Journal: Molecular cell

Article Title: Reaction mechanisms of Pol IV, RDR2 and DCL3 drive RNA channeling in the siRNA-directed DNA methylation pathway

doi: 10.1016/j.molcel.2019.07.008

Figure Lengend Snippet: RNA polymerase activities of Pol IV and RDR2 A. A simplified model for RNA-directed DNA methylation. B. Potential modes of Pol IV-RDR2 cooperation in dsRNA synthesis. C. ). Transcripts resolved by denaturing polyacrylamide gel electrophoresis were visualized by autoradiography. HA-tagged RDR2 purified from a Pol IV mutant background ( nrpd1 ) was tested in lane 2. FLAG-tagged Pol IV purified with associated RDR2 or from a rdr2 null mutant was tested in lanes 4 and 5, respectively. Lanes 1 and 3 are controls testing non-transgenic plant lysates subjected to anti-HA or anti-FLAG affinity purification. D. RDR2 can convert ssRNA into dsRNA in vitro . Purified, recombinant RDR2 was assayed for activity using a 37 nt 5' end-labeled ssRNA template. Reaction products resolved by non-denaturing PAGE were visualized by autoradiography. Lane 1 shows 37 nt ssRNA and 37 bp dsRNA controls. In lanes 2-5, the ssRNA template tested had a 3’OH group. RDR2 was included in all reactions except for lane 2. Reactions of lanes 4 and 5 included free streptavidin or streptavidin-agarose resin. Reactions of lanes 6-8 tested ssRNAs with a biotin group on the 3' terminal nucleotide. Streptavidin or streptavidin-agarose were included in lanes 7 and 8. E. Pol IV transcripts generated from ssDNA templates are sensitive to RNase H. Pol IV-RDR2 was tested in lanes 1 and 2; Pol IV isolated in the rdr2 mutant background was tested in lanes 3 and 4. Transcripts in lanes 2 and 4 were subjected to RNase H treatment. Positions of run-off transcripts and the labeled RNA primer are shown. F. Pol IV transcripts generated from ssDNA templates are sensitive to RNase I. Pol IV-RDR2 was tested in lanes 1 and 3; Pol IV isolated from rdr2 mutant plants was tested in lanes 2 and 4. Transcripts in lanes 3 and 4 were subjected to RNase I treatment.

Article Snippet: RNase sensitivity assays RNase-treated transcription reactions involved incubation with 2.5 units of RNase H (New England Biolabs), 2.5 units of RNase I (Promega), or both enzymes, at 37° C for 30 min. For RNase III tests, 1 unit of enzyme (Epicentre) was added 10 min after addition of RNases I and H. Reactions were stopped by adding SDS to a final concentration of 0.15% (w/v).

Techniques: DNA Methylation Assay, Polyacrylamide Gel Electrophoresis, Autoradiography, Purification, Mutagenesis, Transgenic Assay, Affinity Purification, In Vitro, Recombinant, Activity Assay, Labeling, Generated, Isolation

Journal: Molecular cell

Article Title: Reaction mechanisms of Pol IV, RDR2 and DCL3 drive RNA channeling in the siRNA-directed DNA methylation pathway

doi: 10.1016/j.molcel.2019.07.008

Figure Lengend Snippet: Tests of RNA and DNA parameters for effects on Pol IV termination and RDR2 coupling A. Test of transcript length on Pol IV termination/RDR2 coupling sites. End-labelled RNA primers of 16-28 nt, varying at their 5' ends (see diagram), were used to initiate transcription by Pol IV-RDR2. Duplicate reactions were differentially subjected to RNase I/H digestion. B. Test of displaced nontemplate DNA length on Pol IV termination/RDR2 coupling. Nontemplate DNA strands of 28-53 nt, with variable unpaired 5' end lengths (1-26 nt), were annealed to the T-less template (see diagram) and tested in Pol IV-RDR2 transcription reactions initiated using an RNA primer. In the reactions of lanes 1-6, the primer was 5' end-labelled, allowing Pol IV transcript detection. In the reactions of lanes 7-12 the primer was unlabeled and α- 32 P-ATP was used to body-label second strand RDR2 transcripts. The vertical blue line denotes the 27 basepaired region common to all template-nontemplate combinations. C. Test of the number of basepaired nontemplate nucleotides distal to the Pol IV encounter position. 28 nt nontemplate DNA strands forming 12-27 bp with the T-less template, but varying at their 3 ' ends, were tested in Pol IV-RDR2 transcription reactions initiated using an RNA primer. In the reactions of lanes 1-4, the primer was 5' end-labelled, allowing Pol IV transcripts to be visualized. In the reactions of lanes 5-8 the primer was unlabelled and α- 32 P-ATP was used to body-label RDR2 transcripts. D. Test for effects of template and/or nontemplate DNA cytosine methylation on Pol IV termination. Template and nontemplate strands that were unmethylated (far left lane), or methylated at CG, CHG or CHH (in 5' to 3' orientation) positions indicated in the cartoon at top, were tested in Pol IV-RDR2 transcription reactions initiated using a 5' end-labelled primer to visualize Pol IV transcripts. E. Test of single-stranded hairpin templates for Pol IV termination/RDR2 coupling. The T-less template was lengthened to allow formation of hairpins with 24-30 bp stems, as indicated in the cartoon. Pol IV-RDR2 transcription reactions were initiated using a 5' end-labeled primer to visualize Pol IV transcripts (lanes 1-6) or using an unlabeled primer and α- 32 P-ATP to body-label RDR2 transcripts (lanes 7-12). Pol IV purified from rdr2 null mutant plants was tested in lanes 8, 10 and 12. Reaction products of lanes 2, 4 and 6 were treated with RNases I and H. Images shown are lanes from the same gel and autoradiogram exposure. F. Test for Pol IV transcription from an initiation bubble. Primer-initiated Pol IV-RDR2 transcription reactions were conducted using single-stranded template DNA or template DNA annealed to a partially non-complementary DNA strand, as depicted in the cartoon. The primer was 5' end-labelled to visualize Pol IV transcripts.

Article Snippet: RNase sensitivity assays RNase-treated transcription reactions involved incubation with 2.5 units of RNase H (New England Biolabs), 2.5 units of RNase I (Promega), or both enzymes, at 37° C for 30 min. For RNase III tests, 1 unit of enzyme (Epicentre) was added 10 min after addition of RNases I and H. Reactions were stopped by adding SDS to a final concentration of 0.15% (w/v).

Techniques: Methylation, Labeling, Purification, Mutagenesis

Journal: Molecular cell

Article Title: Reaction mechanisms of Pol IV, RDR2 and DCL3 drive RNA channeling in the siRNA-directed DNA methylation pathway

doi: 10.1016/j.molcel.2019.07.008

Figure Lengend Snippet: Pol IV, RDR2 and DCL3 are sufficient for siRNA biosynthesis in vitro . A. Baculovirus-mediated production of recombinant DCL3. Top panel: Stained SDS-PAGE gel showing protein standards (lane 1), uninfected Sf9 cell lysate (lane 2), DCL3-baculovirus infected cell lysate (lane 3) or affinity purified DCL3-FLAG. Bottom panel: immunoblots using anti-DCL3 or anti-FLAG antibodies. B. siRNA synthesis by Pol IV, RDR2 and DCL3. Template DNA was annealed to a 28 bp nontemplate strand and Pol IV-RDR2 transcription was initiated using an end-labeled RNA primer. The autoradiogram shows untreated (lane 1), RNase I/H (lane 2), RNase III (lane 3) or DCL3-FLAG digested (25-500 ng; lanes 5-9) transcription products. Uninfected Sf9 cell lysate subjected to anti-FLAG affinity purification serves as a control in lane 4. C. Pol IV-RDR2 dsRNA products are diced from either end. Using the T-less template, primer-initiated Pol IV-RDR2 reactions were labeled using end-labeled primer to detect Pol IV strands, using α- 32 P-ATP incorporation to body-label RDR2 strands, or using capping enzyme and α- 32 P- GTP to label RDR2 strand 5’ ends. For each labeling scheme, total transcription products and DCL-diced products are compared. D. Pol IV, RDR2 and DCL3 produce siRNAs from single-stranded M13 bacteriophage DNA. Transcription reactions using α- 32 P-ATP to body-label transcripts were conducted using Pol IV-RDR2 (lanes 5-8) or Pol IV purified from rdr2 . E. .

Article Snippet: RNase sensitivity assays RNase-treated transcription reactions involved incubation with 2.5 units of RNase H (New England Biolabs), 2.5 units of RNase I (Promega), or both enzymes, at 37° C for 30 min. For RNase III tests, 1 unit of enzyme (Epicentre) was added 10 min after addition of RNases I and H. Reactions were stopped by adding SDS to a final concentration of 0.15% (w/v).

Techniques: In Vitro, Recombinant, Staining, SDS Page, Infection, Affinity Purification, Western Blot, Labeling, Purification

Journal: Molecular cell

Article Title: Reaction mechanisms of Pol IV, RDR2 and DCL3 drive RNA channeling in the siRNA-directed DNA methylation pathway

doi: 10.1016/j.molcel.2019.07.008

Figure Lengend Snippet: Tests of second strand synthesis by RDR2. A. Capping assay to test for a 5’ triphosphate on RDR2-dependent transcripts. First strands, synthesized by Pol IV associated with RDR2 (+) or purified from a rdr2 mutant (−), were initiated using an RNA primer with a labeled 5' monophosphate (lanes 1-8) or 5' hydroxyl group (lanes 9-12). In half of the reactions, the template was annealed to the 28 nt nontemplate DNA strand to induce Pol IV termination; remaining reactions lacked the nontemplate strand. Lanes 1-4 differ from lanes 5-8 with respect to RNase I/H treatment. In lanes 9-12, vaccinia virus capping enzyme catalyzed labeling with α- 32 . B. Use of a T-less DNA template and α- 32 P-ATP incorporation to label second strands. All reactions contain Pol IV, associated with RDR2 (+) or purified from an rdr2 for details). Transcripts in lanes 1-4 were initiated using a 5' end-labeled RNA primer to label Pol IV transcripts. In lanes 5-8, RDR2 transcripts were body-labeled using α- 32 .

Article Snippet: RNase sensitivity assays RNase-treated transcription reactions involved incubation with 2.5 units of RNase H (New England Biolabs), 2.5 units of RNase I (Promega), or both enzymes, at 37° C for 30 min. For RNase III tests, 1 unit of enzyme (Epicentre) was added 10 min after addition of RNases I and H. Reactions were stopped by adding SDS to a final concentration of 0.15% (w/v).

Techniques: Synthesized, Purification, Mutagenesis, Labeling

Journal: Journal of Bacteriology

Article Title: 16S rRNA Is Bound to Era of Streptococcus pneumoniae

doi:

Figure Lengend Snippet: Analysis of RNA association with the Era protein of S. pneumoniae. E. coli rRNAs were isolated as described in Materials and Methods. All samples were electrophoresed on a 1.5% agarose gel containing ethidium bromide. Lane 1, DNA standards (100-bp increments from bottom to top; Gibco BRL); lanes 2 and 3, E. coli rRNA untreated and treated with RNase A, respectively; lanes 4 and 5, phenol-chloroform-extracted material from a purified GST-Era protein preparation untreated and treated with RNase A, respectively; lanes 6 and 7, a purified GST-Era protein preparation untreated and treated with RNase A, respectively; lane 8, phenol-chloroform-extracted material from a purified GST-Era protein preparation treated with DNase.

Article Snippet: Purified Era proteins and extracted RNA were treated with 1 mg of RNase A (Boehringer Mannheim, Indianapolis, Ind.) per ml or 0.2 mg of DNase I (Gibco BRL) per ml for 15 min at room temperature prior to electrophoresis.

Techniques: Isolation, Agarose Gel Electrophoresis, Purification

Journal: Journal of Bacteriology

Article Title: 16S rRNA Is Bound to Era of Streptococcus pneumoniae

doi:

Figure Lengend Snippet: Analysis of Era-RNA complex formation in a crude extract of S. pneumoniae by gel filtration column chromatography. A crude extract of S. pneumoniae was prepared, untreated (A) or treated with RNase A (1 mg/ml) (B), and subjected to chromatography on a gel filtration column as described in Materials and Methods. The presence of Era in the fractions collected was detected by Western blotting analysis with polyclonal antibodies prepared against the native Era protein of S. pneumoniae ). The intensity of each band was quantified by scanning as described in Materials and Methods. Lanes 1 and 11, molecular mass markers and purified Era of S. pneumoniae (30 ng), respectively; lanes 2 to 10, fractions 23 to 31, respectively; lanes 12 to 19, fractions 39 to 46, respectively.

Article Snippet: Purified Era proteins and extracted RNA were treated with 1 mg of RNase A (Boehringer Mannheim, Indianapolis, Ind.) per ml or 0.2 mg of DNase I (Gibco BRL) per ml for 15 min at room temperature prior to electrophoresis.

Techniques: Filtration, Column Chromatography, Chromatography, Western Blot, Purification

Journal: Journal of Bacteriology

Article Title: 16S rRNA Is Bound to Era of Streptococcus pneumoniae

doi:

Figure Lengend Snippet: Effects of RNase A and DNase I treatments on the GST-Era GTPase activity of S. pneumoniae . Purified GST-Era protein (200 μg/ml) was treated with RNase A (200 or 800 μg/ml) or DNase I (100 μg/ml) at 37°C for 30 min. The RNase A- or DNase I-treated and untreated (control) GST-Era proteins (10 μg each) were then tested for their GTPase activities at 37°C for 30 min by the HPLC method as described in Materials and Methods. After the RNase A and DNase I treatments, parts of the GST-Era preparations were also analyzed by agarose gel electrophoresis (see Materials and Methods), and RNA was not detectable by ethidium bromide staining (data not shown).

Article Snippet: Purified Era proteins and extracted RNA were treated with 1 mg of RNase A (Boehringer Mannheim, Indianapolis, Ind.) per ml or 0.2 mg of DNase I (Gibco BRL) per ml for 15 min at room temperature prior to electrophoresis.

Techniques: Activity Assay, Purification, High Performance Liquid Chromatography, Agarose Gel Electrophoresis, Staining