DOI: 10.5061/dryad.n5tb2rbx8
title: Translin facilitates RNA polymerase II dissociation and suppresses genome instability during RNase H2- and Dicer-deficiency (data files)

description: The conserved nucleic acid binding protein Translin contributes to
 numerous facets of mammalian biology and genetic diseases. It was first
 identified as a binder of cancer-associated chromosomal translocation
 breakpoint junctions leading to the suggestion that it was involved in
 genetic recombination. With a paralogous partner protein, Trax, Translin
 has subsequently been found to form a hetero-octomeric RNase complex that
 drives some of its functions, including passenger strand removal in RNA
 interference (RNAi).  The Translin-Trax complex also degrades the
 precursors to tumour suppressing microRNAs in cancers deficient for the
 RNase III Dicer. This oncogenic activity has resulted in the Translin-Trax
 complex being explored as a therapeutic target. Additionally, Translin and
 Trax have been implicated a wider range of biological function ranging
 from sleep regulation to telomere transcript control. Here we reveal a
 Trax- and RNAi-independent function for Translin in dissociating RNA
 polymerase II from its genomic template, with loss of Translin function
 resulting in increased transcription-associated recombination and elevated
 genome instability. This provides genetic insight into the longstanding
 question of how Translin might influence chromosomal rearrangements in
 human genetic diseases and provides important functional understanding of
 an oncological therapeutic target.
description: <strong>Microscopic examination
 of mitosis</strong> Single colonies of appropriate strains were
 inoculated into 5 ml YEL containing supplemental adenine (200 mg/ml) and
 incubated with rotation at 30ºC until mid-log phase. Cells were harvested
 in a benchtop microfuge and resuspended in 1 ml 70% ethanol and incubated
 at room temperature for 10 minutes. Cells were re-harvested, and the
 pellets were washed three times with 1 ml phosphate-buffer saline (PBS;
 137 mM NaCl, 2.7 mM KCl, 10 mM
 Na<sub>2</sub>HPO<sub>4</sub>, 1.8 mM
 KH<sub>2</sub>PO<sub>4</sub>). Cell were finally
 resuspended in 100 ml of PBS. 2
 ml of cells
 was mixed with 2 ml DAPI (50 mg/ml)
 on a poly-L-lysine coated slide (Sigma, P8920) and air dried. Coverslip
 and a drop of Vectashield were applied prior to examination under a Zeiss
 Axioskop 2 plus fluorescence microscope. Slides were counted
 blind. <strong>Estimation of mini chromosome
 instability </strong> Ch<sup>16-23R </sup>is a
 derivative of <em>S. pombe </em>Chromosome III and it carries
 the <em>ade6-M216 </em>allele, which interallelically
 complements an <em>ade6-M210</em> allele located on the
 full-length Chromosome III (27-29). Strains containing a mini chromosome
 <em>ade6-M216</em> allele and a full-length chromosome
 <em>ade6-M210</em> allele will be Ade+ (these cells produce
 colonies that are white on YEA plates without supplemental adenine,
 whereas cells that have lost the minichromosome and only carry the
 <em>ade6-M210</em> allele are Ade- and grow as red colonies);
 loss of the mini chromosome will result in Ade-. To calculate the rate of
 minichromosome loss we employed the method of Niwa (2018. <em>Cold
 Spring Harb. Protoc</em>. 2018, 1-4). In brief, appropriate strains
 containing the minichromosome were cultured in liquid EMMG medium
 containing appropriate supplements, but no adenine (for minichromosome
 maintenance selection). Cultures were incubated until late log-phase,
 subjected to serial dilutions, and then plated out onto YEA without
 supplemental adenine to give plates with approximately 100-200 colonies
 per plate. Loss of the minichromosome prior to plating will result in
 colonies that are completely red. Cells that retain the minichromsome in
 both daughter cells after the first mitotic division post-plating (i.e.
 one cell to two cell stage) will either be all white or have red/white
 sectored colonies with the ratio of white being greater than red (the
 latter arising due to minichromosome loss after the first division). Half
 sectored colonies (50% white and 50% red) represent a minichromosome loss
 event in the first division of the single colony forming cell. Counting
 the half-sectored colonies as a fraction of the total number of cells
 gives a relatively accurate approximation of the minichromosome loss rate
 per cell division [see Niwa (2018. <em>Cold Spring Harb.
 Protoc</em>. 2018, 1-4) for further details and limitations]. Colony
 colours were counted to quantify cells that had retained the minichromsome
 at the first post-plating division (white or sectored with the majority
 white), cells that did not contain the minichromosome at plating (totally
 red) and cells that had lost the minchromosome at the first division
 post-plating (half sectored 50:50 red:white). All strains were counted
 blind. Colony
 counts<strong> </strong>were used to gain an approximate value
 for loss rate per cell division using the equation
 <em>N</em><sub>hs</sub>/(<em>N</em><sub>total</sub><em> - N</em><sub>r</sub>), where <em>N</em><sub>hs</sub> = number of half-sectored, <em>N</em><sub>total</sub> = total number of colonies, <em>N</em><sub>r</sub> = total number of all red colonies (Niwa. 2018. <em>Cold Spring Harb. Protoc</em>. 2018, 1-4). <em>P</em> values were calculated using Student’s <em>t</em>-test (two-tailed) and error bars represent standard deviation. <strong>Determination of recombination frequency </strong> Appropriate <em>S. pombe </em>strains containing the plasmid pSRS5 (Pryce  <em>et al</em>., 2009. <em>Proc. Natl. Acad. Sci. USA 106</em>, 4770-4775) were cultured to mid-log phase in liquid EMMG medium containing appropriate supplements. Cells were subjected to serial dilution and plated out on EMMG agar containing appropriate supplements at a dilution that resulted in well-dispersed single colonies following incubation at 30ºC. Colonies were permitted to grow until visible, but not permitted to reach greater than 1 mm in diameter. At this point a minimum of 7 whole colonies were individually picked and inoculated into individual 5 ml volumes of sterile liquid EMMG containing appropriate supplements, ensuring all the cells from the colony were transferred to the liquid medium. Cultures were incubated at 30ºC with shaking until very early stationary phase. Serial dilutions were made for each culture and these were plated onto YEA (dilution range 10<sup>-4</sup> to 10<sup>-6</sup>) and YEA containing 20 mg/ml guanine (pH 6.5) (dilution range neat – 10<sup>-2</sup>), which prevents the uptake of adenine because of purine antagonism (Pryce <em>et al</em>., 2005. <em>Genetics </em>170, 95-106). Plates were incubated at 30ºC for 3 days. All strains were counted blind. The numbers of colony forming units per ml of culture were counted to determine recombination frequencies (Ade+ cells / 10<sup>6</sup> viable cells). The recombination frequency was determined for 7 independent cultures for each strain to be tested and the median value was used for the recombination frequency (to avoid ‘jackpot’ values). This was repeated a minimum of three times for each strain to obtain mean values of independent biological repeats. P values were calculated using Student’s <em>t</em>-test (two-tailed) and error bars represent standard deviation. <strong>Genomic alkali lability assay </strong> Appropriate <em>S. pombe</em> strains were cultured in YEL to mid log phase (~0.5 OD<sub>600</sub>). DNA was extracted using the Epicentre MasterPure Yeast DNA Purification Kit (Cambio; MPY80200). Either KOH or KCl was added to 1 mg of genomic DNA to a final concentration of 0.2 M in 40 ml volumes and incubated at 55ºC for 2 hours. 6X loading buffer (alkaline; AlfaAesar; J62157) was added to the KOH-treated samples and 6X loading dye (non-alkaline; New England Biolabs; B7024s) was added to the KCl-treated samples. Alkali treated samples were loaded onto a 1% alkaline agarose gel (1% agarose, 1 mM EDTA, 50 mM NaOH) and run in alkaline electrophoresis buffer (1 mM EDTA, 50 mM NaOH). Electrophoresis of KCl treated samples was performed using a 1% agarose gel run in tris-borate-EDTA (TBE) buffer (130 mM tris [pH 7.6], 45 mM boric acid, 2.5 mM EDTA). Gels were run at 1 V/cm for 18 hours. Alkaline gels were neutralized by soaking in 1 M tris HCl (pH8.0) for 1 hour prior to staining with SYBR Gold (Thermo Fisher Scientific; S11494) and imaged on a UV transilluminator (BioRad; Molecular Imager Gel Doc XR System). Quantification of undegraded genomic DNA intensity from alkali and native gels was performed using ImageQuant software. Values from the intensity of undegraded genomic DNA from alkali gels were normalized against the values of the chromosomal DNA in the native gel. <em>P</em> values were calculated using one sample Student’s <em>t</em>-test and error bars represent standard deviation. <strong>DNA:RNA immunoprecipitation (DRIP)</strong> DNA extraction was performed as described in Forsdurg and Rhind (2006. <em>Methods Enzymol</em>. 470, 759-795). Genomic DNA was fragmented using <em>Dde</em>I (10 U/µg of DNA) for 2 hours at 37ºC (New England Biolabs; R0175s). DNA samples were divided into two and one sample was treated with RNase H (New England Biolabs; M0297s) for 2 hours at 37ºC, the other sample was left untreated. For DRIP, DNA samples were then incubated overnight at 4ºC in immunoprecipitation (IP) buffer [100 mM MES (pH 6.6), 500 mM NaCl, 0.05% Triton, 2 mg/ml BSA] in the presence of Protein G-coupled Dynabeads (Life Technologies; 10003D) previously incubated with S9.6 antibody (Kerafast; ENH001) according to the manufacturer’s instructions. The beads were then washed three times in IP buffer. After two additional washes in Tris-EDTA (TE) buffer [10 mM tris-HCl (pH 8.0), 1 mM EDTA] the beads were resuspended in 10% Chelex resin (BioRad; 1421253) and incubated at 98ºC for 5 minutes. The mixture was then incubated with 20 mg of proteinase K (Qiagen; 19131) at 43ºC for 30 minutes and then at 98ºC for 5 minutes. After centrifugation for 5 minutes at 6,000 r.p.m. in a benchtop microcentrifuge, DRIP-qPCR was performed using the supernatant.  Real-time PCR was performed using 25 ng of input DNA and 1/20 of the input immunoprecipitated DNA (above) in the presence of GoTaq Green Master Mix (Promega; A6002). Reactions were done in duplicate and standard curves were calculated on serial dilutions (100 ng – 0.1 ng) of input genomic DNA. IP enrichment was calculated relative to RNase H treated IP using the following formula: DRIP enrichment = {[IP amount (ng) (no RNase H) / input amount (ng) (no RNase H)] / [IP amount (ng) (+ RNase H) / input amount (ng) (+ RNase H)]}. The resulting values were then presented as a percentage of the wild-type value. Primer sequences are given in Supplemental Table S2 of the manuscript. Quantification for DRIP-qPCR was accomplished using Ct values and a standard curve of ten-fold dilutions of input genomic DNA from the wild-type strain. Experiments were performed in duplicates. Ct values for DRIP-qPCR were normalized using the Percent Input analysis method which represents the amount of DNA pulled down by using the antibody of interest in the ChIP reaction, relative to the amount of starting material (‘input’ fraction). Experiments were done in triplicate. <em>P</em> values were calculated using Student’s unpaired <em>t</em>-test with Welch’s correction, and error bars represent standard deviation. <strong>Chromatin immunoprecipitation (ChIP)</strong> Appropriate <em>S. pombe</em> strains were cultured in 50 ml of YEL to mid log-phase. Cells were crosslinked with 1% paraformaldehyde solution (Electron Microscopy Sciences; 15714-5) at room temperature for 15 minutes. Reactions were quenched by addition of 2 ml of 2.5 M glycine for 15 minutes at room temperature. Cells were harvested by centrifugation for 5 minutes at 1000 <em>g</em>, washed twice with ice-cold PBS and resuspended in 400 ml of Buffer A [50 mM HEPES (pH 7.5), 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate] supplemented with 1 mM PMSF (Sigma; P78830-1G) and 1 x Halt protease inhibitors (ThermoFisher Scientific; 78430). After an addition of an equal volume of acid washed glass beads (Sigma; G8772-500G), cells were vortexed for 60 minutes at 4ºC using a disruptor genie (Scientific Industries) with a Turbomix attachment. Lysates were recovered from the beads and sonicated using a bath Bioruptor Sonicator (Diagenode) at 30 seconds on followed by 30 seconds off for 10 minutes to obtain chromatin fragments in the range of 200-800 base pairs. The total volume was increased to 1 ml by addition of Buffer A and the sonicate was centrifuged at 4ºC in a benchtop microfuge at 14,000 r.p.m. for 10 minutes. The soluble chromatin was retained. 20 ml of washed Dynabeads M-280 sheep anti-mouse IgG (Thermofisher Scientific; 11201D) were added to the chromatin sample and incubated for 2 hours at 4ºC. 20 ml of the pre-cleared sample was kept for the ‘input’ fraction and the rest was incubated overnight at 4ºC with 2 mg of anti-RNA polymerase II antibody (Abcam; ab5408) or in the absence of antibody. 20 ml of washed Dynabeads were added and after 2 hours at 4ºC they were washed sequentially three times with Buffer A, twice with Buffer A with 500 mM NaCl, twice with 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM tris-HCl (pH8.0) and twice with 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0). The beads and ‘input’ were resuspended in 100 ml of 10% Chelex (BioRad; 1421253) and incubated at 98ºC for 5 minutes. The mixture was then incubated with 20 mg of proteinase K (Qiagen; 19131) at 43ºC for one hour and then at 98ºC for 5 minutes. After centrifugation for 5 minutes at 6,000 r.p.m. in a benchtop microcentrifuge, the supernatant was collected and analyzed by qPCR (below). qPCR was performed using the primers listed in Supplemental Table S2 of the manuscript. Average CT was calculated across technical triplicates for each sample. IP enrichment was calculated as percentage of input (whole cell extract) and presented relative to the wild-type value.
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