The Ccr4-Not complex removes mRNA poly(A) tails to regulate eukaryotic mRNA stability and translation. RNA-binding proteins contribute to specificity by interacting with both Ccr4-Not and target mRNAs, but this is not fully understood. Here, we reconstitute accelerated and selective deadenylation of RNAs containing AU-rich elements (AREs) and Pumilio-response elements (PREs). We find that the fission yeast homologues of Tristetraprolin/TTP and Pumilio/Puf (Zfs1 and Puf3) interact with Ccr4-Not via multiple regions within low-complexity sequences, suggestive of a multipartite interface that extends beyond previously defined interactions. Using a two-color assay to simultaneously monitor poly(A) tail removal from different RNAs, we demonstrate that Puf3 can distinguish between RNAs of very similar sequence. Analysis of binding kinetics reveals that this is primarily due to differences in dissociation rate constants. Consequently, motif quality is a major determinant of mRNA stability for Puf3 targets in vivo and can be used for the prediction of mRNA targets. When a cell needs to make a particular protein, it first copies the instructions from the matching gene into a molecule known as a messenger RNA (or an mRNA for short). The more mRNA copies it makes, the more protein it can produce. A simple way to control protein production is to raise or lower the number of these mRNA messages, and living cells have lots of ways to make this happen. One method involves codes built into the mRNAs themselves. The mRNAs can carry short sequences of genetic letters that can trigger their own destruction. Known as “destabilising motifs”, these sequences attract the attention of a group of proteins called Ccr4-Not. Together these proteins shorten the end of the mRNAs, preparing the molecules for degradation. But how does Ccr4-Not choose which mRNAs to target?Different mRNAs carry different destabilising motifs. This means that when groups of mRNAs all carry the same motif, the cell can destroy them all together. This allows the cell to switch networks of related genes off together without affecting the mRNAs it still needs. What is puzzling is that the destabilising motifs that control different groups of mRNAs can be very similar, and scientists do not yet know how Ccr4-Not can tell the difference, or what triggers it to start breaking down groups of mRNAs. To find out, Webster et al. recreated the system in the laboratory using purified molecules.The test-tube system confirmed previous suggestions that a protein called Puf3 forms a bridge between Ccr4-Not and mRNAs. It acts as a tether, recognising a destabilising motif and linking it to Ccr4-Not. Labelling different mRNAs with two colours of fluorescent dye showed how Puf3 helps the cell to choose which to destroy. Puf3 allows Ccr4-Not to select specific mRNAs from a mixture of molecules. Puf3 could distinguish between mRNAs that differed in a single letter of genetic code. When it matched with the wrong mRNA, it disconnected much faster than when it matched with the right one, preventing Ccr4-Not from linking up.The ability to destroy specific mRNA messages is critical for cell survival. It happens when cells divide, during immune responses such as inflammation, and in early development. Understanding the targets of tethers like Puf3 could help scientists to predict which genes will switch off and when. This could reveal genes that work together, helping to unravel their roles inside cells. Shortening or removal of mRNA poly(A) tails (deadenylation) represses gene expression in eukaryotes. The Ccr4-Not complex is a conserved deadenylase that can initiate cytoplasmic mRNA decay and reduce translation by releasing poly(A)-binding protein (Pab1/PABPC1) . Ccr4-NoRNA-binding proteins such as Pumilio/Puf and Tristetraprolin/TTP can recognize sequence motifs in mRNAs, and also bind Ccr4-Not to target those RNAs for deadenylation . The setRecently, three sequence elements that strongly promote mRNA destabilization were identified in an unbiased screen in zebrafish embryogenesis: the Pumilio-response element (PRE), the AU-rich element (ARE) and the miR-430 seed sequence . PRE- anSaccharomyces cerevisiae (Sc), there are six Pumilio proteins. Of these, ScPuf3 is most closely related to homologs in higher eukaryotes  also contains residues that define this additional selectivity pocket, and is therefore predicted to bind sequences containing an upstream cytosine  do not necessarily regulate mitochondrial function  was identified in unstable mRNAs encoding cytokines and lymphokines in human cells . This seNA motif . An inteS. pombe Ccr4-Not complex after overexpression of the subunits in insect cells  fusion proteins, and intact recombinant seven-subunit eviously . Pull-doeviously . TherefoBy using a short model RNA substrate, deadenylation can be visualized at single-nucleotide resolution, and can be accurately quantified . To inveCcr4-Not removed the poly(A) tails of both Puf3 and Zfs1 substrate RNAs in vitro. Puf3 and Zfs1 each stimulated deadenylation of their target RNAs: In the absence of Puf3, complete removal of the poly(A) tail from PRE-containing RNA occurred in approximately 32 min, whereas in the presence of Puf3 the reaction was complete in less than 2 min . TherefoNon-adenosine residues upstream of the poly(A) tail were also rapidly removed by Ccr4-Not when Puf3 or Zfs1 were present. The exonucleases stop when they reach nucleotides that correspond to the binding site of the protein, suggesting that either the RNA-binding proteins cannot be released by Ccr4-Not, or that deadenylation becomes very slow after release of the RNA binding protein. This is consistent with a model in which the specificity of Ccr4-Not for adenosine is substantially less evident when it is tethered to the RNA substrate for a prolonged period of time . In addiIn the absence of additional protein, Ccr4-Not is approximately 3-fold more active on the ARE-containing substrate than on the PRE-containing substrate . It is gPUM and Zfs1TZF, PUM caused a modest acceleration . Compared to the wild-type proteins, these mutants had a substantially reduced ability to bind their substrate RNAs, and to accelerate deadenylation -selective exonucleases, Caf1 and Ccr4. To determine whether Puf3 and Zfs1 can stimulate deadenylation in the absence of the non-enzymatic subunits, we performed reactions with a purified dimeric subcomplex comprised of Caf1 and Ccr4 . Puf3 acZfs1 did not substantially affect the deadenylation activity of the Caf1-Ccr4 dimeric complex , right. Poly(A)-binding protein (Pab1) contributes to translation initiation, and its release from mRNAs can repress translation . Ccr4-NoElectrophoretic mobility shift assays showed that stoichiometric amounts of Puf3 and Pab1 could be simultaneously loaded onto the RNA substrate . In deadWe previously reported that Caf1 and Ccr4 have similar activities within the purified Ccr4-Not complex . More reUsing our in vitro assays, Puf3 accelerated deadenylation by both Ccr4-inactive and Caf1-inactive complexes . This inHaving established that Puf3 and Zfs1 are capable of accelerating Ccr4-Not activity, we sought to test whether they are also sufficient for substrate-selectivity. We began by examining whether the presence of the target motif recognized by each protein was essential for accelerated deadenylation. The effect of Puf3 and Zfs1 on its own target RNA was compared with its effect on a non-target RNA. For the latter, we used the RNA target of the other protein . We were surprised to find that each protein stimulated deadenylation of an RNA that did not contain its target motif . QuantifSince Puf3 with a mutated RNA-binding domain still stimulates deadenylation, allosteric activation of Ccr4-Not by the Puf3 low complexity region may contribute to this accelerated deadenylation of off-target RNAs . HoweverKD = 1–2 nM). In contrast, Puf3 bound to the off-target ARE sequence with KD of 120 nM, while Zfs1 bound the off-target PRE sequence with KD of 850 nM. In both cases, poly(A) was bound with ~100 fold lower affinity than the target RNA. Hence, both Puf3 and Zfs1 are selective in RNA binding, with >100 fold higher affinity for their target RNA than off-target RNAs. Still, Puf3 and Zfs1 bind off-target sequences with nanomolar affinities and this could account for their abilities to stimulate deadenylation of off-target RNAs.To determine the binding affinities of full-length Puf3 and Zfs1 for the RNA substrates, we performed fluorescence polarization RNA binding assays with RNAs containing the target motif of Puf3 or Zfs1, as well as 30-mer polyadenosine . Each RNWe hypothesized that Puf3 and Zfs1 would select their targets from a mixture of target and non-target RNAs to promote targeted deadenylation. To test this, we developed a system to directly examine selective deadenylation by Ccr4-Not. Two polyadenylated RNA substrates labelled with different fluorophores were combined in stoichiometric quantities before the addition of Ccr4-Not and analysis of deadenylation activity. The same gel could therefore be used to monitor deadenylation of both the fluorescein-labelled Puf3-target substrate, and the Alexa647-labelled Zfs1-target substrate, displayed in blue and red respectively . In thisWe then performed a series of reactions using three different concentrations of Puf3 or Zfs1, and each RNA at 100 nM. With 50 nM Puf3 or Zfs1, deadenylation was accelerated only on the target RNA , middle.To examine RNA binding by Puf3 and Zfs1 under the same conditions, we performed electrophoretic mobility shift assays. Consistent with deadenylation assays, target RNA binding is complete and selective at 250 nM, but is non-selective at 1000 nM . In concS. cerevisiae (Sc) Puf3 differs from a target of ScPuf5/Mpt5 in two nucleotide positions within a core 8-nt motif , both the ScPuf3- and ScPuf5-target RNAs were deadenylated rapidly. We performed RNA EMSAs and found that the ability of Puf3 to stimulate deadenylation correlated with RNA binding: binding to ScPuf5-target RNA occurred only when Puf3 protein was in molar excess to the ScPuf3-target RNA ). We tested whether there is a preference for A or U at position five using the two-color deadenylation assay and found that the ScPuf3-target(5A) RNA was deadenylated in preference to ScPuf3-target(5U) RNA (We next examined whether Puf3 can distinguish between sequences that are even more similar. Within the 8-nucleotide motif recognized by olerated . This isolerated . In the (5U) RNA . Thus, PScPuf3-target(5A), ScPuf3-target(5U), and ScPuf5-target. We measured the rates of association (onk) and dissociation (offk) of fission yeast Puf3PUM binding to each of these, as well as to polyadenosine as a negative control.To gain insight into the selective RNA binding by Puf3 that allows it to distinguish between RNAs of very similar sequences, we analyzed the binding kinetics using SwitchSENSE . SwitchaScPuf3-target(5A) was ~25% faster than with ScPuf3-target(5U) and ~three fold faster than with the ScPuf5-target. Association with polyadenosine was even slower (~seven fold). Thus, RNA sequence affects the rate of Puf3 association. Importantly, however, all sequences were bound rapidly: at a concentration of 20 nM, binding to all RNAs took place on the timescale of seconds  (ScPuf3 to 476 mRNAs using an RNA-tagging method (ScPuf3 regulation therefore remains unclear.We compiled a list of 1331 putative IP-chip) . A more IP-chip) : the larg method . While tg method . The setWe began by computationally identifying the most enriched sequence motif in the complete set of 1331 mRNAs using MEME . This haScPuf3 binding site is within the COX17 mRNA .The best-characterized X17 mRNA . This mRX17 mRNA . Interesition +5 . Having y values   were related to mitochondrial biogenesis , as well as mRNAs related to mitochondrial biogenesis but with motif 4 in the 3ʹ-UTR (33 transcripts). These represent possible false-positives and false-negative predicted targets respectively. mRNAs with motif 1, 2 or 3 that were not related to mitochondrial biogenesis had significantly less ScPuf3 association than those that were related to mitochondria  will detect mRNAs that are not genuine targets. An indication of in vivo dissociation rates of  tagging . In this with it . This st with it . Taken t with it  studies. with it .S. cerevisiae Puf3 demonstrates, however, that accurate prediction may be possible from examination of the 3ʹ-UTR sequence. If, as our data suggests, the function of S. cerevisiae Puf3 is limited to mitochondrial biogenesis,~88% of mRNAs with a high-quality motif are genuine targets. The workflow we have used should therefore be informative in defining the regulatory network of other RNA-binding proteins  in Sf9 cells (Sf9 lysate used for Ccr4-Not expression (S. pombe Pab1 (amino acids 80–653) was prepared as described previously (Intact Ccr4-Not complex was purified as described previously after overexpression of the seven core subunits of the f9 cells . The Cafpression . S. pombeviously .S. pombe Puf3 and Zfs1 were synthesized with codon optimization for E. coli (GenScript). Primers Puf3_Fwd and RBP_Rev were used to amplify the Puf3 sequence, and primers Zfs1_Fwd and RBP_Rev were used to amplify the Zfs1 sequence (Mut: Y449A/Y671A) and the Zfs1 RNA-binding domain (Zfs1Mut: F349A/F387A). Primers used to introduce mutations (Puf3_Quikchange and Zfs1_Quikchange) are listed in DNA sequences encoding full-length sequence . DNA wasE. coli (Thermo Fisher Scientific). Transformed cells were grown at 37°C to an A600 nm of 0.6 before the temperature was reduced to 18°C and protein expression induced by the addition of IPTG to 0.5 mM. Growth was continued for 18 hr before cells were harvested by centrifugation and flash frozen for storage at −80°C. Cells were defrosted and lysed by sonication in lysis buffer  supplemented with protease inhibitor cocktail (Roche) and DNase I (5 μg/ml) (Sigma). The lysate was cleared by centrifugation at 40,000 × g for 30 min at 4°C. Supernatant was applied to amylose affinity resin , and incubated with rotation for 1 hr at 4°C. Resin was separated from lysate with an Econo-Column (Bio-Rad) and washed three times with 50 ml of RBP buffer . Protein was eluted from the resin with RBP buffer supplemented with 50 mM maltose. Sample was loaded onto a HiTrap Q HP 5 ml column , and the protein was eluted with a gradient of 20 column volumes into RBP buffer containing 1 M NaCl. Protein in the elution peak was concentrated to 0.5 ml with an Amicon Ultra concentrator  by centrifugation at 3000 × g at 4°C. Sample was then applied to a Superdex 200 10/300 GL size-exclusion column  equilibrated in RBP buffer. The column was run at 0.5 ml/min at 4°C. Full-length protein eluted at 10–13 ml, typically together with smaller protein species identified by tandem mass spectrometry to be truncated protein lacking C-terminal residues. The sample was applied to a HiTrap Heparin HP 5 ml column , and the protein was eluted with a gradient of 20 column volumes into RBP buffer containing 1 M NaCl. Full-length protein eluted at ~250 mM NaCl, while shorter fragments eluted at 100–200 mM NaCl. Peak fractions were concentrated with an Amicon Ultra 50 kDa MWCO centrifugal concentrator (Millipore) and stored at −80°C.Puf3 and Zfs1 were expressed as N-terminal MBP fusions in BL21 Star (DE3) S. pombe Puf3 was defined as residues 378–714, while the RNA-binding TZF domain of S. pombe Zfs1 was defined as residues 699–769. DNA sequences encoding the RNA-binding domains of Puf3 (Puf3PUM) and Zfs1 (Zfs1TZF) were amplified from vectors encoding full-length proteins using primers Puf3_PUM_Fwd and Puf3_PUM_Rev or Zfs1_TZF_Fwd and Zfs1_TZF_Rev  E. coli (Thermo Fisher Scientific). Expression and cell lysis were performed as described for full-length MBP-Puf3 and MBP-Zfs1. Clarified lysate was applied to glutathione sepharose 4B affinity resin , and incubated with rotation for 1 hr at 4°C. Resin was separated from lysate with an Econo-Column (Bio-Rad) and washed three times with 50 ml of RBP buffer. Protein was eluted from the resin in 20 ml of glutathione elution buffer . The GST tag was cleaved by incubation with 1 mg of 3C protease for 16 hr at 4°C. Cleaved protein was buffer-exchanged into 20 mM Bis-Tris pH 6.5, 50 mM NaCl, 0.1 mM TCEP with a HiPrep 26/10 desalting column . The sample was applied to a Mono S 5/50 GL cation-exchange column  and eluted over 20 column volumes into buffer supplemented to 1 M NaCl. Pure protein eluted at ~200 mM NaCl, and samples were pooled and stored at −80°C.The RNA-binding PUM domain of _TZF_Rev . These w2, 0.1 mM TCEP and added to a final concentration of 100 nM in the reaction. Reactions were performed in 20 mM PIPES pH 6.8, 10 mM KCl, 45 mM NaCl, 2 mM Mg(OAc)2, 0.1 mM TCEP (includes components added with protein factors) at 22°C.Deadenylation activity was measured as previously described . Puf3 anImageJ as described previously . Samples were applied to TBE (Tris-borate-EDTA), 20% polyacrylamide gels containing 7 M urea and run at 400 V in 1 × TBE running buffer. Gels were scanned with a Typhoon FLA-7000 with excitation at 478 nm. Densitometric analysis was performed with eviously . Poly(A)eviously . DeadenyScPuf3(5U)-target-A30 RNAs were synthesized with a 5ʹ-Alexa647 fluorophore label, and Puf5-target-A30 with a 5ʹ-Cy3 fluorophore label (Integrated DNA Technologies). Reactions contained 100 nM of each RNA, mixed prior to addition of protein factors. Gels were scanned twice with a Typhoon FLA-7000 with excitation at 478 nm (fluorescein detection) and either 633 nm (Alexa647 detection) or 573 nm (Cy3 detection). Images were superimposed with color overlay.For RNA competition experiments, Zfs1-target-A30 and 2, 0.1 mM TCEP. For RNA competition experiments, 100 nM of each RNA was mixed prior to addition of protein. The sample was incubated for 15 min at 22°C before the addition of 6 × loading dye (30% glycerol and 0.2% w/v orange G). Samples were loaded onto 6% TBE-polyacrylamide non-denaturing gels and electrophoresis was performed at 100 V in 1 × TBE running buffer. Gels were scanned with a Typhoon FLA-7000 with excitation at 478 nm (fluorescein detection) and 633 nm (Alexa647 detection).Binding reactions (10 μl) were prepared by adding protein at the indicated concentration to RNA (200 nM) in 20 mM PIPES pH 6.8, 10 mM KCl, 90 mM NaCl, 2 mM Mg(OAc)GraphPad Prism 6. Error bars indicate the standard deviation in three replicate measurements.A two-fold protein dilution series was prepared in 20 mM HEPES pH 7.4, 150 mM NaCl. Proteins were incubated for 2 hr at 22°C with 0.2 nM 5ʹ 6-FAM RNA (synthesized by IDT). Fluorescence polarization was measured with a PHERAstar Plus microplate reader (BMG Labtech). Dissociation constants were estimated by non-linear regression in Kinetic measurements were performed using a DRX series instrument with a MPC-48–2-Y1 chip (Dynamic Biosensors). Hybrid oligonucleotides were synthesized (IDT) with the RNA of interest  at the 5PUM (residues 378–714) was applied to the chip in binding buffer  at 20°C with a flow rate of 30 μl/min. For association measurements, a series of protein concentrations was tested: 5, 10, 15, 20, 25, 30, 50, 70 nM for ScPuf3- and ScPuf5-target RNA; 50, 100, 150, 200 nM for poly(A). The dynamic response represents the change in nanolever switch speed on the timescale 0–4 μsec as described previously (Kobs) was estimated by fit of an exponential curve to data points collected with a sampling rate of 1 s with GraphPad Prism 6. Error bars indicate the standard error in three replicate measurements. Association rate (onk) was calculated by linear regression analysis of the protein concentration series.Purified Puf3eviously . The obsPUM was loaded onto RNA as described above. Binding buffer was then flowed across the chip at 20°C with a flow rate of 30 μl/min. Data points from dissociation experiments with ScPuf3-target and ScPuf5-target RNAs were averaged in 10 s intervals to improve the signal-to-noise. Dissociation rates (offk) were estimated by fitting of an exponential function with GraphPad Prism 6.For analysis of dissociation rate, 20 nM Puf3Saccharomyces cerevisiae mRNAs that are candidates for Puf3 regulation were collated from published sources . For this reason, these sequences were treated as equivalent and the mRNAs containing them analyzed as a group. We do not exclude the possibility that differences in ScPuf3 binding exist because statistically evaluation is limited to the number of endogenous genes containing each permutation. Motifs were assigned to the category 3ʹ-UTR if at least part of the motif contained nucleotides 3ʹ of the coding sequence (including the stop codon). Statistical significance was evaluated with a Mann-Whitney rank comparison test in GraphPad Prism.Motifs were identified in mRNA sequences with  v 7.0.6  with thehttp://yeastmine.yeastgenome.org) and the references contained therein.Gene ontology (GO) analysis was performed with PANTHER over-representation test using the dataset released on 2018-02-02 . Compreh 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 three peer reviewers, including Rachel Green as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Kevin Struhl as the Senior Editor.Thank you for submitting your article "The mechanism of Ccr4-Not recruitment to specific mRNAs involves sequence-selective tethering by RNA-binding proteins" for consideration by S. cerevisiae CLIP data is seen to be strength, but the discussion needs some re-working to make connections between the in vitro and in vivo work clearer, and to acknowledge previous in vivo studies in the field that came to related conclusions.We have received reviewer comments and have discussed the manuscript together in coming to a decision. Overall, all three reviewers felt the manuscript detailing how RNA binding protein promote Ccr4-Not mediated deadenylation using a really beautiful in vitro reconstituted system was extremely well executed and brings to light important features of this biological interaction. Importantly, the dependence of the rates of deadenylation on the dissociation rates of the complexes is interesting, and nicely complements previously published in vivo work on this topic . The analysis of already published eLife.We recommend that the manuscript be revised, taking into account the reviewers’ suggestions, and that it will then be appropriate for publication in Reviewer #1:eLife.In this manuscript by Webster et al., the authors describe experiments that define how Ccr4-Not-promoted deadenylation is promoted by RNA binding proteins that recognize specific motifs on the targeted mRNAs. The experiments are entirely performed in vitro with purified proteins and clean kinetically-refined deadenylation assays. The studies show that the fission yeast protein Zfs1 and Puf3 increase the rates of deadenylation on mRNAs that contain a motif that they are known to recognize, and that this recruitment is primarily mediated by the very long disordered regions of these RNA binding proteins (in some opposition to earlier work from Goldstrohm's group). The specificity of the RNA binding protein is shown to be critical, but when concentrations become high, less optimal mRNA targets readily become targets . In the final section of the manuscript, the authors perform a detailed analysis of three different data sets in the literature defining the targets of these mRNA binding proteins in genome-wide experiments. Here the authors are able to better define that specificities of these proteins, and for Puf3, to rationalize how this protein is principally involved in regulating the stability of mRNAs associated with mitochondrial function. This final analysis lends some in vivo depth to the manuscript that raised its significance substantially. In general, the manuscript was very clearly written and the experiments nicely supported the stated conclusions. I recommend the paper for publication in Reviewer #2:S. pombe Pumilio and Tristetraprolin homologs recruit the Ccr4-Not complex to deadenylate mRNAs.This manuscript presents biochemical evidence that the While the biochemical data presented in this manuscript are of high quality, evidences that Pumilio and Tristetraprolin recruit the Ccr4-Not complex to deadenylate mRNAs in other species have been widely documented. In this context, the data described in this manuscript appear mostly confirmatory, even if they are based on a different, more direct, analysis. The conceptual advance brought by this manuscript is thus limited.S. pombe Tristetraprolin interacting with the Ccr4-Not complex is not as well defined as the one described 5 years ago for its human homolog for which structural data were presented). Furthermore, a significant fraction of the manuscript focuses on analysis of published data emanating from S. cerevisiae that are rather descriptive, not validated, and poorly connected to the experimental data presented in the rest of the manuscript.While the manuscript does bring new details, e.g. on the context effect on RNA binding, those are not unexpected and will be of interest only to specialists. Moreover, in some case, specific conclusions remain preliminary , make the data and conclusions compelling.PUM . In those studies, the number of uridines added in tagging very likely is related to PUF-RNA dissociation rates in vivo, which correlates well with whether the mRNAs are in fact regulated by Puf3 in the cell. It is in the authors’ interest to make the point that in vivo studies of tagging led to the suggestion of "sampling" vs. "biological regulation" as a function of off-rates; the convergence of the in vitro and in vivo data is striking.1) A central conclusion is that dissociation rates are a key contributor to rates of deadenylation. This important conclusion complements in vivo "tagging" experiments on Puf3S. pombe Puf3PUM and S. pombe Ccr4-Not. This raises a potential confusion for the reader, in that the computational analyses done later in the paper focus on mRNAs that are bound and controlled in vivo in S. cerevisiae by the S. cerevisiae protein. Unfortunately, this makes some of the discussion of Figure 6 a bit confusing, and needs clarification in the text. It is not that mRNAs that bind S. pombe Puf3 in S. pombe necessarily are mitochondrial in function, a point that is relevant to computational studies of site divergence during evolution .2) The experiments use The data and analyses of Figure 6 certainly are informative, and the issue should be dealt with by clarifications in the Results and Discussion sections.3) I may have missed this, but how do the authors quantify deadenylation "rate" ? Does "rate" refer to moles of adenosine monophosphate released per unit time, or the amount of fully deadenylated product per unit time, or something else?eLife. Your revised article has been favorably evaluated by Kevin Struhl (Senior Editor), a Reviewing Editor, and two reviewers.Thank you for resubmitting your work entitled "The mechanism of Ccr4-Not recruitment to specific mRNAs involves sequence-selective tethering by RNA-binding proteins" for further consideration at eLife.Overall, the reviewers felt that most comments had been adequately addressed. However, one reviewer feels that the title and Abstract of the manuscript must be altered to acknowledge what precisely is novel in the study which is the biochemical discrimination between target and non-target mRNAs. Once this issue has been addressed the manuscript should be ready for publication in Reviewer #2:Passmore and colleagues have revised their manuscript, considering most referees' comments. Minor points have been clarified. In line with editorial recommendation, authors argue that their "major conceptual advance is not that Pumilio and Tristetraprolin proteins promote deadenylation, but an improved understanding of two aspects of this process – (1) how RNA-binding proteins distinguish between target and non-target mRNAs, and (2) how RNA-binding proteins recruit Ccr4-Not to achieve rapid deadenylation".As discussed earlier, our new understanding of interaction of RNA binding proteins with the Ccr4-Not is limited in the context of data already available in the literature (see below). Consistently, the authors indicate that a full understanding of this binding will require further studies. This point remains accessory.Altogether, there is a general agreement that the main new point addressed in this manuscript is how RNA-binding proteins distinguish between target and non-target mRNAs. While the text has been improved to clarify this issue, the manuscript title "The mechanism of Ccr4-Not recruitment to specific mRNAs involves sequence-selective tethering by RNA-binding proteins" and Abstract are still confusing. They don't do justice to the numerous manuscripts and groups that have reported over the last 15 years that a wide range of RNA binding proteins recruit the Ccr4-Not complex to target mRNAs, in several cases in the recent years including structural details . The concept of tethering of the Ccr4-Not complex by RNA binding proteins appears without contest well established.Before publication, I recommend the title to be modified to reflect the new findings reported in the manuscript allowing RNA binding proteins to differentiate target and non-target sequences. The Abstract would also merit clarification.Reviewer #3:The authors have adequately addressed my comments. We recommend that the manuscript be revised, taking into account the reviewers’ suggestion, and that it will then be appropriate for publication in eLife.Reviewer #2:This manuscript presents biochemical evidence that the S. pombe Pumilio and Tristetraprolin homologs recruit the Ccr4-Not complex to deadenylate mRNAs.While the biochemical data presented in this manuscript are of high quality, evidences that Pumilio and Tristetraprolin recruit the Ccr4-Not complex to deadenylate mRNAs in other species have been widely documented. In this context, the data described in this manuscript appear mostly confirmatory, even if they are based on a different, more direct, analysis. The conceptual advance brought by this manuscript is thus limited.In this work, we examined the mechanisms that allow RNA-binding proteins to achieve sequence-selective deadenylation. The major conceptual advance is not that Pumilio and Tristetraprolin proteins promote deadenylation, but an improved understanding of two aspects of this process – (1) how RNA-binding proteins distinguish between target and non-target mRNAs, and (2) how RNA-binding proteins recruit Ccr4-Not to achieve rapid deadenylation.S. pombe were selected for this study precisely because homologs from other organisms have been documented to recruit Ccr4-Not. They are therefore a useful model system to examine the biochemical mechanisms of RNA-binding proteins that recruit Ccr4-Not.The RNA-binding proteins Puf3 and Zfs1 from While the manuscript does bring new details, e.g. on the context effect on RNA binding, those are not unexpected and will be of interest only to specialists. Moreover, in some case, specific conclusions remain preliminary .S. cerevisiae Puf3 in a priority order , we were able to ascertain an improved definition of its mRNA targets, and consequently its cellular role. While we have used Pumilio proteins as a model system, this method of analysis could be applied to any RNA-binding protein.The evidence we present indicates that the kinetics of binding define which RNA sequence motifs are recognized as targets by RNA-binding proteins. We describe this as a ‘priority model’ of RNA binding, which challenges the traditional distinction between specific and non-specific binding. This model is of general importance. By defining the RNA motifs recognized by S. pombe Puf3 and Zfs1 contribute to Ccr4-Not recruitment, where each individual binding site is likely of relatively low affinity.  of ~2 µM. Other parts of TTP are likely to contribute to the interaction with Ccr4-Not). It is likely that these RNA-binding proteins interact with Ccr4-Not through an avidity driven mechanism, and a full understanding of this distributed binding will require studies of larger parts of the proteins.We did not attempt to define the interaction interface of RNA-binding proteins and Ccr4-Not at the resolution of amino acids. Instead, we show that multiple regions within the low-complexity regions of Furthermore, a significant fraction of the manuscript focuses on analysis of published data emanating from S. cerevisiae that are rather descriptive, not validated, and poorly connected to the experimental data presented in the rest of the manuscript.Our analysis of published experimental data served to test whether our model of RNA-binding is important in vivo: do subtle differences in motif quality dictate which mRNAs are targets of Puf3? The ability to predict mRNA targets based only on RNA motif quality had not previously been tested. Our analysis of published experimental data revealed that examining subtle differences in motif quality has substantial predictive power.Reviewer #3:Webster et al. is an important contribution to understanding mechanisms of mRNA control. The use of purified, reconstituted Ccr4-Not and two different RNA-binding proteins, and the care taken in experimental design, as in the dual reporter assays , make the data and conclusions compelling.PUM . In those studies, the number of uridines added in tagging very likely is related to PUF-RNA dissociation rates in vivo, which correlates well with whether the mRNAs are in fact regulated by Puf3 in the cell. It is in the author's interest to make the point that in vivo studies of tagging led to the suggestion of "sampling" vs. "biological regulation" as a function of off-rates; the convergence of the in vitro and in vivo data is striking.1) A central conclusion is that dissociation rates are a key contributor to rates of deadenylation. This important conclusion complements in vivo "tagging" experiments on Puf3In the Discussion we have added the text:ScPuf3 were previously obtained using RNA tagging . […] Taken together, there is a striking convergence between the results of in vitro(this study) and in vivo studies.”“An indication ofin vivo dissociation rates of PUM and S. pombe Ccr4-Not. This raises a potential confusion for the reader, in that the computational analyses done later in the paper focus on mRNAs that are bound and controlled in vivo in S. cerevisiae by the S. cerevisiae protein. Unfortunately, this makes some of the discussion of Figure 6 a bit confusing, and needs clarification in the text. It is not that mRNAs that bind S. pombe Puf3 in S. pombe necessarily are mitochondrial in function, a point that is relevant to computational studies of site divergence during evolution .2) The experiments use S. pombe Puf3The data and analyses of Figure 6 certainly are informative, and the issue should be dealt with by clarifications in the Results and Discussion sections.ScPuf3. This clarifies the text, making it easier to discriminate whether we are discussing budding yeast or fission yeast proteins.We have changed our nomenclature of budding yeast Puf3 to We also now state in the Introduction:S. pombe) do not necessarily regulate mitochondrial function .”“Puf3 homologs in other organisms  I may have missed this, but how do the authors quantify deadenylation "rate" ? Does "rate" refer to moles of adenosine monophosphate released per unit time, or the amount of fully deadenylated product per unit time, or something else?All rates were calculated using densitometric analysis of the signal from 5′-fluorophore labeled RNAs in denaturing polyacrylamide gels that have single-nucleotide resolution. We determined the poly(A) tail length of the most abundant RNA species at each time point and plotted this versus time. The slope is then the average deadenylation rate. The rate is therefore the number of adenosines removed per unit time. This method is described in detail in the reference given . We now add more description in the Materials and methods.In Figure 2B we state in the legend:“The most abundant RNA size decreased linearly with time, and average reaction rates in number of adenosines removed/min were determined by linear regression on triplicate measurements.”This is the same method used in Figure 1—figure supplement 1E and Figure 2—figure supplement 1C, and we have now added this text to those figure legends.Reviewer #2:Passmore and colleagues have revised their manuscript, considering most referees' comments. Minor points have been clarified. In line with editorial recommendation, authors argue that their "major conceptual advance is not that Pumilio and Tristetraprolin proteins promote deadenylation, but an improved understanding of two aspects of this process – (1) how RNA-binding proteins distinguish between target and non-target mRNAs, and (2) how RNA-binding proteins recruit Ccr4-Not to achieve rapid deadenylation".As discussed earlier, our new understanding of interaction of RNA binding proteins with the Ccr4-Not is limited in the context of data already available in the literature (see below). Consistently, the authors indicate that a full understanding of this binding will require further studies. This point remains accessory.Altogether, there is a general agreement that the main new point addressed in this manuscript is how RNA-binding proteins distinguish between target and non-target mRNAs. While the text has been improved to clarify this issue, the manuscript title "The mechanism of Ccr4-Not recruitment to specific mRNAs involves sequence-selective tethering by RNA-binding proteins" and Abstract are still confusing. They don't do justice to the numerous manuscripts and groups that have reported over the last 15 years that a wide range of RNA binding proteins recruit the Ccr4-Not complex to target mRNAs, in several cases in the recent years including structural details . The concept of tethering of the Ccr4-Not complex by RNA binding proteins appears without contest well established.S. pombe Puf3 and Zfs1 bind directly to Ccr4-Not.”We certainly did not intend to diminish previous data that show many details of the interactions of RNA-binding proteins with Ccr4-Not. We cited many of the papers mentioned by the reviewer and discuss these data many times throughout the manuscript. We include descriptions of these in the Introduction and Discussion. In addition, in the Results section when we first describe accelerated deadenylation, we state: “Therefore, like metazoan homologs, To avoid confusion, we made a change to the Results section to further emphasise previous results:“As mentioned above, specific interactions between Ccr4-Not and Pumilio/TTP proteins had been previously described . To dissect the mechanism of accelerated deadenylation and to determine the contributions of different parts of Puf3 and Zfs1, we made a series of mutations and truncations in these proteins, and tested their abilities to stimulate deadenylation.”Before publication, I recommend the title to be modified to reflect the new findings reported in the manuscript allowing RNA binding proteins to differentiate target and non-target sequences. The Abstract would also merit clarification.The title we chose (“The mechanism of Ccr4-Not recruitment to specific mRNAs involves sequence-selective tethering by RNA-binding proteins”) was meant to emphasize the sequence-selective nature of the RNA-binding proteins. In other words, the RNA-binding proteins that interact with Ccr4-Not can differentiate target and non-target sequences.We have now changed the title to: “RNA-binding proteins distinguish between similar sequence motifs to promote targeted deadenylation by Ccr4-Not.”We also modified the Abstract in the revised manuscript.


Efficient RNA extraction methods are needed to study transcript regulation. Such methods must lyse the cell without degrading the genetic material. For cyanobacteria this can be particularly challenging because of the presence of the cyanobacteria cell envelope. The great breath of cyanobacterial shape and size  created a variety of cell lysis methods. However, there is still a lack of reliable techniques for nucleic acid extraction for several types of cyanobacteria.Planktothrix agardhii. Techniques based on the use of beads, sonication, and heat shock appeared to be too soft to break the Planktothrix agardhii cell envelope, whereas techniques based on the use of detergents degraded the cell envelope but also the RNA. Two protocols allowed to successfully obtain good-quality RNA. The first protocol consisted to manually crush the frozen cell pellet with a pestle and the second was based on the use of high-intensity ultra-sonication. When comparing these two, the high-intensity ultra-sonication protocol was less laborious, faster and allowed to extract 3.5 times more RNA compared to the liquid nitrogen pestle protocol. The high-intensity ultra-sonication protocol was then tested on five Planktothrix strains, this protocol allowed to obtain >8.5 μg of RNA for approximatively 3.5 × 108 cells. The extracted RNA were characterized by 260/280 and 260/230 ratio > to 2, indicating that the samples were devoid of contaminant, and RNA Quality Number > to 7, meaning that the integrity of RNA was preserved with this extraction method. In conclusion, the method we developed based on high-intensity ultra-sonication proved its efficacy in the extraction of Planktothrix RNA and could be helpful for other types of samples.Here we designed and tested 15 extraction methods using physical, thermic or chemical stress on the filamentous cyanobacteria  Cyanobacteria are photosynthetic prokaryotes that played a key role on Earth history as they were responsible for the oxygenation of the atmosphere 2.5 billion years ago. Nowadays, these microorganisms and are still essential in ecosystem functioning as they are at the base of the food chain and contribute to primary production . MoreovePlanktothrix because of the recurrent toxicity of Planktothrix bloom [Planktothrix populations synthesize microcystins that are cyclic heptapeptides and potent cyanotoxins. Microcystins act as hepatotoxins, inhibiting the protein phosphatase type 1 and type 2A [Planktothrix genomes have been extensively studied [Planktothrix. These studies have focused on the regulation of genes belonging to the microcystin gene cluster [Planktothrix.Researchers have particularly focused on the genus ix bloom . Some Pl type 2A . Plankto studied –9. Howev cluster ,11 and t cluster . Hence sFluorescence-based quantitative real-time polymerase chain reaction (qPCR) is considered as the gold standard for the measurement of transcript abundance. Nucleic acid extraction is a critical step to ensure reliable measurement of transcript abundance . NucleicPlanktothrix. Therefore, 15 extraction methods were designed based on the existing homogenization methods for cyanobacteria nucleic acid extraction, including Planktothrix. Each of these 15 extraction methods was then tested on P. agardhii (Gomont) Anagnostidis et Komárek 1988. Planktothrix agardhii  are up to 300 μm long and (2.3) 4–6 (9.8) μm wide  7805 were grown in batches in sterile BG11 medium [2/s and a 13:11 h light:dark cycle. It was subcultured every four weeks to promote optimal growth of the cyanobacteria. A volume of 50 mL with an optical density measured at 750 nm (OD750nm) of approximately 0.3, which corresponded to approximatively 3.5 × 108 cells . For the protocol “Vortex 65” (3), the sample was incubated at 65 °C for 12 min, vortexed three times during the incubation period and then centrifuged. For the protocol “Pestle” (4), the pellet was crushed manually with a single-use pestle (Argos Technologies) for approximatively 30 s. In the “Tungsten” protocol (5), the pellet was resuspended in Trizol and transferred into a clean eppendorf tube containing two tungsten carbide beads (3 mm Qiagen). Then, the sample was processed three times for 30 s at 30 kHz with the TissueLyser system (Qiagen). In the protocol “Heat shock” (6), the tube was immerged in liquid nitrogen and then incubated at 95 °C for 5 min; the operation was repeated two times. For the protocol “Liquid nitrogen pestle” (7), the frozen pellet was crushed manually with a single-use pestle three times for 30 s, with the immersion of the tube in liquid nitrogen between each cycle, and then 1 mL of Trizol was added.1 mL of Trizol (Invitrogen) was added to the freeze dried pellet and the tube was vortexed. In the “Beads” protocol (1), the pellet was resuspended in Trizol and was transferred into a clean eppendorf tube containing 300 μL of glass beads (0.1mm diameter). Further, the sample was vortexed three times for 30 s. In the “Sonication” protocol (2), the sample was subjected to 20 min of sonication in an Elmasonic S10-H instrument  was added, the sample was vortexed, incubated for 3 min at room temperature, and was then centrifuged. Volumes of 400 μL of Trizol and 400 μL of chloroform were added and the sample was vortexed. After 3 min of incubation at room temperature, the sample was centrifuged, and the supernatant was transferred into a clean eppendorf tube. After the addition of 200 μL of ethanol, the tube was vortexed for 5 s and incubated 5 min at room temperature. The end of the extraction was realized using a NucleoSpin miRNA kit (Macherey Nagel) according to the manufacturer’s instructions. The large RNA fraction (>200 nucleotides) was recovered in 50 μL RNAse/DNAse free sterile water according to the instructions in the protocol section “Small and large RNA in two fractions”.2O Milli-Q at 4 °C and submitted to High Intensity (HI) ultra-sonication with an ultrasonic Processor  two times for 30 s each at 20% of maximal power. The sample was vortexed after each 30 s pulse.A volume of 1 mL of lysis buffer  was added to the frozen pellets and the tube was vortexed. In the “Beads” protocol (8), the pellet was homogenized in the lysis buffer and transferred into a clean eppendorf tube containing 300 μL of glass beads (0.100 mm diameter). Then, the sample was vortexed three times for 30 s each. In the “Sonication” protocol (9), the sample was subjected to 20 min of sonication in an ultrasonic bath. For the protocol “Vortex” (10), the sample was incubated at room temperature for 12 min and vortexed three times during the incubation time. For the “HI ultra-sonication” (11), the sample was placed in a cup horn  filled with autoclaved HA volume of 1 mL of Tween 20 (0.2% in ML lysis buffer) or 1 mL of Triton X (0.2% in ML lysis buffer) was added to the frozen pellets and the tube was vortexed. Then, the sample was incubated for 30 min at 65 °C (for the protocols “Tween 65” (12) and “Triton 65” (13)) or at 95 °C (for “Tween 95” (14) and “Triton 95” (15)). The sample was vortexed three times during the incubation time.After cell lysis, the sample was vortexed and then centrifuged to allow the cellular debris to settle. The supernatant was transferred into a NucleoSpin Filter and centrifuged to eliminate the remaining cellular debris. Because the pellet formation was sometimes observed in the collection tube, 600 μL of supernatant was transferred into a new tube taking care not to disturb the pellet. After the addition of 225 μL ethanol, the tube was vortexed for 5 s and incubated 5 min at room temperature.The end of the extraction was realized using the NucleoSpin miRNA kit (Macherey Nagel) according to the manufacturer’s instructions. The large RNA fraction (>200 nucleotides) was recovered in 50 μL RNAse/DNAse free sterile water according to the instructions in the protocol section “Small and large RNA in two fractions”.™ Green I Nucleic Acid Gel Stain (Invitrogen) by UV light on an illuminator. To confirm RNA integrity, the samples were analyzed using RNA Nano chips on a Bioanalyzer (Agilent Technologies). RNA quantity and purity was assessed using a Nanodrop Spectrophotometer (ThermoFisher Scientific).RNA integrity was analyzed on 1% (w/v) agarose gel with SYBR™ Reverse Transcriptase was replaced by PCR-grade water for non-retro transcript samples.The first strand of cDNA was synthesized from 1 μg of RNA in a final volume of 20 μL using a High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific) according to manufacturer’s instructions . Reverse Transcription was realized in a GeneTouch Thermal Cycler (BIOER Technology) and the cDNA was stored at -20 °C until it was used in a qPCR reaction. The MultiScribegyrB), the ribonuclease P protein component (rnpA), and the 30S ribosomal protein S12 (rpsL) were the three genes selected to evaluate the suitability of the extraction technic for qPCR analyses. Primer pairs (The DNA gyrase subunit B (er pairs  of 20 bpThe qPCRs were performed in the LightCycler 480 (Roche) following the manufacturer’s instructions . Each 20 μL reaction contained 3 μL of PCR-grade water (Roche), 2 μL of the gene-specific primer pair at a final concentration of 300 nM for each primer, 10 μL of MasterMix (Roche), and 5 μL of cDNA. For non-template controls (NTC), cDNA was replaced by PCR-grade water. For non-retro transcript controls (NRT), the cDNA was replaced by non-retro transcript RNA. Each qPCR was realized in duplicate.http://www.genoscope.cns.fr/agc/microscope).Specificity was checked for each reaction by analyzing the PCR product using melt curve analysis, gel analysis, and sequencing. The dissociation curve of the PCR product was obtained by following the SYBR Green fluorescence level during gradual heating of the PCR products from 65 to 95 °C. In addition, samples were run on 2% (w/v) agarose gel to confirm that the amplicon was of the expected size. Successful amplifications were sent for sequencing and then blasted on the Microbial Genome Annotation and Analysis Platform  is given by the equation: E = 10 (-1/slope) − 1. The theoretical maximum of 1 (or 100%) indicates that the amount of product doubles with each cycle.A standard curve was used to determine the reaction efficiency by producing a 10-fold dilution series over five points from the most concentrated sample. The qPCR reaction was performed for the three primer pairs used in the experiment. LC480 Software (Roche) was used to construct the standard curves by plotting the log of the starting quantity of the template against the CPlanktothrix agardhii . Nevertheless, RNA concentrations were below the detection limit (RNA was not detected for the “Beads” (1), “Sonication” (2), “Vortex 65” (3), “Pestle” (4), “Tungsten” (5), or “Heat shock” (6) of the Trizol-based protocols, nor for the “Beads” (8), “Sonication” (9), or “Vortex” (10) of the Lysis Buffer ML-based protocols. To identify if the unsuccessful step was cell lysis or the separation of the RNA from cellular debris, samples were observed under a microscope after the homogenization step . After ton limit  revealinPlanktothrix, despite the use of high temperature, bead beating, and sonication already successful for Planktothrix agardhii cell lysis [Planktothrix agardhii, a genus yet without mucilaginous envelopes or sheaths, cell lysis in our study could be linked to cell envelop thickness, strain specific-characteristics, or by culture conditions. Moreover, we observed that the addition of Trizol or lysis buffer ML to the frozen pellet led to the formation of highly viscous samples preventing efficient sample homogenization. In the mechanical protocols using glass, tungsten beads, or a pestle, the beads/pestle performed poorly in terms of breaking Planktothrix cells and trichomes. Additionally, extraction methods based on the use of detergents were tested (12 to 15) with nonionic detergents used to solubilize protein membranes, such as Tween 20 and Triton X. These protocols effectively denatured the cell envelope because nucleic acids could be quantified but resulted in low-quality RNA as revealed by the results of electrophoresis analyses , the frozen pellet was crushed with a pestle and the tube was immerged several times in liquid nitrogen between each crushing of the cells to ensure that the sample was still in a frozen state. Electrophoresis  revealedPlanktothrix cell lysis.The “HI ultra-sonication” treatment (11) resulted in high-quality RNA , at a coPlanktothrix cell envelope appeared to be particularly difficult to break. Techniques based on the use of beads  sonication (2 and 9), and heat shock (6) appeared to be too soft to break the Planktothrix cell envelope, whereas techniques based on the use of detergents (12–15) degraded the cell envelope but also the RNA. Hence, particularly strong techniques were required to lyse the cells without degrading the genetic material. In conclusion, of the 15 RNA extraction protocols tested for Planktothrix, only two allowed for the extraction of high-quality RNA: the “Liquid nitrogen pestle” and the “HI ultra-sonication.” When comparing these two, the “Liquid nitrogen pestle” presented some disadvantages linked to the manual nature of cell lysis that did not occur in the “HI ultra-sonication.” Consequently, the “HI ultra-sonication” protocol was selected and further optimized for Planktothrix agardhii RNA extraction.In our study, the The proposed protocol for RNA extraction based on the use of HI ultra-sonication is presented in The 50 mL of culture was filtered through a polycarbonate membrane . The filter was transferred into a 2 mL eppendorf tube containing 1.5 mL of RNAlater (Sigma). The tube was stored at 4 °C overnight and then transferred at -80 °C until RNA extraction. After thawing at room temperature, the filters were transferred to a new 2 mL eppendorf tube, cut in small pieces, and 850 μL ML was added. The tube was briefly vortexed and submitted to HI ultra-sonication with an ultrasonic Processor  two times for 30 s each at 20% of the maximal power. The sample was vortexed after each 30 s pulse. After 5 min of incubation at room temperature, 700 μL of sample was transferred into a NucleoSpin Filter and centrifuged for 1 min. To eliminate all cellular debris that may affect RNA extraction, 600 μL of the supernatant was transferred into a new tube taking care not to disturb the pellet. A volume of 225 μL of ethanol was added, the sample was vortexed 5 s, and incubated 5 min at room temperature. The large RNA fraction (>200 nucleotides) was recovered in 25 μL RNAse/DNAse free sterile water according to the instructions in the protocol section “Small and large RNA in two fractions”.Planktothrix strains: P. agardhii PCC 7805, P. rubescens PCC 7821, P. agardhiii PCC 10110, P. agardhii NIVA CYA 126/8, and P. agardhii NIVA CYA 126/8 ΔmcyD. The RNA concentration, purity and integrity obtained with this protocol for the five strains are presented in This protocol has been tested on five Planktothrix strains tested were > to 2 indicating that the samples are devoid of contaminant. The integrity of RNA was evaluated with the RNA Quality Number (RQN), this number varies from 0  to 10 (intact RNA). A RQN value > to 7 is characteristic of samples of high quality. The mean RQN for the samples extracted with the HI ultra-sonication method were > to 7 for all five strains, meaning that the integrity of RNA was preserved with this extraction method.The use of the HI ultrasonication permitted to extraction RNA at concentrations superior to 300 ng/μL. Moreover, our results indicated that this protocol resulted in high-quality RNA (i.e. RNA purity and RNA integrity). RNA purity was evaluated regarding the 260/280 and the 260/230 absorbance ratio. A 260/280 ratio of ~2.0 is generally accepted as “pure” for RNA. This ratio provides an indication of RNA purity, because the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm alters the ratio. The 260/230 ratio is used as a secondary measure of nucleic acid purity. Generally accepted 260/230 ratio for good quality RNA are > 2.0–2.2. If the ratio is appreciably lower than expected, it may indicate the presence of contaminants which absorb at 230 nm like EDTA, carbohydrates, phenol or guanidine isothiocyanate. In our study, the mean 260/280 and 260/230 ratio measured for all five The new method based on the use of HI ultra-sonication we proposed is rapid and easy to perform and allows for the extraction of RNA with high integrity and high purity, requisite criteria for downstream applications as qPCR.r2) reported in The suitability for qPCR analyses of the extract, obtained with the optimized protocol for RNA extraction based on HI ultra-sonication, was experimentally assessed regarding PCR efficiency and the coefficient of determination  permitted us to validate the primers designed, as well as the suitability of the RNA obtained by the extraction method developed to study the genetic expression of Planktothrix agardhii PCC 7805.To conclude, the assessment of assay performance based on the PCR efficiency and the coefficient of determination Click here for additional data file.S2 FigThe solid line represents the mean cellular concentration (n = 18), .(TIF)Click here for additional data file.S3 Fig(a) 1: RNA ladder; 2–3: Tween 95; 4–5: Triton X 95; 6–7: Tween 65; 8–9: Triton X and (b) 1: RNA ladder; 2–3: Liquid nitrogen pestle; 5–6: HI ultra-sonication.(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.


Caenorhabditis elegans UNC-45 and muscle myosin MHC-B in insect cells. In addition to providing a cellular chaperone assay, the established system enabled us to produce large amounts of functional muscle myosin, as evidenced by a biochemical and structural characterization, and to directly monitor substrate binding to UNC-45. Data from in vitro and cellular chaperone assays, together with crystal structures of binding-deficient UNC-45 mutants, highlight the importance of utilizing a flexible myosin-binding domain. This so-called UCS domain can adopt discrete conformations to efficiently bind and fold substrate. Moreover, our data uncover the molecular basis of temperature-sensitive UNC-45 mutations underlying one of the most prominent motility defects in C. elegans.Myosin is a motor protein that is essential for a variety of processes ranging from intracellular transport to muscle contraction. Folding and assembly of myosin relies on a specific chaperone, UNC-45. To address its substrate-targeting mechanism, we reconstitute the interplay between  Myosin, a motor protein essential for intracellular transport to muscle contraction, requires a chaperone UNC-45 for folding and assembly. Here authors use in vitro reconstitution and structural biology to characterize the interplay between UNC-45 and muscle myosin MHC-B. The myosin superfamily contains about 35 subtypes, among which the class II muscle proteins (myosin II) are the arguably most prominent member driving muscle contraction2. Myosin II molecules found in skeletal and cardiac muscles dimerize via their C-terminal coiled-coil domain and associate with essential and regulatory light-chains to yield the basic hexameric myosin complex. These hexamers further assemble into myosin thick filaments, from where the N-terminal myosin ATPase domains project to interact with the adjacent actin (thin) filaments. Establishing and maintaining the intricate myosin–actin interplay, occurring at the interface of the two differently organized muscle filaments, is a great challenge for the cellular chaperone machinery. Therefore, muscle cells express a vast number of specialized folding and assembly factors that control the expression, folding, assembly and interplay of actin and myosin molecules7. Failure of muscle filament assembly or maintenance leads to severe myopathies9.Myosins are cytoskeletal, molecular motors promoting a variety of mechano-chemical processes in the cellC. elegans, where site-specific temperature-sensitive (ts) mutants revealed the importance of the chaperone for myosin function13. Subsequent studies in C. elegans, Xenopus, Zebrafish, and Drosophila confirmed the role of UNC-45 as a myosin-specific chaperone, promoting the folding of the myosin ATPase domain as well as coordinating the assembly of thick filaments during muscle development19. In higher organisms, UNC-45 is particularly important for the maturation of myosin II in muscle cells22 and, together with Hsp90 (heat shock protein 90) and UFD-2 (ubiquitin fusion degradation 2), maintaining the functionality of myosin filaments during stress situations24. Mechanistic insight into the chaperone function of UNC-45 comes from structural studies revealing its 3-domain architecture25. UNC-45 is composed of an N-terminal TPR (tetratricopeptide repeat) domain mediating the interaction with Hsp70/Hsp90, an elongated UCS (UNC-45/Cro1/She4p) domain at the C-terminus providing a myosin-binding site19, and a central domain aligning the two functional TPR and UCS units to each other. Further analyses of UNC-45 from C. elegans revealed that the chaperone can form a linear protein chain, which constitutes a myosin assembly line licensing Hsp70 and Hsp90 to act in a defined periodicity on myosin heads protruding from the myofilaments26.Among the chaperones expressed in muscle cells, UNC-45 appears to be the key component of the myosin assembly machinery. The UNC-45 protein was initially identified in C. elegans UNC-45 and MHC-B  in a cellular context. Notably, co-expression of UNC-45 allowed production of fully functional MHC-B in large amounts, yielding about 15 mg muscle myosin per liter culture. The recombinant myosin was also key to address the basic mechanistic properties of the UNC-45 chaperone, revealing for example the molecular basis of ts motility defects of mutant worms harboring point-specific UNC-45 mutations. Our data show that these ts mutations affect the myosin-binding capability of UNC-45 rather than its protein stability.To address the myosin targeting mechanism of UNC-45, we reconstitute the chaperone-substrate interplay both in vitro and in vivo. Using insect cells as host system, we monitor the interaction between the C. elegans body wall muscle myosin MHC-B as model system and co-expressed it with different C. elegans chaperones in insect cells  and DAF-21 (Hsp90) are critical for myosin folding and assembly28, we next tested whether co-expression of these chaperones improves the production of the MHC-B motor domain in its soluble form. The experiments revealed that the C. elegans Hsp70 and Hsp90 had only a moderate effect in yielding soluble muscle myosin in insect cells. However, co-expressing UNC-45 strongly increased the amount of the MHC-B motor domain in the soluble fraction of the cell lysate  in the myosin-binding canyon19. Deletion of the UCS domain had a strong impact on UNC-45 chaperone function, as seen by the reduced levels of soluble MHC-B in the insect cell co-expression system  repeat protein structurally related to the UCS domain, suggested that Asn801 of UNC-45 may directly interact with myosin tem Fig. . The sit19. When we assayed the activity of the K82E mutant, which is impaired in Hsp70/90 binding , we observed that it yielded similar levels of soluble myosin as the co-expressed wt UNC-45  have been identified that underlie the so-called uncoordinated (unc) ts-phenotype29. Ts-worms grown at the permissive temperature of 15–18 °C during their larval development show no defects in myosin thick filament formation, while ts-worms grown at the restrictive temperature of 25 °C exhibit motility defects and a distorted sarcomere organization11. Though known for decades, the mechanistic details underlying the ts-phenotype of UNC-45 have remained elusive. Strikingly, in our cellular chaperone assay, all ts-mutations strongly reduced the production of soluble myosin  that still supported survival of insect cells and corresponds to the permissive temperature in growing ts-worms. Also, at 18 °C we observed the same solubility defect for myosin co-expressed with the ts-mutants . Using similar stopped-flow experiments, we carried out a broad kinetic characterization of the MHC-B myosin motor, determining the rate constants of ATP and ADP binding to myosin and the actin:myosin complex, respectively. In conclusion, the kinetic data demonstrate that the MHC-B variant produced in insect cells is a fully functional myosin motor, which exhibits similar kinetic properties to other muscle myosins  among the closest structural homologs of the determined MHC-B structure. The two motor domains are 59% identical and 72% similar  and the molecular details of forming cognate UNC-45/myosin complexes need to be resolved. Instead of applying the rather indirect aggregate prevention assay, we monitored formation of the UNC-45/myosin complex at two different temperatures, 4 and 27 °C, in a size-exclusion chromatography (SEC) based assay. The 4 °C condition served as control temperature, preserving the native state of the myosin substrate , we observed that myosin was destabilized and eluted in the void volume of the SEC column as expected for aberrant proteins. In contrast, all tested UNC-45 variants were stably folded, eluting at the same retention volume as the native chaperone at 4 °C Fig. . When myts-mutations, which are among the most studied motility defects in C. elegans13. Of note, our cellular chaperone assay revealed that the UNC-45 ts-mutations have a negative effect on the myosin folding activity, however, did not resolve whether the ts-mutations directly impair the chaperone activity or exert rather indirect effects, destabilizing for example the UNC-45 protein. With regards to the latter point, we carried out circular dichroism (CD) measurements to determine the melting temperature (Tm) and thus stability of the purified ts-proteins  and determine their structure, as described later.The established in vitro interaction assay furthermore allowed us to address the molecular basis underlying the UNC-45 ins Fig. . The CD ts-mutants showed a reduced ability to form a complex with the unfolded myosin, as judged by the smaller protein amounts co-eluting with the myosin substrate , hydrophobic interface and overall domain organization as the previously characterized wt oligomer. However, when we compared the UCS domains of wt and ts UNC-45, we noted two major differences. First, the ts UCS domains adopt different conformations, as most clearly seen for the C-terminal portion of the UCS domain that moves about 10–15 Å  extending away from the central domain  In the UNC-45 oligomer, the TPR domain is essential to connect neighboring molecules19. As shown in this study, the L121W mutation, which prevents oligomer formation, negatively affects the amount of produced myosin. (2) Our structural data suggest that the TPR domain obtains an additional intra-molecular scaffolding role. It undergoes van-der-Waals contacts with the UCS domain, thereby arranging the myosin binding site to accommodate substrate. Accordingly, formation of UNC-45 oligomers via the TPR domain may be critical to set up a functional UCS myosin-binding site, thus linking UNC-45 oligomerization to its myosin folding activity.The N-terminal TPR domain of UNC-45 is a well-characterized protein–protein interaction domain that serves as the major binding site for Hsp70 and Hsp9059. The present data suggest this concept holds also true for the myosin-binding domain of UNC-45. The UCS domain adopts an extended ARM repeat scaffold that is penetrated by a long canyon serving as protein binding site. Our structural data reveal the capability of the UCS domain to adopt distinct defined conformations. Together, the wt, G427E, L822F, and ∆TPR crystal structures depict four different UCS conformations highlighting the pronounced dynamicity of the myosin-binding domain  with a C-terminal His6-tag. FLAG-Hsp70 (C. elegans HSP-1), FLAG-Hsp90 (C. elegans DAF-21) or UNC-45-Strep were added into the p10 promotor site of the pFastBac DUAL vector. Previously described constructs served as DNA templates19. All primers used are listed in Supplementary Table 6 cells ml−1 were infected with baculovirus (P1 amplification) and incubated for 72 h at 21 °C. The cells were collected by centrifugation, lysed by freeze-thaw, and resuspended in 50 mM Tris, pH 7.5, 300 mM NaCl (buffer A), containing 20 mM imidazole. The cleared cell lysate was loaded onto a NiNTA column  equilibrated with buffer A. After washing the column, myosin and associated UNC-45 were eluted with buffer A, containing 150 mM imidazole. The elution was applied to a Superdex 200 column equilibrated with 20 mM Tris pH 8.0, 150 mM NaCl. For crystallization purposes, this buffer was optimized to 20 mM Tris pH 8.5, 20 mM NaCl.MHC-B (1–790) and NMY-2 (1–796) were cloned from For small scale myosin purifications  cells (Stratagene) induced with IPTG. The three-step purification procedure included NiNTA and anion exchange chromatography and a final SEC step in 20 mM Tris pH 8.0, 150 mM NaCl.UNC-45 point mutants  were generated by site-directed mutagenesis of the wild-type protein in pET21a. All primers used are listed in Supplementary Table 60 with minor modifications. Briefly, no ATP was added at the final step to polymerize actin and residual amounts of nucleotide were removed by sequential centrifugation of actin and dialysis steps. Actin was labeled with pyrene (pyr-actin) as previously described61.Rabbit actin was purified by the method of Lehrer and Kewar24. Briefly, spectra (190–260 nm) of UNC-45 proteins at 0.7 mg ml−1 were recorded on a Chirascan plus CD spectrometer (Applied Photophysics). Data were analyzed with the Global3 evaluation software.CD experiments were essentially performed as previously described24 and bacterial expression constructs described above into a pIDC derivative encoding an N-terminal Twin-Strep-tag (former One-Strep-tag) followed by a 3C protease cleavage site. The MHC-B motor domain (1–790) was cloned into pACEBac1 with a C-terminal fusion to a His6-tag. FLAG-CeHsp90 and FLAG-CeHsp70 were cloned into pACEBac1 derivatives. All primers used are listed in Supplementary Table E. coli DH10EMBacY (Geneva Biotech). Baculovirus production and protein expression was performed as described above. For expressions at 18 °C, cells were harvested after 96 h. High Five cells were harvested, lysed by freeze-thaw and resuspended in 50 mM sodium phosphate buffer pH 8, 300 mM NaCl. Whole cell lysates and cleared cell lysates (supernatant after centrifugation at 21130 g for 30 min at 4 °C) were analyzed by Western blot using anti-FLAG , anti-Penta-His  and anti-CeUNC-45 antibodies 24. For Supplementary Fig. For comparing the effect of different UNC-45 mutants on the solubility of MHC-B, UNC-45 proteins were cloned from previously described constructs6 cells ml−1 were infected with baculovirus (P1 amplification) and incubated for 72 h at 21 °C. A pellet from 7.5 ml expression culture was lysed in 2 ml of 50 mM Tris pH 7.5, 20 mM NaCl containing benzonase. Cleared cell lysate was applied to 100 µl OPUS RoboColumns (Repligen) packed with Strep-Tactin Superflow (IBA Lifesciences) using a Janus Liquid handling system (Perkin Elmer). The resin was washed with 3 ml of 50 mM Tris pH 7.5, 20 mM NaCl and UNC-45 was eluted with 100 µl of 50 mM Tris pH 7.5, 20 mM NaCl, 5 mM d-Desthiobiotin. Elutions were analyzed by western blot using anti-Hsp70 , anti-Hsp90 (Abcam [EPR16621-67] (ab203126), used 1:250) and anti-Strep  antibodies.High Five cells (Expression Systems) at a density of 1 × 10ts-mutant protein) on ice for 60 min. After incubation samples were applied to a S200 PC 3.2 column in 20 mM Tris pH 8.0, 150 mM NaCl. Indicated fractions were analyzed by SDS-PAGE.UNC-45 proteins or BSA and myosin were incubated at a final concentration of 5 µM at 4 °C or 27 °C for 60 min. For the SEC analysis in Supplementary Fig. F-actin was mixed with myosin (plus and minus 30 mM ATP) to a final concentration of 10 and 2.5 µM, respectively. The samples were centrifuged at 150,000 g, for 60 min at 4 °C. Supernatant and pellet fractions were analyzed by SDS-PAGE.Stopped-flow experiments to measure MHC-B single turnover were performed with a TgK stopped-flow apparatus . The experimental data were fitted to a single exponential using Kinetic Studio, and the observed rate analyzed with Prism (GraphPad).63. All measurements were performed at 20 °C in 20 mM MOPS, 25 mM KCl, 5 mM MgCl2, 1 mM NaN3 at pH 7.0, unless indicated otherwise. Rapid-mixing experiments were completed in triplicate using a High-Tec Scientific SF-61 DX2 stopped-flow system. Transient kinetic traces were initially fitted with Kinetic Studio (TgK Scientific) and subsequently plotted with Origin (OriginPro). For the single turnover experiment, 10 µM MHC-B and 5 µM ATP stock solutions (concentration before mixing) were prepared in 20 mM MOPS pH 7, 25 mM KCl, 5 mM MgCl2, 1 mM NaN3. For the actin affinity experiment, the amplitude dependence of the MHC-B concentration was fitted to the standard quadratic equation for a binding isotherm64.α = fraction of actin bound to MHC-B, [M] = concentration of MHC-B, KD = the dissociation constant of MHC-B for actin and [A]0 = concentration of actin. For experiments probing the actin-myosin interaction with ATP and ADP the fluorescence signal for pyrene-labelled actin was recorded, which has an excitation wavelength at 365-nm and the emission was detected after passing through KV389-nm cut-off filter. In the absence of actin, intrinsic tryptophan fluorescence was monitored by excitation at 295-nm and emission detected with a WG-320 cut-off filter.Fast kinetic data was performed as previously described65. In all, 2 µM MHC-B was incubated with 37.5 U ml−1 pyruvate kinase, 42.9 U ml−1 lactate dehydrogenase, 0.25 mM NADH, 15 mM phosphoenolpyruvate, and 2 mM ATP in a buffer containing 20 mM MOPS pH 7.0, 100 mM KCl, 5 mM MgCl2. The absorption at 340 nm was recorded for 30 min using a Pherastar FS plate reader. The experiment was performed in the presence of increasing concentrations of actin (ranging from 0 to 90 µM) and corrected by subtracting the ATPase activity of actin. The Michaelis-Menten equation was fit to the data to determine the maximal activity (Vmax) and the association constant of actin for myosin (Km) using Prism (GraphPad). For the activity measurement of MHC-B produced with different UNC-45 mutants, the experiment was performed with 90 µM actin concentration.ATPase activity was determined by a coupled enzymatic reaction4)2SO4) and 1 µl reservoir solution . Crystals of UNC-45 L822F and UNC-45 ΔTPR were grown in 96-well plates set up with a Mosquito robot (TTP Labtech) in 0.1 M MES/NaOH pH 6.5, 0.2 M ammonium acetate, 30% glycerol ethoxylate and 0.1 M Tris pH 8.5, 32% glycerol ethoxylate, respectively. All crystals were grown by sitting drop vapor diffusion: MHC-B crystals at 4 °C, UNC-45 crystals at 19 °C. MHC-B, UNC-45 L822F and ΔTPR were directly frozen in liquid nitrogen, whereas UNC-45 G427E crystals were shortly incubated in reservoir solution containing 25% ethylene glycol, mounted into 90°-bent loops and frozen in liquid nitrogen as previously described19. Diffraction data were collected at the European Synchrotron Radiation Facility (ESRF) (wavelength: 0.979 Å): UNC-45 G427E anomalous diffraction dataset at beamline ID 29, UNC-45 L822F dataset at beamline ID 14-4; UNC-45 ΔTPR dataset at ID 23-1. Diffraction data for MHC-B were collected at Deutsches Elektronen-Synchrotron (DESY) (wavelength: 0.979 Å), beamline P13. Data were integrated with XDS and scaled with SCALA or XSCALE67.Crystals of MHC-B were grown in 96-well plates set up with a Mosquito robot (TTP Labtech) in 0.1 M MES/imidazole pH 6.5, 10% w/v PEG 4000, 20% v/v glycerol, 0.02 M Morpheus alcohol mix -1,2-propanediol, 0.2 M 2-propanol, 0.2 M 1,4-butanediol, 0.2 M 1,3-propanediol). Crystals of the UNC-45 G427E mutant were obtained by manually mixing 2 µl protein (200 µM) with 0.3 µl additive  with PHASER68. The wild-type UNC-45 (PDB code: 4i2z) structure was split into three parts comprising residues 5–507, 525–760, and 761–930 to create an optimal search model for the MR of the UNC-45 L822F structure. For solution of the UNC-45 ΔTPR structure, the previous UNC-45 structure (PDB code: 4i2z) was divided into two fragments 135–507 and 525–930. Molecule A was clearly defined, while residues 719–828 and 829–930 had to be placed as separate units into the density obtained for molecule B. Initial structural refinement of the UNC-45 L822F and ΔTPR structures was performed with CNS72. Following rebuilding of the structure using COOT and O, the final refinement was performed using PHENIX71. Structure figures were generated with PYMOL73. Data collection and refinement statistics are shown in Supplementary Table The crystal structure of MHC-B was solved by molecular replacement with Phaser using chicken smooth muscle myosin motor domain as search model (PDB code: 5m05)A reporting summary for this Article is available as a Supplementary Information file. Further information on research design is available in the Supplementary InformationReporting SummaryPeer Review FileSource Data


The default mode network (DMN) is often associated with internally-directed cognition, distinct from the constraints of the external environment. However, a recent finding is that the DMN shows strong activation after large task switches during a demanding externally-directed task . Following other proposals, we have suggested that the DMN encodes cognitive or environmental context, and that context representations are momentarily strengthened during large cognitive switches, perhaps so that new activity can be checked against current environmental constraints. An alternative account, consistent with the role of the DMN in episodic memory, might be that switches to a substantially new task increase demands on rule retrieval. To test this alternative, we directly manipulated rule retrieval demands. Contrary to the retrieval account, increased retrieval demand led to reduced DMN activity, accompanied by increased activation in prefrontal and lateral parietal cognitive control areas. Unlike episodic retrieval, with its rich contextual representations, rule retrieval does not drive DMN activity. Accordingly, it cannot explain increased DMN activity during large cognitive switches. •Rule retrieval demand is associated with deactivation of default mode (DM) regions.•Thus DM activity at large task switches is unlikely to be due to rule retrieval.•DM activity in response to task switches may depend on hierarchical task structure. The DMN is one of the best established brain networks, with its emergence consistently replicated through functional connectivity methods . Early rSince these early findings, research has shown overlapping DMN activation in a diverse set of tasks . DMN regEvidence is now emerging, however, to show DMN activation during some kinds of complex, externally-directed activity. In two recent studies , particiHere we test an alternative account of the In the previous studies  effects 22.146 participants , between 18 and 35 years old, were recruited through the Medical Research Council Cognition and Brian Sciences Unit volunteer panel. All participants selected were right handed, native English speakers, with normal or corrected to normal vision, and between 18 and 40 years old. The experiment was con-ducted in accordance with ethics approval granted from the Cambridge Psychology Research Ethics Committee. 4 participants  were excluded from further analysis due to poor task performance (n = 1), excessive motion (n = 2) or mid-task cancellation (n = 1).2.2Task events are illustrated in Each stimulus remained until a button press was made. Participants were asked to respond as quickly as possible without making mistakes. An inter-trial interval (ITI) of 2 s followed each response, with a fixation cross presented in the centre of the screen.The two stimulus domains were presented in pseudorandom order. This generated four conditions that crossed the number of response options (2 or 6) with whether the stimulus domain repeated or switched: 2-choice domain-stay (a 2-choice stimulus following another), 2-choice domain-switch (switch from a 6-choice stimulus to a 2-choice stimulus), 6-choice domain-stay (a 6-choice stimulus following another) and 6-choice domain-switch (switch from a 2-choice stimulus to a 6-choice stimulus). The experiment consisted of a single block of 145 trials. Each block contained 36 trials of each condition, plus the first trial of the block (switch type undefined) which was discarded from further analysis. In The experiment was controlled using Psychophysics Toolbox for MATLAB . Stimuli2.3Participants were carefully pre-trained to ensure good learning of button presses. To encourage separation of the two task domains, participants were first presented with the 2-choice response rules and then asked to repeat them from memory. Next, participants were presented with the 6-choice response rules and asked to repeat them from memory. After learning the 2-choice and 6-choice rules separately, participants were shown one exemplar from each response category in a random order and asked which finger they would use to respond. This process was repeated twice. At the second run-through, all participants got all responses correct. Finally, participants performed a short practice block of 36 trials (9 from each combination of 2/6 choice x domain stay/switch) outside the scanner.2.4Images were acquired using a 3 T S Prisma magnetic resonance imaging (MRI) scanner, fitted with a 32-channel head coil. Functional MRI (fMRI) acquisitions used T2*-weighted multiband Echo-Planar Imaging . T1-weighted multiecho magnetization-prepared rapid gradient-echo (MPRAGE) images were also obtained .2.5Images were preprocessed using automaticanalysis (version 4)  and SPM 2.6http://imaging.mrc-cbu.cam.ac.uk/imaging/Mdsystem using the separate-ROI version and selecting only the frontoparietal ROIs.To stay as close as possible to 2.7fMRI data for each participant were examined using the General Linear Model. Regressors were separately created for each combination of choice number  by domain switch condition  by response switch type . Each regressor was modelled as a rectangular function from stimulus onset to response, convolved with the canonical hemodynamic response function. Given that participant accuracy was very high (mean 98.3%), the original GLM did not exclude error trials. To check for any effect of error trials, in a follow-up GLM (not presented) each error trial was modelled separately and excluded from further analysis. This model produced almost identical results to those presented here.Beta weight images were subtracted for the contrasts 6-choice>2-choice, 2-choice>6-choice, domain switch > domain stay, and domain stay > domain switch. As shown in For the ROI analysis, mean contrast values were extracted from each ROI for each participant using the MarsBaR SPM toolbox , and con33.1Average accuracy was 98.3%, and average RT 0.95s. Independent samples t-tests showed no significant effect of 6-choice domain  (accuracy: t(40) = 1.97, p = 0.055, RT: t(40) = 1.07, p = 0.290) so results are collapsed across these groups.To test for an effect of choice number we performed paired-samples t-tests. To ensure equal numbers of domain switch trials and hand switch trials in 2 and 6 choice measures, we selected the four trial types that were present across both choice levels: domain stay, response stay; domain switch, hand stay; domain stay, hand switch; domain switch, hand switch . We firsTo examine domain switch effects while controlling for response switch and choice number effects, we restricted the analysis to hand switch trials only see . A two-wIn summary, behavioural results show that the manipulation of retrieval difficulty by choice number was effective, and that switches of stimulus domain increased RT in the 2-choice but not the 6-choice conditions.3.2To examine the effects of retrieval difficulty, our primary analysis compared brain activity for 2- and 6-choice tasks. For this purpose, we used average beta values over the four trial types that were present across both choice levels: domain stay, response stay; domain switch, hand stay; domain stay, hand switch; domain switch, hand switch . DiffereResults were clear cut. For DMN regions, activation was greater in 2-choice than in 6-choice. This difference was significant in core and dmPFC subnetworks (core: t(41) = 3.16,p < 0.01, dmPFC: t(41) = 2.70,p < 0.01). In the MTL subnetwork, there was no significant effect of choice number (t(41) = 0.78,p = 0.44). These results rule out the hypothesis that DMN activity increases with the number of choice alternatives during rule retrieval. In contrast, MD regions showed significantly greater activity for the more difficult 6-choice compared to 2-choice trials (t(41) = 3.17,p < 0.01). Additional t-tests revealed significant differences in contrast values between MD network regions and all three DMN subnetworks (core: t(41) = 4.54,p < 0.001; MTL: t(41) = 3.43,p < 0.001; dmPFC: t(41) = 5.16,p < 0.001). A finer breakdown by individual regions within each (sub)network showed largely consistent results, though with variable significance across regions see .We next examined effects of domain switching. For this purpose, we compared mean beta values on domain switch vs domain stay trials, restricting analysis just to hand switch trials see . A two-w3.3At the whole brain level, we found no significant effects of domain switch vs domain stay for either 2 choice or 6 choice tasks, or when combined across choice number.4The primary aim of this study was to test whether increased difficulty of rule retrieval is associated with increased DMN activity. To achieve this, we manipulated the number of alternative rules in a task set. This manipulation had a large effect on behaviour with participants performing significantly slower for 6-choice trials compared to 2-choice trials. However, DMN activity, at least in core and dmPFC regions, showed deactivation with increased retrieval difficulty. This rules out the retrieval difficulty hypothesis and instead matches many cases of deactivation with increasing task difficulty . Other sAs stated in the introduction, DMN activity has been linked to different aspects of retrieval e.g., but espGiven the negative relationship between DMN activity and retrieval demand, in isolation the current results could be interpreted in line with the common view of the DMN as task-negative, showing deactivation with increasing demand of an externally-focused task. However, a large body of work suggests that the relationship between the DMN and task related processes is more complex. For example, during a sustained attention task, DMN activity was positively related to a state of reaction time stability in which participants made fewer errors, however, within this state, further increases in DMN activation were predictive of errors . SimilarA secondary goal of this research was to see whether domain switches cause increased DMN activation in a much simpler task setting than used previously . In thisUnlike core and MTL DMN subnetworks, the dmPFC subnetwork more consistently shows activity decreases with difficulty, even during large domain switches . In the In summary, we find that the difficulty of rule retrieval cannot satisfactorily explain the previous findings of Data and code is available upon direct request to the corresponding author.


When first discovered, mRNA molecules were considered as the simple way through which the information stored in the DNA could be transformed in the real effector molecules, the cellular proteins. This view did not last very long because it became clear very soon that RNA molecules had the ability to determine the way the information was transferred from DNA to the proteins.Today, it is very clear that RNA molecules play a fundamental role in two of the most complex macromolecular machineries found in cells: the spliceosome and the ribosome. Furthermore, the presence in our cells of a vast array of small and large noncoding RNAs (ncRNAs) whose function and regulation are still partially unknown has demonstrated that RNA can actively affect regulatory networks. In particular, the developmental and tissue-specific expression of these ncRNAs can profoundly affect constitutive and alternative mRNA regulatory processes and also participates in the fine tuning of translational processes. As a result of all this complexity, the emerging view is one where RNA molecules occupy a central position in almost all cellular processes within the cell, and only their correct expression can ensure both proper functioning and survival. Unfortunately, the presence of all these very complex regulatory networks also implicates that defects at the level of pre-RNA processing pathways are a major cause of human disease.The purpose of this Research Topic has been to provide an overview of several issues that relate to this issue, with a special focus on new emerging aspects of disease-related RNA connections that range from studying modifications of the RNA molecule itself to the effect of proteins that regulate its processing.N6-methyladenosine (m6A) modifications has been recently suggested to play a critical role in a variety of biological processes and to be especially associated with cancer risk. In their approach, Tang et al. have developed an online database (DRUM) to support the query of disease-associated RNA m6A methylation sites that will help unravel disease mechanisms at the epitranscriptome layer.Regarding the RNA molecule itself, the analysis of RNA Silva et al. In their work, Silva et al. have investigated the importance of TDP-43 in frontotemporal dementia and that mice conditionally expressing this protein recapitulate several core behavioral features of Frontotemporal Dementia/Amyotrophic Lateral Sclerosis (FTD/ALS) spectrum of human pathology. This particular study highlights the importance of RNA binding proteins in neurodegenerative diseases Interestingly, many of these proteins have not yet been characterized very well, despite being known since a long time, and this particular topic has been analyzed by Cappelli et al. with regard to the genes controlled by the two closely related hnRNP Q and R in neuronal cell lines and that were previously shown to affect TDP-43 toxicity in flies and neuronal cell lines  and in targeting a more specialized behavior of RNA binding proteins that is represented by their ability to undergo phase transitions .Finally, much of the excitement that is taking place nowadays with regard to RNA metabolism is targeted at the use of small effectors to recover RNA alterations in a variety of diseases. Therefore, the two final chapters in this research topic look at RNA-based therapeutic strategies both in general terms (Both authors contributed to the writing of this editorial.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.