Suv39h1 mutational studies indicated that both the nucleic acid–binding and H3K9me–binding activities of Suv39h1-CD were crucial for its pericentric heterochromatin assembly. These results suggest that chromatin-bound RNAs contribute to creating SUV39H’s target specificity.SUV39H is the major histone H3 lysine 9 (H3K9)-specific methyltransferase that targets pericentric regions and is crucial for assembling silent heterochromatin. SUV39H recognizes trimethylated H3K9 (H3K9me3) via its chromodomain (CD), and enriched H3K9me3 allows SUV39H to target specific chromosomal regions. However, the detailed targeting mechanisms, especially for naïve chromatin without preexisting H3K9me3, are poorly understood. Here we show that Suv39h1’s CD (Suv39h1-CD) binds nucleic acids, and this binding is important for its function in heterochromatin assembly. Suv39h1-CD had higher binding affinity for RNA than DNA, and its ability to bind nucleic acids was independent of its H3K9me3 recognition. Suv39h1 bound major satellite RNAs DOI:http://dx.doi.org/10.7554/eLife.25317.001 Plants, animals and fungi store much of their DNA tightly packed with proteins in a form named heterochromatin. This arrangement helps to inactivate genes that are not needed in specific cells or at specific times, and provides a way to protect the genetic material from damage. Heterochromatin tends to form when an enzyme called a lysine methyltransferase chemically modifies some of the proteins associated with the DNA, which are known as histones. This enzyme modifies only some of the histones to get the process started, while a second protein then binds to the modified histones and causes more of the DNA to become packaged up as heterochromatin.In 2012, researchers reported that the version of the lysine methyltransferase enzyme from yeast binds to RNA molecules via a portion known as its chromodomain. Moreover, the enzyme needed to bind to RNA to help heterochromatin to form. A similar mechanism also occurs in fruit flies, another organism that is commonly studied in the laboratory. However, it was not clear if it happened in mammals like mice and humans.Now, Shirai, Kawaguchi et al. – who include many of the researchers involved in the 2012 study – report that the corresponding enzyme from mice can also bind to RNA molecules via its chromodomain. Further experiments showed that this activity was closely linked with the enzyme’s ability to target the correct histones and efficiently form heterochromatin. The first experiments were conducted using purified enzymes in the laboratory, while follow-up experiments looked at the enzyme’s activity within mouse cells.Other studies have previously reported that mutant mice lacking the lysine methyltransferase enzyme have defective heterochromatin, tend to die young and have genetic instabilities that are associated with an increased risk of tumors and male infertility. The new findings of Shirai, Kawaguchi et al. reveal that the mechanism behind the establishment of heterochromatin has mostly likely been conserved over a billion years of evolution, which is when yeast and mammals last shared a common ancestor. By revealing more about how mammalian cells can protect their DNA, these new findings could also mark an important step toward understanding and preventing birth defects that are caused when an embryo’s genetic material becomes damaged.DOI:http://dx.doi.org/10.7554/eLife.25317.002 Suv39h1/2 double-knockout (Suv39h dn) cells, the H3K9me3 levels over the pericentromere are severely diminished and HP1 proteins do not accumulate , and developmentally controlled facultative heterochromatin. CH generally consists of repetitive DNA and is transcriptionally silent, and its higher-order chromatin structure can spread, causing the heritable inactivation of genes adjacent to the CH . The estcumulate .Schizosaccharomyces pombe (S. pombe), the RNA interference (RNAi)-dependent pathway is essential for pericentric heterochromatin formation and maintenance, and the process of pericentric transcription is coupled with the targeting of the SUV39H homologue Clr4 to chromatin  and the C-terminal SET domain. The CD functions as a binding module that targets H3K9me3 marks, and the SET domain is responsible for SUV39H’s enzymatic activity. Since the N-terminal CD of Clr4 is required for the efficient spread and stable inheritance of H3K9me3 marks , it has in vivo in a manner dependent on its nucleic acid-binding activity and that knockdown of major satellite RNAs lowered Suv39h1 retention on pericentric heterochromatin. Our data suggest that chromatin-bound RNAs contribute to the targeting and retention of Suv39h1 at specific chromosomal regions.Here we demonstrated that Suv39h1-CD can bind nucleic acids, and that this binding is crucial for Suv39h1’s function. Suv39h-CD prefers to bind ssRNA rather than dsDNA, and Suv39h-CD’s nucleic acid binding is independent of its H3K9me3 recognition. Mutational analyses revealed that both the nucleic acid- and H3K9me3-binding activities of Suv39h1 are important for its induction of heterochromatin assembly. We also showed that Suv39h1 bound major satellite RNA in vitro transcribed, single-stranded 130-nt major satellite RNA (ssRNA) as a probe. GST-fusion proteins containing fission yeast Chp1-CD (GST-Chp1-CD) or GST alone were used as controls . Although nucleosomal DNA might contribute to Suv39h1’s chromatin targeting, the present study focused on Suv39h1-CD’s ssRNA-binding activity.We previously showed that fission yeast Chp1-CD and Clr4-CD bind both ssRNA and double-stranded (ds) DNA . We testACTB ORF ssRNA with a similar affinity as for 130-nt major satellite ssRNA (10) ssRNAs and that part of SUV39H1’s N terminus (residues 1–106) is responsible for this RNA-binding activity ; this was consistent with previous observations , and the CD and RRM cooperate to repress centromeric transcripts . To deteSuv39h dn immortalized mouse embryonic fibroblasts (iMEFs) , methylated H3K9 binding (Y67A), or both (Y67A-4A), and FLAG-tagged wild-type Suv39h1 . We tran (iMEFs) . On day nd day 6 . We monind day 6 .10.7554/Suv39h dn iMEFs rescued pericentric H3K9me3 signals in the DAPI-dense pericentric regions (H3K9me3 foci+), 64.0 ± 7.9% of the cells on day 3 and 97.0 ± 1.3% on day 6 were H3K9me3-positive  activity for free H3, comparable to Suv39h1-WT (Suv39h1 dn iMEFs infected with an empty or expression vector for Suv39h1 proteins (The induction of wild-type Suv39h1 (Suv39h1-WT) expression in positive . Inducinsduction . Suv39h1v39h1-WT . Cell prproteins . Thus, iSuv39h dn iMEFs on day 6 further demonstrated that compared to iMEFs rescued with Suv39h1-WT, iMEFs rescued with the Suv39h1 mutants contained larger cell populations that were negative for both H3K9me3 and HP1β  on day 6 (83.0 ± 4.9%), but the phenotype was rescued only poorly by the Suv39h1 mutants (Suv39h dn iMEFs (~10 times higher than that of WT iMEFs) and introduction of Suv39h1-WT suppressed this up-regulation of major satellite repeats  mutant of Suv39h1  also possesses chromatin-binding activity independent of its HP1 interaction  and insoluble, chromatin-enriched (Ppt) fractions, and endogenous Suv39h1’s chromatin association was evaluated by immunoblotting. We found that most of Suv39h1 was present in the chromatin-enriched fraction . We confhromatin . Intereshromatin , most ofhromatin , suggesthromatin . TogetheSuv39h dn iMEFs clearly indicated that Suv39h1-CD’s H3K9me3- and RNA-binding functions are both critical for establishing H3K9me3 marks in pericentric regions without preexisting H3K9me3 marks. On the other hand, we were still able to induce pericentric H3K9me3 and HP1 accumulation in Suv39h dn iMEFs complemented with 4A, Y67A, or Y67A-4A Suv39h1 mutants  than in those expressing Suv39h1-Y67A (which is deficient in H3K9me3 binding), RNA-binding activity is particularly involved in recruiting Suv39h1 to H3K9me3-free chromosomal regions. Since pericentric regions consist of major satellite repeats, it is possible that nascent transcripts derived from such regions possess specialized properties that are recognized by Suv39h1. Although SUV39H1 is reported to preferentially bind TERRA transcripts derived from telomeric repeats  were PCR-amplified with a T7 promoter sequence, and the resultant PCR fragments were used for in vitro transcription by Thermo T7 RNA polymerases (Toyobo). Transcribed RNAs were purified on denaturing 7 M Urea Polyacrylamide Gel and extracted with Biomasher Ι(Assist). Purified ssRNAs and dsDNAs were labeled with the 5’ EndTag Nucleic Acid Labeling System , and the labeled probes were incubated with GST- or MBP-fused proteins in 10 μl of binding buffer  containing 4 U RNaseOUT (Invitrogen). The fluorescent dye can be conjugated with dsDNA at the single or both 5’-ends, and the number of conjugated dye could affect the probe migration in gels, resulting in appearance of doublet bands. Binding reactions were carried out on ice for 30 min. For titration EMSAs, serially diluted proteins (diluted 3:5 or 1:2) were used. To examine the relationship between RNA binding and H3K9me3 recognition, an equimolar amount of H3K9me3 peptide was added to the binding reactions. The reaction samples were loaded onto a 5% polyacrylamide gel with 0.5 × Tris-borate EDTA buffer (TBE), and the labeled probes were detected with a Typhoon 9400 . The unbound fractions were measured with ImageMaster 1D software . Igor Pro software  was used for curve fitting.EMSAs were performed as described previously  using a Suv39h dn iMEFs (AB_259529), anti-FLAG-M2-HRP , anti-α-tubulin , anti-Suv39h1 , anti-H3 , anti-H3K9me3 (AB_843158), and anti-GST .Eco-Pack 2–293 cells (Clontech) and 293T cells (gifted from Dr. Yao ) were grdn iMEFs  were gro_2616099), anti-HPDNA fragments encoding Suv39h1-CD (residues 39–105 or 39–96) were PCR-amplified from pCAG-IRES-Puro-FLAG-Suv39h1 . The ampWT/mut-IRES-GFP or pMCs-GFP-Suv39h1WT/mut. The resultant retrovirus supernatants were used to infect control or suv39h dn iMEF cells  were transfected with pMCs-FLAG-Suv39h1EF cells . To obtaCells cultured on chamber slides  were fixed in 4% paraformaldehyde for 8 min at room temperature. The fixed cells were permeabilized with 0.1% Triton X-PBS for 15 min at room temperature and blocked with 3% BSA in 0.1% Tween-20 TBS (3% BSA-TTBS) for 15 min at room temperature. Next, the cells were incubated with primary antibodies diluted in 3% BSA-TTBS for 1 hr at room temperature, washed twice with PBS, and incubated with secondary antibodies conjugated with Alexa568, 647 Fluor (Invitrogen) diluted in 3% BSA-TTBS for 1 hr at room temperature. The nuclei were counterstained with DAPI and observed under a confocal microscope  at the Support Unit for Bio-Material Analysis in the RIKEN BSI Research Center (RRC).iMEFs in eight 150 mm dishes (~80% confluent) were fixed with 1% formaldehyde for 10 min at room temperature. The harvested cells were lysed in SDS lysis buffer , and the cleared cell lysates were sonicated with a Bioruptor (Cosmo Bio). The samples were diluted with RIP dilution buffer  supplemented with 40 U/ml RNase inhibitor  and were immunoprecipitated with anti-FLAG M2 antibody–conjugated Dynabeads (Invitrogen) at 4°C overnight. The beads were washed with ice-cold low-salt buffer , high-salt buffer , LiCl buffer , and diethylpyrocarbonate (DEPC)-treated water. The protein–RNA complexes were eluted with RIP elution buffer  and treated with 0.8 µg/ml Proteinase K for 1 hr at 42°C, followed by de-crosslinking at 65°C for at least 4 hr. Immunoprecipitated RNAs were further purified with TRIzol LS Reagent (Invitrogen) and treated with 2 U of DNase Turbo (Ambion) in 50 µl reaction buffer for 20 min at 37°C. The immunoprecipitated RNAs were extracted with phenol/chloroform (pH 5.2) and recovered by adding an equal volume of 2-propanol and 1/10 vol of 3 M sodium acetate (pH 5.2) using 1 µl of ethachinmate (Nippon Gene) as a carrier. The precipitated RNAs were dissolved in 50 μl of DEPC-treated water. Samples were denatured for 15 min at 65°C in loading buffer  before dot-blot analysis. For the quantitative RIP experiment, the dissolved RNAs were further treated with 2 U of DNase Turbo in 50 µl reaction buffer for 20 min at 37°C, extracted with phenol/chloroform (pH 5.2), and recovered by adding an equal volume of 2-propanol and 1/10 vol of 3 M sodium acetate (pH 5.2). cDNA was synthesized by SuperScript III reverse transcriptase (Invitrogen) with random hexamers. Quantitative real-time PCR was performed by the Fast SYBR Green Master Mix (Applied Biosystems) and the StepOnePlus Real Time PCR system (Applied Biosystems).2, 100 mM NaCl, 1% Triton-X100, 10% glycerol, and 1 mM sodium fluoride). After 10 min incubation on ice, the lysates were centrifuged at 14,010 ×g for 10 min at 4°C to separate supernatant and chromatin-enriched pellet fractions. Whole cell lysates and supernatant and pellet fractions were resuspended in SDS-sample buffer and sonicated using a Bioruptor (Diagenode) to reduce viscosity before SDS-PAGE analysis. Proteins in each fraction were analyzed by western blotting.Cells were harvested by trypsin treatment, washed twice with ice-cold PBS, and resuspended in lysis buffer . Two images were collected at a 5 s interval , a circle (2.55 μm diameter) covering a heterochromatin region was bleached , and 25 images were further acquired every 5 s using the original settings. Fluorescence intensities were measured using the ImageJ 1.47v program (http://rsb.info.nih.gov/ij/). The net intensities of the bleached and unbleached heterochromatin regions were obtained by background subtraction in each time frame. The intensity of the bleached heterochromatin was normalized to that of the unbleached heterochromatin, and the intensity relative to the original intensity before bleaching was plotted. The recovery curves from single cells were individually fitted to single exponential kinetics to obtain the half-time (t1/2) and mobile fraction (P(1-expt-k), where R = relative intensity, P = plateau value, k = association constant, and t = time. The images obtained before bleaching were also used to measure the heterochromatin:euchromatin ratio.FRAP was performed as described previously  using a fraction , using ITo confirm the enrichment of GFP-Suv39h and its mutants in Hoechst-dense heterochromatin, iMEFs were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 10 min and stained with 100 ng/ml Hoechst33342  for 1 hr before collecting images with a confocal microscope , with a PlanApoN 60× OSC NA = 1.4 oil-immersion objective lens .6 ~cells) were suspended in 100 μl MEF II nucleofector solution (Lonza) and then mixed with LNA-DNA GapmeRs ). Transfection was carried out in an electroporation cuvette using the Nucleofector instrument (Lonza). The transfected cells were transferred to fresh medium and incubated at 37°C for 24 hr. Then these cells were changed into fresh medium and incubated at 37°C for 24 hr, followed by harvesting cells for RNA preparation or FRAP analysis.We obtained locked nucleic acid (LNA)-DNA GapmeRs from Exiqon. An LNA-DNA GapmeR for major satellite RNA was previously reported . The seq4 pH 7.0, 150 mM NaCl, 1 mM MgCl2, and 0.1% blocking reagent) at 55°C and twice with a second-wash buffer  at room temperature. Images were captured with a LAS-3000 Mini (Fujifilm).Immunoprecipitated RNA or DNA samples were blotted onto a Hybond N+ nylon membrane  by Bio-Dot (Biorad) according to the manufacturer’s instructions. The membrane was rinsed with 10X SSC (1.5 M NaCl and 150 mM sodium citrate) and crosslinked with a UV Stratalinker (Stratagene). The membrane was pre-hybridized at 55°C for at least 15 min and hybridized with a probe at 55°C overnight. PCR-amplified major satellite or 18S rRNA probes were labeled and hybridized using the Alkphos Direct Labeling and Detection System  according to the manufacturer’s instructions. The membrane was washed three times with a first-wash buffer  cells. Cells were lysed by sonication , and proteins in the lysates were purified using Glutathione Sepharose  according to the manufacturer’s instructions. To produce maltose binding protein (MBP)-fused recombinant Suv39h1 (MBP-Suv39h1) proteins, the corresponding Suv39h1 cDNAs were introduced into the pMAL-c2X vector (NEB). Proteins expressed in the E. coli strain BL21 (DE3) were purified using Amylose Resin (NEB: E8021S) according to the manufacturer’s instructions. The GST- or MBP-fused recombinant proteins were further purified by anion-exchange chromatography . The eluted material was dialyzed against phosphate-buffered saline (PBS) with 10% glycerol, divided into aliquots, and stored at −80°C until use.A recombinant GST-fusion protein was expressed in 4 pH 6.8 and 10 mM NaCl). A typical titration consisted of injecting 10 μl aliquots of the ligand STGGKAPRKQLAY) into the protein sample at 3 min intervals to ensure that the titration peak returned to the baseline. The ITC data were analyzed using the Origin program.ITC was performed on a MicroCal VP-ITC calorimeter  at 20°C. The proteins were dissolved in phosphate buffer (20 mM KPOPeptide pull-down assays were performed as described previously . GST-CD 5 cells) were harvested and re-suspended in nuclear isolation buffer (Sigma). The chromatin was fragmented for 7 min at 37°C, and diluted in ChIP immunoprecipitation buffer . Lysate mixture was incubated with anti-H3K9me3 (2F3) coupled with 10 μl of mouse IgG Dynabeads for over night at 4°C. The immune complexes were sequentially washed in low salt wash buffer , and high salt wash buffer . The immune complex was purified with 1.8×volume of Ampure XP DNA purification beads (Agencourt), then analyzed by real-time PCR using primers reported as specific for major satellite DNA , Major Satellite ChIP R (5’- CATATTCCAGGTCCTTCAGTGTGC-3’), Gapdh ChIP F (5’- ATCCTGTAGGCCAGGTGATG-3’), Gapdh ChIP R (5’- AGGCTCAAGGGCTTTTAAGG-3’), Gapdh RT-PCR F (5’- ATGAATACGGCTACAGCAACAGG-3’), Gapdh RT-PCR R (5’- CTCTTGCTCAGTGTCCTTGCTG-3’), NEAT1-6 F (5’- TGCTTACACGGCTTGTTCAG-3’), NEAT1-6 R (5’- AACTCCAAGGTCCCTGTCCT-3’).Suv39h dn iMEFs were seeded onto a 10 cm dish the day before the experiment. Cells were labeled with 10 µM CellTrace Far Red (Thermo Fisher) on the dish for 20 min at 37°C, then washed twice in medium and returned to the incubator for 10 min at 37°C. Then, these labeled Suv39h dn iMEFs were infected with a retrovirus containing pMCs-FLAG-Suv39h1WT/mut-IRES-GFP, and incubated for 3 days at 37°C. Analysis was performed on 10,000 cells/ GFPdull+ sample using FACS.wt/mut were lysed in RIPA buffer  containing 1 mM PMSF, and were subjected to sonication on ice. FLAG-tagged Suv39h1wt/mut proteins were purified with anti-FLAG M2 affinity gel.48 hr after transfection, 293T cells expressing FLAG-tagged Suv39h1in vitro histone methyltransferase assay was performed as described previously , is a member of our Board of Reviewing Editors. The reviewers have opted to remain anonymous.Thank you for submitting your article "Impact of nucleic acid and methylated H3K9 binding activities of Suv39h1 in its heterochromatin assembly" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This study explores the possible function of an RNA binding activity in the chromo domain of the mouse Suv39h1 in its heterochromatin localization. The authors demonstrate that the chromo domain of Suv39h1 binds to single stranded RNA (ssRNA), has higher affinity for ssRNA than dsDNA, generate mutations in the chromo domain that abolish RNA binding without affecting the association of the chromo domain with H3K9me3 peptides, show that Suv39h1 associates with major satellite RNA in vivo, and that RNA binding appears to affect the stable association of Suv39h1 with heterochromatic foci. The experiments in the paper are well executed and support the main conclusion regarding the effect of RNA binding on the stable association of at least Suv39h1-EGFP (which is somewhat overexpressed) with heterochromatin. Non-specific affinity for RNA (or DNA) may potentially play a role in heterochromatin stability. However, the functional significance of the observed non-specific RNA binding (effects on silencing) is not established and additional work is required to rule out alternative explanations prior to publication.Essential revisions:1) The authors present some data on the effect of the RNA binding mutations on the levels of H3K9me3 and HP1 β at heterochromatic foci. Here the effect is weak, 2-fold or less reduction in intensity in the mutants . They sh2) 3) Subsection “Suv39h1-CD's nucleic acid binding and methylated H3K9 recognition/binding are both crucial for Suv39h1-mediated pericentric heterochromatin assembly”/4) 5) Subsection “Suv39h1-CD’s nucleic acid binding is required for its interaction with major satellite RNAs in vivo”/A) What happens to RIP of Y67A and Y67A-4A? A prediction of the proposed model is that combining RNA- and H3K9me binding mutations in the same protein should reduce the ability of Suv39h1 to interact with MSR RNAs more than either of the single mutants. For better quantification, it is suggested to consider performing qRT-PCR reactions and normalize all RIP signals to input RNA.B) These RIP experiments could be performed from nuclear and cytoplasmic fractions to ask whether Suv39h1 interaction with RNA occurs in nucleus and requires H3K9me3 (in case of Y67A).in vitro”/in vitro binding assay results. 6) Subsection “Full-length Suv39h1 exhibits diminished nucleic-acid binding activity in vitro (7) in vitro , display8) 9) Showing that RNaseH / A treatment has a similar effect to the Suv39h1 nucleic acid binding mutation in the FRAP analyses would make the results more convincing that the RNA is a key contributor to Suv39h1 stability on chromatin. Essential revisions:1) The authors present some data on the effect of the RNA binding mutations on the levels of H3K9me3 and HP1 β at heterochromatic foci. Here the effect is weak, 2-fold or less reduction in intensity in the mutants . They shSuv39h dn iMEFs that were infected with an empty or expression vector for Suv39h1 (WT and mutants) on day 14. As reported previously , MSRs were derepressed in Suv39h dn iMEFs (~10 times higher than that of WT iMEFs) and introduction of WT Suv39h1 suppressed the up-regulation of MSR expression. Mutant Suv39h1 proteins  also decreased MSR expression, but their effects were milder than that of WT, which is consistent with their phenotypes of pericentric accumulation of H3K9me3 and HP1β. Furthermore, among these mutants, Suv39h1-Y67A-4A exhibited the weakest effect, which is also consistent with the H3K9me3/HP1β IF staining and GFP-Suv39h1 FRAP results.Thank you for this comment. Since Suv39h is mostly dispensable for silencing of retrotransposons in iMEFs , we focused on major satellite repeats (MSRs). We compared MSR expression levels in wild-type (WT) and We included this data in new For H3K9me3 levels, rather than the crude assay involving intensity of staining at heterochromatic foci, the authors should perform ChIP.Suv39h dn iMEFs infected with an empty or expression vector for FLAG-tagged Suv39h1 (WT and mutants). As shown in new Suv39h dn iMEFs and rescued by FLAG-tagged Suv39h1-WT expression. The expression of Suv39h1 mutant Y67A, 4A and Y67A-4A also recovered H3K9me3 levels on day 6 after transduction up the similar levels to that of Suv39h1-WT. Among them, Suv39h1-Y67A-4A showed the weakest recovery, which is consistent with the observations of MSR RT-qPCR, H3K9me3/HP1β IF staining, and GFP-Suv39h1 FRAP analyses.We performed H3K9me3 ChIP-qPCR analysis on MSRs in We included this data in new 2) We have prepared double stranded MSR RNA and performed EMSA using GST-Suv39h1-CD. As shown in new 3) Subsection “Suv39h1-CD's nucleic acid binding and methylated H3K9 recognition/binding are both crucial for Suv39h1-mediated pericentric heterochromatin assembly”/in vitro and in vivo labeling of cells to trace multiple generations using dye dilution by flow cytometry.Again, thank you for such a valuable comment. for this purpose, we performed celltrace™ (thermo fisher scientific) staining analysis, which is well established method for https://www.thermofisher.com/order/catalog/product/c34564Suv39h dn imefs were labeled with celltrace™ far red, infected with retroviruses for gfp-suv39h1 wt and mutant expression, grown for 3 days, and subjected for facs analysis. far red signal intensities were measured in the gated fraction based on gfp expression level, as demonstrated for the rescue experiments shown in suv39h dn imefs transduced with different constructs, indicating that the numbers of cell division during 3 days after the virus infection were not much different among them. it is therefore likely that the mutants’ slower heterochromatin re-establishment is not due to the defect or delay in cell cycle progression.We include this data in new 4) Since Chp1-CD does not bind to unmodified H3 peptide  Subsection “Suv39h1-CD’s nucleic acid binding is required for its interaction with major satellite RNAs in vivo”/A) What happens to RIP of Y67A and Y67A-4A? A prediction of the proposed model is that combining RNA- and H3K9me binding mutations in the same protein should reduce the ability of Suv39h1 to interact with MSR RNAs more than either of the single mutants. For better quantification, it is suggested to consider performing qRT-PCR reactions and normalize all RIP signals to input RNA.in vivo”, last paragraph).As suggested by reviewers, we have performed RIP experiments using cells expressing wild-type or mutant Suv39h1 and examined interaction with MSR RNAs by RT-qPCR. We have confirmed that Suv39h1-WT stably bound MSR ssRNAs and that this interaction was impaired for Suv39h1-4A . We founB) These RIP experiments could be performed from nuclear and cytoplasmic fractions to ask whether Suv39h1 interaction with RNA occurs in nucleus and requires H3K9me3 (in case of Y67A).in vivo”, last paragraph).In the RIP experiments, cells were fixed with 1% formaldehyde before harvest and we have no experience to fractionate cells after fixation. To address whether Suv39h1 interaction with RNA occurs in nucleus, we conducted chromatin fractionation assay without fixation and examined Suv39h1’s chromatin association. We found that Suv39h1 was predominantly detected in the chromatin-enriched precipitated fractions and only negligibly in the soluble fractions containing cytoplasmic proteins new . While S6) Subsection “Full-length Suv39h1 exhibits diminished nucleic-acid binding activity in vitro”/in vitro”).We thank the reviewer for raising this point. One of the obstacles that hampered our EMSAs using full-length Suv39h1 proteins was their low solubility: most of recombinant Suv39h1 proteins went into insoluble fractions, and we had to use a harsh condition to obtain sufficient amount of full-length Suv39h1. We speculate that this procedure might compromise the function of full length Suv39h1 and affect the EMSA experiments. We noticed, however, that the solubility of full-length Suv39h1 was dramatically improved when it was expressed with a maltose binding protein (MBP). Using this system, we have prepared MBP alone, MBP-fused Suv39h1-CD, and full-length Suv39h1 WT and mutant (4A) new  and cond7) in vivo context. Alternatively, impacts on mut1 on Suv39h1-CD’s RNA-binding might be compensated by in vitro condition such as GST’s dimerization property. In either case, we believe our in vitro RNA binding data still maintains our hypothesis.We thank the reviewer for raising this important point. Yes, the mut1’s behavior in the FRAP experiment  seems to8) Based on the reviewer’s suggestion, we revised Original: Revised: Regarding the statement in the original text, we rephrased the text to be more precise.“GFP-Suv39h1-4A, -Y67A, and -Y67A-4A also formed foci on Hoechst-dense regions . However9) Showing that RNaseH / A treatment has a similar effect to the Suv39h1 nucleic acid binding mutation in the FRAP analyses would make the results more convincing that the RNA is a key contributor to Suv39h1 stability on chromatin.Suv39h dn iMEFs and enhanced the mobility of GFP-Suv39h1 on pericentric heterochromatin. This data further supports that MSR RNA contributes to Suv39h1 stability on pericentric regions.We have tried RNaseH/A treatments by permeabilized cells, but it has been difficult to interpret the results. Therefore, we performed knock down experiments to reduce major satellite repeat (MSR) transcripts using locked nucleic acid (LNA)-DNA gapmers . Introduction of MSR-specific LNA-DNA gapmers significantly decreased MSR transcripts in We added this data as new Neat1 but not MSRs transcripts, but did not significantly enhance dynamics of GFP-Suv39h1 on heterochromatin, indicating the specificity of effects of MSRs specific LNA-DNA gapmer. However, due to the time limit, we could only complete this experiment including the cells treated with Neat1 specific LNA-DNA gapmers once. Therefore, we provide this data (In this RNA KD experiment, we also included nuclear non-coding RNA, Neat1 specific LNA-DNA gapmers as a negative control. Neat1 specific LNA-DNA gapmers decreased his data  only for


The mobile genetic element s2m has been described in several families of single-stranded RNA viruses. The function remains elusive, but an increasing number of s2m-containing sequences are being deposited in publicly available databases. Currently, more than 700 coronavirus sequences containing s2m can be found in GenBank, including the severe acute respiratory syndrome (SARS) coronavirus genome. This is an updated review of the pattern of s2m in coronaviruses, the possible functional implications and the evolutionary history. The genomes of single-stranded RNA (ssRNA) viruses have complex secondary structures, maintained both through conventional and non-Watson-Crick base-pairing ,2. CoronAstroviridae, Caliciviridae, Picornaviridae and Coronaviridae [A 43-nucleotide genetic element with a highly conserved secondary (as well as primary and tertiary) structure has been described in four different families of positive-sense ssRNA viruses, aviridae . The preaviridae . The 3′ aviridae ,7,8, butaviridae . The funaviridae  and RNA aviridae . In coroaviridae . The presence of s2m near the 3′ end of some coronavirus genomes has been documented previously ,11, but A total of 20,068 coronavirus nucleotide sequences have been deposited in the current version of Genbank, and when translated into amino acid sequences, the complete ORF1ab polyprotein sequence could be found for 1113 of these entries. A phylogenetic analysis was performed using aligned ORF1ab sequences , and numTorovirus genus as well as the recently described Bafinivirus genus [To get an overview of the current status for s2m in coronaviruses, all coronavirus nucleotide sequences were screened for the presence of the s2m motif using the strategy described by Tengs et al. . The conus genus , albeit Three phylogenetic clusters were found to contain s2m . All of Looking at topology and branch lengths, branch 1  represenExcluding sequences with ambiguous bases, a total of 37 different s2m genotypes were found within the coronavirus sequences . The greFor a mobile genetic element to be successful, especially in the context of rapidly evolving genomes such as those of RNA viruses, it must offer an immediate selective advantage. s2m viruses infect higher vertebrates, making the permissive host species a lot more closely related than the viruses. The observation that s2m can apparently be transferred between unrelated viruses and remain functional (under selection to maintain sequence and structure) suggests strongly that the target for s2m is host-specific and not viral. As there are no conserved elements flanking the s2m motif in viral genomes, it is also plausible that the element has an ‘autonomous’ function, independent of other viral genes, transcripts or genome secondary/tertiary structures. Somewhat surprisingly, the acquisition of s2m only has a subtle effect; exchanging the (non-s2m-containing) 3′-end of a murine coronavirus (MCV) with the 3′-end of a SARS virus did not appear to have any dramatic consequences , but 3′-The folding of s2m is quite similar to the hairpin structures formed by microRNAs that are involved in RNAi-associated gene regulation, and s2m could potentially be involved in gene silencing. In vertebrates, pre-microRNA transcripts are generated in the nucleus and processed by the nuclear protein DGCR8 and the enzyme Drosha . ProcessObservations arguing against an RNAi-like function would be that the stem region of s2m is a bit short compared to standard processed RNAi molecules . We werAnother hypothesis that would be compatible with the general assumptions regarding s2m functionality is that s2m is involved in the protection of the virus genome from being degraded by host ribonucleases. Coronaviruses are positive-sense ssRNA viruses with non-segmented genomes that are polyadenylated and capped. The genomes may serve as mRNA for the translation of viral polyproteins and contain two overlapping reading frames that encode precursor polyproteins pp1a and pp1ab through a frameshifting mechanism . After iFor some positive-strand ssRNA viruses, genome circularization has been shown to be essential for virus replication . RNA cirIn order to elucidate the function of s2m, it seems imperative to establish a reverse genetics system for an s2m-containing viral strain. A good system could be an astrovirus-derived infectious construct, as there already are protocols described for the design of astrovirus cDNA clones that lead to the production of infectious viral particles . Targete


The expression of the compact mammalian mitochondrial genome requires transcription, RNA processing, translation and RNA decay, much like the more complex chromosomal systems, and here we use it as a model system to understand the fundamental aspects of gene expression. Here we combine RNase footprinting with PAR-CLIP at unprecedented depth to reveal the importance of RNA–protein interactions in dictating RNA folding within the mitochondrial transcriptome. We show that LRPPRC, in complex with its protein partner SLIRP, binds throughout the mitochondrial transcriptome, with a preference for mRNAs, and its loss affects the entire secondary structure and stability of the transcriptome. We demonstrate that the LRPPRC–SLIRP complex is a global RNA chaperone that stabilizes RNA structures to expose the required sites for translation, stabilization, and polyadenylation. Our findings reveal a general mechanism where extensive RNA–protein interactions ensure that RNA is accessible for its biological functions. The mitochondrial genome, being compressed to 16 kb, is an attractive model system to investigate how RNA-binding proteins chaperone mRNA lifecycles. Here the authors use RNase footprinting and PAR-CLIP to show that the LRPPRC–SLIRP complex stabilizes mRNA structures to expose sites required for translation and polyadenylation. The importance of nuclear and mtRBPs is exemplified by the number of different disorders caused by mutations in genes encoding these proteins, including neurological conditions, metabolic diseases, and cancer4. Recent efforts have focused on transcriptome-wide identification of new RBPs in different cell types and organisms, as well as validation of already known RBPs or discovery of those with dual functions5. Consequently, an increasing number of these proteins have been assigned to different families/classes of RBPs, thought to play a role in RNA metabolism based on their sequence or fold homologies. Although we have insight into the RNA targets of a few well-characterized RBPs, the targets of most RBPs identified by high-throughput techniques are not known. Considering that RBPs coat RNAs extensively and thereby regulate their stability, translation and localization, it is essential to understand not only how they bind but more importantly the transcriptome-wide consequences of their loss in cells and organisms. This has been investigated at a genome level where loss of transcription factors, histones or histone modifying enzymes affect genome organization and transcription6, but this is lacking for post-transcriptional regulation of gene expression by RBPs both in the cytosol and mitochondria. Currently, the major goals in this field are the in-depth identification of RNA targets for RBPs and particularly characterizing their mechanism of action in vivo. Our study provides a model of transcriptome-wide consequences of mtRBP loss on RNA accessibility and secondary structure, revealing the critical role of a RBP as a RNA chaperone.RNA-binding proteins (RBPs) regulate the lifecycles of RNAs from transcription to degradation and as such are important modulators of gene expression. This is particularly evident in mitochondria, where gene expression is predominantly regulated by nuclear-encoded mitochondria localized RBPs (mtRBPs)7. Although the RNA targets of PUF proteins have been identified and validated biochemically, structurally and in vivo, the physiological targets of PPR proteins have remained largely unknown, limiting our understanding of their mechanistic roles in organelle gene expression and energy metabolism9. This is particularly true for the mammalian PPR proteins, where there are only seven PPR domain proteins that are all localized to mitochondria10. Cellular and in vivo studies have identified that these proteins regulate different aspects of mitochondrial RNA metabolism; from transcription via the mitochondrial RNA polymerase (POLRMT)12, to RNA processing 19, maturation and stability 21, and protein synthesis 23. Although the RNA recognition code for plant PPR proteins has been identified computationally and using in vitro approaches24 and based on these findings artificial PPR proteins have been designed to modulate gene expression25, the RNA targets of mammalian PPR proteins remain elusive.The sequence specific RNA-binding repeat proteins, including the Pumilio and FBF homology (PUF), and pentatricopeptide repeat (PPR) proteins, have been of particular interest because of their modular recognition of their RNA targets and their potential use as designer RBPs27. This differs from nuclear gene transcription and requires several unique processing and maturation steps to form functional RNAs, all of which depend on mtRBPs. In nearly all cases, genes encoding protein or rRNA are interspersed by one or more tRNAs, which act as “punctuation” marks for processing28, recruiting the RNase P complex or RNase Z that carry out the cleavage at the 5′ end or 3′ end of tRNAs, respectively29. Processing is followed by maturation of the RNAs by a range of mtRBP, including, the LRPPRC–SLIRP complex, poly(A) polymerase, methylases, tRNA modifying enzymes, and the CCA-addition enzyme, all necessary for the assembly of the rRNAs into mitochondrial ribosomes and translation of the mRNAs using tRNAs (reviewed in ref. 2).Here we use the mitochondrial transcriptome as a compact model to investigate and identify RNA–protein interactions at an unprecedented depth that has not been possible for the nuclear transcriptome. Mitochondrial genes coding for proteins and tRNAs are located on both the heavy and light strands of the mtDNA, and are transcribed as large polycistronic transcripts covering almost the entire length of each strand30. LRPPRC was identified when a mutation in its gene was shown to cause a rare French-Canadian variant of Leigh syndrome with a cytochrome c oxidase deficiency31. LRPPRC is one of the longest PPR proteins known with 30–33 PPRs and does not contain any other functional domains32. LRPPRC forms a stable complex with SLIRP21 via a PPR–RRM protein interface32, where SLIRP acts to protect LRPPRC from degradation in vivo30. Both proteins within the complex are required to regulate the rate of mitochondrial protein synthesis and the stability of the poly(A) tails of mitochondrial mRNAs20; however, the binding sites of the LRPPRC–SLIRP complex and its molecular role are not known.To understand the role of the LRPPRC–SLIRP RNA–protein complex in vivo, we sought to identify its RNA targets by photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) and high-throughput RNase footprinting of mitochondrial transcriptomes from mice where each of the genes coding for these proteins are knocked outHere we have investigated transcriptome-wide footprints of both LRPPRC and SLIRP, and the changes in molecular interactions and RNA secondary structure in their absence. We identify that single strand regions on mitochondrial mRNAs are the primary targets of the LRPPRC–SLIRP complex in vivo, indicating the role of this complex in RNA remodeling required to enable polyadenylation and translation. Furthermore we show transcriptome-wide changes in RNA interactions in the absence of these proteins, indicating that the LRPPRC–SLIRP complex acts as a global mt-RNA chaperone required to relax secondary structures. Our findings reveal the role of RBPs as chaperones for the lifecycles of mitochondrial mRNAs and provide insight into their recognition modes by investigating them at a much greater depth than previously possible.Lrpprc or whole body Slirp knockout mice, where these proteins are lost  was calculated based on the central RNase accessibility relative to the flanking regions in both controls  and knockout  data sets. A significant increase in the F score in the KO relative to the WT data sets enabled us to identify 178 different footprints for LRPPRC that were dispersed throughout mt-RNAs, the majority concentrated on mRNAs and to a lesser degree in rRNAs  and its incorporation into de novo transcripts, followed by cross-linking and immunoprecipitation, enabled us to map the precise LRPPRC-binding sites by identifying sites with thymidine (T)-to-cytidine (C) transition rates indicative of cross-linking in the sequenced data set. The specificity of LRPPRC binding was established when compared to a control PAR-CLIP experiment that did not result in specific alignment to the mitochondrial transcriptome  and the 5′ ends of the Atp6 or Nd4 ORFs, suggesting that LRPPRC may be required to expose the internal start and stop codons of these transcripts to the ribosome for translation initiation or termination. Interestingly, we found LRPPRC-binding sites within the 16S rRNA that were concentrated predominantly at either end of this transcript, suggesting that because of its affinity for transcript ends LRPPRC could facilitate rRNA processing, maturation and ribosome assembly. LRPPRC binding may facilitate the processing of the 16S rRNA from the RNA19 precursor transcript since loss of LRPPRC only decreases the levels of tRNALeu that is also contained within this precursor transcript20.We performed PAR-CLIP on mouse embryonic fibroblast (MEF) cells isolated from mt-Co3 mRNA identified both in the footprinting and PAR-CLIP data sets  using targets identified in both the footprinting and PAR-CLIP analyses. We show that the mouse LRPPRC–SLIRP protein complex has a high affinity for a region within the ets Fig.  and thatets Fig.  as previNext we defined a set of high confidence direct RNA targets of LRPPRC by selecting a set of sites that overlap between our footprinting and PAR-CLIP data sets Fig. . The ovemt-Nd5 mRNA in the Lrpprc KO mice that may be a result of a change in secondary RNA structure or caused by another RBP that associates with this mRNA in the absence of LRPPRC  polymerase (MTPAP) to facilitate polyadenylation. Evidence for this comes from in vitro experiments, where addition of LRPPRC increased the processivity of MTPAP36 and that loss of LRPPRC causes a polyadenylation defect20.LRPPRC-binding sites predominantly decorated the mRNAs and resulted in disruption of local mitochondrial RNA secondary structures. This suggests that relaxing the RNA structure by LRPPRC–SLIRP at these sites facilitates the coordinated translation and stability of mRNAs via polyadenylation20. Furthermore, it is possible that LRPPRC’s chaperone activity might assist in the production of tRNAs. Since mRNAs are typically flanked by tRNAs, LRPPRC binding at the termini of mRNAs could prevent these flanking regions from interfering with tRNA folding, processing or modification. Indeed, previous analyses of the steady-state levels of tRNAs found that a proportion were altered in the absence of LRPPRC20.The mechanisms by which mitochondrial ribosomes recognize mRNAs and initiate translation are not currently understood. The start codons of mitochondrial mRNAs are typically found at the extreme 5′ ends with little or no 5′-UTR sequence. Our observation that the RNA chaperone, LRPPRC–SLIRP, relaxes RNA structures of mRNAs, may indicate that the mitochondrial ribosome requires the start codons to be exposed in order to initiate translation37. This might be important for LRPPRC’s ability to bind many diverse RNAs, since only a subset of repeats would need to recognize individual RNA bases in order to achieve sufficient binding affinity for each individual RNA target. Nevertheless, the specificity of LRPPRC’s PPRs is still important to target critical regions of the transcriptome, as exemplified by the co-variation we observed between LRPPRC and its RNA targets through the evolution of human and mouse.LRPPRC has more PPRs than almost any other reported PPR protein, containing 33 predicted PPRs. Although structural information is only available for a few PPR proteins to date, it appears that they can often interact with their RNA targets in a binding mode where each PPR recognizes a single nucleotide base38, as an ensemble they likely act as LRPPRC does in the mitochondrial transcriptome to coat RNAs and assist in their folding. Therefore, the binding of LRPPRC across the mitochondrial transcriptome represents a minimal system to study the importance of ATP-independent protein-mediated RNA folding. The massively deep coverage of the mitochondrial transcriptome in our sequencing experiments has enabled us to elucidate the in vivo binding modes of the LRPPRC–SLIRP complex and establish a model approach of how to investigate the effects of RBP loss on a global transcriptome-wide level.Here we have shown that loss of a global RNA chaperone can lead to remodeling of secondary RNA structures as well as other RNA–protein interactions. Studies of the RNA-binding sites of eukaryotic nuclear and cytosolic RBPs have revealed sparse distributions of binding sites across individual mRNAs. However, given that there are an estimated 1542 human RBPsLrpprc and Slirp knockout transgenic mice on a C57BL/6 N background were housed in standard cages (45 × 29 × 12 cm) under a 12-h light/dark schedule (lights on 7 am to 7 pm) in controlled environmental conditions of 22 ± 2 °C and 50 + 10% relative humidity and fed a normal chow diet and water were provided ad libitum. LRPPRC loss is embryonic lethal therefore there are no full-body Lrpprc knockout mice and we used heart and skeletal muscle-specific knockout mice20. Also, SLIRP loss is not embryonic lethal and consequently we used hearts from the full-body Slirp knockout mice30. All mice used in this study were 12-week-old males. The study was approved by the Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein–Westfalen and performed in accordance with the recommendations and guidelines of the Federation of European Laboratory Animal Science Associations (FELASA).39. Hearts or livers were cut and washed three times with ice cold PBS, and once with mitochondrial isolation buffer (MIB) containing 310 mM sucrose, 10 mM Tris-HCl and 0.05% BSA (w/v) by centrifugation at 4500 g for 1 min at 4 °C. Heart pieces were homogenized in 5 ml of fresh MIB using a Potter S pestle (Sartorius). The homogenate was centrifuged at 1000×g for 10 min at 4 °C and the supernatant was centrifuged at 4500×g for 15 min at 4 °C to isolate mitochondria. Crude mitochondrial pellets were suspended in MIB supplemented with 1x Complete EDTA-free protease inhibitor cocktail (Roche). Protein concentration was determined by the Bradford or BCA method using BSA as a standard. Mitochondria (2 mg ml−1) were lysed by addition of 200 µl of lysis buffer . The concentration of the RNase A (10 U µl−1), RNase T1 (0.1 U µl−1) or RNase If (0.01 U µl−1) were optimized and added to each mitochondrial lysate or purified RNA to generate 15–55 nt size fragments. All incubations were carried out at 37 °C for up to 30 min and reactions were ended by addition of 700 µl Qiazol, followed by RNA isolation using the miRNeasy Mini Kit (Qiagen). We used mitochondria lysed and mock treated as controls for the footprinting assay.Mitochondria were isolated from homogenized hearts and isolated by differential centrifugation30, in Odyssey Blocking Buffer (Li-Cor). IR Dye 800CW Goat Anti-Rabbit IgG or IRDye 680LT Goat Anti-Mouse IgG (Li-Cor) secondary antibodies were used and the immunoblots were visualized using an Odyssey Infrared Imaging System (Li-Cor).Immunoblotting was carried out on mitochondrial proteins isolated from mouse hearts using a rabbit polyclonal antibody against LRPPRC  and in-house monoclonal antibody directed against mouse SLIRP (diluted 1:200)RNA concentration, purity and integrity were confirmed by BioAnalyser (Agilent). The libraries were constructed using the Illumina TruSeq Small RNA Sample Prep Kit and deep sequencing of the mitochondrial small RNAs was performed by Australian Genomic Research Facility  on an Illumina GAII (Illumina) according to the manufacturer’s instructions with one modification, sample isolation from the PAGE gel after adaptor ligation was performed with a modified set of size markers to facilitate capture of small RNAs between 15–55 nt.40 v2.2.9 with a seed length of 10 and reporting up to 20 alignments per read (–L10 –k 20). Paired alignments with multiply mapping reads that aligned once to the mitochondrial genome and at least once to a NUMT region were rescued. All properly paired alignments to the mitochondrial genome with an observed template length of 15–35 nt were retained and a subtractive alignment against nuclear tRNA and Illumina contaminant sequences was performed. Strand-specific fragment BED files were created and 5′ coverage profiles normalized to sequenced library size were produced with BEDtools41 v2.26.0. Footprints were identified as previously described33 with some modifications reported here. The RNase accessibility of each base, i, in the mitochondrial genome was quantified according to its C score, defined as:i+1, T1i+1, Ifi+1 and untreatedi+1 represent the normalized 5′ coverage of the nucleotide immediately downstream of the inspected position in RNase A-, T1-, and If-treated and untreated samples, respectively. The footprint detection algorithm searches for a span of between 8 and 40 nucleotides with an average C score lower than the flanking left and right three nucleotides, the central footprinting region, and calculates its F score:C, L and R are the average C score of the central, left flanking and right flanking regions, respectively. We initially searched for footprints in the control data and for each footprint we calculated its F score(control) and the F score of the equivalent region in the knockout sample, F score(experiment), and the log2-transformed fold change of F scores (knockout/control). We also searched for new RBP interactions or sites of decreased RNase accessibility by searching for footprints in the experimental data initially, before calculating F scores and extracting the data from the equivalent region in the control sample. To estimate if the F score fold change of a footprint is significant, we built an empirical null model by shuffling the C score of both samples within the mitochondrial genome 1000 times and for each locus, calculating an F score fold change from each pair of the shuffled data sets. For the candidate footprint loci, the central footprint region was required to have a C score(experiment) greater than its C score(control), and the F score fold change was filtered to achieve an expected 5% false discovery rate (FDR) relative to a score obtained from random shuffling 1000 times42. The propensity for secondary structure was quantified according to the ratio of double-stranded RNase cleavage over single-stranded, its R score:Technical replicates were pooled and sequenced reads trimmed of adapter sequences with cutadapt v1.10, using default parameters, and aligned to the mouse genome (mm10) with Bowtie243, and the log2 fold change of R scores calculated as:Violin plots were generated with the vioplot packageControl footprint analyses of the cytoplasmic 28S rRNA were carried out in an identical manner, except alignment was performed to the GENCODE vM14 reference transcript set combined with the 28S rRNA sequence downloaded from GenBank (NR_003279.1), and no significant footprints were identified (Supplementary Data Lrpprc–FLAG transgenic mice20 were maintained in DMEM medium (Thermo Fisher Scientific) supplemented with GlutaMAX-I, 10% fetal bovine serum, 1% penicillin-streptomycin and 1× MEM non-essential amino acids. HeLa Tet-On cells stably transformed with pTRE-hLRPPRC-FLAG20 were maintained in DMEM medium supplemented with GlutaMAX-I, 10% fetal bovine serum, 1% penicillin-streptomycin and 200 μg ml−1 hygromycin44. All cells were tested and found to be free of mycoplasma. Induction of LRPPRC-FLAG expression was achieved by addition of 2 μg ml−1 doxycycline to the incubation medium for 72 h. PAR-CLIP was performed according to Spitzer et al.45, with the exception that cells were lysed in lysis buffer (10 mM Tris-HCl (pH 7.5), 260 mM sucrose, 100 mM KCl, 20 mM MgCl2, 1% digitonin) in the presence of RNase inhibitor (New England Biolabs) and protease inhibitors (Roche). Lysates were clarified by centrifugation and diluted in lysis buffer without digitonin until the final digitonin concentration was 0.2%. Diluted lysates were incubated with anti-FLAG M2 magnetic beads for 2.5 h at 4 °C. Beads were washed in lysis buffer with 0.1% digitonin and then in lysis buffer without digitonin. RNA library preparation and sequencing was performed by Vertis Biotechnologie  using unique molecular identifiers to identify PCR duplicates by incorporating 10 random bases in the 5′-adapter, according to Kivioja et al.46.MEFs from 47 v1.10, requiring a minimum length after trimming of 14 nt. As PAR-CLIP libraries detect cross-linked regions utilizing photoactivatable ribonucleosides, the reads will contain a large number of sequence mismatches relative to the reference. To prevent misalignment of mitochondrial reads to nuclear mitochondrial sequences, the mouse and human genome reference sequence (mm10 and hg38) was masked for these regions based on the mm9 and hg19 NumtS tracks from UCSC, with coordinates converted to mm10 and hg38 with UCSC liftOver48. Trimmed reads were aligned to the Numts-masked genome sequence with Bowtie49 v1.12 (–k 1 –n 2 –M 100 –chunkmbs 512 –best –strata). Cross-linked regions were identified with BMix50, using default parameters . MEME51 4.11.2 was used to search for ungapped motifs within the binding sites identified by BMix . Strand-specific coverage profiles were generated with BEDtools v2.26.041. To assess the regions of the mitochondrial genome that were found to be overlapped by both PAR-CLIP and RNase footprinting, the mitochondrial genome was binned into 50 nt windows with BEDtools v2.26.041. The number of PAR-CLIP sites or RNase footprints overlapping each region was calculated and the significance of the number of sites overlapped by both was tested with a hypergeometic test.The PAR-CLIP libraries were prepared using indexed adapter containing an 8 bp barcode followed by 8 random bases to allow multiplexing and unambiguous detection of PCR duplicates. Following de-multiplexing and de-duplication, adapter sequences were removed from PAR-CLIP reads with cutadapt32. Codon-optimized ORFs corresponding to the mature forms of human and mouse LRPPRC or SLIRP were expressed in Rosetta 2 cells  by induction with 0.5 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) at 30 °C for 16 h in Enpresso B media (Biosilta). After lysis, the proteins were purified over a His-Select Ni2+ (Sigma-Aldrich) resin and dialyzed against H-0.2  after the addition of TEV protease at a 1:50 protease:protein ratio. Further purification was conducted over a heparin column equilibrated in H-0.2. After washing with H-0.2, the proteins were eluted with H-0.2 buffer containing 600 mM NaCl and purified to homogeneity over a HiLoad 16/60 Superdex 200 gel filtration column  in buffer H-0.2 lacking glycerol.Recombinant human and mouse LRPPRC and SLIRP proteins were expressed and purified as follows:32. Purified proteins were incubated at room temperature for 30 min with fluorescein labeled RNA oligonucleotides (Dharmacon) in 10 mM HEPES (pH 8.0), 1 mM EDTA, 50 mM KCl, 2 mM DTT, 0.1 mg ml−1 fatty acid-free BSA, and 0.02% Tween-20. Reactions were analyzed by 10% PAGE in TAE and fluorescence was detected using a Typhoon FLA 9500 biomolecular imager (GE Lifesciences). RNA chaperone activity was assayed according to Rajkowitsch et al.51. Briefly, the following oligoribonucleotide 21R+: 5′-Cy3-AUGUGGAAAAUCUCUAGCAGU-3′ and its complement 21R−: 5′-Cy5-ACUGCUAGAGAUUUUCCACAU-3′ (Dharmacon) were hybridized in the presence or absence of 1 μM of mouse LRPPRC–SLIRP complex. Aliquots were removed at specific time points, mixed on ice with stop buffer and separated on a 20% native polyacrylamide gel at 4 °C. Fluorescence was detected with a Typhoon FLA 9500 biomolecular imager (GE Lifesciences).RNA electrophoretic mobility shift assays were performed as follows:mt-Nd1, mt-Cyb and mt-Nd2 was examined using a modified 3′-RACE method52, where the Terminator nuclease treatment was omitted, since mitochondrial RNAs are not capped, and a universal or poly(A)-specific anchor primers were used in place of the “U-select primer”, since no poly(U) polymerase was present in our samples. The following gene-specific primers were used to identify the lengths of 3′-end polyadenylated products from mt-Nd1: 5′-GCGGGAGTACCACCATACAT-3′; mt-Nd2: 5′-TCCACCCTAGCTATCATAAGCA-3′; and mt-Cyb: 5′-GCCAACTAGCCTCCATCTCA-3′.Polyadenylation of Lrpprc knockout mouse hearts on an Illumina HiSeq platform, according to the Illumina Tru-Seq protocol. We used random hexamer primers for cDNA library generation and carried out cytoplasmic rRNA depletion using the Ribo-Zero rRNA removal kit. Sequenced reads were aligned against the mouse transcriptome with RSEM v1.2.31 (–bowtie2 –strandedness reverse –paired-end) using the GENCODE vM13 transcript set combined with custom mitochondrial rRNA and mRNA sequences with an additional 50 adenine residues added to the 3′-ends to allow mapping of polyA tails. Gene-level counts were imported and analyzed for differential expression with tximport v1.4.053 and DESeq2 v1.16.1. Coverage profiles for each mitochondrial transcript were generated from full fragment BED files with BEDtools v2.26.041.RNA sequencing was performed on total RNA from four control and four All the data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Information. All the sequencing data generated in this study are available at the NCBI Gene Expression Omnibus . The additional data related to this paper may be requested from the authors.Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5


Self-assembling peptide hydrogels offer a novel 3-dimensional platform for many applications in cell culture and tissue engineering but are not compatible with current methods of RNA isolation; owing to interactions between RNA and the biomaterial. This study investigates the use of two techniques based on two different basic extraction principles: solution-based extraction and direct solid-state binding of RNA respectively, to extract RNA from cells encapsulated in four β-sheet forming self-assembling peptide hydrogels with varying net positive charge. RNA-peptide fibril interactions, rather than RNA-peptide molecular complexing, were found to interfere with the extraction process resulting in low yields. A column-based approach relying on RNA-specific binding was shown to be more suited to extracting RNA with higher purity from these peptide hydrogels owing to its reliance on strong specific RNA binding interactions which compete directly with RNA-peptide fibril interactions. In order to reduce the amount of fibrils present and improve RNA yields a broad spectrum enzyme solution—pronase—was used to partially digest the hydrogels before RNA extraction. This pre-treatment was shown to significantly increase the yield of RNA extracted, allowing downstream RT-qPCR to be performed. In the past decade a variety of designs have emerged that allow the synthesis of short peptides that self-assemble into fibrillar structures that, above a critical gelation concentration (CGC), associate and/or entangle to form 3D, percolated networks swollen by water; hence hydrogel.[In the past two decades, significant efforts have been made to design 3-dimensional scaffolds that mimic the extracellular matrix (ECM) for a variety of biomedical applications, such as tissue engineering and regenerative medicine. One such class of material are hydrogels, as these highly hydrated materials can be engineered to mimic the cellular niche., 2 A varhydrogel., 5 TheseZhang’s design has been the basis of many self-assembling peptide variants, some of which include: EAK16,, 6 KLD12To determine the suitability of 3D matrices / scaffolds for cell culture, it is important to understand how the biomaterial influences cell behaviour. The biological response of cells to their surroundings is mirrored by changes in gene expression which can be observed using various techniques, including: northern blotting, quantitative polymerase chain reaction (qPCR) and microarray analysis. Regardless of the technique, it is crucial to first isolate good quality RNA. Most commercially available kits for RNA extraction rely on one of two basic principles, either: 1) solution-based, phase-separation using guanidinium thiocyanate, phenol and chloroform; or 2) direct, solid-state binding of RNA to a silica membrane within spin columns under conditions of high ionic strength. The firsIsolating sufficient amounts of good quality RNA from cells encapsulated within hydrogels has proven difficult with gel contaminants hindering downstream applications, such as reverse-transcription-qPCR (RT-qPCR)., 21 Opti® (Sigma-Aldrich); and one column-based, RNeasy Mini Kit® (Qiagen). The effect of first enzymatically degrading the hydrogels prior to RNA extraction was also investigated. The extracted RNA was evaluated in terms of: yield and purity, using UV spectroscopy; integrity, using electrophoresis; and quality, using RT-qPCR analysis of common housekeeping genes to assess its suitability in downstream applications.Typically β-sheet forming self-assembling peptides tend to contain multiple protonated residues. The overall charge carried by the peptide/peptide fibre is related to the excess cationic residues present in the sequence. If the peptide contains the same number of cationic (e.g: lysine and arginine) and anionic (e.g.: glutamic acid and aspartic acid) residues then the peptide/peptide fibre will be neutral at pH 7. Alternatively if there are a higher number of cationic residues then the peptide/peptide fibre will carry a net positive charge. It is therefore of little surprise that these hydrogels also interfere with the RNA extraction process. In the present study we compare and optimise the extraction of RNA from human endothelial kidney cells (HEK293) encapsulated in four different β-sheet forming self-assembling peptide-based hydrogels with varying net positive charges: PGD-Alpha1 , PGD-Alpha2 (medium net positive charge), PGD-AlphaProB (high net positive charge) and PGD-AlphaProC (high net positive charge) at pH 7. These peptide hydrogels were selected because they offer a range of mechanical properties and relative net positive charges at neutral pH which we have shown recently can affect cell behaviour. This setDesign Technologies : PGD-Alpha1 , PGD-Alpha2 (medium net positive charge at pH 7), PGD-AlphaProB (high net positive charge at pH 7) and PGD-AlphaProC (high net positive charge at pH 7). Peptide solutions corresponding to each hydrogel formulation for electrophoresis were kindly supplied by PeptiGelDesign Technologies. Additional information on the hydrogels is available on request to PeptiGelDesign Technologies (www.peptigeldesign.com).All materials and reagents were purchased from Thermo Fisher Scientific , unless stated otherwise. The following four peptide hydrogels were purchased from PeptiGel®; Gilson, Bedfordshire, UK) into 24-well cell culture inserts , to obtain a final cylindrical hydrogel with a height of ~1.5 mm. Cells were cultured to 80% confluence, detached using TrypLE cell dissociation reagent and re-suspended to achieve a stock solution of 400 × 106 cells mL−1. A 10 μl aliquot of the cell stock solution was physically mixed into the hydrogel using the tip of a 20 μL pipette, thereby producing a hydrogel of 100 μL containing ~ 4 × 106 cells. Media was then placed in and around the insert and the hydrogels were placed in an incubator at 37 °C in a 95% humidified atmosphere (20% O2) with 5% CO2. By incubating the hydrogels with media all hydrogels were subsequently buffered to pH 7. For cell-only controls, cells were treated as above but re-suspended in 90 μL of Dulbecco’s phosphate-buffered saline (dPBS)  with no media incubation.Human endothelial kidney cells (HEK293)  were maintained under standard cell culture conditions in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin solution and MEM non-essential amino acids solution (1 ×). At 80%–90% of confluence, cells were sub-cultured using TrypLE cell dissociation reagent, transferred to a Falcon tube and centrifuged to remove the TrypLE solution. The cell pellet was then re-suspended in fresh medium and adjusted to the required cell concentration before being re-seeded onto tissue culture plastic. For 3D cell encapsulation, 90 μL of each hydrogel was first pipetted using a precision positive displacement microliter pipette  or TRI Reagent® . Briefly, the hydrogels were seeded with ~ 4 × 106 cells and incubated for 30 minutes. The cell culture medium was then removed before each hydrogel was washed three times in dPBS for 5 minutes. When using the kits alone (TRI Reagent method and RNeasy Mini Kit (MK) method), the hydrogels were then suspended directly in either 1 mL of TRI Reagent® or 350 μL of RNeasy Lysis Buffer® and homogenised at speed 6 (out of 10) for 15 seconds using an automated homogeniser . RNA was then isolated according to manufacturer’s instructions. For the combined TRI reagent® and RNeasy Mini Kit® method (TRI Reagent + RNeasy MK method), following the washing step the hydrogels were homogenised, as described, in 1 mL of TRI Reagent®. Then 0.2 mL of chloroform was added and the solution was incubated at room temperature for 15 minutes. The solution was then centrifuged at 12,000 × g for 15 minutes at 4 °C. The aqueous phase was then removed and combined with an equal volume of 70% ethanol and then applied to the RNeasy MK spin columns, as per manufacturer’s recommendations. For all methods, the RNA was eluted in 30 μL of RNase-free water. For the enzymatic degradation, each hydrogel was washed as previously mentioned, removed from the insert and placed in a microcentrifuge tube. Then, 4 volumes of pronase solution (10 mg mL−1 stock solution prepared in DNase and RNase-free water) were added to the hydrogel, triturated and placed in a 37 °C water bath for 5 minutes with gentle agitation every minute. The hydrogel solution was then added directly to the RNeasy® lysis buffer, homogenised and extracted as described above.RNA was extracted using the following commercial kits: RNeasy Mini Kit6 cells and re-suspended in 30 μL of water. The concentrations of RNA extracted from all samples were calculated using UV spectroscopy by measuring the absorbance at 260 nm (A260). The value was calculated using the Lambert-Beer law based on absorbance at 260 nm (1 A260 unit = 40 mg mL−1 RNA). For each sample, a 1 μL aliquot was taken and measured using a spectrophotometer . From the absorbance spectra (220–350 nm), the A260/A280 and A260/A230 ratios were used to assess the purity of RNA. Ratio values of ≥ 1.8 for A260/A280 and ≥ 2 for A260/A230 are an acceptable indicator of good quality RNA.[260/A220 ratio was used to estimate the level of peptide contamination. Three independent samples were measured with the data shown as mean ± standard error of mean (SEM). To determine RNA integrity, each RNA sample (100 μg) was run on a TapeStation automated electrophoresis analysis system . Separation profiles composed of intact 28S (4.5 kb), 18S (1.9 kb) and small RNA species were compared. The RNA integrity number (RIN) values were determined using the TapeStation analysis software. Three independent samples were measured for each sample.For all samples and controls, RNA was extracted from 4 × 10® method, described previously. Then 1 μg of total RNA was incubated with solutions of each peptide at different molar concentrations, before then being run on a non-denaturing agarose (0.8%) gel. Briefly, 5 mM of lyophilized peptide, corresponding to each hydrogel formulation, was dissolved in RNase-free water, sonicated for 1 hour and left overnight at 4 °C. A serial dilution was created for each peptide sequence by diluting the samples 1:5 with RNase-free water. Then, equal volumes of RNA and peptide solution were combined to create a 30 μL solution at the following peptide concentrations: 2.5 mM, 0.5 mM, 0.1 mM, 0.02 mM and 0.004 mM; each containing 1 μg of RNA. The RNA-peptide solutions were incubated on ice for 30 minutes, mixed with DNA loading buffer (6 ×) and loaded into the well. Gel electrophoresis was performed at 75 V for 40 minutes in 0.5 × TBE running buffer. For reference, an RNA-only sample was run to show the expected bands and relative intensities (positive control). A peptide-only control, at the highest concentration (2.5 mM), was also run (negative control). The agarose gel was prepared in 0.5 × TBE running buffer using molecular grade agarose . The agarose gel and running buffer were diluted using diethyl pyrocarbonate (DEPC)-treated water. RNA was stained with SYBER® Safe  which was incorporated into the gel before casting. The gel was imaged using a Chemidoc imaging system . All equipment was washed according to the following protocol: wash with 1% sodium dodecyl sulphate (SDS) in distilled water, then with 100% ethanol, then incubate with 3% hydrogen peroxide (H2O2) in distilled water for 10 minutes and finally rinse with DEPC-treated water.RNA was isolated from HEK293 cells using the TRI Reagent®/ TRI Reagent®) or 1 μg (post-enzymatic degradation) of RNA from each sample was converted into cDNA. Briefly, total RNA was treated with amplification grade DNase I  and transcribed into cDNA using a high capacity RNA-to-cDNA kit, supplemented with RNase inhibitor, as per manufacturer’s instructions. For qPCR, cDNA was diluted 1: 1 (0.5 μg) or 1:3 (1 μg) with RNase-free water and the Brilliant III Ultra-Fast SYBR® QPCR kit  was used following a two-step RT-qPCR protocol: enzyme activation for 3 minutes at 95 °C, followed by 40 cycles of denaturation for 5 seconds and annealing/elongation for 25 seconds at 60 °C. All reactions were performed in triplicate using pre-designed primers  for 5 common housekeeping genes: GAPDH, encoding glyceraldehyde 3-phosphate dehydrogenase; RPL13A, encoding ribosomal protein L13A; ACTB, encoding β-actin; B2M, encoding β2-microglobulin; and RRN18s, encoding 18S ribosomal RNA. All qPCR reactions were performed on an ABI 7500 Fast System (Applied Biosystems). Threshold cycle (Ct) values were determined by using the Sequence Detection System software. The expected product sizes were confirmed by melting temperature (Tm) analysis in the range 60–95 °C; all samples produced identical, overlapping melting curves with a single peak at the expected Tm value.For RT-qPCR, total RNA was isolated, as previously described, and either 0.5 μg , 5 mM cysteine HCl; Pronase: 10 mg mL−1 in HPLC grade H2O; Proteinase K: 400 μg mL−1 in 50 mM TrisHCl and 2.5 mM CaCl2 and Thermolysin: 1 mg mL−1 in 50 mM TrisHCl and 0.5 mM CaCl2. Trypsin was used as supplied by the manufacturers. Peptide hydrogels (250 μL) were pipetted into 12-well ThinCerts™, covered with medium (as used in cell culture experiments) and allowed to incubate for 24 hours in a 37 °C / 5% CO2 humidified incubator. Hydrogels were removed from the inserts and added to 1 mL of enzyme solution. Samples were incubated at 37 °C and triturated with a Pasteur pipette for 10 seconds every minute. Hydrogel samples were collected after 5 minutes of incubation with the enzyme and were diluted to 1mg/ml−1 using H2O:CH3CN (1:1 v/v) HPLC grade solvent mixture (80% H2O / 20% CH3CN with 1% TFA). HPLC was used to measure the amount of non-degraded peptide in each sample. An analytical scale Phenomenex Jupiter 4 μm Proteo column 90 A° (250 × 4.66 mm) was used with a flow rate of 1 mL min−1. The column was equilibrated in 90% H2O / 10% CH3CN with 0.05% TFA, followed by a 200 μL sample injection. The elution gradient used went from 90% H2O / 10% CH3CN to 30% H2O / 70% CH3CN  over 45 minutes. The percentage of non-degraded peptide was calculated using the following equation:AUPt and AUPc are the areas under the HPLC peaks, as calculated by Chromeleon software, for the sample treated 5 minutes with the enzyme and the untreated control sample, respectively.[Enzymes were prepared as per manufacturer recommendations at the following concentrations: Papain: 6.25 mg mLectively. At leastP ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001, compared to the control. For hydrogel degradation, the mean and SEM were calculated from three independent repeats and statistical significance was determined using the Kruskal-Wallis H-test.The RNA yield and purity were calculated from three independent samples, and the RT-qPCR experiments were also carried out from three independent samples and run in triplicate. The mean and SEM were calculated from the three independent repeats, where the averages of the RT-qPCR triplicates were used for each biological repeat. To compare the mean values, a two sample t-test was performed and statistical significance was determined when: *, β-sheet forming self-assembling peptides typically contain polar residues that will carry positive (e.g: lysine & arginine) or negative (e.g.: aspartic acid & glutamic acid) charges around neutral pH. In addition, if unprotected and unmodified these peptides will also have a carboxyl and an amino end-group, which will also carry a negative and a positive charge at pH 7, respectively. As a result, if the number of positively charged groups is higher than the number of negatively charged groups on the peptide, then both the peptide monomer and its fibrillar aggregate (peptide fibril) will carry a net positive charge around neutral pH. Therefore, the peptide and/or peptide fibril can interact with the negatively charged RNA and interfere with its extraction process. To demonstrate that RNA interacts electrostatically with the peptide and/or peptide fibril of our hydrogels, gel electrophoresis was performed on RNA that had been pre-incubated with solutions of peptide from each hydrogel formulation at different molar concentrations . The hypothesis being; if the peptide and/or peptide fibrils interact with RNA then it will affect the RNA mobility and cause a shift in the expected band sizes. As such, RNA-peptide samples were run on a non-denaturing agarose gel and the separation profiles were compared to an RNA-only control. As can be seen from RNA-peptide molecular complexing through electrostatic interactions has been observed with signal peptides, which are characterised by having a positively charged region. The fact® (Sigma-Aldrich), and one column-based, RNeasy Mini Kit® (Qiagen). Three different protocols were used: 1) TRI Reagent method (TRI Reagent® kit only), 2) RNeasy MK method (RNeasy Mini Kit® only) and 3) TRI Reagent + RNeasy MK method (TRI Reagent® kit followed by RNeasy Mini Kit®). This latter method was designed to test whether the RNeasy MK method could improve the purity of RNA extracted using the TRI Reagent method. Cells were encapsulated within the peptide hydrogels that were then allowed to set in media for 30 minutes. This time was selected in order to minimise changes in cell gene expression between the hydrogels. Cell-only 3D controls (cells suspended in PBS for 30 min.) were carried out for each extraction method. Both TRI Reagent and RNeasy MK methods were capable of isolating comparable concentrations of pure RNA from the controls. As expected for the TRI Reagent + RNeasy MK method a lower concentration of pure RNA was extracted compared to either method used separately , PGD-AlphaProC (266 ± 112 ng μL−1) and PGD-AlphaProB (290 ± 74 ng μL−1). The lowest yield of RNA was obtained for PGD-Alpha1 (108 ± 31 ng μL−1), approximately half the concentration obtained from the other three hydrogels. In all cases, the RNA concentrations were much lower than that of the cell-only control (733 ± 34 ng μL−1). For the RNeasy MK method, there was little difference between the amount of RNA extracted from PGD-AlphaProC (535 ± 53 ng μL−1) and PGD-AlphaProB (549 ± 54 ng μL−1), although the concentration of RNA obtained was almost double that of the TRI Reagent method. No RNA was extracted from PGD-Alpha2 when using RNeasy MK method. For PGD-Alpha1 the concentration of RNA obtained (161 ± 57 ng μL−1) was similar to the amount extracted using the TRI Reagent method but much lower than that extracted from PGD-AlphaProC and PGD-AlphaProB. For all four hydrogels the concentration of RNA extracted using the RNeasy MK method was lower than the cell-only control (862 ± 45 ng μL−1). For the TRI Reagent + RNeasy MK method, as expected the relative concentration of RNA obtained for each hydrogel was related to the amount of RNA that could be extracted when using the TRI Reagent method. Except for PGD-AlphaProB, for which no statistically significant difference could be observed, the concentration of RNA obtained was much lower using the TRI Reagent + RNeasy MK method than the TRI Reagent method only.UV absorbance at 260 nm  was used260/A280 and A260/A230 . In this case electrostatic interactions play a key role in the separation process. When using this method, the purity of RNA extracted from cells encapsulated in the two systems carrying the highest positive net charge—PGD-AlphaProB and PGD-AlphaProC—was comparable to the cell-only control and suggests a stronger binding affinity of RNA to the matrix rather than to the peptide fibrils. However, the lower yields compared to the cell-only control still suggests that some RNA is lost and washed away; most likely a result of peptide fibrils interfering with the binding of RNA to the silica matrix. For PGD-Alpha1, the neutral peptide, contaminated RNA was extracted. The AFor PGD-AlphaProB, comparably pure RNA was also extracted using the TRI Reagent + RNeasy MK method. For PGD-AlphaProC, the prior use of the TRI Reagent method resulted in contamination that was not observed when using the RNeasy MK method alone. Since RNA extracted from PGD-Alpha1, PGD-Alpha2 and PGD-AlphaProC using the TRI Reagent + RNeasy MK method still contained contaminants, and also resulted in lower RNA yields, it was not investigated further.To examine the integrity of the RNA extracted using the TRI Reagent and RNeasy MK methods, samples were analysed using electrophoresis . All conTo assess the quality of RNA, cDNA was prepared from all samples of RNA extracted using both methods (TRI Reagent and RNeasy MK). The expression levels  of two commonly used endogenous control genes; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and ribosomal protein L13A (RPL13A), were analysed using RT-qPCR and compared to values obtained from the cell-only controls . A low CRNA extracted from cells encapsulated in PGD-AlphaProB and PGD-AlphaProC showed no difference in amplification efficiency compared to the controls for either extraction method. This data indicates that, in both cases, the amount of RNA extracted and its purity was high enough to perform RT-qPCR. For PGD-Alpha1, Ct values similar to the control and < 20 were obtained only when RNA was extracted using the RNeasy MK method. For RNA extracted from PGD-Alpha1 using the TRI Reagent method, a Ct value > 20 was obtained; confirming that the concentration of RNA for this sample was an overestimate and so less RNA was converted into cDNA for use in the RT-qPCR reaction. Finally for PGD-Alpha2, the Ct values obtained for RNA extracted using the TRI Reagent method were similar to the control sample. No results are shown for PGD-Alpha2 using the RNeasy MK method as no RNA could be extracted. These results suggest, in particular for the TRI Reagent method, that the peptide carried through the extraction process does not interfere with RT-qPCR reaction suggesting, as above, that there is no or weak complexation at the molecular level between the RNA and these peptides. These findings also clearly indicate that the amount of RNA extracted, but more importantly the purity of RNA is critical in generating reliable and reproducible RT-qPCR data.As discussed above, we hypothesised that the interaction between RNA and the peptide fibrils was the main reason for the low RNA yields obtained. In an effort to reduce the amount of fibrils present in the samples, enzymes were used to attempt to proteolytically degrade the peptide monomers corresponding to each of the four hydrogels investigated.260 absorbance values–Five commercially available enzymes / enzyme mixtures: papain, pronase, proteinase K, thermolysin and trypsin, were tested for their ability to digest each of the four peptide hydrogels. The fraction of non-degraded peptide monomer was estimated after 5 minutes of digestion  and was The RNA integrity was once again assessed by electrophoresis . For allFinally the suitability of the RNA isolated from these hydrogels, following enzymatic digestion, for RT-qPCR was evaluated by comparing the expression levels of five common housekeeping genes encoding: glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L13a (RPL13A), β-actin (ACTB), β2 microglobulin (B2M) and 18S ribosomal RNA (RRN18s).All five primer sets produced equally low Ct values for the control and each of the four peptide hydrogels . The melWe have investigated the effect of RNA-peptide interactions on RNA extraction protocols. We have shown that RNA interactions with peptide fibrils interfere with the extraction process, rather than molecular complexing of RNA and peptide monomer. Moreover, the amount of RNA extracted was critical to afford good quality RT-qPCR data. However, the contamination of RNA by small amounts of peptide carried through the extraction process did not interfere with the enzymes involved in cDNA synthesis nor RT-qPCR. Our results also show that methods based on solid-state binding of RNA are more suited to the extraction of RNA from β-sheet forming self-assembling peptide hydrogels, for this strong binding competes directly with the RNA-peptide fibril interactions. Pre-digestion of these hydrogels using a broad spectrum enzyme—pronase—was shown to significantly improve the extracted RNA yields for all four hydrogel formulations and makes this approach potentially applicable more universally across peptide-based hydrogels. The ability to extract high yields of high purity RNA is a key step towards the general use of these materials in the biological and medical fields and therefore to the fulfilment of their full potential.


APOBEC3A and APOBEC3G cytidine deaminases inhibit viruses and endogenous retrotransposons. We recently demonstrated the novel cellular C-to-U RNA editing function of APOBEC3A and APOBEC3G. Both enzymes deaminate single-stranded DNAs at multiple TC or CC nucleotide sequences, but edit only a select set of RNAs, often at a single TC or CC nucleotide sequence. To examine the specific site preference for APOBEC3A and -3G-mediated RNA editing, we performed mutagenesis studies of the endogenous cellular RNA substrates of both proteins. We demonstrate that both enzymes prefer RNA substrates that have a predicted stem-loop with the reactive C at the 3′-end of the loop. The size of the loop, the nucleotides immediately 5′ to the target cytosine and stability of the stem have a major impact on the level of RNA editing. Our findings show that both sequence and secondary structure are preferred for RNA editing by APOBEC3A and -3G, and suggest an explanation for substrate and site-specificity of RNA editing by APOBEC3A and -3G enzymes. The histidine and cysteine residues coordinate Zn2 + (The APOBEC3 (A3) family of cytidine deaminases restricts endogenous retroelements and exogenous viruses and therefore plays an important role in the vertebrate innate immune system . Recent te Zn2 + , and thete Zn2 + .A3 proteins can bind to both ssDNA and ssRNA oligonucleotides . Howeverin vitro, whereas DNA editing is non-specific and occurs at multiple TC nucleotides (edited C underlined) (APOBEC3A (A3A) is highly expressed in monocytes and macrophages and its expression is upregulated on treatment with interferon-α . We receerlined) . More reerlined) . Intereserlined) . In the erlined) . These berlined) . HoweverCell cultures of primary monocyte-enriched PBMCs, exposure to hypoxia (1% oxygen) and interferon type 1 were performed as previously described .Plasmid constructs for expression of human A3A cDNA, for the generation of C-terminal Myc-DDK-tagged A3A and A3G, pcDNA 3.1(+) vector (used as an empty vector control) were obtained from sources mentioned in The TLA-HEK293T human embryonic kidney cells (293T cells)  were transfected with plasmid DNA using the jetPRIME (Polyplus-transfection) reagent as per the manufacturer’s instructions. The transfection efficiency was 60%–80% as assessed by fluorescent microscopy of cells that were transfected with the pLemiR plasmid  for expression of a red fluorescent protein. Cells were harvested 2 days following transfection.E. coli  transformed with a bacterial expression construct for C-terminal His6-tagged WT A3A was grown in Luria broth at 37 °C. The cells were induced for expression of the recombinant protein with 0.3 mM isopropyl β-D-1-thiogalactopyranoside and cultured overnight at 18 °C. A3A protein was purified from the lysates by affinity chromatography using the Ni-NTA His bind Resin (EMD Millipore).  The concentrated protein was stored in 25 mM Tris (pH 8.0) with 50 mM NaCl, 1 mM DTT, 5% v/v glycerol and 0.02% w/v sodium azide at −80 °C.The WT A3A was purified as described in SDHB, TMEM109 and APP RNAs were folded using the Mfold nucleic acid folding program (PRPSAP2 RNA were folded using both Mfold and RNAfold 2.3.2 (18 nucleotides (with 7 nucleotides flanking on each side of the tetra-loop sequence) of WT  program . 18 nuclld 2.3.2 . No optiSDHB , TMEM109 and APP RNAs were amplified using oligonucleotide primers listed in SDHB RNAs were generated from the 1.1 kb complete SDHB ORF encoding plasmid  following site-directed mutagenesis and XhoI linearization of the plasmid DNA. Sanger sequencing was performed on all DNA templates to confirm the desired mutations, which were then in vitro transcribed to generate RNAs using reagents and methods provided with the MEGAscript or MEGAshortscript T7 Transcription Kit . RNAs isolated from the transcription reaction were treated with DNAse I  and their integrity was verified by electrophoresis on an agarose gel.The DNA templates for generating WT and mutant In vitro RNA-editing assay with purified APOBEC3A contained 1–10 µM APOBEC3A, 50 pg of synthetic RNAs, 10 mM Tris (pH 8.0), 50 mM KCl and 10 µM ZnCl2. The reactions were incubated for 2 h at 37 °C. RNA was purified from the reactions using TRIzol  as per the manufacturer’s instructions and reverse transcribed to generate cDNAs as described previously  was assessed by allele-specific AS-RT–qPCR as described previously (r = 0.94) between SDHB 136C>U RNA editing level measurements obtained by AS-RT-qPCR and Sanger sequencing, although estimates obtained by Sanger sequencing were somewhat higher (slope of correlation = 0.71). Thus, it is possible that true editing levels in SDHB constructs M2, M8, M9 and M10, which were estimated by Sanger sequencing  in the PRPSAP2 expression plasmid . Total RNA was isolated and RT-PCR was performed using a PRPSAP2-specific forward primer and a vector specific reverse primer complementary to the DDK tag sequence  for the WT and mutant cDNAs generated from RNAs are listed and underlined in C tetra-loop flanked by an average of three palindromic nucleotides (C (N is any nucleotide) flanked by an average of four palindromic nucleotides (C1QA mRNA (rs172378) markedly increased C>U RNA editing three nucleotides upstream of the polymorphism (C1QA mRNA (rs172378) is predicted to increase the stem length and subsequently the stem stability of a putative stem-loop structure, resulting in increased RNA editing. While the CCCCCUCGG(a/a) (expressed SNP variation in lower case) sequence shows 11% and 21% editing in 2 donors, CCCCCUCGG(a/g) increased the average editing to 40% when monocyte-enriched peripheral blood mononuclear cells (MEPs) were exposed to hypoxia/IFN-1 SDHB , TMEM109 APP PRPSAP2 . TMEM109 is predicted to form a hepta-loop with a four bp stem and a ΔG value of −1.7 kcal/mol. To test the importance of stem-loop structures for A3A and A3G-mediated RNA editing, we created various mutations (see methods) in the putative loop and stem regions of A3A substrates SDHB, APP and TMEM109 and the A3G substrate PRPSAP2   and one , R222W)  for furthe Mfold  or RNAfohe Mfold  nucleic AP2 RNAs . The pre PRPSAP2 –1D and aquencing . In eithC) in A3A and A3G substrates. C>U editing sites are most commonly present within a CCAUCG sequence motif in ssRNA A3A substrates (C>CC) in the predicted loop region of the SDHB RNA , increased editing to 364% (C [A/G] sequence and therefore prefer C at −1 position (C>GC) in the A3G substrate PRPSAP2 RNA loop markedly reduced RNA editing to 15% as compared to WT (We tested the importance of the −1 nucleotide (nt)  of the PRPSAP2 RNA abolished A3G-mediated RNA editing  in A3A substrates is occupied by a G base-paired with C or in some cases A, which was substituted with A in M8 (37% editing) and C in M9 (17% editing)  . Hence, B mutant  increaseB mutant . These rPRPSAP2 and the A3A substrate TMEM109. Interestingly, weakening the putative stem by substituting two G-C base pairs with A-U base pairs in PRPSAP2 RNA only affected RNA editing slightly (80%)  , M6P. As, the Mfold program predicts a ΔG value of −5.2 kcal/mol for TMEM109 M1T structure  and changing CC>GC in PRPSAP2 RNA (A3G substrate) markedly reduces or abolishes RNA editing by these enzymes respectively, thus highlighting the importance of the −1 nt in the loop  to convert adenosine to inosine. Secondary structure in the form of internal loops, bulges and mismatches in the dsRNAs dictate site-specificity in these enzymes resulting in the editing of a few adenosines as compared with long (>100 bp) dsRNA substrates, in which more than half of the adenosines are edited . The sitficiency . Althougficiency . Also, tficiency .34) of the tRNAArg2 anticodon stem-loop and involves an induced fit of the RNA stem-loop into an inflexible protein cleft  has the active site characteristic of the cytidine deaminases and its mechanism of reaction is analogous to that of cytidine deaminases . TadA dein cleft . Site-spin cleft . Further by TadA . Another by TadA . Recentle of A3A . A crystC1QA (rs172378) has been previously linked to an increased risk of disease severity and nephritis in systemic lupus erythematosus , our findings provide evidence that the G allele of rs172378 may alter the secondary structure of mRNA to favor a stronger stem and thereby increase the RNA editing level. This alteration in the predicted stem-loop structure may in turn affect mRNA stability, turnover or translatability  and −4C>G  (mutated residue in lower case)  that causes a missense alteration in PRPSAP2. Mutations in the APP gene have been linked to Alzheimer’s disease. When we change −1C>T , this synonymous mutation increases the editing level of the missense RNA alteration  by 264%  on the level of RNA editing. The G allele of a common A/G synonymous SNP in ematosus . We obseematosus . RNA ediematosus . Althougtability . Further by 264% , M1A. ThRNA editing is a mechanism to diversify information encoded by a gene and of regulation of gene expression. Our work provides the first experimental information on how stem-loop structures of endogenous RNA substrates may be preferred for site-specific editing mediated by A3A and A3G cytidine deaminases that are highly expressed in innate immune cells. These enzymes have hundreds of substrates and a single synonymous mutation altering the secondary structure in the substrate RNA could have consequences on the resulting protein product. It is possible that other APOBEC3 enzymes may prefer stem-loop structures, pending the discovery of their RNA editing function. Thus, this study provides the basis for future structural and functional studies.10.7717/peerj.4136/supp-1Table S1Click here for additional data file.10.7717/peerj.4136/supp-2Table S2Click here for additional data file.10.7717/peerj.4136/supp-3Figure S1The predicted ΔG values are below each RNA structure.Click here for additional data file.10.7717/peerj.4136/supp-4Data S1Click here for additional data file.