rtcb  (New England Biolabs)

Journal: bioRxiv

Article Title: Small circRNAs with self-cleaving ribozymes are frequently expressed in metazoan transcriptomes

doi: 10.1101/721605

Figure Lengend Snippet: Circularization of Mexican axolotl self-cleaved retrozyme RNA. Purified linear monomeric RNA resulting from double self-cleavage of DRtz353 (see Figure 4C ) was run directly in a denaturing gel (lane 1), after 1h incubation with RtcB ligase in its corresponding buffer (lane 2), in RtcB buffer without protein ligase (lane 3), and in 50 mM Mg 2+ buffer (lane 4). Circular RNAs can be readily detected in lane 2 (up to 80% circularization for the 350 nt monomer) and lane 4 (3% self-circularization for the 350 nt monomer). Minor fractions of linear and circular dimeric RNAs are also indicated (lane 2).

Article Snippet: For the assays of RtcB ligation, 1 to 10 ng of gel-purified retrozyme RNA were ligated using RtcB ligase in its corresponding buffer for 1 h at 37 °C (New England Biolabs).

Techniques: Purification, Incubation

Journal: Nature biotechnology

Article Title: Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts

doi: 10.1038/s41587-019-0090-6

Figure Lengend Snippet: Tornado expression system generates circular RNA a , Ribozymes efficiently self-cleave during transcription reactions.. The construct containing Twister P1 and Twister P3 U2A ribozymes was transcribed in vitro and quenched with urea before running on denaturing PAGE and visualizing RNA. Fully cleaved products and the side products of cleavage accumulate efficiently and rapidly after transcription. b , Fully-cleaved products of transcription in a contain appropriate ends for circularization by the endogenous ligase, RtcB. We excised the fully-cleaved RNA from a and performed an RtcB ligation reactions. RtcB treatment produces a shift in gel mobility that is not observed without ligation or with pre-treatment with T4 PNK. This shift in gel mobility suggests that the fully-cleaved RNA contains the appropriate ends for ligation. Staining of the gel with DFHBI-1T and comparison of fluorescence relative to SYBR Gold signal demonstrates that circular Broccoli is brighter than linear Broccoli. c , Twister-based ribozyme-assisted circular RNA (racRNA) expression generates significantly higher levels of circular RNA than the previous circular RNA expressing system. HEK293T cells expressed racRNA Broccoli from a variety of racRNA expression systems (see Fig. 1 ) with different combinations of 5’ and 3’ ribozymes and were compared to expression using the tricY system. Cells were treated with actinomycin D (ActD) for 6 h to observe the drop in RNA levels after new RNA synthesis was inhibited. The Twister P1 and Twister P3 U2A construct, dubbed “Tornado”, expresses high levels of Broccoli RNA that exhibit high stability, characteristic of circRNA. d , Tornado-expressed RNA is decisively circular. DNA-directed cleavage by RNase H of a linear RNA produces two bands, each of expected size given the transcript length and probe site. The identical treatment of the same sequence expressed from Tornado produces a single band similar in size to the uncleaved transcribed sample.

Article Snippet: 10 pmol of this purified T4-PNK-treated RNA or of the gel purified RNA was ligated using RtcB Ligase (New England Biolabs M0458) for 1 h at 37 °C.

Techniques: Expressing, Construct, In Vitro, Polyacrylamide Gel Electrophoresis, Ligation, Staining, Fluorescence, Sequencing

Journal: Cell reports

Article Title: ELAC1 Repairs tRNAs Cleaved during Ribosome-Associated Quality Control

doi: 10.1016/j.celrep.2020.01.082

Figure Lengend Snippet: Requirements of Repairing ANKZF1-Cleaved tRNAs for CCA Addition (A) During ribosome-associated quality control (RQC), stalled ribosomes are dissociated to 60S and 40S ribosomal subunits. The 60S-peptidyl-tRNA complex is recognized by NEMF (teal) and Listerin (orange), which mediates polyubiquitination (Ub) of the nascent protein. ANKZF1 (purple) cleavage of the peptidyl-tRNA generates a ΔCCA tRNA with a 2’,3’-cyclic phosphate (2’,3’ > p) on the discriminator base (N 73 ) that must be resolved before TRNT1 is able to add back the 3’CCA nucleotides (red). (B) A total of 2 ng/μL of in - vitro -transcribed radiolabeled leucyl-tRNA lacking the 3’CCA nucleotides appended to the hepatitis delta virus ribozyme (ΔCCA-HDV) was incubated with nothing to maintain a 2’,3’ > p ( > p), T4 PNK to generate 2’ and 3’ hydroxyl groups, CNP to generate a 2’ phosphate (2’-p), or RtcB to generate a 3’ phosphate (3’-p) on N 73 . The ΔCCA tRNA products were incubated with 100 nM TRNT1 and nucleotides directly (lanes 2–5) or after treatment with calf intestinal alkaline phosphatase (CIP) (lanes 6–8). The reactions were analyzed by SDS-PAGE and autoradiography to assay for the addition of 3’CCA nucleotides producing full-length (FL; red arrowheads) tRNA. Yellow arrowheads denote slower migration of ΔCCA tRNAs without a 2’,3’ > p. (C) Coomassie stain (top) of flow-through (FT) or salt-eluted fractions of reticulocyte lysate from heparin resin. Individual fractions were incubated with radiolabeled ΔCCA-HDV and TRNT1 to assay for FL tRNA production (bottom). Fractions with peak activity (red arrowheads) were pooled for further analyses. (D) Immunoblot of ELAC1 distribution through the fractions shown in (C). .

Article Snippet: RtcB reactions were performed with 20 ng/μL of radiolabeled ΔCCA-HDV, 0.1 mM GTP, and 0.75 μM RtcB (NEB) in the supplied buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 , 10 mM DTT).

Techniques: In Vitro, Incubation, SDS Page, Autoradiography, Migration, Staining, Activity Assay