TRAM, a conserved domain among RNA modification proteins ubiquitously distributed in organisms, occurs as an individual protein in most archaeal phyla and has a structural similarity to Csp proteins, yet its biological functions remain unknown. Through physiological and biochemical studies on four TRAM proteins from a cold adaptive archaeon Methanolobus psychrophilus R15, this work demonstrated that TRAM is an archaeal Csp and exhibits RNA chaperone activity. Three TRAM encoding genes  exhibited remarkable cold-shock induced transcription and were preferentially translated at lower temperature (18°C), while the fourth (Mpsy_2002) was constitutively expressed. They were all able to complement the cspABGE mutant of Escherichia coli BX04 that does not grow in cold temperatures and showed transcriptional antitermination. TRAM3066 (gene product of Mpsy_3066) and TRAM2002 (gene product of Mpsy_2002) displayed sequence-non-specific RNA but not DNA binding activity, and TRAM3066 assisted RNases in degradation of structured RNA, thus validating the RNA chaperone activity of TRAMs. Given the chaperone activity, TRAM is predicted to function beyond a Csp.Cold shock proteins (Csps) enable organisms to acclimate to and survive in cold environments and the bacterial CspA family exerts the cold protection via its RNA chaperone activity. However, most Archaea do not contain orthologs to the bacterial  Escherichia coli contains nine Csp proteins, among which CspA, CspB, CspG, and CspE are essential for cold acclimation of the bacterium. Typically, the cspA gene does not express at mesophilic temperatures but is remarkably induced by cold shock, and the level is reduced after the organism acclimates to the cold  are essential for organisms to grow and survive in cold environments. These proteins enable organisms to acclimate to downshifts in temperature by destabilization of cold-induced misfolded RNAs that may cause premature transcription termination, inefficient RNA turnover, and reduced translation. Two categories of bacterial Csps are classified in terms of their working mechanisms, the cold shock protein A (CspA) family of proteins functions mainly as RNA chaperones by affecting transcription or translation of a nascent transcript , while tthe cold . Differeld shock . Caulobary phase . This inE. coli cells when encountering temperature downshifts and assist the bacterium in acclimation to cold. Through RNA chaperone activity, the CspA family proteins prevent or melt incorrect RNA duplexes formed under lower temperatures, which would hinder efficient translation and regular turnover of a transcript.The most intensively studied CspA family are basic small proteins of approximately 70 amino acid residues, and the native folds contain an OB (oligonucleotide binding)-fold for binding RNA . They accspE is shown to affect the transcription of genes containing promoter-proximal elements that induce premature transcription termination. E. coli CspC and CspE stabilize the RpoS transcript, a sigma factor that specifically exerts roles under adverse conditions  broth.Strains used in this study and their characteristics are listed in Supplementary Table Methanolobus psychrophilus R15 is preserved in our laboratory and cultured at 18°C in mineral medium containing 20 mM trimethylamine, as previously described  values. To estimate the copy numbers of the transcripts, the expression plasmids pIN-0643, pIN-2002, pIN-3043, and pIN-3066, constructed in the following section, were serially diluted to generate a calibration curve by PCR amplification using the corresponding primer pairs  at 37°C for 5 h. RNA-dependent DNA synthesis was performed using the Moloney murine leukemia virus reverse transcriptase  according to the manufacturer’s protocol with random primers (Promega) and 2 μg of DNA-free RNA as the template. The generated cDNA was used as the template for quantitative PCR (qPCR). Real-time qPCR was conducted using the Eppendorf Mastercycler system  with 25 μL of SYBR green Premix (TaKaRa) and corresponding primers listed in Supplementary Table Mpsy_0643, Mpsy_2002, Mpsy_3043, and Mpsy_3066, were PCR-amplified using the genomic DNA of M. psychrophilus R15 as a template and the corresponding primer pairs . The plates were incubated for 2–5 days at 37°C, 22°C and 18°C, respectively.To test the archaeal TRAMs for cold protection, the open reading frames of the four TRAMs, E. coli BX04, IPTG-induced cultures for 2 h at 37°C were pelleted by centrifugation at 12,000 g at 4°C for 5 min and lysed by ultrasonication at 240 W using a cycle of 3 s sonication and 3 s pause for 15 min on ice. The total cellular proteins were electrophoresed on Tricine-SDS-PAGE and quantified by Western blot as described below.To examine the expression of TRAM proteins in Mpsy_0643, Mpsy_2002, Mpsy_3043, and Mpsy_3066, respectively. After digestion with NcoI/XhoI, the PCR products were cloned into the same sites of the expression plasmid pET28a , resulting in p28a-0643, p28a-2002, p28a-3043, and p28a-3066, respectively. Using the same approach, E. coli cspA was cloned into the expression plasmids as p28a-cspA. The pET28a-derived plasmids were each transformed into E. coli BL21 (DE3) pLysS . The E. coli BL21 (DE3) pLysS strains that each harbors these plasmids were cultivated at 37°C in LB medium containing 50 μg/ml kanamycin. When growth reached an OD600 of 0.4–0.6, 0.5 mM IPTG was added. IPTG-induced cultures were allowed to grow for additional 16 h at 22°C, and cells were harvested from 1 L culture by centrifugation, resuspended in 30 ml of lysis buffer (pH 8.0) containing 20 mM Tris-HCl, 1 M NaCl and 10% glycerol and lysed by ultrasonication at 300 W using a cycle of 3 s sonication and 3 s pause for 30 min on ice. After cell debris was removed by centrifugation, the supernatant was mixed with 0.5% (V/V) polyethyleneimine to remove any contaminated nucleic acids. The mixture was centrifuged at 10,000 g for 30 min at the same temperature, and proteins in the supernatant were collected by precipitation with (NH4)2SO4 powder to a final 70% saturation. The protein precipitation was dissolved in 15–20 ml of binding buffer  and dialyzed overnight at 4°C against the same buffer in 3.5 kDa-pore size dialysis tubing . The dialyzed mixture was centrifuged, and the supernatant was passed through a 0.22-μm filter. The His6-tagged recombinant proteins were purified by passage on a HisTrap HP affinity column , followed by HiTrap Q HP anion exchange column  and gel filtration chromatography through a Hiload 16/600 Superdex 75 pg column . Proteins were concentrated with Amicon Ultrafra-15 concentrators (Millipore) when necessary. The purified proteins were examined by Tricine-SDS-PAGE, and the concentrations were determined using a bicinchoninic acid (BCA) protein concentration assay kit . The purity of proteins (>99%) and residual nucleic acid were confirmed using a NanoDrop ND-100 Spectrophotometer. To identify the purified proteins, they were digested with trypsin in gels according to the method of For overexpression of the His6-tagged recombinant proteins, primer pairs P27/P28, P29/P30, P31/P32, and P33/P34 , and lysed by ultrasonication at 240 W in a cycle of 3 s sonication and 3 s pause for 15 min on ice. All manipulations were conducted following standard protocols  were used to wash the membrane with gentle shaking for 5 min, and 0.2% glutaraldehyde diluted in 1 × TBS was used to wash the membrane for 45 min. The TRAM3066-specific peptide and TRM2002 total protein-generated antibodies were prepared by the MBL . The secondary antibody was purchased from Easybio .A total of 25 ml of each cold-shocked R15 culture, which was cultured for different durations, was harvested at 12,000 rotocols  with a fThe density of the Western blotting protein band was determined using Bio-Rad Quantity One , and the amount of protein was calculated by referring to the gray value of the overexpressed protein.in vitro transcription using the MEGA shortscript T7 Kit  and primer pairs (P35–P47) listed in Supplementary Table in vitro transcription products were purified on 7% urea-PAGE using a ZR small-RNA PAGE Recovery Kit (Zymo Research), and the concentrations were determined using NanoDrop ND-100 Spectrophotometer. All in vitro transcribed RNAs were 3′-end biotinylated using a 3′-End Biotinylation Kit .The Pentaprobes (PP) library developed by 2, 10 mM KCl, 1 mM DTT, and 10% of glycerol), biotin-labeled RNA substrate, and purified recombinant proteins. After incubation at 25°C for 15 min, the reaction mixtures were loaded onto a 6% polyacrylamide gel and electrophoresed under 100 V for 1 h in 0.5 × TBE running buffer . Free RNA and RNA-protein complexes in gels were transferred to a nylon membrane at 380 mA for 45 min using a Trans-Blot transfer . After cross-linking using a GS Gene LinkerTM UV Chamber  for 2 min, a Chemiluminescent Nucleic Acid Detection Module kit (Thermo Scientific) was used to detect chemiluminescence by exposure on a Tanon-5200 Multi instrument .An RNA binding assay was performed in a 20-μl reaction mixture containing binding buffer  was first conditioned with 3–5 injections (10 ml/min) of 1 M NaCl in 50 mM NaOH until a stable baseline was obtained. The chip cells were washed twice with 0.05% SDS in buffer I  for 3 min. The biotinylated single-stranded oligonucleotides (SPR linker) were diluted to 10 μM in buffer II  and immobilized on a streptavidin-coated sensor chip SA at a flow rate of 5 ml/min for 10 min. Fifty mM NaOH was injected at 5 μl/min to remove unbound SPR linker until the response units (RU) reached 150. The A18C3 probe and Pentaprobe RNAs containing A18C3 tail were immobilized to SPR linkers in different flow cells by manual injection at a flow rate of 1 ml/min until the RU reached a stable state. TRAMs were diluted to 0 to 100 μM by a twofold dilution series with buffer I and injected using the K-inject command at room temperature. CspA and BSA were included as positive and negative controls, respectively. Buffer I (30 ml/min) was used as the running buffer. At the end of each cycle, chip cells were washed with 0.1% SDS to remove bound protein. Data analysis was performed using the BIA evaluation 3.0 program .A surface plasmon resonance (SPR) assay was performed to determine the binding affinities of TRAM3066 and TRAM2002 to ssRNAs on a BIAcore 3000 system  as described previously  with minin vivo nucleic acid melting activity of TRAMs, each of Mpsy_0643, Mpsy_2002, Mpsy_3043, and Mpsy_3066 cloned pINIII plasmids was transformed into E. coli RL211. The transformants were cultured overnight in LB complemented with 100 μg/ml ampicillin and diluted 100-fold in fresh medium. When the culture grew to an OD600 of 1.0, 6 μl cultures were spotted onto LB plates containing 100 μg/ml ampicillin, with or without 0.2 mM IPTG and 30 μg/ml chloramphenicol, and grown for 2–3 days. E. coli cspE and pINIII were used as positive and negative controls, respectively. Expression of TRAM proteins in strain RL211 was determined using the same procedure as in strain BX04.To determine the 32P] ATP  using T4 Polynucleotide Kinase  according to the manufacturer’s instruction. Radiolabelled PP6 was denatured at 85°C for 5 min and subject to a 30 min temperature-gradient descent till 10°C. 100 fmol of labeled PP6 was mixed with 1,000 pmol TRAM3066 protein in binding buffer . Various concentrations of RNase A  or RNase T1  were added to the PP6-TRAM3066 mixture immediately and incubated at room temperature for 10 min. Ten microliters mixture was loaded onto a 6% or 10% polyacrylamide gel for RNase A and RNase T1 assay, respectively, and electrophoresed at 150 V for 50 min at room temperature. The polyacrylamide gel was vacuum-dried immediately and visualized using autoradiography.The PP6 RNA substrate was labeled with [γ-Methanolobus psychrophilus R15 contains four paralogous genes encoding small TRAM proteins of approximately 7 kDa. Because transcriptomic assay showed that three  of four TRAM genes, excluding Mpsy_2002, exhibit elevated transcription . This is consistent with the transcriptomic assay (Mpsy_0643), TRAM2002 (Mpsy_2002), TRAM3043 (Mpsy_3043), and TRAM3066 (Mpsy_3066).cription  at loweric assay . HereaftFigure 1B) but only weakly to TRAM0643 and TRAM3066 . Using this antibody, Western blot detected two proteins in the 30°C culture and three in the 18°C culture . Using the purified protein as a reference, the amount of TRAM3066 in the 18°C culture was quantified as less than 5 ng per 100 μg total proteins. While, the density of arrow 3 pointed-band was obviously more intensive than that of 5 ng purified TRAM3066, therefore, we predict this band contained both TRAM0643 and TRAM3066. As the TRAM2002 antibody detected the additional smaller protein(s) in the 18°C culture, both TRAM0643 and TRAM3066 are assumed to be cold-adaptive proteins and reduce translation at higher temperature to a low level below the Western blot detection limit. A few hours of cold shock at 4°C appeared to induce the translation of TRAM3066, but not TRAM0643 . In summary, there are two types of archaeal TRAMs, one represented by TRAM2002 constitutively expresses in response to temperature shift; another type is induced by low temperatures and thus acts as a Csp. Considering the estimated cellular abundance, the constitutively expressed TRAM2002  and cold-induced TRAM3043  , respectively, in the cold and could function mainly as Csps.To detect TRAM protein abundance in cold-shocked cells, we first produced two types of antibodies against TRAM2002 protein and TRAM3066-specific peptide. The antibody raised by TRAM2002 recognized all four TRAMs  in the three cold-induced TRAMs  and TRAM3066 (approximately 5.1-fold higher protein content) were more induced by IPTG than the other two proteins (2.1-fold). This highly increased abundance of the two TRAMs did not hurt the E. coli, though Western blot detected relatively higher protein content for TRAM0643 than for TRAM2002 and TRAM3043 in BX04 cell  by referenced to the purified four TRAMs . Collectively, the four archaeal TRAMs, with slightly varied behavior between TRAM0643/3043/3066 and TRAM2002, are cold-adaptive proteins similar as the bacterial Csps.In addition to cold-induced expression, the archaeal TRAM protein shows a similar tertiary OB-fold structure to the bacterial Csps, possessing five main β-strands . In the absence of an RNA chaperone to melt the terminator, cat is not expressed, and the cells are sensitive to chloramphenicol. However, if the archaeal TRAMs can melt the RNA structure in the terminator, the downstream cat gene is expressed, and the strain becomes resistant to chloramphenicol. Upon transformation with each of the four TRAM encoding genes , the RL211 strain became chloramphenicol-resistant in the absence of IPTG , and 12 RNA Pentaprobes (PP) which are approximately 100 nt in length and encompass all possible 5 nt RNA sequences . Using the antibody generated from TRAM3066, the RNA-bound protein was confirmed as TRAM3066 . Low affinities to RNA substrates PP1 and PP10 at KD (equilibrium dissociation constant) values ranging from 9 to 34 μM were determined for TRAM3066 and 12 to 89 μM for TRAM2002. These values were comparable but lower than those for CspA under the same condition. However, neither TRAM3066 nor TRAM2002 showed binding to ssDNA or dsDNA even up to a ratio of 105 protein per RNA molecule (data not shown).Next, a SPR assay was employed to determine the RNA affinities of the two TRAMs more quantitatively . For example, neither 6.25 pg of RNase A nor TRAM3066 alone degraded PP6 , but addition of TRAM3066 promoted degradation of two-thirds of PP6 by the same amount of RNase A . Similarly, TRAM3066 facilitated RNase T1 to hydrolyse PP6 . This is most likely attributed to TRAM3066 in melting the RNA secondary structure, making it more susceptible to RNase hydrolysis. This confirms the RNA chaperone activity of the archaeal TRAM.To further validate the RNA duplex melting activity of the archaeal TRAMs, TRAM3066 was selected to test its ability to assist RNase A and RNase T1 degradation of a highly structured RNA PP6, referencing the role of oli CspA . The 92-in vivo cold-induced expression and complementation of the cold-growth defect of Csp-deficient E. coli BX04, demonstrates that the single-domain TRAM acts as a Csp in methanogenic archaea. Three of the four TRAM genes of M. psychrophilus R15 are expressed either only at, or with increased expression upon reduced temperature, e.g., 18°C, and cold shock treatment induced transcription and slightly induced translation. Similar to bacterial Csps, TRAMs exhibit typical RNA chaperone activity in transcriptional antitermination, assisting RNases to degrade structured RNA and binding RNA irrespective of sequence, but with lower affinity. Given the wide distribution of TRAM across archaeal phyla  rarely presents at 37°C and is highly induced by cold shock, while type II  occurs at both temperatures . It was determined that six residues, Trp11, Phe18, Phe20, Phe31, His33, and Phe34 in CspA and CspE are key to the RNA binding activity .Though similar to the bacterial Csps in RNA chaperone activity, the archaeal TRAMs appear to have a lower RNA affinity than CspA determined in both rEMSA and SPR assays ( melting . While T melting , and twoE. coli (Methanococcus maripaludis remarkably retarded the growth. Further study of the working mechanisms of TRAM is ongoing.In addition to cold protection, bacterial Csps display additional functions such as antioxidative stress, stationary growth and anti-antibiotic activity . Given tE. coli . In our Because TRAM is a conserved domain among RNA modification proteins that are ubiquitously distributed in organisms, the findings in this work would shed light on understanding the TRAM domain in rRNA and ribosomal protein methylation in other organisms.XD and JL designed the study. BZ, LY, LZ, LQ and JL performed experiments. XD, JL, and BZ analyzed data. XD and BZ wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Heterochromatin formed by the SUV39 histone methyltransferases represses transcription from repetitive DNA sequences and ensures genomic stability. How SUV39 enzymes localize to their target genomic loci remains unclear. Here, we demonstrate that chromatin-associated RNA contributes to the stable association of SUV39H1 with constitutive heterochromatin in human cells. We find that RNA associated with mitotic chromosomes is concentrated at pericentric heterochromatin, and is encoded, in part, by repetitive α-satellite sequences, which are retained in cis at their transcription sites. Purified SUV39H1 directly binds nucleic acids through its chromodomain; and in cells, SUV39H1 associates with α-satellite RNA transcripts. Furthermore, nucleic acid binding mutants destabilize the association of SUV39H1 with chromatin in mitotic and interphase cells – effects that can be recapitulated by RNase treatment or RNA polymerase inhibition – and cause defects in heterochromatin function. Collectively, our findings uncover a previously unrealized function for chromatin-associated RNA in regulating constitutive heterochromatin in human cells.DOI:http://dx.doi.org/10.7554/eLife.25299.001 Each cell in a human body contains the same DNA sequence, which serves as a set of instructions for how the body should develop and operate. However, only certain sections of DNA are “active” at any particular time and in any given type of cell. When a section of DNA is active, cells make many copies of it using a molecule called RNA. When a section of DNA in inactive, very little RNA is made. Some sections of DNA must always be kept inactive to avoid damaging the cell.DNA is packaged around proteins called histones, and enzymes that modify histones control which sections of DNA are switched on or off. One such modifying enzyme, called SUV39H1, is important for inactivating sections of DNA that could cause harm to the cell if they are active. Previous studies showed that the loss of SUV39H1 and related proteins cause abnormalities and cancer in mice. However, it is not clear how this enzyme identifies and inactivates the DNA it needs to target.Johnson, Yewdell et al. studied SUV39H1 in human cells. The experiments show that RNA binds to the SUV39H1 enzyme and controls how it interacts with DNA. Specifically, Johnson, Yewdell et al. found that sections of DNA that are inactive can still make a small amount of RNA, and that this RNA tethers SUV39H1 to the DNA to keep the DNA switched off. Mutant forms of SUV39H1 that are unable to interact with RNA fall off the DNA, which allows DNA sequences that are normally switched off to become active.The findings of Johnson, Yewdell et al. reveal a new role for RNAs in regulating whether DNA is switched on or off. The next step is to determine whether other enzymes that can also modify histones use the same mechanism to activate or inactivate DNA. Differences in how the activity of DNA is regulated between individuals plays a crucial role in generating the diversity we see in nature. Therefore, this work helps us to understand our basic biology and may provide new opportunities for treating disease.DOI:http://dx.doi.org/10.7554/eLife.25299.002 Drosophila melanogaster (The histone methyltransferase Su(var)3–9 was first identified in forward genetic screens as a suppressor of position effect variegation in nogaster . Previounogaster . This fanogaster , as wellnogaster . SUV39 pnogaster . HP1 pronogaster .SUV39H1 and H3K9me3 are predominately associated with constitutive heterochromatin, which represses ‘selfish’ genetic elements and repetitive DNA to promote genomic stability . In manyOct4 locus  , centrifA previous study demonstrated that transcription by RNA polymerase II (Pol II) can occur from the core centromere regions of human mitotic chromosomes . This RNTo determine which RNA polymerase transcribes the pericentric RNA, we added different polymerase inhibitors to cells during EU treatment. We measured a reduction of approximately 50% in pericentric RNA signal after treatment with α-amanitin or triptolide – which preferentially inhibit Pol II – but observed a complete loss of signal after treatment with actinomycin D or CX-5461 – which preferentially inhibit Pol I, and show no detectable Pol II inhibition . Thus, mOur data does not exclude the possibility that some Pol II-dependent transcription is also occurring at the core centromere, as has been previously reported in human cells . In thatTo investigate the cell cycle timing of pericentric RNA synthesis, we either labeled cells continuously prior to mitosis, or for a short pulse followed by washout prior to mitosis. We found that continuous EU labeling for 8 or 4 hr before mitosis led to pericentric EU-RNA signal, but a 4 hr EU pulse followed by a 4 hr chase resulted in no EU-RNA signal on mitotic chromosomes . This suThe finding that RNA is bound to the pericentric regions of human mitotic chromosomes is intriguing in light of previous observations that direct RNA binding by the heterochromatin factors Polycomb Repressive Complex 2 (PRC2) and HP1α is important for their proper localization and function . BecauseSUV39H1 binds directly to HP1 proteins , and mouE. coli. Because we were unable to purify full-length SUV39H1 protein free of degradation products, we purified truncations of SUV39H1 containing either the N-terminal extension alone (amino acids 1–41), the chromodomain alone (amino acids 42–106), the N-terminal extension plus the chromodomain (amino acids 1–106), or the C-terminus consisting of the pre-SET, SET, and post-SET domains (amino acids 107–412) (d) of 0.15 ± 0.01 µM and 2.31 ± 0.17 µM, respectively. The N-terminal extension alone bound to RNA with a significantly reduced Kd (51 ± 30 µM) compared to the truncations containing the chromodomain. Excess unlabeled RNA competed with radiolabeled RNA for binding to SUV39H1  with a random 19-mer ssRNA oligonucleotide and MBP-tagged human SUV39H1 protein purified from 107–412) . We foun107–412) . The add SUV39H1 , confirm SUV39H1 , and tha SUV39H1 .10.7554/E. coli MBP sequence to generate each nucleic acid type  alter the binding of SUV39H1 to H3K9me3 . We found that α-satellite RNA was enriched in the WT SUV39H1 IP 13 ± 2 fold when compared to GFP alone  ; indicatR55A mutant in the same experiments (52 ± 9% of WT)   . This in42 ± 5%) . Taken tPrevious studies found that HP1α localization at heterochromatin is sensitive to RNase treatment in both mouse interphase cells and on human mitotic chromosomes . We alsoR55A, SUV39H1R84A, SUV39H1F43A, and SUV39H1W64A using fluorescence recovery after photobleaching (FRAP)  . After pg (FRAP) , with a g (FRAP) . The mobg (FRAP) , consistg (FRAP) . The aroP)  that was greater than WT SUV39H1, but less than the aromatic cage mutants SUV39H1W64A and SUV39H1F43A , an H3K9me3 binding mutation (F43A) or both (R55A/F43A) . We madeSeveral models have been proposed for how chromatin-associated RNAs regulate the organization and transcription of the genome . One emeDrosophila cells, where SAT III RNA transcribed from the X chromosome localizes to all centromeres in trans  also exhibits relatively promiscuous, length-dependent, sequence-independent RNA binding; though specificity for certain RNA sequences can be observed in vivo and in vitro , 2015. IBy generating point mutations that separate the nucleic acid binding and H3K9me3 binding functions of the SUV39H1 chromodomain, we assessed the individual contributions of these two interactions on SUV39H1 localization and function. We show for the first time that direct nucleic acid binding contributes to SUV39H1 localization by stabilizing its interaction with heterochromatin, in both interphase and mitotic human cells  and 7, aIt is important to note that because of the high conservation between SUV39H1 and HP1α aromatic cages , it has It remains a major outstanding question how SUV39H1 specifically recognizes its genomic sites of action. Although it had been originally proposed that HP1 proteins directly recruit SUV39H1 , subsequHere, we identify both RNA and H3K9me2/3 as main determinants of SUV39H1 localization. Importantly, the RNA-dependent mechanism for SUV39 function appears to be present in both human and mouse cells  suggestiWe propose a model in which SUV39H1 engages chromatin through several different interactions that in combination define its genomic distribution . When diHeLa, U2OS, HFF, HT1080, Huh-7, and primary fibroblasts were grown in Dulbecco’s Modified Eagle Medium (DMEM), hTERT-RPE cells were grown in DMEM/F12 media, and DLD-1 cells were grown in RPMI 1640 media. HeLa cells and U2OS cells were acquired from the ATCC. We obtained DLD-1 human colorectal cancer cells from Dr. Daniele Fachinetti and Dr. Don Cleveland, primary fibroblasts from Dr. Paul Khavari, HFF (human foreskin fibroblast) cells from Dr. Matthew Bogyo, HT-1080 fibrosarcoma cells from Dr. Jianghong Rao, hTERT RPE-1  cells from Dr. Tim Stearns, Huh-7 human hepatoma cells from Dr. Peter Sarnow, and Flp-In T-REx HeLa cells from Dr. Pat Brown. These cells were not independently authenticated. Mycoplasma contamination was monitored frequently by cytoplasmic DAPI staining. All cells were grown under American Tissue Culture Collection (ATCC) standard conditions. All media was purchased from Thermo Fisher and supplemented with 10% vol/vol FBS and 100 U/ml penicillin and streptomycin.Flp-In T-REx HeLa cell lines expressing SUV39H1-GFP proteins were made by co-transfecting pOG44 Flp-recombinase expression vector and pcDNA5/FRT/TO tet inducible Flp-In cloning vector (Invitrogen) containing SUV39H1-GFP sequences  using Fugene 6 (Promega). After 48–72 hr, cells were selected with 350 μg/mL hygromycin B (Invitrogen) and 15 μg/mL blasticidin S (Invivogen) until visible colonies formed, then the entire selected population of cells was saved.SUV39 DKO cells were generated essentially as described . BrieflyDLD-1 cell lines with point mutations in the endogenous SUV39H1 locus were generated by first making a SUV39H2 KO cell line , then performing further genome editing by co-transfecting pX458 (pSpCas9(BB)−2A-GFP) with a SUV39H1-targeting gRNA  and a puc18 homology arm construct containing desired point mutations  with Fugene HD. After 4–5 days, GFP-positive cells were single-cell sorted into 96-well plates using a Sony SH800 cell sorter. Clonal populations were expanded and screened by western blotting for SUV39H1. Mutations in SUV39H1 were confirmed by purifying genomic DNA, PCR amplifying the targeted locus, and analyzing the amplicons by Sanger sequencing.∧5 cells/mL. 100 μL cells were spun onto no. 1.5 glass coverslips using a Shandon Cytospin 4 at 2000 rpm for 5 min. Coverslips were hydrated with KCM buffer , then cells were permeabilized for 10 min with KCM buffer with 0.5% Triton X-100. Coverslips were blocked with KCM + 2% BSA for 30 min, then stained with specific antibodies diluted in KCM + 2% BSA, 30 min each. After staining, cells were washed with KCM buffer and fixed with 3.7% formaldehyde in KCM for 10 min. After fixation, cells were washed with PBS, stained with 10 μg/mL Hoescht for 10 min, washed with PBS + 0.1% Triton X-100, washed with PBS, then mounted onto microscope slides.For mitotic spreads, cells were grown to about 70% confluency in T-25 or T-75 flasks, then mitotic cells were selected by shaking off less adherent cells. Released cells were pelleted, resuspended in 100 μL 75 mM KCl, then counted on a hemocytometer and diluted with more 75 mM KCl to 2 × 10For staining interphase DLD-1 cells, cells were plated on glass coverslips, fixed and permeabilized with 1% formaldehyde, 0.5% Triton X-100, 1X PBS for 10 min, then re-permeabilized with 0.5% Triton X-100, 1X PBS for 10 min. Cells were blocked in antibody dilution buffer  for 30 min, then stained with primary and secondary antibodies diluted in AbDil for 30 min each. Cells were then stained with 10 μg/mL Hoechst in Abdil for 10 min, washed with PBS + 0.2% Triton X-100, washed with PBS, then mounted onto slides and imaged.2 in experiments with RNase H and RNase III). Plus inhibitor (+inhibitor) controls also containing 4 U/μL each of RNaseOUT (Invitrogen) and RNase inhibitor (Ambion AM2684).We used the following antibodies for immunofluorescence: mouse anti-HEC1 , rabbit anti-CENP-A-Alexa647 , rabbit anti-GFP-Alexa488 , rabbit anti-HP1α , rabbit anti-H3K9me3 , human anti-centromere protein . All secondary antibodies were obtained from Invitrogen and diluted 1:1000. For experiments where SUV39H1-GFP localization was assessed, expression of WT or mutant versions of SUV39H1-GFP was induced in HeLa Flp-In T-REx cells with 1 μg/mL doxycycline for 6 hr before mitotic shake off. To visualize RNA by EU labeling, cells were incubated with 0.5 mM EU (synthesized as previously described ) for 4–1Stacks of fixed spread mitotic chromosomes and interphase nuclei were acquired at 0.2 μm axial steps using a motorized stage mounted on an Olympus IX70 microscope, using a 100X, 1.4 NA PlanApo objective and a CoolSnap-HQ CCD camera (Photometrics) on a DeltaVision Spectris system (Applied Precision). Displayed images are maximum intensity projections of raw or deconvolved z-stacks.Fluorescent signal at pericentric regions of mitotic chromosomes or at centromeres in interphase nuclei was quantified using custom software. Pericentromere finder : centrom4 cells/mL, then swelled at 37°C for 15 min. 500 μL cells were cytospun onto RNase AWAY treated slides . Cells were permeabilized in KCM buffer for 5 min, fixed in 4% PFA in PBS for 10 min, then blocked for 15 min  and stained with specific antibodies (described above) in block. Cells were permeabilized again in CSK buffer/2 mM RVC(Ribonucleoside Vanadyl Complex)/0.5% Triton X-100, 10 min on ice, then fixed again in 4% PFA in PBS for 10 min. Slides were dehydrated with an ethanol series , then air-dried at 37°C. After precipitating and denaturing, probes were hybridized in hybridization mix (+RVC) at 37°C overnight in a humid chamber . Slides were then washed with 2X SSC/0.05% Tween/formamide (same formamide percentages used in hybridization mix), pH 7 at 37°C, 3 times 5 min each -- then with 2X SSC/0.05% Tween, at 37°C, 3 times 5 min each – then 1X SSC/0.05% Tween, at room temp, 10 min – then 4X SSC/0.1% Tween, at room temp, 5 min. For biotin-16-dUTP labeled probes, after post-hybridization washes, slides were blocked in blocking buffer (PBS + 0.5% Triton X-100 + 1% BSA + 0.02% sodium azide) and biotin was detected with Alexa Fluor 488 streptavidin diluted in blocking buffer for 2 hr at room temperature. Slides were then washed with 4X SSC/0.1% Tween three times at room temp, 5 min each. All slides were stained with DAPI, coverslips were mounted, and images were acquired as described above. CSK buffer: 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM PIPES pH 6.8. Probe hybridization mix: 50% formamide (65–68% for repetitive), 10% dextran sulfate, 2X SSC, 1% Tween-20. RNase-treated controls slides were incubated with Ambion RNase cocktail (7.5 μL/mL KCM buffer) at 37°C for 2 hr up to overnight before hybridization.Mitotic DLD-1 cells were selected by mitotic shake off, washed with PBS, resuspended in 75 mM KCl to 5.8 × 10DNA FISH was performed as above, with the following differences: slides were fixed in 10% formalin for 10 min at room temp, permeabilized in KCM for 10 min, denatured in 70% formamide, 2X SSC, pH 7 at 70°C for 3 min, then hybridized overnight.2 area for at least 4 hr at −80° C or overnight at −20° C. Before use, probes were pelleted at maximum speed in an Eppendorf microcentrifuge, washed with 70% ethanol, resuspended in 65% formamide hybridization mix, and denatured at 74°C for 10 min.All probes were labeled by nick translation of plasmids either indirectly with Roche biotin-16-dUTP and subsequently detected with AlexaFluor 488 streptavidin or directly with AlexaFluor 488-dUTP. D1Z5-specific RNA FISH probes were generated from the pSD1-1 plasmid, which contains a single copy of the 1.9 kb D1Z5 higher order repeat (HOR). Probes recognizing chromosome 13 and chromosome 21 α-satellite arrays were generated using a template PCR amplified from human genomic DNA with the following primers: α-satellite I (ASON 614): 5’-CTTGCTAGCAATCTGCAAGTGG-3’, and α-satellite II (ASON 615): 5’-CTTGTCGACTACAAAAAGAGTG-3’. β-satellite is detected with nick-translated plasmid pβ4, which recognizes β-satellite sequences on the acrocentric chromosomes . Satellite III is detected with nick-translated plasmid pTRS-47, which recognizes Satellite III subfamily sequences on chromosomes 14 and 22. pSD1-1, pβ4, and pTRS-47 plasmids were a gift from the lab of Dr. Huntington Willard. 500 ng of 488 dUTP-labeled probes were precipitated with 0.5 uL salmon sperm DNA (10 mg/mL stock) and 2.5 volumes 100% EtOH for each 18 × 18 cmThe ‘RGB profile plot’ ImageJ plugin was used for line scan analysis of RNA FISH images.E. coli and grown in 2–6 L of 2X YT at 37°C to an OD of 0.6–0.8. Cells were induced with 0.3 mM IPTG for 4–6 hr at room temperature. Cells were harvested, frozen in liquid nitrogen and stored at −80°C.All purified SUV39H1 proteins used in this study were expressed as N-terminal Maltose Binding Protein (MBP) fusions. pMAL-c2X (NEB) expression vectors, carrying sequences encoding truncated versions of human SUV39H1, were transformed into BL21 E. coli culture was resuspended in 20 mL of ice cold 4X lysis buffer . Lysates were sonicated and centrifuged at 96,000Xg for 1 hr at 4°C. Supernatants were passed over 2–5 mL of either amylose resin (NEB) or glutathione agarose (Sigma Aldrich), followed by washing with approximately 10 column volumes of 4X lysis buffer, 30 column volumes of 1X lysis buffer , and five column volumes of 1X lysis buffer without Triton X-100 at 4°C. Protein was eluted with approximately five col. volumes of 1X lysis buffer without Triton X-100 plus either 10 mM maltose or 5 mM glutathione. MBP fusions were dialyzed into 20 mM Tris-HCl pH 7.4, 50 mM NaCl, 10% glycerol, 5 mM 2-mercaptoethanol. For alanine scanning point mutants, frozen pellets from 100 mL cultures were thawed and resuspended in 5 mL of 4X lysis buffer. Lysates were sonicated, divided into 5 × 1 mL aliquots and spun at approximately 20,000Xg for 20 min at 4°C. Supernatants were combined and incubated with 500 μL of amylose resin (NEB) for 30 min at 4°C. The amylose resin was washed 3 times with 1 mL of 4X lysis buffer, 3 times with 1 mL of 1X lysis buffer without Triton X-100, and 3 times with 20 mM Tris-HCl pH 7.4, 50 mM NaCl, 10% glycerol, 5 mM 2-mercaptoethanol. Protein was eluted with 750 μL of 20 mM Tris-HCl pH 7.4, 50 mM NaCl, 10% glycerol, 5 mM 2-mercaptoethanol, 10 mM maltose for 30 min at 4°C. Amylose resin was pelleted by spinning at 20,000Xg for 2 min, and supernatants were collected. To remove contaminating RNases, proteins were loaded onto a 1 or 5 mL HiTrap SP sepharose FF column (GE) using an AKTA FPLC. The flowthrough fraction was taken and concentrated using Amicon Ultra 30 kDa centrifugal filters (EMD Millipore). Protein concentrations were quantified by Bradford assay and A280 measurement using a NanoDrop 1000 spectrophotometer. The absence of co-purifying nucleic acids was monitored by ensuring the absorbance ratio at 260 nm/280 nm was less than 0.7.Each liter of pelleted RNA and DNA probes ≤50 bp were ordered from Integrated DNA Technologies.Sense 19mer,5’-AUAUGGGAACCACUGAUCC-3’;antisense 19mer,5’-GGGAUCAGUGGUUCCCAUA-3’;sense MBP 1–50,5’-ACCAAAAUCGAAGAAGGUAAACUGGUAAUCUGGAUUAACGGCGAUAAAGG-3’;antisense MBP 1–50,5’-CCUUUAUCGCCGUUAAUCCAGAUUACCAGUUUACCUUCUUCGAUUUUGGU-3’.To synthesize 180 bases of α-satellite or β-actin ssRNA, cDNAs were obtained from HeLa cell total RNA using the SuperScript III First-Strand Synthesis System (Life Technologies) according to the manufacturer’s instructions, and the following primers:α-satellite forward,5’-CTTGCTAGCAATCTGCAAGTGG-3’;antisense β-actin,5’-CGTAGATGGGCACAGTGTGG-3’.cDNA was amplified into dsDNA using Phusion high-fidelity DNA polymerase (NEB) and the following primer pairs; α-satellite forward (sequence above) andα-satellite reverse +T7,5’- GTAATACGACTCACTATAGGGcttgtcgactacaaaaagagtg-3’;antisense β-actin (sequence above) and sense β-actin + T7,5’- GTAATACGACTCACTATAGGGAGGCCCCCCTGAACCCCAAG-3’.dsDNA was used for in vitro transcription as described below.32P (Perkin Elmer) and polynucleotide kinase (NEB). Approximately 15 pmol of ATP γ-32P was added to 5–10 pmol of nucleic acid, polynucleotide kinase, and 10X reaction buffer, and incubated for 75 min at 37°C. The polynucleotide kinase was subsequently deactivated by incubating at 68°C for 20 min. Free ATP γ-32P was separated from labeled nucleic acids using Micro Bio-spin Columns with Bio-Gel P-6 (Bio Rad) according to the manufacturers protocol, and exchanged into 20 mM Tris-HCl pH 7.4, 50 mM NaCl, 10% glycerol in DEPC H2O. Labeled nucleic acids were diluted into approximately 1.5 mLs of Tris-HCl pH 7.4, 50 mM NaCl, 10% glycerol in DEPC H2O to a final concentration of approximately 1–5 nM. MBP 1–50 nucleic acids were further purified by native gel electrophoresis . Bands were identified using HyBlot CL Autoradiography Film (Denville Scientific), excised and eluted for 3 hr at 25°C into 1 mL of 20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 10% glycerol in DEPC H2O at a final concentration of ≤1 nM. α-satellite and β-actin ssRNAs were further purified by denaturing gel electrophoresis . Bands were identified using HyBlot CL Autoradiography Film (Denville Scientific), excised and eluted for 3 hr at 25°C into 1 ml DEPC H2O with the addition of 160 units of RNase Inhibitor . RNA was concentrated using Amicon Ultra 30 kDa centrifugal filters and washed 3 times with 500 μL DEPC H2O. RNA was resuspended in 1 mL of 20 mM Tris-HCl pH 7.4, 50 mM NaCl, 10% glycerol, made with DEPC-treated water at a final concentration approximately ≤1 nM.Nucleic acids were end-labeled using ATP γ-32P.RNA was transcribed from α-satellite and β-actin dsDNA  using the MEGAscript T7 Transcription Kit (Life Technologies) according to the manufacturers protocol, with the addition of 40 units of RNase Inhibitor . Transcribed RNA was loaded onto a 1% agarose gel to verify lengths, and RNA concentrations were measured using a NanoDrop 1000 spectrophotometer. Approximately 2 μg of RNA was desphosphorylated for 60 min at 37°C using Antarctic Phosphatase (NEB) with the addition of 40 units of RNase Inhibitor  in preparation for end-labeled using ATP γ-2O) and kept on ice until mixed with RNA. Proteins were mixed with 32P-labeled nucleic acids and approximately 4 units RNase Inhibitor , and allowed to equilibrate to at room temperature for 10 min before loading onto a native gel  buffered with 0.5X TBE. Gel electrophoresis was carried out for 1 hr at 25°C at 10 mAmps. Gels were exposed to phosphorimaging screens for approximately 1 hr before signal acquisition was performed using a Typhoon 9400 Variable Mode Imager (GE). Gels were quantified using FIJI software, and binding curves, dissociation constants, and standard error were plotted and calculated using Graphpad Prism software.Purified proteins and nucleic acids were thawed at room temperature and kept on ice. Proteins were serially diluted into binding buffer  of approximately 60 bp, spanning a 5’ AscI site, the SUV39H1 chromodomain (residues 42–106), and a 3’ PacI site. Following PCR assembly, DNA was purified using Agencourt AMPure XP beads. 40 μL of each PCR product was mixed with 72 μL Ampure beads and incubated for 5 min at 25°C. Beads were collected using a 24-well magnetic stand for 5 min, and washed twice with 200 μL 70% ethanol. Beads were air-dried for 20 min at 25°C, and DNA was eluted with 40 μL of H2O) and kept on ice until mixed with RNA. Proteins were mixed with 32P-labeled sense 19mer RNA and approximately 4 units RNase Inhibitor , and allowed to equilibrate to at room temperature for 10 min. 4 μL of each binding reaction was dotted onto a nitrocellulose membrane. The membrane was washed several times with binding buffer without glycerol, and membranes were exposed to phosphorimaging screens and imaged using a Typhoon 9400 Variable Mode Imager (GE). Gels were quantified using FIJI software, and binding curves, dissociation constants, and standard error were plotted and calculated using Graphpad Prism.Purified proteins and nucleic acids were thawed at room temperature and kept on ice. Proteins were serially diluted into binding buffer  were diluted to 40 μM in peptide binding buffer . 5 μL of SUV39H1 42–106  was mixed with 1 μL peptide  and 44 μL peptide binding buffer and incubated for 45 min at 4°C. 20 μL of Dynabeads M-280 Strepavidin (Life Technologies) were washed 3 times with 1 mL of peptide binding buffer, added to the binding reaction and incubated for 45 min at 4°C. Beads were washed 4 times with 1 mL of peptide binding buffer and eluted with 100 μL of protein SDS sample buffer . 20 μL was loaded onto a 12.5% polyacrylamide SDS denaturing gel and transferred onto a PVDF membrane for western blotting with anti-MBP antibody. Bands were quantified using FIJI software and p-values for an unpaired t-test were calculated using Graphpad Prism software.The structures of the chromodomains of human HP1α bound to H3K9me3  PDB ID: PDB ID:. Residue14C-SAM (Perkin Elmer), ±124 μg/mL β-actin RNA , in activity buffer  in a final volume of 20 μL. Reactions proceeded at 30°C for the indicated time, then samples were boiled in SDS-PAGE loading buffer for 5 min and saved at −20°C. Samples were run on a 20% SDS-PAGE gel; the gel was fixed, Coomassie stained, and dried; and methylation was detected with a phosphorimaging screen scanned on a Typhoon 9400 Variable Mode Imager (GE).150 μg/mL full-length SUV39H1 proteins were incubated with 70 μg/mL Xenopus H3/H4 tetramer , 0.8 μCiRabbit anti-MBP, anti-GFP, and anti-HP1α polyclonal antibodies were generated by Cocalico Biologicals and purified from rabbit serum. All primary antibodies were incubated for 1 hr (except for anti-SUV39H1 which was incubated overnight) at the following dilutions: rabbit anti-MBP , rabbit anti-GFP , rabbit anti-HP1α , mouse anti-SUV39H1 , rabbit anti-H3K9me3 , rabbit anti-histone H4 , and mouse monoclonal anti-tubulin .To western blot for endogenous SUV39H1 in DLD-1 cells, nuclear lysates were prepared to enrich for the SUV39H1 containing fraction. Cells were incubated in hypotonic buffer  for 10 min on ice, dounced 10 times with a Kontes glass dounce (B pestle), nuclei were pelleted at 5000xg for 10 min, then lysed in DLB . If neceAll buffers listed here are described in t) were determined using Sequence Detection Systems software. For each primer set, standard curves of diluted cDNA were used to assess the exponential amplification (expAmp) of the amplicon. The signal in each experimental sample was determined by signal = expAmp-Ct. For all α-satellite measurements, ‘-RT’ signal was subtracted from ‘+RT’ signal, as the repetitive nature of α-satellite DNA leads to high background. α-satellite signal was then divided by the β-actin or GAPDH signal to get final α-satellite normalized signal. For IP enrichment values, the α-satellite normalized signal for the IP was then divided by the α-satellite normalized signal of the input lysate.RNA was reversed transcribed into cDNA using the SuperScript III First-Strand Synthesis System (Life Technologies 18080–051) per manufacturer’s instructions, using the following RT primers: α-satellite I (ASON 614): 5’-CTTGCTAGCAATCTGCAAGTGG-3’, β-actin rev (ASON 2430): 5’-ATGTCCACGTCACACTTCAT-3’, and GAPDH rev: 5’-CCTGCTTCACCACCTTCTT-3’. Quantitative PCR (qPCR) was then performed with the same reverse primers plus the following forward primers: α-satellite II (ASON 615): 5’-CTTGTCGACTACAAAAAGAGTG-3’, β-actin fwd (ASON 2392): 5’-AGAGCTACGAGCTGCCTGAC-3’, and GAPDH fwd: 5’-AGATCATCAGCAATGCCTCC-3’. qPCR mix recipe: 1X Phusion HF buffer (NEB), 3% DMSO, 200 µM dNTPs, 1X ROX reference dye (Life Technologies), 1X SYBR green I (Invitrogen S-7563), Phusion polymerase, and 100 nM each primer. The following programs were used: α-satellite , β-actin . qPCR was performed on a 7900HT Fast Real-Time PCR System (Applied Biosystems), and the number of cycles to reach the automatically set threshold  fwd (ASON 1991): CCTTCGATAGCTCAGCTGGTAGAGpre-tRNA(tyr) rev (ASON 1990): AAAAAACCGCACTTGTCTCCTTCGGAPDH pre-mRNA fwd (ASON 4619): CATGCCTTCTTGCCTCTTGTGAPDH pre-mRNA rev (ASON 4620): TGAGGTCAATGAAGGGGTCA45S pre-rRNA fwd (ASON 1891): CCTGCTGTTCTCTCGCGCGTCCGAG45S pre-rRNA rev (ASON 1892): AACGCCTGACACGCACGGCACGGAGFor total RNA-seq analysis of control and SUV39 DKO HeLa and DLD-1 cells, total RNA was purified using TriPure, then treated with 10 units of TURBO DNase (Life Technologies) at 37°C for 30 min per the manufacturer’s instructions. RNA was re-purified with TriPure, resuspended in DEPC-treated water, and quality checked by Bioanalyzer (Agilent). RNA was then analyzed either by RT-qPCR to measure α-satellite RNA levels as described above, or a cDNA library was generated for RNA-Seq analysis. For RNA-Seq, ribosomal RNAs were first depleted using a NEBnext rRNA depletion kit (New England Biolabs), RNA was purified using Agencourt Ampure XP beads (Beckman Coulter), then cDNAs were generated, amplified, and indexed with the ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre) per the manufacturer’s instructions. Indexed libraries were quantified by Bioanalyzer and qPCR, pooled, and sequenced on a NextSeq 500 (Illumina).For analysis of repetitive RNAs, raw fastq files were adapter trimmed and converted to fasta format. The fasta files were then split into smaller files of 1,000,000 sequences each. All smaller fasta files of each original fasta file were analyzed by RepeatMasker 4.0.3 using the human species database . The RepAnalysis of non-repetitive RNA was performed by aligning reads  to the UCSC hg38 build  using tophat2 (v 2.2.9). The aligned reads were annotated and quantified using Cufflinks (v 2.2.1).Flp-In HeLa cells containing WT or mutant SUV39H1-GFP were grown on glass-bottom chambered coverglass (LabTek) under standard conditions. SUV39H1-GFP expression was induced by adding 1 µg/mL doxycycline six hours before data collection. For drug treatment experiments, cells were treated with 1 μM CX-5461 (Millipore), 50 μg/mL α-amanitin (Santa Cruz Biotech), or 1 μM triptolide (Selleckchem) for 2 hr before image acquisition. Triptolide and α-amanitin experiments were done in cells induced with 50 ng/mL doxycycline for 24 hr. Immediately before the experiment, the media was exchanged to pH-indicator free media . Microscopy was performed with a Nikon Eclipse Ti at 100x magnification. Photobleaching was performed using an Andor Mosaic II patterned illumination system coupled to a 450 mW 405 nm laser. Photobleaching was performed using 100% power for one second using a circular spot with a diameter of ~3 µm. Images of the recovery were collected, and intensity profiles of the bleaching area and of the whole nucleus were calculated using ImageJ 2.0. Intensity profiles were normalized using the following formula:t. The intensity at time zero is (0)I , (bleach)I is the intensity immediately after bleaching, and (t)I is the intensity at subsequent time intervals. The normalized intensity traces were then analyzed for each cell using the following single-exponential function:Where ROI and total are the intensities of the photobleaching area and whole nucleus, respectively, at either time zero or at time max is the plateau of the recovery curve and t1/2 is the halftime for recovery, in seconds. The amplitude of the plateau is equivalent to the fraction of mobile protein.where Y In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Jessica Tyler as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "RNA-dependent stabilization of SUV39H1 at constitutive heterochromatin" 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 role of RNA in localization of the human SUV39H1 to chromatin. The authors perform a careful series of experiments showing that the stable association of a moderately overexpressed SUV39H1-EGFP protein with heterochromatic foci is RNase-sensitive. The authors further show that major satellite repeats are transcribed and give rise to RNAs that remain associated with mitotic chromosomes and are present in SUV39H1 immunoprecipitates. Using purified SUV39H1, the authors demonstrate that the enzyme binds to single stranded RNA of any sequence and identify specific mutations in the chromo domain of SUV39H1 that disrupt RNA binding without affecting its interaction with H3K9me3 peptides.Finally, the authors demonstrate that the RNA binding mutations affect the stable binding of SUV39H1 to chromatin in vivo. Importantly, when the chromo domain RNA binding defective mutants are introduced into knockout cells, their weak association with centromeric heterochromatin is not further reduced by RNase treatment, providing strong support for the idea that specific interactions between the SUV39H1 chromo domain and RNA stabilize its association with heterochromatin. The experiments in the paper are straightforward and support the main conclusion of the paper. The model that the authors present, suggesting that non-specific RNA binding works alongside H3K9me3 to stabilize the association of SUV39h1 with chromatin, is reasonable and potentially important.However, what is lacking in the study is any demonstration that the RNA binding activity described by authors contributes to the silencing function of SUV39H1 in vivo. This seems like a major shortcoming that needs to be addressed prior to publication.Essential revisions:1) A demonstration of functional importance of the RNA binding needs to be performed. This should be tested by introducing wildtype versus RNA-binding mutant SUV39H1 into their SUV39H1/H2 DKO cells and seeing what happens to H3K9me levels and what happens to Silencing of major satellite repeats and other transposons?2) The authors make the intriguing observation that transcription of α satellite RNA is not eliminated by inhibition of RNA pol II, but is eliminated by the inhibition of both RNA pol I and pol II (actinomycine D treatment). It would be very surprising if this were in fact the case. Does pol I localize to α repeats (by ChIP)? I think that the more likely explanation might be that their α-amanitin treatment is not inhibiting pol II completely. You need to show data that more convincingly clarify whether only PolII transcription is occurring, or better evidence for Pol I transcription at the α satellites.3) 4) Comparing 5) A prediction of the proposed model is that loss of CD-mediated nucleic acid and H3K9me binding together should reduce SUV39H1 interaction with α-satellite RNAs more than either mutation alone. This can be done by creating a protein carrying a nucleic acid-binding mutation (R55A/R84A) with an H3K9me-binding mutation (W64/F43) (or a SET domain mutation in a Suv39H1/H2 mutant cell line). RIP experiments with these mutants will permit the authors to test this prediction directly. Either these experiments should be done or the model toned down.6) Other revisions:1) 2) For 3)Subsection “RNA associates with the pericentric regions of human mitotic chromosomes”, last paragraph: Are there other genomic regions where the RNA is associated with mitotic chromosomes in cis? If yes, then what are those euchromatic/heterochromatic regions?4) 5) Subsection “A model for the RNA-dependent stabilization of SUV39H1 on chromatin”, first paragraph. The discussion of the pombe HP1/Swi6 and its requirement for Clr4-dependent H3K9me is incorrect. Even though this was shown in the referenced paper, subsequent work using the complete deletion of swi6  have shown that loss of Swi6 has little impact on Clr4-dependent H3K9me .6) 7) Subsection “SUV39H1 depends on direct RNA binding for its stable association with heterochromatin”, first paragraph. Please keep mobile/immobile percentages consistent between wt and mutant proteins. For example, convert 42% immobile fraction to 58% mobile considering the mobile fraction of mutants is indicated. Essential revisions:1) A demonstration of functional importance of the RNA binding needs to be performed. This should be tested by introducing wildtype versus RNA-binding mutant SUV39H1 into their SUV39H1/H2 DKO cells and seeing what happens to H3K9me levels and what happens to Silencing of major satellite repeats and other transposons?We agree with this suggestion, and have now assessed the functions of the SUV39H1 nucliec acid binding mutants on the levels of H3K9me3 and on transcription of α-satellite sequences. To do this, we used CRISPR/Cas9 to make individual nucleic acid binding and H3K9me3 binding mutations in the endogenous SUV39H1 gene. We performed this gene editing in SUV39H2 knockout cells so that the activities of SUV39H2 did not obscure the analysis of mutant phenotypes. The results, shown in the new 2) The authors make the intriguing observation that transcription of α satellite RNA is not eliminated by inhibition of RNA pol II, but is eliminated by the inhibition of both RNA pol I and pol II (actinomycine D treatment). It would be very surprising if this were in fact the case. Does pol I localize to α repeats (by ChIP)? I think that the more likely explanation might be that their α-amanitin treatment is not inhibiting pol II completely. You need to show data that more convincingly clarify whether only PolII transcription is occurring, or better evidence for Pol I transcription at the α satellites.We agree that incomplete Pol II inhibition could explain the EU-RNA signal on chromosomes after α-amanitin treatment. Because we were unable to find a Pol I antibody suitable for ChIP, we addressed this issue by performing a new set of experiments using a more diverse panel of RNA polymerase inhibitors – including triptolide, that exhibits much faster Pol II inhibition kinetics than α-amanitin  – and by providing a more quantitative assessment of EU-RNA levels and RNA polymerase inhibition specificity new . We find3) We agree that the R55A and R84A mutations likely disrupt binding to all nucleic acid substrates, including RNA and DNA. Therefore, we have changed our language in the manuscript to refer to these as nucleic acid binding mutants. One important note is that the treatment of mitotic chromosomes with RNase A, or treatment of interphase cells with RNA polymerase inhibitors, recapitulates defects of the nucleic acid binding mutants in these assays, indicating that the relevant binding substrate for SUV39H1 localization on chromosomes is likely RNA. Thus, in cases where we can ascribe the primary defect we observe in the mutants to RNA binding, we have maintained this phrase.4) Comparing We acknowledge that the R55A mutation appears to cause a slight but insignificant decrease in SUV39H1 binding to H3K9me3. However, if the R55A mutation were really disrupting H3K9me3 binding, we would expect this to have a measurable effect on SUV39H1 localization. This is clearly not the case, as the localization defects of the R55A mutant – observed by IF on mitotic chromosomes and by FRAP in interphase cells – quantitatively matches the localization defects of R84A , 7, whic5) A prediction of the proposed model is that loss of CD-mediated nucleic acid and H3K9me binding together should reduce SUV39H1 interaction with α-satellite RNAs more than either mutation alone. This can be done by creating a protein carrying a nucleic acid-binding mutation (R55A/R84A) with an H3K9me-binding mutation (W64/F43) (or a SET domain mutation in a Suv39H1/H2 mutant cell line). RIP experiments with these mutants will permit the authors to test this prediction directly. Either these experiments should be done or the model toned down.We agree that a prediction of our model is that breaking nucleic acid binding and H3K9me3 binding together should have a larger effect on SUV39H1 function than breaking either of these interactions in isolation. To test this, we have performed new experiments new  that dem6) We have included a new supplemental figure  showing Other revisions:1) We have now included data showing α-satellite RNA FISH signal on HeLa mitotic chromosomes new . Potenti2) For We agree this in an important consideration, and have purified MBP-SUV39H1 1-41 and measured its binding to the 19mer ssRNA updated . We foun3)Subsection “RNA associates with the pericentric regions of human mitotic chromosomes”, last paragraph: Are there other genomic regions where the RNA is associated with mitotic chromosomes in cis? If yes, then what are those euchromatic/heterochromatic regions?http://biorxiv.org/content/early/2017/03/20/118786), and we plan to apply this technique to mitotic cell populations. However, we feel that a comprehensive characterization of other genomic regions that might have cis-associated noncoding RNAs is outside the scope of this manuscript. We plan to explore this in a future study using the new methods we are currently developing.We are also very interested in the RNAs bound to mitotic chromosomes, and we hope to further explore the identities and functions of these RNA in the future. We have begun preliminary experiments purifying mitotic chromosomes from human cells in the hopes of analyzing bound RNAs by RNA-seq. In addition, our lab has developed a new technique, ChAR-seq, to map chromatin-associated RNAs genome-wide  (4) We have included a new supplemental figure comparing the ability of SUV39H1 mutants to methylate histone H3/H4 tetramer in vitro . The dat5) Subsection “A model for the RNA-dependent stabilization of SUV39H1 on chromatin”, first paragraph. The discussion of the pombe HP1/Swi6 and its requirement for Clr4-dependent H3K9me is incorrect. Even though this was shown in the referenced paper, subsequent work using the complete deletion of swi6  have shown that loss of Swi6 has little impact on Clr4-dependent H3K9me .We thank the reviewers for alerting us to this important point. We have now corrected this sentence to more accurately describe the data in the literature.6) We have now included error bars for the SUV39H1 WT measurement and calculate p values to describe HP1α localization defects in SUV39H1 mutants compared to WT.7) Subsection “SUV39H1 depends on direct RNA binding for its stable association with heterochromatin”, first paragraph. Please keep mobile/immobile percentages consistent between wt and mutant proteins. For example, convert 42% immobile fraction to 58% mobile considering the mobile fraction of mutants is indicated.We have now changed all FRAP descriptions in the text to consistently refer to the mobile fraction.


The RNA-binding protein TRBP is a central component of the Dicer complex. Despite a decade of biochemical and structural studies, the essential functionality of TRBP in microRNA (miRNA) biogenesis remains unknown. Here we show that TRBP is an integral cofactor for time-efficient Dicer processing in RNA-crowded environments. We competed for Dicer processing of pre-miRNA with a large amount of cellular RNA species and found that Dicer-TRBP, but not Dicer alone, remains resilient. To apprehend the mechanism of this substrate selectivity, we use single-molecule fluorescence. The real-time observation reveals that TRBP acts as a gatekeeper, precluding Dicer from engaging with pre-miRNA-like substrates. TRBP acquires the selectivity using the PAZ domain of Dicer, whereas Dicer moderates the RNA-binding affinity of TRBP for fast turnover. This coordinated action between TRBP and Dicer accomplishes an efficient way of discarding pre-miRNA-like substrates. The RNA binding protein TRBP is a component of the Dicer complex but its role in microRNA biogenesis remains poorly understood. Here the authors use a crowded RNA environment and single-molecule imaging to show that TRBP acts as a gatekeeper to prevent Dicer engagement with pre miRNA-like substrates. MicroRNA (miRNA) is small non-coding RNA that is ubiquitously expressed in higher eukaryotes. MiRNA biogenesis occurs through a series of enzymatic processing steps, including the cleavage of hairpin RNA (precursor miRNA or pre-miRNA) by Dicer proteins13457897111113Here we demonstrate that TRBP is a critical factor that assists Dicer to effectively find and cleave pre-miRNA among a large amount of cellular RNAs. We used biochemical and single-molecule fluorescence techniques to reveal how TRBP coordinates pre-miRNA recognition, helping Dicer to discriminate pre-miRNA-like species. Our study suggests that TRBP recruits double-stranded RNA substrates regardless of the end structure of the RNA. TRBP positions the 3′-end of RNA to the PAZ domain of Dicer to verify the authenticity of the substrate. Non-canonical substrates lacking the 2-nt 3′-overhang are quickly released by TRBP, whereas canonical pre-miRNA is transferred to Dicer for cleavage. This selective loading by Dicer-TRBP promotes the efficient RNA processing in the RNA-crowded environment.3′2nt) with micromolar competitor RNAs . The first two domains (domains 1 and 2) mediate an interaction with dsRNA molecules, while domain 3 anchors TRBP to Dicer and other cofactors111617In vivo biotinylated Dicer complexes were pulled down using Flag-beads, and Dicer immunoprecipitates (IPs) were immobilized on a surface via biotin–NeutrAvidin conjugation , a technique that we previously developedjugation . In vivoactivity . When we3′2nt) per field of view. When an equivalent amount of IPs were used, Dicer lacking TRBP showed one order of magnitude fewer stably bound pre-let-7a-13′2nt (58±13 binding) compared with Dicer associated with TRBP  and counted the number of encounters between surface-immobilized Dicer complexes and pre-let-7a-13′2nt . The truncation of TRBP's RNA-binding domain 1 (TRBP D2–D3) and both RNA-binding domains 1–2 (TRBP D3) reduced the pre-miRNA-binding affinity by 29.0±4.9% and 83.0±3.9%, respectively, when the competitor was added . In the time traces, we noticed distinctively different interactions characterized by two dwell times when both TN (R2=0.956) but instead followed a double-exponential decay . This result indicates that Dicer-TRBP exhibits two different binding modes for RNA recognition. A half of the interactions (51.9±8.2%) was short-lived . The total number of binding events was substantially reduced by the competitor RNAs when Dicer was not associated with FL-TRBP , consist3′2nt-biotin) was labelled with Cy3 (green) and was flowed into an imaging chamber. To prevent the biotinylated RNA from being immobilized, unbound NeutrAvidin was saturated with 1 mM free biotin (3′2nt) with Cy5 (red) was added together at the same concentration as a reference. The docking events of these two substrates to Dicer-TRBP were recorded simultaneously using two laser beams (532 and 633 nm). The docking of pre-let-7a-13′2nt-biotin was 3.1 times less frequently observed  compared with that of the standard construct . To find out how Dicer probes pre-miRNA-like substrates when it is not associated with TRBP, we determined the RNA-binding kinetics of Dicer alone using pre-let-7a-13′2nt-biotin. Strikingly, compared with Dicer-TRBP, Dicer alone exhibited a five times larger number of binding on this pre-miRNA-like substrate , which is 1 nt shorter than the overhang in our standard construct -tagged recombinant TRBP proteins on a quartz surface via an anti-glutathione-S-transferase (GST) antibody  within a few seconds. If the RNA has the canonical 3′ 2-nt overhang, the RNA is transferred to a more stable binding mode (ii). The RNA-binding affinity of Dicer was reduced when FL-TRBP was not associated . We specKD=0.24 nM)Our single-molecule data provide insights into how TRBP and Dicer coordinate each other for RNA recognition . TRBP, ain vitro observation in vivo, for example, by extending previous TRBP depletion experiments1133Why does RNA processing demand this intertwined coordination between Dicer and TRBP? Theoretical work has suggested that accurate target recognition can be time-efficient if two different modes of binding are coupled: a rapid mode in which a protein binds molecules promiscuously, which is followed by a subsequent slower but specific recognition mode312. Before transfection, the cells were split into 10 cm cell culture dishes at 25% confluence. Then, 24 h after seeding, the plasmids were transfected using the CaPO4 methodin vivo biotinylation of human Dicer, an additional plasmid coding for the BirA enzyme was co-transfected. After 5 h, the medium was exchanged with fresh DMEM containing 1 μg ml−1 biotin (Sigma-Aldrich), and the transfected cells were incubated for an additional 48 h to allow protein expression and in vivo biotinylation. Mycoplasma tests showed that our cells are not contaminated .Human embryonic kidney cells  were grown in Dulbecco's Modified Eagle's Medium  supplemented with 10% fetal bovine serum  at 37 °C and 5% COg at 4 °C for 5 min to form cell pellets. After discarding the PBS supernatant, the cell pellets were kept frozen at −80 °C for long-term storage. For immediate lysis of freshly collected cells, the cell pellets were frozen for 30 min or longer at −80 °C for consistency.Before collecting the cells, the DMEM was removed and the cells were washed with pre-chilled phosphate-buffered saline . The cells were transferred to 15 ml tubes and centrifuged at 300g at 4 °C for 20 min) to remove cell debris (pellet). The recovered cell extract (supernatant) was used in tandem purification steps.Before cell lysis, the cells were thawed on ice for over 30 min and subsequently resuspended in buffer D (20 mM Tris (pH 8.0), 200 mM KCl and 0.2 mM EDTA). Lysis was carried out by carefully passing the cells 10 times through a needle  while avoiding formation of air bubbles using a 1 ml syringe. After cell lysis, the lysate was centrifuged twice  at 30 °C for 90 min.For immunoprecipitation of FLAG-tagged proteins (wild type and TN Dicer), 1 mg of total protein in the cell extract was incubated 60 min with 2.5 μl of anti-FLAG antibody-conjugated agarose beads  under gentle agitation at 4 °C. After the incubation, the beads were gently washed five times with buffer D and resuspended in 10 μl of buffer D. In cases when the lysis buffer was alternatively used, the buffer was replaced with buffer D at the last step. For the tandem purification, proteins were eluted out of the beads by site-specific cleavage with tobacco etch virus (TEV) protease  and blocked with 5% skim milk (BD Difco). The membrane was exposed to primary antibodies in a 3% BSA solution at room temperature for 1 h or at 4 °C overnight. For chemiluminescence detection, horseradish peroxidase-conjugated secondary antibodies or streptavidin-horseradish peroxidase were incubated at room temperature for 30 min. Chemiluminescence was detected by incubating the membrane with the ECL solution . The chemiluminescent blots were imaged with ChemiDoc MP imager (Bio-Rad). The antibodies and their dilution factors are listed in L-lysine-coated glass coverslips. Forty-eight hours after the transfection, cells were fixed with methanol during 10 min at −20 °C, and washed with pre-chilled PBS twice. Blocking and antibodies hybridization were performed in PBS containing 3% fetal calf serum and 0.1% Triton X-100. After 1 h of incubation with fluorophore-coupled antibodies (Transfected cells were grown on poly-tibodies  at room −1 Ribonuclease inhibitor (Takara), 250–1,000 pM of internally Cy5-labelled pre-miRNA and 3 μl of the purified human Dicer. The reaction mixture was incubated at 37 °C for different incubation times. The RNA was purified from the reaction mixture by phenol extraction and separated on 10% urea polyacrylamide gel and was scanned with a Typhoon phosphorimager .Dicer cleavage reactions were performed in a total volume of 20 μl in 10 mM Tris (pH 8.0), 0.1 mM EDTA, 100 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol, 1 U ml−1), 3 μl 0.1% BSA, 5 μl 10 × ligation buffer provided and 19 μl H2O at 16 °C for 24 h. After ethanol precipitation, the RNA was purified with 12.5% urea polyacrylamide gel. The RNA strands were labelled with the NHS-ester form of Cy dyes  at an ∼100% efficiency3′2nt constructs labelled at a different position and compared the cleavage efficiency  of the microfluidic chamber was coated with poly-ethyleneglycol . A fraction of the poly-ethyleneglycol had biotin at the end . NeutrAvidin was layered on the surface via conjugation with the biotin. The details can be found elsewhere−1, Invitrogen) was incubated for 1 min in the chamber. After washing unbound NeutrAvidin away with 100 μl buffer D, biotinylated Dicer IPs (20 μl) was incubated for 1 min in the chamber. After washing the unbound proteins away with 100 μl buffer D, 200 nM dye-labelled pre-miRNA was injected in the imaging buffer unless otherwise specified. The imaging buffer consisted of buffer D (20 mM Tris (pH 8.0), 200 mM KCl and 0.2 mM EDTA (pH 8.0)), an oxygen scavenging system (0.8% glucose (v/v), 0.1 mg ml−1 glucose oxidase (Sigma-Aldrich), 17 μg μl−1 catalase (Roche)) to reduce photobleaching and 1 mM Trolox (Sigma-Aldrich) to reduce photoblinking of the dyes2nt-biotin) to the surface , the chamber was washed with 100 μl of buffer T50 (10 mM Tris (pH 8.0), 50 mM NaCl). After washing, two fluorescently labelled RNA substrates  were introduced into the microfluidic chamber. After 5 min of incubation, the unbound substrate was washed away with 100 μl of imaging buffer D and data were obtained from 10 fields of view. For each construct this procedure was repeated with three individual dilutions on three different slides.To reach an accurate 1:1 ratio between the concentrations of two RNA samples in two-colour measurements, we measured sub-nanomolar concentrations of RNA using single-molecule fluorescence. We adsorbed RNA molecules to a positively charged surface as follows. KOH-etched quartz slides were coated with a layer of positively charged poly-The fluorescent label Cy3 was imaged using prism-type total internal reflection microscopy at an excitation at 532 nm . Cy5 was excited by a 633 nm HeNe laser . When obtaining the time traces, we excited Cy3 and Cy5 molecules with 532 and 633 nm laser light sources, respectively, as weakly as possible to minimize Cy3 and Cy5 photobleaching during our observation. Under this imaging condition, only a minor fraction of the time traces were affected by Cy3 or Cy5 photobleaching or photoblinking during the first few minutes of imaging . Despiteλcutoff=645 nm, Chroma) and imaged onto two halves of an electron-multiplying charge-coupled device camera . A series of charge-coupled device images were acquired with in-house software written in Visual C++ with a time resolution of 0.3 s. The CCD images illustrated the binding events over 25 × 25 μm2 field of view.Fluorescence signals from single molecules were collected with a × 60 water-immersion objective  with an inverted microscope . Scattering of the 532 nm laser beam was blocked with a 550 nm long-pass filter . When the 633 nm laser was used, 633 nm laser scattering was blocked with a notch filter . Subsequently, the signals from Cy3 and Cy5 were spectrally split with a dichroic mirror  and analysed with Matlab (MathWorks) and Origin (OriginLab Corporation). To systematically select single-molecule fluorescence signals of Cy3 and Cy5 from the acquired images, we used an algorithm written in IDL that looked for fluorescence spots with a defined Gaussian profile and with signals above a threshold. This algorithm was effective in differentiating non-specific signals from other sources.A dwell-time distribution was fitted by either a single-exponential decay curve The data and the computer codes that support the findings of this study are available from the authors on request.How to cite this article: Fareh, M. et al. TRBP ensures efficient Dicer processing of precursor microRNA in RNA-crowded environments. Nat. Commun.7, 13694 doi: 10.1038/ncomms13694 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-10 and Supplementary Tables 1-2The movie displays a two-color competition assay (obtained with a time resolution 300 msec). The standard pre-let-7a-13' 2nt was labeled with Cy5 (right). A biotin group was attached to the 3' overhang of Cy3-labeled pre-let-7a-1 . The movie was obtained by flow measurement in a pre-steady-state (0 min) and in a steady-state condition (80 min). 200 pM of both RNA substrates was used.


NPs from all strains of influenza A viruses contain two nuclear localization signals (NLSs): a well-studied monopartite NLS1 and a less-characterized NLS2, thought to be bipartite. Through site-directed mutagenesis and functional analysis, we found that NLS2 is also monopartite and is indispensable for viral infection. Atomic structures of importin α bound to two variants of NLS2 revealed NLS2 primarily binds the major-NLS binding site of importin α, unlike NLS1 that associates with the minor NLS-pocket. Though peptides corresponding to NLS1 and NLS2 bind weakly to importin α, the two NLSs synergize in the context of the full length NP to confer high avidity for importin α7, explaining why the virus efficiently replicates in the respiratory tract that exhibits high levels of this isoform. This study, the first to functionally characterize NLS2, demonstrates NLS2 plays an important and unexpected role in influenza A virus infection. We propose NLS1 and NLS2 form a bipartite NLS  Thus, an antiviral targeting NP is likely to be effective against a wide variety of different influenza viruses. The primary function of NP is to encapsidate the viral genome, consisting of eight segments of single-stranded negative-sense RNA, into viral ribonucleoprotein complexes (vRNPs)1. The RNA segments range in length from 890 to 2,341 nucleotides and contain approximately one NP per 24 nucleotides of RNA or between 3 to 8 dozen copies of NP per RNA segment3. During the early stages of influenza infection, vRNPs are released in the cytoplasm and imported into the nucleus of infected cells for viral replication4. The nuclear import of vRNPs is mediated by NP5. After initial vRNA transcription, NP is synthesized in in the cytoplasm of infected cells as early as 2 h after infection6 and then enters the nucleus to help in the replication of full-length viral genome segments7 and to assemble progeny vRNPs4.Influenza A virus is a major worldwide threat to human health with limited antiviral therapeutics in place. Currently, only two classes of influenza antiviral drugs are available, which target viral surface proteins. Unlike surface proteins, which are highly variable between strains and mutate easily, the nucleoprotein (NP) of influenza A virus is a highly conserved protein with roles in several stages of the influenza infectious cycle9. NLSs are recognized by soluble nuclear transport receptors of the importin β/karyopherin β superfamily10. The best-characterized NLSs, often referred to as “classical” NLSs, contain either monopartite or bipartite stretches of basic amino acids that are recognized by the adapter importin α, which binds the receptor importin β/karyopherin β1 to transport the trimeric NLS-cargo:importin α/β complex into the nucleus11. NPs from all strains of influenza A viruses contain two NLSs  on the import cargoLSs Fig. : NLS1 at14. It contains two basic amino acid clusters separated by a 13 amino acid linker region  were infected with influenza A virus (strain A/X-31 H3N2). As a negative and positive control, cells expressing 5GFP or 5GFP-NLS1 were used, respectively. 5GFP was used because oligomers with four or less tandem copies of GFP freely diffuse into the nucleus, while 5GFP is excludedLS1 Fig. . SimilarTo determine whether the reduction in viral production in cells expressing 5GFP-NLS2 was due to competition of 5GFP-NLS2 with NP nuclear import during infection, cells expressing 5GFP-NLS2, 5GFP-NLS1, or 5GFP were infected with influenza A virus and NP was immunostained and localized by confocal microscopy at 2 and 10 h post-infection (p.i.). At 2 h p.i. there was no significant difference in the cellular localization of NP for all conditions Fig. . However14. A functional bipartite NLS contains two interdependent clusters of basic amino acids both of which are essential for nuclear import25. To address the contribution of the two clusters of basic residues, two 5GFP-NLS2 constructs were created in which the basic residues were substituted with alanine (A): A1 mutant with two A substitutions at the N-terminal cluster and A2 mutant with three A substitutions at the C-terminal cluster  significantly decreased the nuclear accumulation of the chimeric protein Fig. , a chimeNLS2 is highly conserved between different strains of influenza A, with the noticeable difference that avian and highly pathogenic influenza A strains have an arginine at residue 214 of NP (NLS2-R) rather than a lysine (NLS2-K), as in seasonal strains. To evaluate whether this single basic amino acid difference influences nuclear import, a 5GFP-NLS2 construct with K214→ R mutation Fig. , K214R w26 (ΔIBB-importin α1) with peptides spanning NLS2-K (198-KRGINDRNFWRGENGRKTR−216) and NLS2-R (198-KRGINDRNFWRGENGRRTR-216). The crystal structures of ΔIBB-importin α1:NLS2-K and α1:NLS2-R complexes were solved by molecular replacement and refined to an Rwork/free of 17.4/20.6% and 18.0/21.0%, at 2.25 and 2.10 Å resolution, respectively  and a minor (Arm repeats 7–8) NLS-binding pocket, each with five points of contact for the NLS side chains, referred to as P1-P5 and P1′-P5′, respectively28. Clear electron density for NLS2-K/R was observed at the major NLS site of importin α , but is nearly half than the B-factor at the minor NLS-binding site (120.0 Å2), where the electron density is very weak . Thus, NLS2 is a genuine monopartite signal that targets the major NLS-binding site of importin α.To visualize the interaction of NLS2 with importin α, we co-crystallized mouse importin α1 lacking the N-terminal importin-β-binding domainn α Figs  and 4A,Bn α Figs  and 4C. n α Figs  at the mn α Figs  and 4C. eak Fig. . Similar2 is occupied by R213 in both NP-NLS2-K and NP-NLS2-R, in stark contrast to all known NLSs that have a K at this position8. The lack of K at P2 in NLS2-K is likely compensated by K214, at position P3, which provides a register for correct positioning of the NLS at the major NLS-binding pocket. However, in NLS-2R both P2 and P3 are occupied by arginine residues (R213 at P2 and R214 at P3 position) (Table 2 and P3 are not likely to make a significant energetic contribution to the interaction with importin α. Position P1 lacks a side chain (G211), and T215 at position P4 coordinates a water molecule through the hydroxyl group. It is possible that phosphorylation of this residue enhances binding interaction with importin α, promoting nuclear import, as observed for certain phospho-NLSs29. Finally, position P5 is occupied by R216, which is sandwiched between W142 and W184  that also binds only at the major NLS-binding site but nevertheless has a lysine at P531, appears to be energetically very important. In summary, based on the lack of a lysine at position P2, we predict NLS2 of NP is a weak monopartite NLS.To decipher why an R at position 214 results in decreased association with importin α and reduced nuclear import of 5GFP as compared to NLS-K Fig. , we carrn) Table , possibl184 Fig. . Here th16 prompted us to measure the binding affinities of influenza NLS1 and NLS2 for importin α in solution. Using nano Isothermal Titration Calorimetry (nano-ITC), we measured the heat released upon titration of increasing concentrations of NLS1 and NLS2 peptides inside a cuvette containing purified ∆IBB-importin α1. For NLS1, we observed a saturable endothermic reaction, which saturated within 15–16 injections after the NLS concentration in the cuvette was ~140 µM. Binding data were fit using a one binding site model yielding an equilibrium dissociation constant (Kd) equal to 4.9 ± 0.4 µM  and NP were incubated at physiological concentration  and after reaching equilibrium, the complex was immobilized on glutathione beads, washed to remove unbound NP and eluted with reduced glutathione. Identical elution volumes were analyzed by SDS-PAGE and quantified in triplicates. Three NP mutants were also tested for binding to importin α isoforms: two NP mutants carrying Ala-mutations at P2′/P3′ and P2/P3 of NLS1 and NLS2, respectively, and one double mutant carrying mutations at both NLS1 (P2′/P3′) and NLS2 (P2/P3). Interestingly, we found that NP interacts with importins α1, α3, and α5, and to greatest extent with importin α7  and importin β (3 μΜ)  Fig. . We founμΜ) Fig. . The preμΜ) Fig. , suggest36. To this end, a detail understanding of the nuclear import signals of NP and host importins implicated in the nuclear import of the influenza genome is of paramount importance.Nuclear import of the influenza A viral genome is emerging as an important pharmacological target for the development of novel antiviral drugs that bears significant promise over classical vaccines and the current anti-influenza drugs19. The strong interaction between NP and the host importin α is however incompatible with the weak micromolar binding affinity of NLS1 for importin α  and NLS2 (Kd = 72.4 ± 10.0 µM) reported in this paper are significantly lower than those measured using fluorescence-depolarization39 or surface-immobilized NLSs40, but nonetheless consistent with published ITC studies that employed short NLS-peptides43. Both Kds are significantly higher than the physiological concentration of importin α (~1 μM34), suggesting neither NLSs are sufficient to promote NP nuclear import.in trans like a bipartite NLS that forms only in the tertiary (or quaternary) structure of NP  and NLS2-K214 (at P2) is about 30 Å, which is roughly equal to the distance between NLS1 and NLS2 in the 3D-structure of monomeric NP, although the N-terminal NLS1 was not visible in the crystal structure of NP44 and was computationally modelled. This model predicts that the overall avidity of NP for importin α is determined by the product of the Kds of each NLS for importin α45, explaining how two weak NLSs can function as a potent import signal if simultaneously bound to importin α. It is possible NP developed two weak NLSs, each bearing only 2–3 basic residues rather than 4–5 like most classical NLSs, in order to prevent non-specific association with cellular and viral RNA that could prevent association with importins and thus nuclear translocation. The conservation of multiple weak NLSs complementing in trans to increase the avidity for importin α was also proposed for the tripartite terminase complex of certain herpesviruses43 and may represent a recurring theme in virology.We propose NLS1 and NLS2 function  NP Fig.  and inte NP Fig. . The dis46 but opens up like a bipartite NLS in its auto-inhibited conformation bound to the Arm-core28. Likewise, NF-κB p65 NLS, also within a helical region in complex with IκBα, functions like a classical importin α-dependent NLS47. Interestingly, removing NLS2 does not disrupt NLS1, but possibly slows down nuclear import, which is dominated by kinetics48. This is not readily appreciated in vitro, where disruption of NLS2 had little effect on nuclear accumulation of NP measured using a nuclear import assay in digitonin permeabilized cells21. However, in vivo, where the concentration of NP is typically low and NLS1 is outweighed by a plethora of much stronger cellular NLS-cargos, the synergy of two NLSs is likely essential for infection, especially in cells that have lower concentrations of importin α7, the isoform to which NP binds with the highest avidity  equilibrated in Gel Filtration (G.F.) buffer . Purified ΔIBB-importin α isoforms were concentrated to 20 mg/ml using a Millipore concentrator (cut-off 10 kDa). Expression and purification of human importin β and Ran loaded with GDP (RanGDP) or nonhydrolyzable GTP (RanGppNHp) were carried out as described53. Peptides encompassing influenza NLS1 (1-MASQGTKRSYEQM-13), NLS2-K (198-KRGINDRNFWRGENGRKTR-216) and NLS2-R (198-KRGINDRNFWRGENGRRTR-216) were custom synthesized (GenScript) and purified at 95% homogeneity by reverse phase chromatography. Influenza A virus nucleoprotein and mutants were expressed in Escherichia coli BL21 (DE3) cells and purified using a previously reported procedure44.Importin α isoforms α1, α3, α5 and α7 lacking the N-terminal IBB-domain were expressed in bacteria and purified as previously described2 in Dulbecco’s modified Eagel’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% L-glutamine, and 1% sodium pyruvate. HeLa cells grown as monolayers on glass microscope coverslips were transfected with 5GFP or 5GFP-NLSs plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction.HeLa cells were maintained at 37 °C and 5% COTwenty-four h after transfection the cells were fixed with 3% paraformaldehyde in phosphate buffered saline (PBS) for 15 minutes at room temperature. Cells were then washed with PBS three times, fixed with 3% paraformaldehyde (PFA), and the coverslips were mounted onto microscope slides. Samples were visualized using a Fluoview FV1000 confocal laser-scanning microscope (Olympus).HeLa cells were seeded on glass microscope coverslips and transfected with 5GFP or 5GFP-NLSs using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. Twenty-four h post transfection, cells were infected with purified influenza A virus ; Charles River Laboratories) at a multiplicity of infection (MOI) of 4 in DMEM supplemented with 0.2% FBS. Cells were incubated for 15 minutes at 4 °C to allow the virus to bind to the cell surface. Cells were then moved to 37 °C for 1 h to allow virus internalization. After this incubation period, a mild acidic wash  was performed to exclude the delayed uptake of attached, but not internalized virus particles. Subsequently, cells were incubated at 37 °C in DMEM supplemented with 2% FBS for 1 h and 9 h, respectively. Cells were then washed with PBS three times, fixed with 3% PFA for 15 minutes, permeabilized with 0.2% Triton X-100 in PBS for 5 minutes, and incubated with PBS containing 1% bovine serum albumin (BSA) and 10% goat serum for 1 h at room temperature. Cells were then incubated with mouse NP monoclonal antibody (1:1000 dilution)  for 1 h at 37 °C, washed with PBS containing 2.5% BSA, and incubated with goat anti-mouse conjugated with Alexa Fluor 568 (1:2000 dilution) for 45 minutes at room temperature. Coverslips were mounted in Prolong Gold antifade reagent containing DAPI. Samples were visualized using an Olympus Fluoview FV1000 laser-scanning microscope.5 cells/well 48 h before plaque assay. Supernatants containing virus progeny were serially diluted using 10-fold dilutions. Two hundred µl of each dilution was added to each well in triplicates. The plates were incubated at room temperature for 1 h on a shaker. After two times wash with PBS, 2 ml of nutrient agar overlay  was added to each well and the cells were incubated at 37 °C in 5% CO2 for 72 h. Next, cells were fixed with 4% PFA and stained with 1% crystal violet in 20% methanol. The plaques were counted and used for viral titer calculation. The virus titers were expressed as plaque forming units (PFU)/ml = [(numbers of plaques per well) X dilution]/(inoculum volume).Supernatant were obtained from infected cells after 24 h infection and used to evaluate virus titers. Madin-Darby canine kidney (MDCK) cells were seeded in 6-well plates at a density of 7 × 1050. Briefly, the mean intensity of a defined area in the nucleus was measured and divided by the mean intensity of the same size area in the cytoplasm from the same cell using ImageJ . The fluorescence of the nuclear envelope was not included in the quantification. After correction for background fluorescence, the results were expressed as the ratio of nuclear to cytoplasmic fluorescence (Fn/c). Data was obtained from a total of 85–100 cells per experiment from three independent experiments. Results were analyzed by One-way ANOVA followed by Tukey’s test using GraphPad Prism . All data are represented as the mean value ± standard error of the mean and p < 0.05 were considered significant.To quantify the nuclear import of chimeric proteins, the ratio of the nuclear to cytoplasmic fluorescence signal was determined as previously described−1 and 0.7 µl (3-fold molar excess) of peptide with an equal volume of 0.1 M HEPES buffer (pH 6.0), 0.6 M sodium citrate tribasic ehydrate, 10 mM β-mercaptoethanol and equilibrated against 600 µl of the same precipitant, at 18 °C. Crystals were harvested in nylon cryo-loops, cryo-protected with 27% ethylene glycol and plunged in liquid nitrogen. Crystals were diffracted at LS-CAT Beamline 21-ID-F at Argonne Photon Source on a MARMOSAIC 225 CCD detector and at beamline 14–1 at SSRL on a Rayonix MX325 CCD detector. Data indexing, integration and scaling were carried out with the HKL200054. Initial phases were obtained by molecular replacement using Phaser55 and PDB entry 3Q5U as a search model. Atomic models were built using Coot56 and refined using phenix.refine57. Data collection and refinement statistics are summarized in Table 58.Crystals of ∆IBB-importin α1 bound to NLS2 were obtained by mixing 2.3 µl of gel filtration-purified importin α1 at 18.0 mg ml43. Influenza NLS1 and NLS2-K peptides were dissolved in G.F. buffer between 600–850 μM and injected in 2 μl increments into a calorimetric cell containing 195 µl of ΔIBB-importin α1 at 120 μM. The spacing between injections was 300 seconds. Titration data were analyzed using the NanoAnalyze data analysis software (TA Instruments). Heats of dilution were determined from control experiments with the ITC buffer and subtracted prior to curve fitting using a single set of binding sites model.ITC experiments were carried out at 25 °C using a nano-ITC calorimeter (TA Instruments)34 of purified GST-importin α isoforms (1 μM) (with or without IBB), importin β (3 μM), RanGppNHp or RanGDP (5 μM) and influenza NP (0.75 µM). Pull-downs with different isoforms in Fig. 59 was used to quantify all the bands and data are presented as pixel ratios. Briefly, the pixel density of each NP/mutant band in Fig. 60.All pull-downs were carried out using physiological concentrationsSupplementary information


Application of apomixis, or asexual seed formation, in crop breeding would allow rapid fixation of complex traits, economizing improved crop delivery. Identification of apomixis genes is confounded by the polyploid nature, high genome complexity and lack of genomic sequence integration with reproductive tissue transcriptomes in most apomicts.Hieracium subgenus Pilosella (Asteraceae) which incorporates characterized sexual, apomictic and mutant apomict plants exhibiting reversion to sexual reproduction. Apomicts develop additional female gametogenic cells that suppress the sexual pathway in ovules. Disrupting small RNA pathways in sexual Arabidopsis also induces extra female gametogenic cells; therefore, the resource was used to examine if changes in small RNA pathways correlate with apomixis initiation. An initial characterization of small RNA pathway genes within Hieracium was undertaken, and ovary-expressed ARGONAUTE genes were identified and cloned. Comparisons of whole ovary transcriptomes from mutant apomicts, relative to the parental apomict, revealed that differentially expressed genes were enriched for processes involved in small RNA biogenesis and chromatin silencing. Small RNA profiles within mutant ovaries did not reveal large-scale alterations in composition or length distributions; however, a small number of differentially expressed, putative small RNA targets were identified.A genomic and transcriptomic resource was developed for Hieracium resource represents a substantial contribution towards the investigation of early sexual and apomictic female gamete development, and the generation of new candidate genes and markers. Observed changes in small RNA targets and biogenesis pathways within sexual and apomictic ovaries will underlie future functional research into apomixis initiation in Hieracium.The established The online version of this article (doi:10.1186/s12915-016-0311-0) contains supplementary material, which is available to authorized users. Apomixis describes a suite of reproductive processes in flowering plants that result in asexual seed formation, and subsequently give rise to progeny which are clones of the maternal parent . ContrarPennisetum that stimulates fertilization-independent embryogenesis )[http://www.pymol.org/).Models of the DB:4OLB]), 77. TheDB:4OLB]), followeDB:4OLB]). The aliDB:4OLB]). StereocDB:4OLB]). Z-scoreDB:4OLB]). StructuDB:4OLB]), where 7