Sample Prep Kit, and then subjected to deep sequencing with Illumina HiSeq 2000 at Genergy . Stranded RNA-seq reads were mapped to the Arabidopsis genome annotation (TAIR 10) with Cufflinks 2 [dbr1-2 or Col-0 and q-values smaller than 0.05 were designated deregulated genes. The "bedtools genomecov" command of bedtools [dbr1-2 profiles and had multiple test corrected P-values of smaller than 0.05, calculated with edgeR [Total RNA isolated from Col-0 and flinks 2 . CuffDifbedtools  was usedth edgeR , were deLariat RNAs across the branch site were detected by RT-PCR as described . Total RThe corresponding plasmids of DBR1-pGEX2TK and HYL1 (D1D2)-pMAL were introduced into the expression host BL21-CodonPlus (DE3). Cells was grown to an OD600 of 0.4–0.6, and expression of GST or MBP fused proteins were induced by addition of 0.1 mM IPTG and incubation at 16°C for overnight. Cells carrying the pGEX2TK or pMAL empty vectors were also grown and induced as controls. Cell pellets were resuspended in lysis buffer and purified according to the recommended protocols of Glutathione Sepharose4B resin (for GST) or the amylose resin (for MBP), respectively.N. benthamiana. Leaves were ground in liquid nitrogen and homogenized in lysis buffer (50 mM Tris_HCl (pH 8.0), 150 mM NaCl, 0.2% Nonidet P-40, 2 mM DTT, 5% glycerol, proteinase inhibitor) and centrifuged for 15 min at 13200 rpm. The lysate was incubated with GFP-Trap agarose beads (Chromotek) for 2 h. The immune complexes were then washed with lysis buffer. Proteins retained on the beads were resolved on SDS-PAGE. Anti-GFP and anti-Flag antibodies were used to detect DCL1, and DBR1, HYL1 or SE, respectively, by using western blot analysis.35S::DCL1-YFP with 35S::DBR1-Flag, 35S::HYL1-Flag, and 35S::SE-Flag, respectively, were co-expressed in The data reported in this paper have been deposited in the SRA database of National Center for Biotechnology Information with accession No. SRP062035 for small RNA seq and RNA-seq, and accession No. SRP063916 for RIP-seq.S1 FigMus musculus, NP_113580.2), Human , Fish , Plant , Fly , Worm , and fission yeast , were aligned using ClustalW. Identical residues are highlighted in yellow. Asterisk indicates the Gly that was mutated to Arg (G135R) in dbr1-2. (B) The protein structure of DBR1. The closed triangle indicates the T-DNA insertion site in dbr1-1 and dbr1-3. The arrow indicates the location of the mutation of glycine to arginine in dbr1-2. MPE indicates the metallophosphoesterase domain, LRL indicates the lariat recognition loop, and CTD indicates a novel C-terminal domain. (C) Allelic analysis of dbr1-2 and dbr1-3. A small field was shown for 3-week-old growing F1 progeny, and dbr1-2-like plants are highlighted by red circles. (D) Western blot to determine DBR1 levels in wild type (Col-0) and dbr1-2 using anti-DBR1 antibody. # indicates the endogenous DBR1 in Col-0 and dbr1-2, and * indicates DBR1-Flag and DBR1-GFP in 35S::DBR1-Flag or 35S::DBR1-GFP transgenic plants, respectively. Notably, the loading amount in Col-0 and dbr1-2 is half of that of two transgenic plants. Hsc70 served as a loading control.(A) Partial amino acid sequence alignment of DBR1 from various species. DBR1 from Mouse ((TIF)Click here for additional data file.S2 Figdbr1-2. Hsc70 was the loading control. (C) and (D) ChIP performed with Col-0 (dark gray bars) or dbr1-2 (light gray bars) with anti-NRPB2 antibody. SE was calculated from three biological replicates. (E) Representative GUS staining images of MIR172a::GUS and MIR390b::GUS transgenic plants in Col-0 and dbr1-2 backgrounds. (F) RT-qPCR analysis of the transcript levels of GUS, pri-miR172a, and pri-miR390b in the indicated plants. SE was calculated from three biological replicates.(A) RT-qPCR analysis of mRNA levels of miRNA biogenesis genes. SE was calculated from three biological replicates. (B) Western blot to determine levels of DCL1, HYL1, SE, AGO1, and NRPB2 in Col-0 and (TIF)Click here for additional data file.S3 Figdbr1-2 inflorescences using anti-DBR1 antibody (light gray bars) or no antibody (dark gray bars) as the negative control. RNA was immunoprecipitated from inflorescences of dbr1-2. The amount of pri-miRNAs was determined by RT-qPCR and normalized to the input. UBQ5 was used as a negative control. Lariat RNAs served as positive controls. Error bars show SE calculated from three biological replicates. (B) Co-immunoprecipitation between DCL1 and DBR1, SE, and HYL1. DCL1-YFP was co-expressed with DBR1-Flag, SE-Flag, or HYL1-Flag in N. benthamiana. HYL1-Flag and SE-Flag were used as positive controls. (C) Schematic flow of RNA-seq analysis. 5 μg total RNA were digested by Dnase I, and then RNA were removed ribosomal RNA using Ribo-Zero kit. The half amount of RNAs were treated by RNase R as R(+) RNA to remove linear RNAs, the other half amount of RNAs were used as R(-) RNA. Those RNA fragments with size larger than 100 nt were used for library construction and sequencing. (D) Chromas figures of partial sequences from lariat7 and lariat24a by Sanger sequencing. PCR products of lariat7 and lariat24a from (A) RIP performed with (TIF)Click here for additional data file.S4 Fig(A) Recombinant proteins of MBP, MBP-HYL1 , GST, GST-DBR1. (B) R-EMSA to determine if above recombinant proteins binding Biotin-labeled pri-miR167b. The arrow indicates the HYL1-pri-miR167b complex. (C) R-EMSA to determine if linear RNAs compete for HYL1 binding to pri-miR167b. Cold GAPDH single-stranded linear RNA transcribed by in vitro transcription using the T7 promoter was gradually increased in the reaction, and cold pri-miR167b with corresponding concentrations was used as the positive control. The arrow indicates the HYL1-pri-miR167b complex. (D) R-EMSA to determine if circular RNAs compete for HYL1 binding, using wild type or a dbr1 yeast strain. Experiments were performed as dbr1 yeast cells. The arrow indicates the HYL1-pri-miR167b complex. (E) Hybridization intensities were quantified and normalized to the controls (lane 1 in D), and are shown in the line graph. Bars represent the average normalized intensity of three biological replicates.(TIF)Click here for additional data file.S5 Fig(A) Strategy to over-express lariat42 in tobacco leaves. The intron sequence (indicated as lariat42-OE) was inserted into the split YFP and transiently expressed in tobacco leaves. (B) YFP signals were shown in tobacco leaves infiltrated by the above plasmid. Bright dots indicate YFP signals in nuclei. (C) RT-PCR analysis to detect the expression of lariat42. Total RNA from tobacco leaves infiltrated by the above plasmid or the blank control was used for cDNA synthesis, and lariat42 was amplified to indicate the level of lariat42, Actin7 as the loading control. (D) Northern blot analysis of miR167 in the control and lariat42-OE. U6 was used as a loading control.(TIF)Click here for additional data file.S6 FigN. benthamiana leaves. Representative images were shown. (D-E) Three additional representative images of lariat24a (D) and two additional representative images of lariat41 (E) were shown. Scale bars = 10 μm.(A) Subcellular localization of DBR1 and DBR1-2. 35S::DBR1-YFP and 35S::DBR1-2-YFP were transiently expressed in tobacco leaves and fluorescence signals were observed after 48 hr. (B) Strategy to visualize lariat RNAs in live cells. The MS2 sequence (indicated as stem-loops) was inserted into the lariat41-located intron. A co-expressed GFP-tagged MS2-CP protein was used to visualize lariat41. Grey boxes indicate exons, and lines indicate the intron. (C) HYL1-RFP alone or MS2-CP-GFP and local24-MS2 were transiently expressed in (TIF)Click here for additional data file.S1 Tabledbr1-2/Col-0, from two biological replicates. The first sheet lists 57 miRNAs that were significantly reduced in the replicate 1 of dbr1-2, the second sheet lists 56 miRNAs that were significantly reduced in the replicate 2 of dbr1-2, the third sheet lists 54 miRNAs that were commonly reduced in the two replicates of dbr1-2, the fourth sheet lists the information of genome structure of 54 commonly reduced miRNAs in dbr1-2, and the fifth sheet lists miRNAs co-regulated by both DBR1 and HYL1.More than 100 miRNAs with more than 100 RPTM were identified in small RNA sequencing libraries, and are listed with information of their ID, normalized reads , and log2 ratio of (XLSX)Click here for additional data file.S2 Tabledbr1-2 mutant. The genomic location, strand information, binding capacity (RPKM), and log2 ratios of dbr1-2/Col-0 from DCL1 and HYL1 RIP-seq were listed in the first and second sheets of the table.There were 55 and 48 miRNA genes with log2 fold changes of smaller than -1 or larger than 1 identified as deregulated in binding to DCL1 and HYL1, respectively. Most of them, i.e. 47 in the DCL1- and 29 in the HYL1- RIP-seq profiles, had less occupancy of DCL1 and HYL1 in the (XLSX)Click here for additional data file.S3 Tabledbr1-2 in RNase R (-) libraries, the second sheet lists the 1534 intron RNAs whose average abundance in dbr1-2_RNase R (+) libraries are ≥ 5 from 1560 introns, and the third sheet lists the 360 introns whose average abundance values in Col_RNase R (-) libraries are ≥ 5 RPKM from the 1534 introns on the second sheet.The genomic location, strand information, intron ID, and normalized abundance (RPKM) in different RNA-seq libraries, log fold change (logFC), log count per million sequencing reads (logCPM), P-value and False Discovery Rate (FDR) are listed. The intron ID is named according to the accession ID of the corresponding gene, followed by "I" and the intron sequential number. The first sheet lists all 1560 intron RNAs that have significantly different expression levels between Col-0 and (XLSX)Click here for additional data file.S4 Table(DOC)Click here for additional data file.


The genome of hepatitis delta virus (HDV) is a 1.7-kb single-stranded circular RNA that folds into an unbranched rod-like structure and has ribozyme activity. HDV redirects host RNA polymerase(s) (RNAP) to perform viral RNA-directed RNA transcription. RNA recombination is known to contribute to the genetic heterogeneity of HDV, but its molecular mechanism is poorly understood. Here, we established a whole-genome HDV-1/HDV-4 recombination map using two cloned sequences coexisting in cultured cells. Our functional analyses of the resulting chimeric delta antigens  and recombinant genomes provide insights into how recombination promotes the genotypic and phenotypic diversity of HDV. Our examination of crossover distribution and subsequent mutagenesis analyses demonstrated that ribozyme activity on HDV genome, which is required for viral replication, also contributes to the generation of an inter-clade junction. These data provide circumstantial evidence supporting our contention that HDV RNA recombination occurs via a replication-dependent mechanism. Furthermore, we identify an intrinsic asymmetric bulge on the HDV genome, which appears to promote recombination events in the vicinity. We therefore propose a mammalian RNAP-driven and viral-RNA-structure-promoted template-switching mechanism for HDV genetic recombination. The present findings improve our understanding of the capacities of the host RNAP beyond typical DNA-directed transcription. In vivo, HDV RNA replication is considered to involve a double rolling-circle mechanism in which the genomic and antigenomic RNAs are synthesized as multimers and can be processed to unit-length circular RNAs  and pSVL-D2IIb using primer pairs 18/55’ and 88/78, respectively. The overlapping PCR fragments were joined by OE-PCR using primer pair 77/78. The resulting PCR product was gel-purified and cloned into pCR3.1. The generated plasmid was subjected to sequencing confirmation of the correct sequence and proper orientation, and designated pCR3.1- Sm4125/1471. Similar OE-PCR strategy was also used to generate chimeric HDAgs Sm-1101/1164 and Sm-4101/1161, which had a crossover mapped to nt 1255-1274, and control Sm-148/544 and Sm-448/541, which had a junction that was not identified in the inter-clade recombination map. The utilized templates and primer pairs are summarized in Six plasmids encoding HDV-1/HDV-4 chimeric S-HDAgs were constructed. The S-HDAg ORFs contained in pSVL-Drs 77/78 , and theIn vitro-transcribed monomeric HDV-1 RNAs were used as the hybridization probes for NB. A previous study showed that the HDV-4 RNA could be identified with an HDV-1 RNA probe under the utilized hybridization and washing conditions [COS7 monkey kidney cells were plated in six-well (35-mm-diameter) dishes and transfected with 4 μg of DNA per well, using the Lipofectamine reagent (Invitrogen), as described elsewhere . At 6 danditions . All expin vitro transcription. COS7-SmT1 cells were co-transfected with in vitro-transcribed HDV-1 GA and HDV-4 genomic RNA [The expression vector, pECE-GA, which was kindly provided by Dr. Michael Lai , consists of a dimer of HDV-1 cDNA carrying a mutation 3’ of the genomic self-cleavage site . The Pstomic RNA , and theomic RNA , 53. Theomic RNA . The for2M1, -D2d1, -D2d3, and -D2M2, as appropriate. Similarly, pCR3.1-D2IIb was replicating HDV constructs that expressed the genomic dimer RNA of WT HDV-4. Our results revealed that all of the mutants were replication-competent, albeit with different replication abilities (2IIb (expressing HDV-4 genome), and RNA samples were harvested at 6 days post-transfection. A constant amount of the HDV-4 genome expression plasmid was co-transfected with varied amounts of the HDV-1 mutants to achieve similar band intensities between the parental sequences, as judged by RT-PCR-XhoI-RFLP of the region amplified by primers 18/55’ (nt 886-1308) [A C-to-T base substitution at nt 1169, which disrupted the recombination junction spanning nt 1157-1205 into two smaller domains, was introduced into the HDV-1 cDNA using a single-step, single-primer PCR-based mutagenic technique . pG4Z-D1bilities . These H86-1308) . pCR3.1


We discuss how this assay can be used with short synthetic RNAs, as well as longer RNA substrates generated using in vitro transcription. Importantly, quantitation of the reactions allows detailed analyses of deadenylation in the presence and absence of accessory factors, leading to new insights into targeted mRNA decay.Poly(A) tails are found at the 3′ end of almost every eukaryotic mRNA and are important for the stability of mRNAs and their translation into proteins. Thus, removal of the poly(A) tail, a process called deadenylation, is critical for regulation of gene expression. Most deadenylation enzymes are components of large multi-protein complexes. Here, we describe an  Removal of the poly(A) tail from an mRNA, a process called deadenylation, is a critical and conserved process in eukaryotic mRNA decay The poly(A) tail is shortened in the 3′ to 5′ direction by the activity of adenosine-selective exonuclease enzymes. Several proteins have been identified that catalyze this reaction. The most highly conserved are Ccr4/CNOT6, Caf1/CNOT7, and Pan2 in vivo. Finally, the proteins involved in mRNA decay often play multiple roles in this process. For example, Ccr4-Not and its accessory factors can inhibit gene expression by catalyzing deadenylation, recruiting decapping stimulators Given the central role of deadenylation in post-transcriptional regulation of gene expression, factors that control its rate are of great importance to many biological processes. For example, the control of the cell cycle, embryogenesis and the inflammatory response are known to depend on targeted deadenylation activity in vitro system reconstituted from purified factors. Previously, the activities of individual nuclease enzymes of Ccr4-Not have been examined For these reasons, it is advantageous to directly characterize deadenylation using an in vitro analyses. The larger quantities obtained from a recombinant system permit more stringent purification, which minimizes co-purification with contaminating RNases and endogenous factors that may regulate the deadenylation activity. Furthermore, we have found that the constituent subunits of recombinant protein complexes are purified in stoichiometric ratios that likely match the physiological compositions We recently reported methods for the purification of recombinant Pan2-Pan3 in vitro. By examining reactions performed with polyadenylated RNA substrates of uniform length, the sequential removal of adenosine nucleotides can be visualized at high resolution by denaturing polyacrylamide gel electrophoresis. This allows quantitation of the reaction rate. We provide a detailed rationale for the experimental setup, as well as key considerations necessary to obtain reliable and accurate results. While the deadenylation activity of Ccr4-Not is shown here as an example, the techniques described could aid in high-resolution analysis of the RNase or DNase activities of a variety of other enzymes.Here, we describe a method for analyzing the deadenylation activities of the Ccr4-Not and Pan2-Pan3 deadenylation complexes 2in vitro  tail, substrates containing ∼20 non-poly(A) nucleotides upstream of 10–30 contiguous adenosines have been used 20A10-30 can be commercially synthesized to reconstitute and characterize targeted deadenylation in vitro. For example, by adding a specific mRNA destabilizing element  to a short synthetic RNA with an A30 tail, we could reconstitute targeted deadenylation by Ccr4-Not with the adaptor protein Mmi1 Furthermore, sequence motifs can be incorporated into the upstream non-poly(A) sequence. RNA-binding proteins that regulate deadenylation typically bind to short sequence motifs 4–10 nucleotides in length. The sequences bound by many of these proteins have been defined 3.2S. cerevisiae and 200–250 adenosines in higher eukaryotes in vitro assay with long poly(A) tails may therefore better resemble the entire deadenylation process that occurs in the cell. In addition, sequences that regulate deadenylation in cis, such as motifs recognized by Pumilio proteins, are often distributed throughout 3′ UTRs, and can be separated from the start of the poly(A) tail by hundreds of nucleotides While short model substrates provide practical benefits, the use of longer substrates more accurately mimics native mRNAs. Transcripts are exported from the nucleus with poly(A) tails of 70–80 adenosines in in vitro transcription reactions using T7 RNA polymerase and a DNA template encoding the desired RNA sequence 1.Poly(A) polymerase enzymes are commercially available, and can be used to add a non-templated poly(A) tail to RNA 2.in vitro transcription using an encoded downstream ribozyme. While the more commonly used hammerhead ribozyme has a requirement for upstream sequence features that prevents its use in preparing terminal poly(A), the HDV ribozyme does not A defined RNA 3′ end can be generated after 3.in vitro transcription. First, a DNA plasmid containing a T7 RNA polymerase promoter and an encoded poly(A) sequence is made (We use DNA templates terminating in an encoded 3′ poly(A) sequence generated from a plasmid treated with a Type IIS restriction enzyme (such as BsaI), which cleaves outside an asymmetric recognition site. This strategy yields RNAs with poly(A) tails of uniform length by  is made . A restr is made . SimilarLong RNAs are prepared by Once the transcript is synthesized, it should be purified. This is best carried out by separating the desired transcript from other contaminating nucleic acids on a denaturing polyacrylamide gel, excising the band containing the desired transcript, and eluting the RNA, e.g. by electroelution.in vitro transcription. This heterogeneity in 3′ ends results in asynchronous reactions that are difficult to quantitate. A second major limitation of using long RNAs is their reduced resolution on denaturing gels: while shorter RNAs (≤50 nt) can be analyzed at single nucleotide resolution, longer RNAs require longer gels, lower percentage acrylamide and increased electrophoresis durations. All of these factors reduce the resolution that can be achieved. Thirdly, long UTRs have an increased propensity to become structured. Re-folding techniques such as heat treatment may be required to mimic structures within natural mRNAs, remove unwanted secondary structures, and achieve conformational homogeneity While longer RNAs produced in this manner may be necessary for investigating certain processes, there are several limitations to their use. Firstly, run-off transcription reactions yield RNAs with heterogeneous 3′ ends, including truncated products with shorter poly(A) tails and RNAs containing non-templated terminal nucleotides 3.3The method of RNA visualization is another important consideration in the design of substrates for deadenylation reactions. Fluorophores can be incorporated at the 5′ end of short chemically synthesized RNAs. 5-carboxyfluorescein (5-FAM) or 6-carboxyfluorescein (6-FAM) are often used, and are available from many commercial suppliers. 3′ end-labeling or internal-labeling with fluorophores may interfere with the deadenylation reaction, and so should be avoided.in vitro transcription can be 5′ end-labelled with radioactive ATP(32P) using T4 polynucleotide kinase RNAs generated by 44.11.A buffer to maintain the pH between 6.5 and 8.5.2.Monovalent salts. We have found that the activity of the Ccr4-Not complex is highly sensitive to salt concentration  are dependent on the presence of divalent cations, typically included at 1–2 mM as magnesium acetate or chloride salt 4.Reducing agents such as DTT and TCEP – typically at a concentration of 0.1–1 mM.An optimal deadenylation reaction should include:Other potential additives include detergents , spermidine, BSA, and glycerol 4.2in vitro deadenylation reactions with recombinant Caf1 or Ccr4 The concentration of enzymes, and their abundance relative to that of the RNA substrate, must also be considered. We found that in the absence of additional factors, it takes approximately 30 min for Ccr4-Not (100 nM) to fully deadenylate A30 RNA (200 nM) in our chosen buffer conditions  at saturating RNA concentrations would be better suited for measurements of kinetic parameters.The assays described here do not use a large excess of substrate over enzyme which would be required for determination of V4.3The absolute quantity of RNA used in the reaction depends on the method of visualization. In general, 0.8 pmol of synthetic RNA labelled with 6-FAM provides adequate signal to measure the intensity of each RNA product band  or fluorescence polarization. It may also be informative to perform a series of deadenylation reactions with varying concentrations of the accessory factor.The sensitivity of the deadenylation rate to the concentration of monovalent salts makes it imperative that controls are performed appropriately, i.e. by adding an equal volume of storage buffer to negative control reactions. A difference in salt concentration as small as 10 mM could produce a change large enough to obscure the effect of the accessory factor  tail with up to three Pab1 molecules on each A80 tail Poly(A)-binding protein (Pab1/PABPC) binds to poly(A) RNA with high affinity 51.2+ to prevent metal-dependent hydrolysis. Heating the RNA-buffer mixture to 95 °C and cooling it rapidly on ice may be required to eliminate unwanted RNA secondary structure. Following cooling, the magnesium salt can be added to the desired concentration. An aliquot should be collected at this point to be run in the first lane of the gel. This serves as a control for the initial integrity of the RNA sample at time zero.Mix the RNA with buffer solution at the desired concentration (e.g. 200 nM), but in the absence of Mg2.If additional factors such as Pab1 are to be tested, we recommend adding these to the RNA and allowing the binding equilibrium to be reached before the start of the reaction.3.The reaction is initiated by the addition of enzyme at the desired concentration. We normally add 5 μl of Ccr4-Not at 1 μM to 45 μl RNA solution to obtain a final enzyme concentration of 100 nM. The reaction must be mixed thoroughly. Incomplete mixing can lead to high local concentrations of enzyme, and result in the rapid deadenylation of a subset of the RNA. This could be incorrectly interpreted as processive deadenylation. The reaction rate is temperature-dependent, and incubation is typically performed at 20–37 °C.4.Aliquots of the reaction (e.g. 4 μl) are collected at a series of time points. By adding these to a solution of denaturing loading dye , the reaction is stopped. We typically use 12 time points over 1–2 h to obtain enough data points for accurate quantification of deadenylation. Once added to loading dye, the samples can be briefly heated  to ensure complete protein denaturation and elimination of RNA structure. Samples can be analyzed immediately by gel electrophoresis  are needed to achieve single-nucleotide resolution for longer RNAs, greater than 50 nucleotides. Deadenylation of a substrate with an A60 tail is shown here as an example  final concentration) and TEMED (0.5 μl/ml of gel solution). Due to safety considerations, we recommend the use of commercially-available pre-made acrylamide solutions rather than making solutions from solid acrylamide. After polymerization, remove the comb, and wash all wells thoroughly with TBE running buffer using a syringe. This should be repeated immediately before samples are loaded for maximum resolution and to avoid streaking of the RNA caused by excess urea and unpolymerized acrylamide.The gel can be pre-run for 20 min prior to loading, although this isn’t always required for short unstructured RNAs. Load the gel and include appropriate size markers. We prepare a control RNA lacking a poly(A) tail to define the anticipated end-point of the reaction. A size marker can be generated by alkaline hydrolysis of the substrate, which has the advantage of providing single-nucleotide resolution Next, the gel is imaged. If the RNA substrate was labelled with a fluorophore, it can be scanned directly (e.g. using a Typhoon imager [GE]) after electrophoresis with fluorescence laser excitation at the appropriate wavelengths (for 6-FAM this is 470–490 nm). Gels containing RNA that is not labelled can be stained with SYBR Green II according to the manufacturers’ instructions. We find that it is important to wash the gels 5–10 times with 1 × TBE buffer after staining to minimize the background signal. Gels containing radiolabeled RNA are exposed to a PhosphorImager screen.To maximize signal and avoid visible air bubbles, gels should be removed from the glass plates used for electrophoresis. Select a gain value that does not result in saturated pixels. If necessary, a neutral density filter can be used to dampen the signal and ensure detection is within the linear range of the detector. A complete absence of bands upon scanning indicates that too little RNA has been loaded on the gel, or the staining/phosphorimaging protocol has not been performed correctly. If RNA is only visible in the sample collected before addition of the enzyme, the enzyme may contain RNase contaminants that have digested the non-poly(A) portion of the RNA substrate. An RNA ladder can ensure that the separation range and gel running parameters are appropriate.7In some cases, resolving the products of deadenylation at high resolution is not necessary: reaction rates can be estimated from the change in the poly(A) tail length without complete separation of RNA fragments if appropriate size markers are included on the gel D [31]. NFor reactions that proceed linearly, with no substantial changes in rate based on the length of the poly(A) tail, the average rate of the reaction can be obtained by plotting the size of the most abundant RNA species. We refer to this as the modal poly(A) tail length. To determine this, a densitometric profile of each lane of the gel is generated using the ‘Gels’ tool in ImageJ A. If theImportantly, the migration position of bands in electrophoretic gels do not linearly correlate with RNA size. While not essential to the calculation of the deadenylation rate, a plot can be generated that adjusts for this, and represents poly(A) tail length in a linear fashion B. To do in vitro deadenylation reactions are highly reproducible, and the standard deviation of reaction rates calculated from triplicate experiments is typically low.Once the modal poly(A) tail length has been calculated for each time point, a plot can be generated for the length of the poly(A) tail with respect to time C. If appWhile plotting the modal poly(A) tail length works well for reactions that are distributive, it should not be used for highly processive reactions where the enzyme undergoes multiple turnover events without dissociating from the RNA. In this case, fully deadenylated RNA accumulates while RNA with an intact poly(A) tail is still present in the reaction. As a result, RNAs with intermediate tail lengths are not observed and deadenylation reactions can be analyzed by calculating the rate at which fully deadenylated RNA accumulates. First, invert the contrast to display white bands on a black background using ImageJ. Draw a box around the area of the gel that includes completely deadenylated RNA. It is important that this also includes all bands that correspond to RNA fragments generated by digestion of the non-poly(A) sequence, as shown in Differentiating between distributive versus processive enzymatic activity is only valid under conditions when the substrate is saturating with respect to the enzyme. To achieve this, the substrate concentration must be significantly greater than enzyme concentration in the reaction mix.t-test can be applied to test for statistical significance. These statistical tests should be used to compare a particular reaction condition with a negative control, e.g. a reaction where the accessory factor is added should be compared to a reaction where that factor is omitted (and buffer is added instead).To establish the statistical significance of assays, each should be performed at least in triplicate in order to determine the error related to the experiment, such as electrophoretic artifacts and the low signal to noise ratio in densitometric analyses. For each data point, error bars indicating standard deviation should be plotted. If a linear fit is applied, R-squared values for the best fit straight line can be provided. Residuals can also be plotted to ensure that a linear fit is justified. Finally, when comparing deadenylase activity under different conditions, such as in the presence of accessory factors, standard statistical analyses such as the two-sample 8in vitro deadenylation catalyzed by the Ccr4-Not and Pan2-Pan3 complexes depends on pH, magnesium concentration, salt concentration, and temperature. It has previously been reported that 0.1 mM magnesium yields highest activity for isolated Ccr4 from S. cerevisiaeH. sapiens ortholog CNOT6L The rate of 20A30 RNA  vs non-A upstream ‘UTR’ sequences. Other methods could be valuable for evaluating the kinetics of deadenylation, e.g. colorimetric detection of AMP release or FRET-based detection In this report, we describe a PAGE-based method for characterizing deadenylation activity in vitro system could also aid in the analysis of the activities of other RNase or DNase enzymes.A growing number of protein factors and RNA features have been implicated in the control of deadenylation, making validation of these findings and exploration of the mechanisms by which they occur of increasing importance. Our reaction setup allows the rigorous analysis of deadenylation systems with high temporal and nucleotide resolution. In the future, this


Previously, analyses of RNA secondary structures suggested that some RNAs resembling ancestral (t)RNAs formed recently de novo, other parasitic sequences cluster with rRNAs. Here positive control analyses of additional RNA secondary structures confirm ancestral and de novo statuses of RNA grouped according to secondary structure. Viroids with branched stems resemble de novo RNAs, rod-shaped viroids resemble rRNA secondary structures, independently of GC contents. 5′ UTR leading regions of West Nile and Dengue flavivirid viruses resemble de novo and rRNA structures, respectively. An RNA homologous with Megavirus, Dengue and West Nile genomes, copperhead snake microsatellites and levant cotton repeats, not templated by Mimivirus' genome, persists throughout Mimivirus' infection. Its secondary structure clusters with candidate de novo RNAs. The saltatory phyletic distribution and secondary structure of Mimivirus' peculiar RNA suggest occasional template-free polymerization of this sequence, rather than noncanonical transcriptions .We examine the hypothesis that  Diverse numbers of simple organic compounds spontaneously self-organized at life's origins. This system crystallized around the ribonucleic-protein system forming the main organizational building blocks of known organisms Szostak, . Then prOther molecules  presumably subsisted mainly as parasites and occasionally contributing new, sometimes functional parts. Persistence of the cooperative system implies integrating new molecules with new functions, while channeling most resources to critical components such as ribosomes. Nowadays, ribosomes compete with parasitic elements that hijack the cell's integrated cooperative system . Correlation analyses plot each of the four variables 1–4 of Y for one of the new RNAs analyzed here as a function of corresponding variables 1–4 of X for each of previously analyzed RNAs  have P < 0.05. There are 24 × 13 = 312 comparisons with D1; only two (0.64%) have P < 0.05. No comparison between the 24 simulation-generated RNAs with D2 has P < 0.05. Hence our interpretation of B1 as representing secondary structures formed by ancestral RNAs agrees with the independent approach developed by Demongeot and Moreira . This result is statistically significant also after considering multiple testing, using the Benjamini-Hochberg correction for false discovery rates that accounts for the number of tests done , have P < 0.05.Evidence for recent vs. precellular origins of viroids are equivocal. Potentially, results of previous classifications of viroid secondary structures might be biased by including in analyses viroids with high GC contents and forming complex branching secondary structures. Analyses here include ten viroids forming rod-like secondary structures with GC contents ranging from 35 to 61%. All cluster with D1 and D2  with P < 0.05]. The 10 submarine MITE sequences integrated in Pandoravirus' protein coding genes cluster slightly more frequently with D2 than D1, as compared to the remaining submarine MITEs . Hence most Pandoravirus submarine MITEs cluster with D2 , resembling RNAs from the main tRNA-rRNA axis. Under this scenario, similarities with D1 (characterized by retroviruses and 23S rRNA subunits) would result from chance or secondary convergences. However, this interpretation does not account for additional lateral transfers: many genes of giant viruses originate from lateral transfers, mainly from bacteria sharing their habitat in their amoeban host  and aligned these with Acanthamoeba polyphaga Mimivirus' complete genome (NC_014649). The amoeba's mitogenome includes four rRNA genes—5S, 16S, and 18S rRNAs—and a 23S-like sequence . Most Pandoravirus MITE sequences resemble D2, characterized by 16S rRNA  and some Mimivirus sequences resembling mitochondrial rRNAs cluster within D2 . Overall results indicate the hypothesized link between viral RNAs and rRNAs.Analysis of conserved secondary structures formed by 3′ and 5′ RNA structures in four flavivirid viruses  includes numerous short RNAs that are not homologous to Mimivirus' genome. Here we focus on a 42-nucleotide-long sequence (5′-GAGACACGCAACAGGGGATAGGCAAGGCACACAGGGGATAGG-3′) because this sequence also occurs according to Blastn in diverse taxa: Megavirus, Dengue and West Nile genomes, copperhead snake microsatellites and levant cotton repeats. This Mimivirus RNA matches the giant virus Megavirus terra1  and some  genomes of Dengue and West Nile viruses (both are Flaviviridae). In these Flaviviridae, the sequence is inserted in (or close to) the 5′ UTR leading region. The e value of alignments between Mimivirus' RNA and the flavivirid 5′ UTR sequences is 2 × 10−11. This RNA is detected by Blastn  in Mimivirus' transcriptome throughout amoeban infection: −15, 0, 90, 180, 360a,b, 540, and 720 min after infection. This RNA does not map on any region of Mimivirus' genome.Mimivirus' transcriptome [data from  eligmann , with fueligmann .5). Table P = 0.066). Similarly, mean lengths of swinger reads correlate positively for data produced by 454 and SOLID . Combining P-values from these two tests using Fisher's method for combining P values  yields a chisquare statistic with 2 × k degrees of freedoms with a combined P = 0.034. Hence results from both sequencing methods are overall congruent, swinger RNAs are not artifacts resulting from massive sequencing technologies.Our preliminary explorations of the kinetic data of the transcriptome of the giant virus Mimivirus . Hence the simple approach used here could be applied to study closely related clouds of secondary structures formed by a given RNA. It could also be adapted to discriminate further between similar secondary structures by including additional variables, such as GC contents in internal vs. external loops, and angular rotation between stem branches.Overall our results indicate that the versatility of RNA structures enable for functional novelties. Their physicochemical properties are also compatible with this role in primitive protolife organic conditions.Interpretations of a previous classification of RNA secondary structures formed by a variety of RNAs . Secondary structures formed by a sequence synthesized template free (hence de novo) cluster with B2.Cluster B2 was previously interpreted as representing de novo RNAs). Hence results suggest that some viroids are recent, rodshaped ones are presumably ancient RNAs.Rodshaped viroids from a wide range of GC contents belong independently of GC contents to D1 and D2, two clusters characterized by rRNAs and parasitic sequences. Viroids forming secondary structures characterized by more complex branching patterns belong to cluster B2 (putative Rickettsia genomes. This fits previous grouping of secondary structures formed by parasitic sequences with 23S and 16S rRNAs (clusters D1 and D2).Presumed parasitic palindromic sequences from Pandoravirus (MITE submarine family) resemble cluster D2. D2 is characterized by16S rRNA subunits and Rickettsial palindromic elements that parasitize de novo origins for viral sequences resembling rRNAs. These results are based on relatively weak similarities and hence can only be considered as preliminary. Nevertheless, they suggest that viruses produce rRNA-like sequences, in line with the prediction that the study of giant viruses will ‘change current conceptions of life, diversity and evolution’ . Overall, results hint that parasitic RNAs form rRNA-like secondary structures, and template free polymerizations apparently enrich genomes with new RNA/DNA sequences.HS and DR designed the study and analyses.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hordeum vulgare L.) is an important tissue to extract RNA and analyze expression levels of genes that control aspects of germination. These tissues are rich in polysaccharides and most methods for RNA extraction are not suitable to handle the excess polysaccharides. Here, we compare the current methods for RNA extraction applicable to germinated barley tissue. We found that although some of these standard methods produced high-quality RNA, the process of extraction was drastically slow, mostly because the frozen seed tissue powder from liquid N2 grinding became recalcitrant to buffer mixing. Our suggested modifications to the protocols removed the need for liquid N2 grinding and significantly increased the output efficiency of RNA extraction. Our modified protocol has applications in other cereal tissues rich in polysaccharides, including oat.Germinated seed from cereal crops including barley ( Seed germination is a complex process wherein the seed absorbs moisture, breaks dormancy, breaks down the endosperm, and allows for the emergence of the radicle from the embryo. In barley, an understanding of germination is highly emphasized because of its role in the economically important malting process. Germination and malt production involve dynamic changes to enzymatic activity, such as to the glucanases, amylases, and proteinases as physiological and chemical changes are made to the starchy endosperm ,3,4,5,6.2, produces RNA of high quality, and is much faster than the source methods.Here, we investigated several extraction protocols developed for ample high-quality RNA for analysis of gene expression from seed, including cereal crop seed ,11,12,13We initially tested three standard protocols for high quality RNA extraction from germinated barley seeds. Protocols included (i) the GeneJET plant RNA minikit ; (ii) the TRIzol reagent-based protocol ; and (iii) the Li and Trick  protocolThe GeneJet minikit failed to extract RNA due to homogenate solidification that prevented phase separation. Despite following the manufacturer’s protocol for handling polyphenols, terpenoids or polysaccharides in samples, homogenate remained solidified B. The st2 to grind samples is one commonality among these published methods. However, we found that the frozen powder resulting from liquid N2 grinding became recalcitrant to the buffer even after prolonged and vigorous vortexing. In addition, the grinding step is slow and adds a freeze/thaw component to the procedure that could degrade RNA. We circumvented this time-consuming step by grinding the soft sprouted seed tissue at room temperature directly in the extraction buffer. Although the quality and yield of RNA resulting from the standard vs. modified method of Li and Trick [2 successfully inhibited RNase, as evidenced by RNA integrity (The use of liquid Nnd Trick  (or of Wnd Trick ) were nond Trick  protocolntegrity . DNase Integrity . Also, nntegrity B.We conducted quantitative real-time PCR (qRT-PCR) assays using cDNA synthesized from RNA extracted by the modified Li and Trick method, using several gene-specific primer sets relevant to barley malting quality characteristics. As expected, each primer pair amplified a single DNA fragment with the correct band size by reverse transcription PCR (RT-PCR) A, and pr2 are the simplest and fastest for a large number of germinated individual barley seed. These two protocols are nearly equivalent. Under either modified method, the resulting RNA samples are of sufficient quality for expression analysis of genes involved in germination and malting quality. This protocol was also effective for extracting RNA of germinated oat seed (data not shown) and will likely be effective for RNA extraction from germinated seed of other cereals that contain high amounts of polysaccharides.In conclusion, either the Li and Trick  or Wang Prior to RNA extraction, all glassware, plastic pestles, and stainless-steel ware were rinsed with nuclease-free water and oven-dried. Other associated lab supplies, including microcentrifuge tubes, used in RNA extraction were purchased as nuclease-free.Extraction buffer 1: 100 mM Tris (pH 8.0), 150 mM LiCl, 50 mM EDTA (Ethylenediaminetetraacetic) acid, 1.5% sodium dodecyl sulfate (SDS), and 1.5% β-mercaptoethanol.Extraction buffer 2: 70% guanidinium sulfate (w/v) , 750 mM sodium citrate (incorrect concentration*), 10% lauryl sarcosine (incorrect concentration*), and 2 M sodium acetate (incorrect concentration*).  of Li and Trick  indicate.Correct extraction buffer 2: 4.2 M guanidine isothiocyanate, 25 mM sodium citrate, 0.5% lauryl sarcosine, and 1.0 M sodium acetate .*(1)A single 3- to 4-day sprouted barley or oat seed was added to a 1.5 mL microcentrifuge tube with 400 μL of extraction buffer 1 and ground in solution with an RNase sterile micropestle.(2)g for 30 min at 4 °C.An amount of 250 μL of a phenol-chloroform mixture  was added and centrifuged immediately at 21,000× (3)Approximately 250 μL of the top phase was transferred to a new tube.(4)An equal volume of extraction buffer 2 was added, the tube was inverted to mix and incubated for 10 min at room temperature.(5)An amount of 200 μL chloroform-isoamyl alcohol (24:1) was added and centrifuged as above.(6)The supernatant was removed and added to 300 μL isopropanol and 250 μL 1.2 M sodium chloride in a new tube.(7)g for 30 min at 4 °C.The solution was mixed by inversion and placed on ice for 15 min, then centrifuged at 21,000× (8)The supernatant was decanted and the RNA pellet was carefully washed with 400 μL cold 70% ethanol.(9)The RNA pellet was dried for 10–15 min in a laminar flow hood.(10)The RNA was dissolved in 50 μL of nuclease-free water and used in downstream applications.Refer to Wang et al.  for protFor spectrophotometric measurements, RNA concentration and purity were determined using a Take3 Micro-Volume PlateMicroplate with an Epoch UV-VIS spectrophotometer . For DNase digestion of RNA samples to remove contaminating gDNA and subsequent LiCl precipitation, the concentration was adjusted to 20–25 μg of total RNA in 50 μL nuclease-free water. Then, 6 μL 10× DNase I reaction buffer and 10 units total of DNase I  were added. Samples were incubated for 60 min at room temperature. An amount of 60 μL of 5 M lithium chloride solution was added prior to incubation for 1 h to overnight at −20 °C. After centrifugation as above, samples were washed with 400 μL of cold 70% ethanol. The resulting DNA-free RNA preparations were air-dried as above, and resuspended in 40 μL of nuclease-free water. RNA was subjected to spectrophotometric measurements and agarose gel electrophoresis both before and after DNA-free treatments.α-amyl1, alpha-amylase1 (M17126), (AACGAGAGCAAGCTGCAAAT/ATGAGGTTCCCCACATCGTA); B-Hord, B-Hordein (DQ267479), (AACCCCAAAAACAGTTGCAG/TGTGTGGTACCTGCTGTGGT); Cellulose synthase A6, CesA6 (AY483155),(CAACAGCGGTTACCAATCCT/CCCTTGAGGAAGGGGTAGAG); Cp3, Cysteine protease (AB377533), (TGCTGCATCTACGAGTACGG/ATGAGGGCAGCAGCTGTAGT).cDNA was synthesized, and RT-PCR and qRT-PCR were performed as described by Rashid and Deyholos . The fol


Bamboo mosaic virus (satBaMV), the only satRNAs of the genus Potexvirus, have become one of the models of how satRNAs can modulate HV replication and virus-induced symptoms. In this review, we summarize the molecular mechanisms underlying the interaction of interfering satBaMV and BaMV. Like other satRNAs, satBaMV mimics the secondary structures of 5′- and 3′-untranslated regions (UTRs) of BaMV as a molecular pretender. However, a conserved apical hairpin stem loop (AHSL) in the 5′-UTR of satBaMV was found as the key determinant for downregulating BaMV replication. In particular, two unique nucleotides (C60 and C83) in the AHSL of satBaMVs determine the satBaMV interference ability by competing for the replication machinery. Thus, transgenic plants expressing interfering satBaMV could confer resistance to BaMV, and interfering satBaMV could be used as biological-control agent. Unlike two major anti-viral mechanisms, RNA silencing and salicylic acid-mediated immunity, our findings in plants by in vivo competition assay and RNA deep sequencing suggested replication competition is involved in this transgenic satBaMV-mediated BaMV interference. We propose how a single nucleotide of satBaMV can make a great change in BaMV pathogenicity and the underlying mechanism.Satellite RNAs (satRNAs) are sub-viral agents that may interact with their cognate helper virus (HV) and host plant synergistically and/or antagonistically. SatRNAs totally depend on the HV for replication, so satRNAs and HV usually evolve similar secondary or tertiary RNA structures that are recognized by a replication complex, although satRNAs and HV do not share an appreciable sequence homology. The satRNAs of  SatRNA mimicry is mostly conserved in higher-order RNA structures. As well, satRNAs may adopt different mimicry at different stages of virus infection such as replication and translation (Satellite RNAs (satRNAs) are short RNA molecules that share no or little sequence homology to their cognate helper virus (HV) but totally depend on the HV for replication, encapsidation and efficient movement . The homnslation .Bamboo mosaic virus (BaMV), Cucumber mosaic virus (CMV), Peanut stunt virus (PSV), Grapevine fanleaf virus, Artichoke mottled crinkle virus, Cymbidium ringspot virus (CymRSV), Tobacco ringspot virus (TobRSV), and Groundnut rosette virus (GRV)  .Satellite RNAs of BaMV (satBaMVs) are well studied. Natural isolates of satBaMVs have been collected from BaMV-infected symptomatic bamboo plants worldwide to analyze the genetic evolution and phylogeny of satBaMVs . The mimIn this review, we focus on studies of interfering satBaMVs and a possible mechanism of satBaMVs interfering in BaMV infection.Bamboo mosaic virus is a single-stranded positive-sense RNA virus containing five open reading frames (ORFs) that belongs to the genus Potexvirus of the family Alphaflexiviridae .iviridae . ORF1 eniviridae , helicasiviridae  and RNA-iviridae . ORF2 toiviridae , 2006. Oiviridae  and sympiviridae  . P20 is not essential for satBaMV replication  and all populations from Bambusa vulgaris. All satBaMVs in clades II and III are derived almost entirely from Ma bamboo from the Taipei Botanical Garden in Taiwan and B. vulgaris in India, respectively , but the secondary structures greatly differed, as revealed by enzymatic probing with RNases A, T1, T2, and V1. The secondary structures of the non-interfering BSF4 5′-UTR contain a large stem loop (LSL) and a small stem loop (SSL)  and negative (-) strands without altering satBaMV level in a plants . Moreovea plants . Furtherm quinoa . Thus, t81UGC83) in IL-1 was found in all natural interfering satBaMVs, whereas a less-conserved AHSL structure or identical AHSL structure but with different sequence (81UGU83) in IL-1 was found in non-interfering satBaMVs .On further analyzing the RNA secondary structure of natural satBaMV isolates, an identical AHSL structure was found shared by all natural interfering satBaMVs. SatBaMV mutants that swap the AHSL region of BSF4 and BSL6 revealed that the AHSL in the 5′-UTR is the determinant of satBaMV-mediated BaMV interference . To furtsatBaMVs . Furtherhenotype . AnotherMV level . Thus, b215, C286 and A330 of WLM2-satCMV could independently affect necrosis induction with different CMV strains  are essential for satBaMV-mediated BaMV interference   of satRNAs (sat-sRNAs) can target HV and induce silencing of HV for CMV ; the satRNA silencing is the major antiviral defense mechanism operating in a sequence-specific manner in plants . In geneFigure 1B), BaMV-sRNAs and satBaMV-sRNAs of BSF4 and BSL6 generated from this region are extremely low in number  of satBaMV 5′-UTR are critical for the interfering satBaMV reducing BaMV level and infection in plants. This AHSL structure and the critical nucleotide C in IL-1 is conserved in the BaMV 5′-UTR and also important for replication. Moreover, interfering satBaMV dose-dependently reduces BaMV level. Thus, interfering satBaMV-reduced BaMV level competes for the replication complex.The AHSL secondary structure and two unique nucleotides  genomic and sub-genomic RNA level without affecting (-) sub-genomic RNA level. As well, the reduced (+) genomic RNA level is greater than the (+) sub-genomic RNA level  is the most privileged. Cytosine can be easily transformed into uracil via deamination. However, m5C cannot be converted to uracil. Cellular RNAs containing m5C include transfer RNA (tRNA), ribosomal RNA, mRNA and non-coding RNA in both eukaryotes and prokaryotes - and (-)-strand level. Bisulfite sequencing  or specific satBaMV-sRNAs may likely target the host gene, which is important for BaMV replication or essential effectors of the host innate immune system other than RNA silencing. Thus, the involvement of RNA silencing in BSL6-mediated interference remains an open question. It could be evaluated by using plant mutants defective in key components of RNA silencing or plants overexpressing VSRs and further analyzing satBaMV-induced specific satBaMV-sRNAs, miRNAs and other types of host endogenous small RNAs. The mechanism underlying interfering satBaMV reducing BaMV level and host symptom development remains a fascinating question requiring long-term study.Drafting the article: K-YL, Critical revision of the article: N-SL and final approval of the version to be published: K-YL and N-SL.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Because RNAP grip to template suggests a sticky translocation mechanism, and because grip is enhanced by increasing K+ and Mg2+concentration, biochemical assays are consistent with a conformational change that drives forward translocation as observed in simulations. Mutational analysis of the bridge helix indicates that 778-GARKGL-783 (Escherichia coli numbering) is a homeostatic hinge that undergoes multiple bends to compensate for complex conformational dynamics during phosphodiester bond formation and translocation.Based on molecular dynamics simulations and functional studies, a conformational mechanism is posited for forward translocation by RNA polymerase (RNAP). In a simulation of a ternary elongation complex, the clamp and downstream cleft were observed to close. Hinges within the bridge helix and trigger loop supported generation of translocation force against the RNA–DNA hybrid resulting in opening of the furthest upstream i−8 RNA–DNA bp, establishing conditions for RNAP sliding. The β flap tip helix and the most N-terminal β′ Zn finger engage the RNA, indicating a path of RNA threading out of the exit channel. Because the β flap tip connects to the RNAP active site through the β subunit double-Ψ–β-barrel and the associated sandwich barrel hybrid motif , the RNAP active site is coupled to the RNA exit channel and to the translocation of RNA–DNA. Using an exonuclease III assay to monitor translocation of RNAP elongation complexes, we show that K Multi-subunit RNA polymerases (RNAPs) are of the two double-Ψ-β-barrel (DPBB) type.+ (40 versus 120 mM) and Mg2+ (0 versus 5 mM) concentrations are found to enhance RNAP grip for RNA–DNA to modulate translocation.RNAP translocation has been described as an unrestrained thermal ratchet with rapid and reversible transitions between the pre-translocated and post-translocated registers.Thermus thermophilus (Tt) RNAP, the PDB 2O5J structure appears to have a closed trigger loop.Saccharomyces cerevisiae RNAP II, the PDB 2E2H structure appears to represent a nearly closed, catalytic conformation, but the trigger loop in 2E2H is not as fully closed or as helical as in 2O5J.All atom molecular dynamics simulations can potentially give insight into protein conformations that are not fully represented by available x-ray crystal structures. Many structures of RNAP ternary elongation complexes (TECs) are available, but only a small number are reported with a closed trigger loop conformation, which is thought to be the catalytic form. For Two major bend points have been identified on the long bridge α-helix.2 RNA end can differentially affect TEC translocation. Buffer conditions and the RNA 3′-end, therefore, can be altered to tune sliding of the RNAP TEC. These assays demonstrate that RNAP has significant grip for RNA–DNA, which can be increased by increasing K+ and Mg2+concentration. K+ may indirectly affect grip by affecting RNA–DNA melting at the RNA 3′-end. Mg2+, by contrast, appears directly to enhance grip, i.e., by bridging interactions of aspartic acid with phosphates. RNAP grip for the TEC is consistent with a conformational model for TEC sliding during translocation.To better understand translocation, the effects of monovalent and divalent ions on RNAP TEC sliding were analyzed. Also, a 3′-OH and a 3′-HAll atom molecular dynamics simulation was used to analyze translocation by Tt RNAP  and 2. BThe conformational change depicted in the simulation is also shown in three movies. Movie 1 indicates the dynamics and bending of the trigger loop. Movie 2 shows the remodeling of the RNA exit channel and the repositioning of the flap tip helix and Zn finger. Movie 3 shows the closing of the downstream cleft. This is a large conformational change that extends throughout RNAP. In the current simulations, clamp closure and RNAP grip for RNA–DNA is neither abetted nor restrained by elongation factors.Escherichia coli (Ec) RNAP dynamics and function, a number of substitutions were generated in and around the N-terminal bridge helix hinge, which cooperates with the two trigger loop GXP hinges. Both the GEPG and GLP trigger loop hinges are very active in the simulation shown (Movie 1), and the GLP hinge bends, apparently generating translocation force on the RNA–DNA hybrid. To better understand 32P-labeled RNA was extended from the G8 position by addition of 200 μM NTPs. To gain insight into the bridge helix N-terminal hinge, transcription assays are shown . TECs weβ′ R780 is highly conserved  and surrN-terminal hinge substitutions also strongly affect transcriptional fidelity . Here, m2. 2. It was found that omitting MgCl2 during TEC assembly caused increased slippage of TECs, particularly for β′ K781Q/R/E substitutions.2  TEC is not observed, indicating that termination occurs at the HPR3→HPR4 transition.2 see , and rigee 2 see . When TEtracking  was more2 chain terminator is present to block incorporation of CTP (the next accurately templated NTP). For the wild-type RNAP, in the absence of CTP, the pre-translocated TEC is mostly detected over the post-translocated TEC. This result is expected because during the exonuclease III digestion, the TEC is likely to return to the pre-translocated position within 3 seconds even if resting TECs are mostly post-translocated. In the presence of CTP, the post-translocated TEC is strongly stabilized over the pre-translocated TEC, as expected. In the presence of CTP, the pre-translocated TEC is observed because of the long times of exonuclease III digestion relative to reverse translocation rates. Backtracked states and hyper-translocated states are evident and vary according to the substitution that is mapped. Wild-type RNAP, R780K, R780Q, G782A, G778A and A779V respond strongly and appropriately to addition of CTP, as expected. None of these RNAPs shows a strong defect in transcription   and C. F2 chain terminator is present to block ATP (the next accurately templated NTP) incorporation. Mapping the upstream boundary, ATP strongly stabilizes the post-translocated register and suppresses hyper-translocation. Little effect is observed on pre-translocated or backtracked states. Once again, wild-type R780K/Q, G782A, G778A and A779V, with minor transcriptional defects, are strongly stabilized by ATP in the post-translocated register, as expected. Strongly defective, K781A and double substitution G778A/G782A do not appear to be stabilized by ATP binding, consistent with the KD measurements shown in For mapping RNAP excursions downstream  and D, tSome of the K781 substitutions appeared to be particularly unstable for in vitro TEC assembly, so K781A/G/D/R/E/Q were compared to wild-type K781 RNAP for their ability to form TECs stable or unstable to repeated washing . TECs im2+ gave similar results in exonuclease III experiments to TECs assembled in the presence of 5 mM Mg2+ (our standard procedure), but TECs assembled in the absence of Mg2+ had slightly altered but useful properties , termination is so evident . H777A, just N-terminal to the 778-GARKGL-783 hinge, is slow in elongation (∼3-fold down compared to wild-type RNAP). K789A is very similar to wild type for transcriptional activity and NTP loading. Although proline substitution is generally disruptive of helices, G794P is only about 2-fold down for transcriptional activity compared to wild-type RNAP and is only somewhat defective for NTP loading. Y795A shows no obvious defect in transcription or stable NTP loading. Substitutions in the more C-terminal bridge helix 794-GY-795 hinge, therefore, are very similar to wild type, showing the primary importance of the N-terminal 778-GARKGL-783 hinge compared to the C-terminal 794-GY-795 hinge for RNAP elongation.Using molecular dynamics simulation and biochemical approaches, we support the idea that hinges on the bridge helix and the trigger loop support a broad conformational change associated with forward translocation. In this and previous work, we indicate that the bridge helix N-terminal hinge GARKGL and two GXP hinges in the trigger loop helices GEPG and GLP are important.Mutagenesis of the bridge helix GARKGL indicates that this is a complex, homeostatic hinge, with two glycine bend points. Either one of the glycines supports much of the necessary hinge dynamics, but mutation of both is much more strongly defective in transcription. The arginine mounted within the hinge forms a “ball in socket” arrangement in the β fork, coupling bridge helix hinge and fork dynamics. Substitution of this arginine generated a fast mutant R780K, a reasonably neutral mutation R780Q and a very strongly defective mutant R780A. The methyl side chain of R780A is not charged and is too short to penetrate the β fork. In the adjacent residue, K781 substitutions have varied and interesting properties. K781E/R/Q substitutions tend to hyper-translocate and terminate and induce instability of TECs to washing in 1 M KCl. For bacterial RNAP, the lysine side chain flexibility appears important to support hinge homeostasis. We posit that K781 makes alternate contacts to β D546 on the fork and to β′ D785 on the bridge helix. Because arginine is much less flexible than lysine, K781R appears to become stuck making just one contact or the other, disrupting the hinge homeostasis, i.e., by forcing the hinge too far in one or the other direction. The analysis here supports the idea of a complex homeostatic hinge with primary G778 and G782 bend points. The complex hinge is coupled to the fork through R780 and tuned by alternating ionic contacts of β′ K781 to β D546 and β′ D785. Because the trigger loop N-terminal helix is closely coupled to the bridge helix, one major function of the GARKGL N-terminal hinge appears to be to maintain bridge helix-trigger loop coupling during catalysis and translocation. Simulations indicate that the trigger loop may overtighten to support chemistry and/or bond completion  base pair. The bridge helix hinge GARKGL and the trigger loop hinge GEPG help to compensate for the greater GLP hinge bending. Forward translocation force generated in part by trigger loop overtightening then appears to open the furthest upstream i−8 RNA–DNA bp, setting up the possibility of forward TEC sliding. Simultaneously, the RNAP cleft and clamp close, indicating that RNAP may undergo a large conformational change during translocation. The RNA exit channel is also remodeled during simulation so that the most N-terminal β′ Zn finger and the β flap tip helix engage strongly with the exiting RNA ; Movie 2+, Mg2+ and the RNA 3′-OH in regulating the sliding and grip of RNAP on DNA and RNA–DNA. Increased K+ suppresses backtracking and hyper-translocation. The effects of K+ on backtracking and hyper-translocation are posited to be primarily due to KCl inhibition of melting the RNA–DNA hybrid at the RNAP active site. Mg2+ appears to enhance the grip of RNAP on the RNA–DNA hybrid. When assembled in the absence of Mg2+, TECs are less stable. This is particularly true of K781E/R/Q substitutions, which tend to slide forward, hyper-translocate and, depending on conditions, terminate. Interestingly, termination due to hyper-translocation appears to occur at the HPR3→HPR4 transition, because HPR4 and further hyper-translocated TECs are not observed in exonuclease III mapping experiments, and HPR3 accumulates at 120 mM KCl but decays sharply from 3–20 seconds at 40 mM KCl, a condition that promotes TEC sliding.In this paper, we posit a conformational model for RNAP translocation. Such a model is consistent with a fairly stiff translocation ratchet, as opposed to a rapidly oscillating ratchet. Using variations in exonuclease III mapping procedures, we show effects of K2+ may bridge ionic contacts, for instance, with aspartic acid residues, to DNA and RNA phosphates. We note that, when Mg2+ is added back to TECs, the Mg2+-mediated grip on DNA is not re-established immediately, as indicated by exonuclease III mapping experiments in which TECs can continue to slide even after Mg2+ re-addition . Error is standard error. Data were fit using the program Origin.Transcription rate was measured at multiple NTP concentrations. A graph of rate versus NTP concentration was fit to the equation rate = The competition fidelity assay was run as previously described.32P on the strand to be attacked with exonuclease III. The unlabeled DNA strand was blocked by incorporation of a 3′-thio-NMP for digestion by exonuclease III, so only the 32P-labeled DNA strand was a substrate for exonuclease III. The 3′ ends of probes (Mapping was done as previously described.f probes  were neaf probes  indicate2 chain terminator) is assigned as the post-translocated register, and other registers are assigned by comparison. This is an interpretation of the data with some potential limitations, and exonuclease III mapping data have been interpreted in slightly different ways by others. However, expressing the data in this manner makes it more intuitive for the reader to interpret. Digestions were done in excess exonuclease III in order to maximize exonuclease III rates. DNA templates were designed to maximize exonuclease III rates of attack on the RNAP boundary. For instance, exonuclease III prefers to hydrolyze CMP from the DNA 3′-end compared to other NMPs and DNA strands are designed for rapid digestion.Assignments of translocation states are made in the following way. The state most strongly stabilized by addition of the accurately loaded NTP (in the presence of a 3′-H