Computational protocol: Quantitative analysis of processive RNA degradation by the archaeal RNA exosome

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Protocol publication

[…] An amount of 120 μM Csl4-exosome (Csl4:Rrp41:Rrp42)3 or its Y70ARrp42 mutant (= 27 g/l) were incubated with 400 μM RNA (3.3-fold excess, 6-mer CCCCUC) for 10 min on ice. Protein:RNA complexes were crystallized by sitting drop vapour diffusion technique by mixing 1 μl protein and 1 μl of reservoir solution (0.1 M NaAcetate, pH 4.6, 30% 3-Methyl-1,5-pentadiol (MPD), 100 mM NaCl) at 20°C. Datasets were recorded at the ID-14-2 beamline (ESRF, Grenoble, France) to 2.4 Å (wild-type exosome) and at the PX I beamline (SLS, Villigen, Switzerland) to 3.0 Å (Y70ARrp42 mutant) and processed with X-ray Detector Software (XDS) (). A model of the apo-Csl4-exosome complex () was used as a search model for molecular replacement using PHASER (). Refinement to 2.4 Å and 3.0 Å, respectively was performed with CNS () and PHENIX (). In the additional electron density RNA nucleotides were positioned using COOT (). Refinement of the complete complexes was followed by iterative cycles of manual model completion with COOT and positional and B-factor refinement with CNS (Supplementary Table S1). [...] For small angle X-ray scattering (SAXS) studies, the (Rrp41:Rrp42:Rrp4)3 complex was purified as described above. To purify the exosome with endogenously bound Escherichia coli RNA the protocol was modified as follows: RNA was not washed off with high salt, and in all buffers the salt concentration was 250 mM or lower. After the Ni-NTA affinity chromatography, the complex was loaded on an anion exchange column to remove unbound nucleic acids and the procedure was repeated to assure the total removal of free RNA. Not until only one distinct peak was eluted, the fractions were pooled, concentrated and flash frozen. The apo-complex was measured at 5, 10 and 15 mg/ml and the RNA complex was concentrated to an absorption at 280 nm of A280 = 55 and measured in a 1: 0, 1: 1 and 1: 2 dilution to evaluate the concentration dependency of scattering. Both complexes did not show concentration dependent aggregation and were not affected by long exposure to high-energy X-rays. SAXS data collection was performed in 20 mM Tris pH 7.4 and 200 mM NaCl buffer at the SIBYLS beamline (Advanced Light Source, Berkeley, CA, USA) (). The radius of gyration was calculated using the Guinier plot in the linear region (constraint: s · Rg <1.3) and the calculation of the pair distribution function was done with GNOM within PRIMUS (). Ab initio modelling of the solution structures was done with GASBORp () and more than 10 identically calculated models were aligned and averaged using DAMAVER and SUPCOMB (). For analysis of the bound RNA, the protein was separated from the RNA by running the complex on a denaturing 6 M urea and 20% polyacrylamide gel and elution of the RNA from the gel. The pelleted RNA was sent to Vertis Biotechnologie AG, where the sample was poly(A)-tailed using poly(A) polymerase followed by ligation of an RNA adapter to the 5′-phosphate of the small RNAs. First-strand cDNA synthesis was then performed using an oligo(dT)-adapter primer and M-MLV H- reverse transcriptase. The resulting cDNAs were PCR-amplified to 20–30 ng/μl in 19 cycles using standard Taq DNA polymerase. We cloned the cDNA products with EcoRV into pET21 vectors, transformed and amplified the plasmids and isolated and sequenced single clones. […]

Pipeline specifications

Software tools XDS, CNS, Coot, ATSAS
Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma