Communication between the 5′ and 3′ ends of a eukaryotic messenger RNA (mRNA) or viral genomic RNA is a ubiquitous and important strategy used to regulate gene expression. Although the canonical interaction between initiation factor proteins at the 5′ end of an mRNA and proteins bound to the polyadenylate tail at the 3′ end is well known, in fact there are many other strategies used in diverse ways. These strategies can involve “non-canonical” proteins, RNA structures, and direct RNA-RNA base-pairing between distal elements to achieve 5′-to-3′ communication. Likewise, the communication induced by these interactions influences a variety of processes linked to the use and fate of the RNA that contains them. Recent studies are revealing how dynamic these interactions are, possibly changing in response to cellular conditions or to link various phases of the mRNA’s life, from translation to decay. Thus, 5′-to-3′ communication is about more than just making a closed circle; the RNA elements and associated proteins are key players in controlling gene expression at the post-transcriptional level. Also important for regulation, the vast majority of mature eukaryotic mRNAs have a modified nucleotide  on their 5′ terminus and a polyadenylate (poly[A]) tail on their 3′ end (Messenger RNAs (mRNAs) provide the template for synthesis of proteins; by definition, they contain an open reading frame (ORF) that encodes an amino acid sequence. However, encoding a protein is not enough: the use and fate of specific eukaryotic mRNAs must be controlled within the overall strategy used by cells to regulate gene expression. Much of the regulatory power is conferred by essential 3′ end . These fIn this review, we discuss various strategies used by mRNAs and viral RNAs to enable communication between their 5′ and 3′ ends. We present these interactions in the context of the ways that they direct RNA function, including driving efficient translation, regulating mRNA stability and turnover, and promoting viral replication. We do not attempt a comprehensive assessment of all related literature, but rather present some illustrative examples to show how evolution has crafted a variety of ways to confer 5′-to-3′ communication, how useful this strategy is, and the dynamic nature of these interactions. We also highlight a few emerging ideas and areas that remain mysterious, encouraging further investigation.1, continued experimentation supports the existence of this communication. To explain how 5′-to-3′ communication can occur, we first outline the canonical initiation mechanism. The eukaryotic initiation factor (eIF) 4F complex, which contains the cap-binding protein eIF4E and scaffold protein eIF4G, binds at the 5′ end4. Bound eIF4F recruits the 43S complex, which contains the 40S ribosomal subunit3. This complex then scans the 5′ UTR to find the appropriate start codon4, where GTP hydrolysis, release of eIFs, and 60S subunit joining create a translationally competent 80S ribosome7 (reviewed inProbably the most well-known use of 5′-to-3′ communication is to promote or regulate translation of eukaryotic mRNAs. Although some have urged caution in broadly accepting that the 3′ end of an mRNA affects initiation from the 5′ end14. The model is that eIF4F binds to the cap at the 5′ end while multiple poly(A)-binding proteins (PABPs) bind the poly(A) tail and eIF4G, physically linking the 5′ and 3′ ends  either up- or down-regulate translation of an mRNA message by modulating the 5′-to-3′ communication22.A ubiquitous and important form of mRNA 5′-to-3′ communication depends on the 5′ cap and poly(A) tail . The pre3′ ends 15,16. 23, and it is proposed that the physical proximity between the 5′ and 3′ ends favors the transfer of terminating ribosomes from the 3′ end of the ORF to reinitiate at the 5′ end of the mRNA24. One piece of evidence in support of this model comes from the observation that ribosomes within mature polysomes have a rate of exchange with surrounding free ribosomes that is slow enough to suggest reinitiation on an mRNA25. In addition, the 5′-to-3′ interaction may serve as a quality-control mechanism to favor translation of full-length, mature mRNAs26.The PABP-eIF4G interaction within a single mRNA is the “closed loop model”in trans stimulates translation of a capped and non-polyadenylated mRNA to a similar degree as when the poly(A) tail is presentin cis; this may be due to increased affinity of eIF4E for the cap27. Also, in a series of recent publications, it has been observed that mRNAs go through changes in their global architectures over time and as the number of loaded ribosomes increases—first forming circles, then “double-row” structures, then complex “helical” structures where the ends may no longer interact—and there is evidence of similarly dynamic mRNA “closed-loops” in living cells30. Even more interesting, when an mRNA is altered to prevent the PABP-eIF4G interaction, only initial rounds of translation initiation were slowed; the formation of higher-order polysome structures eventually occurred31. This suggests that the PABP-eIF4G interaction might be important for “kick-starting” the initial rounds of translation but is less important during steady-state translation on large polysomes, where other forces dictate higher-order architecture. This in turn suggests a dynamic model for 5′-to-3′ communication linked to the maturation of polysomes and perhaps other yet-to-be-determined signals. Clearly, there is much more to be learned even in regard to the canonical eIF4G-PABP interaction, especially surrounding events in live cells and how this relates to other events in the mRNA life, such as decay.The canonical PABP-eIF4G circularization strategy appears to be a generally important mechanism, but care must be taken not to oversimplify its details or nuances. For example, the synergistic increase in translation efficiency conferred by the cap and poly(A) tail is not strictly contingent upon circularization. Specifically, addition of a poly(A) RNA to translationally competent extract32. The poliovirus IRES RNA directly binds eIF4G, which could interact with PABP bound to the poly(A) tail -binding protein (PCBP) bound to the 5′ cloverleaf structure, upstream of the IRES, and PABP bound to the 3′ poly(A) tail  tail, within the 3′ UTR, can enhance translation in the absence of the poly(A) tail, perhaps by using other proteins40. Thus, even within this one IRES-containing virus, there may be multiple ways that 5′-to-3′ end communication can be achieved. In addition, viral RNAs with a 5′ viral protein of the genome (VPg) in place of a 5′ cap structure, such as members of theCalciviridae family, might use VPg recruitment of the eIF4F complex  to mediate end-to-end communication41. These different strategies may be used during different stages of viral infection, suggesting temporally dynamic interactions that respond to changing cellular conditions and help coordinate different viral processes, such as replication (discussed in more depth later in this review).Exploration of 5′-to-3′ communication starts with the canonical PABP-eIF4G interaction  but doesA) tail 33–36. A) tail 37–39. without needing PABP42. In this case (and many others), the precise mechanism of 5′-to-3′ communication is mysterious. Another intriguing example is the cellular mRNA for serine hydroxymethyltransferase 1, which does not require eIF4G or PABP. Rather, proposed structures in the 5′ and 3′ UTRs bind to heterogeneous nuclear ribonucleoprotein H2 (hnRNP H2) and CUG-binding protein 1 (CUGBP1), respectively, which mediate IRES-driven translation initiation via direct protein-protein contact during states of decreased cap-driven translation initiation  tail buttiation 43.46. Whereas earlier studies concluded that NSP3 was not involved in translation based on a partial knockdown47, another interpretation of these data is that NSP3 is a potent translation enhancer, such that a 10-fold decrease in the amount of NSP3 levels still supports viral protein synthesis48. Likewise, the cellular mRNAs that encode for histones have a highly conserved stem-loop structure at their 3′ ends rather than a poly(A) tail. This structure binds stem-loop-binding protein , which facilities interactions between initiation factors at the 5′ end49. Clearly, the advantageous effect of 5′-to-3′ communication can be achieved with a variety of combinations of mRNA intrinsic elements , and RNA structure), used with canonical factors (eIF4G and PABP), or several “non-canonical” proteins, or both. These mRNAs and viral RNAs illustrate how, despite the existence of the general eIF4G-PABP strategies, there are idiosyncratic methods of achieving 5′-to-3′ communication that add layers of regulatory complexity, which may be linked to cell type, cell conditions, and so on. No doubt there are many more strategies to be discovered and understood.Although IRESs are logical places to use alternative modes of 5′-to-3′ communication, non-canonical mechanisms are also found in viral and cellular transcripts that do not contain IRESs. Rotaviruses are capped but do not have a poly(A) tail; rather, viral non-structural protein 3 (NSP3) binds near the 3′ end and interacts with eIF4G 43–46. 51; therefore, we present just a few examples.The above examples illustrate how 5′ and 3′ ends communicate through various combinations of RNA-encoded signals and proteins. Interesting variations on this theme are RNA signals near the 3′ end of an RNA that bind translation-essential proteins to be used at the 5′ end . ExampleBarley yellow dwarf virus (BYDV), whose 3′ UTR contains a proposed cruciform-like secondary structure referred to as a BYDV-like element (BTE). One stem-loop of the BTE can base-pair to five nucleotides in a short 5′ UTR hairpin, creating a “kissing” interaction53. Because the BTE binds to eIF4G directly, this provides a way to deliver eIF4G to the 5′ UTR55 and also possibly binds directly to the 18S rRNA through base-pairing to recruit the ribosome to the 5′ end , which functionally replace a cap structure by directly binding eIF4E60. Although the high-resolution structure of a PTE-eIF4E complex has not been solved, it has been proposed that these RNAs form a compact fold in which a conserved guanine base is extruded from the structure to be recognized by the initiation factor61. Consistent with the trend of many 3′ CITEs, there are sequences in the PTE that are complementary to sequences in the 5′ end, and these are likely part of the mechanism for bringing eIF4E to the 5′ end to be used in translation initiation.CITEs generally eschew the use of proteins to achieve long-range communication, relying instead on direct RNA-RNA base-pairing between distal sequences. This streamlined approach often consists of a hairpin structure in the 3′ UTR that is complementary to four to eight bases in another short hairpin structure in the 5′ UTR  CITEs found in the genome of several viruses, including68. This shortening is accompanied by the loss of bound PABP, disrupting the 5′-to-3′ communication, likely loss of eIF4E affinity for the cap, enzymatic decapping, and degradation by the 5′-to-3′ exonuclease Xrn170. In a less used deadenylation-independent decay pathway, a stem-loop structure in the 3′ UTR of the mRNA binds a factor that enhances decapping (Edc3) at the 5′ end (and subsequent degradation)68. The fact that translation and mRNA decay are both influenced by 5′-to-3′ communication connects and coordinates these processes within an overall regulatory strategy.Thus far, we have focused on the use of 5′-to-3′ communication in translation, but in fact the m7G cap structure and poly(A) tail serve more than one purpose: recruitment of translation machinery for protein synthesis and also protection against degradation of the RNA by exonucleases  RNAs and ensures that only full-length poliovirus RNAs are replicated72. A different mechanism with a similar outcome is found in Dengue virus (a flavivirus). In Dengue, direct base-pairing that forms between sequences in the 5′ and 3′ UTRs of the viral RNA do not appear to have a role in directing translation but are important for viral replication74. Specifically, a 5′ stem-loop functions as a promotor to bind the RNAP and the base-pairing then is thought to deliver the RNAP to the 3′ UTR to commence minus-strand synthesis76.Unlike mRNAs, the genomic RNAs of positive-sense single-stranded viruses must be replicated to make a negative-sense RNA intermediate that serves as a template for many copies of the positive-sense RNA. Communication between the 5′ and 3′ ends is essential for this, and several strategies exist to achieve it. Again with poliovirus as an example, the RNA-dependent RNA polymerase (RNAP) must initiate minus-strand synthesis from the poly(A) tail. To do this, the cloverleaf RNA structure within the 5′ end of the viral RNA binds to PCBP and also to RNAP; both interact with poly(A)-binding protein 1, which binds the poly(A) tail78. This is important because the translation and replication machinery would clash should they both simultaneously use the same copy of viral RNA. This again underscores a common theme: that 5′ and 3′ communication strategies are dynamic and diverse players in regulating the use and fate of the associated RNA.A general theme in many viral RNAs is that 5′-to-3′ communication plays a role in both translation and replication. Dynamic changes of the interactions between the 5′ and 3′ ends  have been proposed as mechanisms to organize these processeset al. used a high-throughput approach to identify thousands of non-canoncial translation initiation signals in both viral and human RNAs located throughout the RNA; although interactions between these and the 5′ end have not been shown, these discoveries suggest that many more examples of long-range communication are still to be discovered57. Also, there is much to be learned about how these interactions may be affected by changing cellular conditions, concentration of factors or RNA, cell type, time of viral infection, and so on. Even in the case of the canonical PABP-eIF4G interaction, quantitative assessment of the affinities, conformational changes and binding dynamics, and how these relate to the maturation of polysomes and transitions between different phases of the mRNA’s life cycle remain areas ripe for more exploration. Overall, there is much to learn about roles of higher-order architecture and the dynamics of these architectures. Happily, this suggests that many more exciting discoveries are on the horizon.In the preceding text, we have illustrated the diverse interaction strategies in which eukaryotic cellular mRNAs and some viral RNAs can promote communication between the 5′ and 3′ ends and how this relates to a number of different processes. Although there are common mechanisms by which communication is achieved, there are no “hard and fast” rules; examples of RNA-RNA, RNA-protein, and protein-protein interactions abound, and these examples use canonical factors as well as more specialized proteins. In some cases, the strategy appears to be idiosyncratic to a particular mRNA or virus, consistent with hundreds of millions of years of evolutionary tinkering and fine-tuning. Although many strategies have been described, it is clear that there are many others remaining to be discovered. Very recently, Weingarten-Gabbey


Nevertheless, some miRNAs were preferentially or exclusively isolated by either of the methods. Trizol® LS resulted in very low purity RNA which affected RT-qPCR efficiency. In general, miRCURY™ biofluids kit efficiently extracted miRNAs from plasma. A careful selection of the RNA isolation method and the consideration of the type and size of input material are highly recommended to avoid biased results.MicroRNAs (miRNAs) have emerged as promising cancer biomarkers. However, exploiting their informative potential requires careful optimization of their detection. Here, we compared the efficiency of commonly used RNA extraction kits in miRNA recovery from cells, plasma and urine/plasma-derived exosomes, using single-gene RT-qPCR and miRNA profiling. We used increasing amounts of starting material to investigate the impact of the input material size on miRNA extraction. We showed that miRNA recovery was largely influenced by the isolation method and by the amount of input material. In particular, the miRCURY™ kit provided highly pure RNA. However, its columns poorly recovered miRNAs from limiting amounts of cells and plasma, and rapidly saturated by large RNA species and plasma components, thus impeding miRNA recovery from high input amounts. Overall, the miRNeasy MicroRNAs (miRNAs) are small non-coding RNAs that downregulate gene expression through their binding to 3′UTR target mRNAs1Since their discovery in bodily fluids, inside membrane-vesicles  or bound to proteins and lipoproteins3561Indeed, several inconsistencies have been observed among the studies and dissimilar results could be explained in part by differences in methodological parameters91011121RNA extraction methods can be roughly classified into three main groups: 1) the phenol-based techniques that rely on the use of organic solvents, phase separation, and recovery of RNA by precipitation, 2) the combined phenol and column-based techniques that utilize phenol and chloroform to separate RNA from other constituents, and a column for RNA adsorption, 3) the column-based techniques that use a phenol-free lysis buffer and a column for RNA recovery8et al.® Serum/Plasma kits were comparably efficient in recovering miRNA spike-in controls from plasma samples, whereas McAlexander M et al.® Serum/Plasma kit versus miRCURY™ biofluids kit. On a different note, Cheng L et al. demonstrated that the miRNeasy® kit was slightly more efficient than the miRCURY™ kit in extracting miRNAs from urinary exosomes® and the mirVana™ kits were the most performant ones for maximizing RNA extraction from urinary exosomes isolated by ultracentrifugation®Several efforts have been made towards the comparison and the optimization of extraction methods for miRNA141516171857® LS), two column- (miRCURY™ RNA Isolation and miRCURY™ biofluids kits) and a combined phenol- and column-based (miRNeasy® Mini kit) RNA isolation techniques. Besides, we investigated the impact of the amount of starting material on the efficiency of miRNA extraction by using variable numbers of cells and different volumes of plasma. This is of particular importance since the use of “unlimited” quantity of material from cells in culture may not yield results that mirror analyses performed with limited-size clinical samples. Besides, some measurements are not feasible with clinical specimens; this is for example the case of plasma RNA quantification with NanoDrop spectrophotometer. In such situations, a fixed volume of input material rather than an equal quantity of RNA should be usedHere, we have compared commonly used RNA extraction methods for their efficiency in miRNA isolation from different sample types and varying amounts of input material. The assessment of miRNA recovery has been evaluated by capillary electrophoresis and by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) through single gene examination and a broad miRNA profiling. In particular we chose to evaluate a phenol- , medium (200 × 103) or high (800 × 103) cell numbers .We first evaluated the efficiency of Trizol® and miRCURY™ kits yielded similar RNA quantities suggesting that they were equally efficient in total RNA extraction from cells in culture . However with medium and low cell numbers, the efficacy of Trizol® LS seemed increased. This apparent improvement likely resulted from a contamination by residual phenol that induced an erroneous overestimation of RNA quantities, due to the absorbance peak at 270 nm . All three extraction methods yielded high RIN values , indicating a high quality RNA. Importantly, the presence of MS2 decreased the RIN to 6.5 in the 25c samples. Nevertheless, this low RIN should not be interpreted as an RNA degradation, since it is due to MS2 itself that changes the RNA electropherogram profile ,® and miRCURY™ versus Trizol® LS. At low cell density, the A260/A280 ratio was low for all extraction methods (A260/A280 <1.8). This ratio raised above 1.8 only after the addition of MS2 RNA, indicating that the low RNA concentrations obtained with 25 × 103 cells, rather than a considerable protein contamination, were the main reason behind the low A260/A280 ratio.RNA purity was assessed by measuring the absorbance at 260 nm, 280 nm and 230 nm as described in the Methods. As expected, when the number of starting material increased, the purity of the sample increased as well, due to a higher RNA absorbance peak at 260 nm, independently of the extraction method . At medi® LS and miRNeasy® extracts for all starting cell numbers, indicating an important presence of contaminants that absorb at 230 nm  and RNA with acceptable purity (A260/230 ≥1.6) at medium cell density, the A260/A230 was substantially low in Trizolt 230 nm . Indeed,iRNeasy® . The guaiRNeasy® . Importa® LS, miRNeasy®, and miRCURY™ kits in downstream RT-qPCR experiments for miRNA detection, using either a fixed volume of eluted RNA  or a fixed amount of RNA (5 ng) as input for RT reaction.We investigated the performances of Trizol® and miRCURY™ kits resulted in equal detection of mir-106a and mir-222 , whereas Trizol® LS extraction yielded significantly higher Cq values (P < 0.001) suggesting a lower recovery of miRNAs and/or a poor PCR efficiency due to phenol contamination  (® and miRCURY™ increased (P < 0.001), suggesting a better detection of mir-106a, mir-222, and mir-141 with miRNeasy®. Whilst miRCURY™ outperformed Trizol® LS at low cell density, Trizol® LS permitted a better detection of miRNAs at high cell density, albeit less efficiently than miRNeasy®. The results obtained with MS2-supplemented conditions were comparable to those obtained without MS2 (mainly with medium and high cell numbers), for each of miRNeasy® and miRCURY™.With a fixed RNA volume and a low cell density, miRNeasymination . When thiRNeasy) . At high® and miRCURY™ kits using 100, 1000 and 10 × 103 cells as starting material. Given the poor microRNA recovery observed with Trizol® LS isolation from 25 × 103 cells, this condition was not tested with fewer cell numbers. When the starting cell number is very low (100 and 1000 cells), miRNeasy® kit permitted a better detection of mir-106a and mir-222 when compared to miRCURY™ kit, independently of MS2 supplementation  is commonly used as a housekeeping gene to normalize miRNA expression in cells, we wished to know whether its detection is affected by the RNA extraction method, similarly to miRNAs. Our data show no or small, albeit statistically significant, fluctuations in U6 recovery (mainly a decreased recovery with Trizol® LS) across the different techniques and with all the quantities of starting material investigated  simultaneous variations of miRNAs and U6 Cq values, (2) the presence of MS2 RNA quantified together with cellular RNA, or (3) the presence of phenol in some samples which may alter the RNA quantification.In order to analyze if differences in miRNA detection can also be observed when a fixed amount rather than a fixed volume of input RNA is used, RT-qPCR experiments were performed with 5 ng of RNA to detect the expression of mir-106a, mir-222 and U6 snRNA. As shown in ® isolation technique outperforms Trizol® LS and miRCURY™ specifically in the detection of miRNAs by RT-qPCR. Our findings imply that miRNA recovery may be affected by the amount of input material.Altogether, these data demonstrate that miRNeasyTo better understand how the amounts of starting material may influence miRNA yields, we analyzed the efficiency of miRNA recovery by each of the methods using increasing number of starting material and a fixed volume of eluted RNA.3 to 200 × 103, the Cq values should decrease and the theoretical mir-106a and mir-222 Cq difference should be of 1.38 . The best Cq differences between these two cell densities were obtained with miRCURY™ extraction (ΔCq (25c–200c) = 1.342 ± 0.488 and 1.646 ± 0.633 for mir-106a and mir-222, respectively) , the miRCURY™ kit resulted in very low ΔCq , when compared to Trizol® LS and miRNeasy® . Whereas the experimental ΔCq values were similar in the absence or presence of MS2 for miRNeasy® protocol, the addition of MS2 worsened miRNA detection when using miRCURY™ kit with 25 × 103 cells (When the cell number increases from 25 × 10ctively) , suggestiRNeasy® , suggest03 cells .®, the recovery of miRNAs with miRCURY™ decreases dramatically when increasing the amount of input material . This observation implies a saturation of the column by RNA species or by protein-induced column clogging.These data suggest that, contrarily to miRNeasy® extraction, for both low and high starting cell numbers. At low cell density, the recovery of small RNAs was better with miRCURY™ than with Trizol® LS. However, the concentration and percentage of miRNAs in this fraction were lower.In order to investigate whether the differences observed in the gene-specific miRNA RT-qPCR data result from variations in the extraction efficiency of small RNAs, we used the Agilent Small RNA kit to quantify each of these fractions. This kit analyses small RNAs in the 6 to 150 nucleotide (nt)- size range and detects miRNAs in the 10 to 40-nt range. As shown in ® and miRCURY™ to extract small RNAs were comparable at low cell density , at high cell density, the miRNeasy® kit showed a better small RNA isolation ability . These differences are illustrated by the small RNA electropherogram overlays shown in ® and miRCURY™, respectively).Whilst the abilities of miRNeasy® kit permits a better isolation of small RNAs including miRNAs compared to Trizol® LS and miRCURY™, and that small RNA extraction efficiency of miRCURY™ kit decreases when the starting cell number increases, ultimately resulting in reduced miRNA recovery.These data demonstrate that miRNeasy® microRNA assay to quantify the amounts of small RNAs in the 800c samples. The concentrations of the so-called “small RNAs” were 51.6 ± 26.5 ng/μl, 101.7 ± 50.0 ng/μl and 177.2 ± 82.3 ng/μl for Trizol® LS, miRNeasy®and miRCURY™, respectively. Curiously, the small RNA fraction, as evaluated by the Qubit® microRNA assay, represented up to 50% of total RNA in the samples. These data are in contradiction with the results obtained with the Agilent Small RNA kit and imply that other majority RNA or DNA species may also be detected by the Qubit® microRNA assay. Since both miRNeasy® and miRCURY™ kits remove most of the DNA present in the samples, a DNA contamination cannot explain the high values observed. On the contrary, despite its selectivity for small RNAs, the Qubit® microRNA assay can also detect 20 to 30% of large RNAs, including ribosomal RNAs (rRNA) and messenger RNAs (mRNA), as described by the manufacturer in its Bioprobes@70 journal. Therefore, these data suggest that miRCURY™ kit extracts more large RNA species than miRNeasy® kit. Since the total RNA yield did not differ between both kits . In contrast, the miRCURY™ biofluids and the miRNeasy® kits showed comparable miRNA recovery . With 100 μL of plasma, miRNeasy® and miRCURY™ biofluids kits demonstrated equal efficiencies in miRNA recovery and outperformed miRCURY™ kit (P < 0.001). With 200 μL of plasma, the recovery of miRNAs continued to increase with both miRNeasy® and miRCURY™ biofluids . Nevertheless, contrarily to miRCURY™ biofluids, the recovery of miRNAs (with the exception of mir-222) with miRNeasy® tended to reach a plateau, as the recovered amount of miRNAs was not anymore proportional to the input volume of plasma, thus suggesting a slight column saturation. It is important to mention that these results were obtained from 3 plasma samples. In these experiments, miRCURY™ biofluids permitted a slightly better miRNA detection than miRNeasy® from 200 μL of plasma. However, when these data are combined with 3 additional plasma samples to yield the results presented in ® are not anymore significant. The efficacy of miRCURY™ kit in miRNA recovery was not satisfactory with any of the starting plasma volumes.Exogenous spike-in controls are often used to monitor the efficiency of RNA extraction. Therefore, we assessed the recovery, by each of the RNA isolation methods, of the exogenous control cel-mir-39-3p spiked into 200 μL of plasma. As shown in ® and miRCURY™ kits was also evaluated in exosomes from urine samples. In order to exclude inter-individual variabilities due to differences in water excretion into urine, we chose to represent individual data from urinary exosomes obtained from 3 healthy donors, rather than the mean ± SD of the data. For each of the 5 miRNAs analyzed, miRNeasy® permitted a better miRNA detection in 2 out of 3 samples, whereas similar recovery by the two kits was observed in the third sample . Importantly, in the set of common miRNAs, the expression levels of miRNAs isolated with miRNeasy® were in general higher than those observed with miRCURY™, with Cq differences exceeding 5 in some cases , and thus may differently affect the adsorption of miRNAs to the columns of each kit. Accordingly, we noticed that the best miRNA recovery from 200 μL-plasma was observed with either miRNeasy kit or miRCURY™ biofluids kit. The low performance of miRCURY™ kit in miRNA recovery from 50 μL to 200 μL of plasma suggests poor column adsorption ability, carryover of blood-borne RT-PCR inhibitors, and/or column clogging. Besides, in accordance with previous findings8et al. described an optimized protocol for the analysis of miRNA from plasma- and serum-derived extracellular vesicles/exosomes using the miRNeasy® kit251227® and the miRCURY™ kits is still to be investigated.The superior performance of miRNeasy® compared to miRCURY™, using single RT-qPCR miRNA assays. Whereas miRNA profiling using TLDA confirmed the overall superiority of miRNeasy® in isolating miRNA from plasma, plasma exosomes and urinary exosomes, it also showed that some selected miRNAs can be preferentially or exclusively extracted by either of the methods. Differences in miRNA profiling have been previously observed when comparing the NucleoSpin® miRNA plasma and the miRNeasy® kits in TLDA assays®, mirVana™ (Life technologies) and miRNeasy® kits in miRNA microarraysOur results demonstrated that higher amounts of miRNAs can be detected with miRNeasyet al., Trizol® and miRNeasy® were less efficient than miRCURY™ for total RNA isolation from cells, whereas all three methods extracted small RNAs, including miRNAs, with equal ability® are equally efficient in total RNA isolation and are superior to Trizol® LS, whereas miRNeasy® resulted in the highest small RNA, including miRNA, yield. These discrepancies may generate, at least in part, from the different amounts of cells used in both studies. Moreover, with the miRNeasy® kit, we opted to use Phase Lock Gel (PLG) for the collection of the aqueous phase in order to increase the volume retrieved while getting a better RNA purity. This may also explain the higher RNA yield we have obtained compared to the study of Eldh M et al. Interestingly Trizol® LS permitted the highest enrichment of miRNA in the small RNA fraction when the extractions were done with a small cell number  in the extraction of miRNA from serum® LS versus miRCURY™ extraction, in contrast to the results obtained with a low cell number.In the study of Eldh M ® kit displayed a better performance as for miRNA detection by RT-qPCR, despite a less optimal RNA purity. However, one should keep in mind that this conclusion is peculiar to miRNA isolation and should not be considered as a general statement relative to any other RNA species. The miRCURY biofluids kit demonstrated satisfactory results in miRNA isolation from 100 μL and 200 μL of plasma. Concerning the phenol-based method of Trizol® LS, it resulted in RNA extracts with very low purity which affected RT-qPCR efficiency, albeit acceptable results were obtained with high input material. Nevertheless, we do not recommend this method for miRNA studies owing to (1) the reproducibility issues that may arise from the poor sample purity, (2) the previously reported selective loss of small RNA molecules with low GC contentIn conclusion, we have shown that the column-based method of the miRCURY™ kit has the advantage of providing highly pure RNA; However its sub-optimal column capacity has the inconveniency of poor miRNA recovery when using very low amounts of cells and plasma and rapid saturation by large RNA species and plasma components, thus hindering the isolation of miRNAs. On the contrary, the combined phenol and column-based method of the miRNeasy® kit is efficient in extracting miRNAs from cells in culture, plasma and biofluid-derived exosomes. However, due to the simplicity of its execution, rapidity and phenol-free procedure, one may prefer to use miRCURY™ biofluids for the isolation of miRNA from 100 μL and 200 μL of plasma. Last but not least, it is of major importance to strictly adhere to the same RNA technique AND to the same amount of input material throughout the entire study in order to have comparable measurements.Finally, when performing miRNA analysis, one should always consider the issues that may arise from the use of random amounts of starting material. Hence it is highly advisable to carefully choose the RNA isolation method according to the type and quantity of input material, and to the downstream experimental requirements. In this regard, we showed that miRNeasy2.A549 cells were cultured in DMEM medium , containing 10% fetal bovine serum and were incubated at 37 °C in an atmosphere of 5% COThe subjects were healthy donors who signed an informed consent according to the Declaration of Helsinki. All the experimental protocols were approved by the national research ethics committee in Luxembourg “Comité National d’Ethique de Recherche” (CNER), and the study was authorized by the national commission for data protection “Commission Nationale pour la Protection des Données” (CNPD). Peripheral blood samples (n = 6) were drawn into EDTA-containing tubes and centrifuged at 1000 g for 10 min at 4 °C. The supernatants were then centrifuged at 2000 g for 10 min at 4 °C to remove debris. The plasma supernatants were aliquoted in 2-mL tubes and stored at -80 °C.Midstream spot urine (n = 3) (50 to 100 mL) was collected in sterile containers supplemented with a protease inhibitor cocktail  and immediately processed for exosome isolation.Four mL of plasma were diluted 5 times with phosphate buffered saline (PBS), then centrifuged at 10,000 g for 50 min at 4 °C to remove large extracellular vesicles. The supernatant was sequentially passed through 1.6- and 0.2-μm pore-sized filters to remove remaining debris and large particles. The diluted plasma was equally split in two tubes and ultracentrifuged at 110,000 g for 1 h 30 min at 4 °C to collect exosomes. Exosomes were then washed with PBS, pelleted again at 110,000 g for 1 h 30 min at 4 °C and resuspended in PBS.Urinary exosomes were isolated as previously described323 (25c), 200 × 103 (200c) and 800 × 103 (800c) cells , from 50 μl, 100 μl and 200 μl of plasma, and from biofluid-derived exosomes, using Trizol® LS reagent , miRNeasy® Mini kit , miRCURY™ RNA Isolation and miRCURY™ biofluids kits  as indicated in the manuscript. RNA isolation was performed as recommended by the manufacturers. MS2 RNA  was added as a carrier for RNA extraction from plasma, biofluid-derived exosomes, and, when indicated, from A549 cells. When specified, 25 fmol of the spike-in control Caenorhabditis elegans cel-mir-39-3p  were added per extraction into the lysis solution of each method prior to the combination with 200 μl of plasma. MS2 was mixed with the QIAzol  and with Exiqon kits lysis solutions prior to the addition of the samples. The quantities of MS2 used were 0.64 μg/sample of A549 cells and urinary exosomes, and 0.96 μg/sample of plasma and plasma exosomes. A “no cell” condition containing 0.64 μg of MS2 and subjected to the same miRNeasy® protocol used for A549 cells has been included. Phase Lock Gel™ (PLG) tubes  were used with the miRNeasy®protocol for the recovery of the aqueous phase. RNA was eluted in 50 μL of RNase-free water and stored at −80 °C.Total RNA was isolated from 100, 1000, 10 × 10® microRNA assay  on the Qubit® 2.0 Fluorometer. For Agilent Small RNA assay, 1 μL of RNA from the stock solution or from equally diluted samples was used. For Qubit® microRNA assay, 10 μL of equally diluted RNA samples were used. Electropherograms were visualized using the Agilent 2100 Expert software (Agilent technologies).Total RNA was quantified using NanoDrop ND-1000 spectrophotometer . RNA purity was assessed by checking the absorbance at 260 nm, 280 nm and 230 nm. An A260/A280 ratio <1.8 denotes protein contamination and an A260/A230 <2 indicates presence of phenol, chaotropic salts (guanidinium thiocyanate) or proteins. RNA integrity was analyzed with the Agilent RNA 6000 Nano or Pico kits  on the Agilent Bioanalyzer 2100  and evaluated based on the RNA Integrity Number (RIN) that classifies the RNA as very degraded (RIN = 1) to intact (RIN = 10). The quantity and the profile of small RNAs were assessed by capillary electrophoresis and fluorescence, using the Agilent Small RNA kit on the Agilent Bioanalyzer 2100, and the Qubit® MicroRNA Assays. Briefly, 2 μL of diluted (or 2.5 μL of undiluted) RNA solution (see below for further details) or 5 ng of RNA were used in a 7.5 μL-RT reaction. Real-time PCR reactions were performed with 0.67 μL of the RT product in 10 μL-PCR reaction volume and done in triplicate using the ViiATM 7 Real-Time PCR System . RT and PCR reactions were performed following the programs recommended by the TaqMan® MicroRNA assays.The detection of miRNAs was performed by RT-qPCR using TaqManFor RT-qPCR experiments using RNA from plasma, plasma exosomes and urinary exosomes, 2.5 μL of RNA samples were used in the RT reactions.For RT-qPCR experiments using RNA from A549 cells: When a constant amount of RNA was used, 5 ng were included in the RT reaction. When a fixed volume of RNA was used, 2 μL of diluted or 2.5 μL of undiluted RNA solution were used according to the following considerations:3 cells as input amounts, 2.5 μL of undiluted RNA solution were used in the RT reactions.When using 100, 1000 and 10 × 103, 200 × 103 and 800 × 103 cells as input amounts, for mir-106a, mir-222 and U6 snRNA analyses, in order to decrease the influence of PCR inhibitory factors (such as phenol) that may be found in some RNA samples, and to follow the instructions of the RT kit as for the RNA quantity to use, we first diluted the RNA samples as follows :When using 25 × 10® MicroRNA RT kit is 0.5 to 5 ng of RNA/7.5 μl of RT reaction, the RNA samples were first diluted  to a maximal concentration of 2.5 ng/μl; 2 μl of the diluted samples were used in the 7.5 μl-RT reaction volume. The mean dilution factors were then 21.7, 66.7 and 150 for the 25c, 200c and 800c series, respectively. When taking into account that the 800c series contains 4 times more cells than the 200c series which contains 8 times more cells than the 25c series, the RT-qPCR results obtained with a fixed RNA volume reflect data from: 800c samples 1.78 times more concentrated than the 200c samples, and 200c samples 2.6 times more concentrated than the 25c samples. Assuming a PCR amplification efficiency of 100%, 1.78 and 2.6 fold differences in starting template concentration should give cycle threshold (Cq) differences of 0.83 between the 800c and the 200c samples (20.83 = 1.78), and 1.38 between the 200c and the 25c samples (21.38 = 2.6).Since the recommended input RNA quantity to be used with the TaqMan2 = 4) between these too conditions.Since mir-141 is expressed at very low levels in A549 cells, the 2 μl of diluted RNA resulted in undetermined Cq; hence we used 2.5 μl of undiluted RNA from the 25c, 200c and 800c samples as starting material for mir-141 detection. The PCR reactions performed with the 25c samples generated Cq > 35 and thus were not considered. As the 2.5 μl of undiluted 800c RNA samples contain 4 times more starting material than the 2.5 μl of undiluted 200c RNA samples, the theoretical mir-141 Cq difference should be of 2 (2ΔΔCq−) is the normalized gene expression (2ΔCq−) in the sample divided by the normalized gene expression (2ΔCq−) in the control, as indicated in the manuscript.The data were analyzed by the ΔΔCq method. The fold change  as described by the manufacturer. Briefly, 3 μL of RNA extracts were reverse-transcribed using Megaplex RT primer pools A and B in a final volume of 7.5 μL. cDNA targets were then preamplified using 2.5 μL of the RT product in a 25 μL-preamplification reaction. Nine μL of 4 time-diluted preamplification products were included into the 900 μL-PCR reaction required for each TaqMan® MicroRNA Array. Real-time PCR was performed using a ViiATM 7 Real-Time PCR System . Only miRNAs with Cq < 35 were considered as detected.A broad miRNA profiling of plasma and biofluid-derived exosomes covering 756 miRNAs was carried out using TaqManΔCq− values. A P-value < 0.05 was considered statistically3132Statistical analysis was carried out with the R software, using a two-way ANOVA model with experiment and extraction method as factors, followed by Fisher’s least significant difference (LSD) test. The p values of the fold change were calculated based on a Student’s t-test of the replicate 2How to cite this article: El-Khoury, V. et al. Assessing cellular and circulating miRNA recovery: the impact of the RNA isolation method and the quantity of input material. Sci. Rep.6, 19529; doi: 10.1038/srep19529 (2016).


Biodiversity patterns across the marine tropics have intrigued evolutionary biologists and ecologists alike. Tropical coral reefs host 1/3 of all marine species of fish on 0.1% of the ocean’s surface. Yet our understanding of how mechanistic processes have underpinned the generation of this diversity is limited. However, it has become clear that the biogeographic history of the marine tropics has played an important role in shaping the diversity of tropical reef fishes we see today. In the last decade, molecular phylogenies and age estimation techniques have provided a temporal framework in which the ancestral biogeographic origins of reef fish lineages have been inferred, but few have included fully sampled phylogenies or made inferences at a global scale. We are currently at a point where new sequencing technologies are accelerating the reconstruction and the resolution of the Fish Tree of Life. How will a complete phylogeny of fishes benefit the study of biodiversity in the tropics? Here, I review the literature concerning the evolutionary history of reef-associated fishes from a biogeographic perspective. I summarize the major biogeographic and climatic events over the last 65 million years that have regionalized the tropical marine belt and what effect they have had on the molecular record of fishes and global biodiversity patterns. By examining recent phylogenetic trees of major reef associated groups, I identify gaps to be filled in order to obtain a clearer picture of the origins of coral reef fish assemblages. Finally, I discuss questions that remain to be answered and new approaches to uncover the mechanistic processes that underpin the evolution of biodiversity on coral reefs. Figure 1A). This region has also been called several other names , or the extent of their geographic range  identifies those groups that are characteristic of a modern reef assemblage , regardless of geographic location . While the majority of major lineages and genera are sampled within these phylogenetic studies, the level at which species within these lineages are sampled varies dramatically . Those families that have more completely sampled phylogenies have achieved it through the combination of multiple sequence datasets and the use of supermatrix phylogenetic methods. The combination of datasets for the butterflyfish family Chaetodontidae , depending on the core aim of the study. The chronogram of Table 1). These large-scale phylogenetic studies employing supermatrix methods have also allowed the identification of the closest sister families to prominent reef fish families. However, disagreement among these large phylogenies still exists for some families. For example, the closest sister group to the Labridae changes from being the family Centrogenyidae  with some possibly being older than the IAA hotspot , the older ages of extant species challenged the early suggestion that sea level fluctuations during the Pleistocene was a major factor in the origin of modern coral reef assemblages . It is likely that when the gaps in taxonomic sampling of these reef fish families are filled, and cryptic species are identified, the inclusion of unsampled lineages closer to the present may enhance the role played by speciation in the Pleistocene and the importance of peripheral locations in promoting biodiversity. Processes at work in the Miocene appear to be the main source of origination of modern reef fish biodiversity patterns, while processes maintaining this pattern are prominent from the Pliocene/Pleistocene. However, to gain a clearer picture of the magnitude of these processes across the marine tropics, studies with a biogeographic focus have been important.The majority of extant coral reef fishes examined by  hotspot . While semblages . For reeemblages . Pleistoemblages , peripheemblages , and moremblages . Pleistoemblages , which d1; IUCN red list2; the Global Biodiversity Information Facility3; Map of Life4; and Ocean Biogeographic Information System5. A recent effort to construct a global database for tropical reef fishes has resulted in over 6300 records for reef fishes across 169 locations . These data are likely to include the majority, if not all reef associated members of these families. In combination with a fully sampled phylogeny of reef fishes, these geographic data would allow us to tease apart some of the questions that have been partially answered so far regarded the origins of tropical biodiversity. Unfortunately, fully sampled phylogenies for important groups are still out of reach. The incomplete and clade biased phylogenetic sampling also translates into a bias in geographic sampling . Those charismatic families such as the Labridae and Chaetodontidae that have been the focus of several papers from a variety of research groups have more even phylogenetic sampling across biogeographic ecoregions, with over 50% of taxa present in each region represented in a published phylogeny . A sampling bias can be observed among ocean basins, and among families, where some families  have higher phylogenetic sampling in Indo-Pacific locations, whereas others  show higher phylogenetic sampling in the Atlantic locations. Overall, the families Apogonidae, Blenniidae, Mullidae and Carangidae show concerning levels of lower phylogenetic resolution across tropical reef habitats  with many regions showing below 10% phylogenetic sampling of ecoregion assemblages. Nonetheless, the geographic data available, regardless of its sampling in phylogenetic trees, have been fruitful in delineating biogeographic regions across the marine tropics.Of the valid nominal species in the nine reef fish families, these geographic checklists  include Figure 1C), each with varying degrees of structure within those delineated regions and provinces , however these data are still from incompletely sampled phylogenies.Albeit incomplete, dated phylogenies combined with biogeographic distributions can detect the initial origins of ancestral reef fish lineages, their extinction and survival with shifting centers of biodiversity, and proliferation within expanding habitat. From these patterns it is possible to identify temporal and spatial variation in rates of speciation, extinction and dispersal and how this variation has resulted in the current biodiversity gradient. Measuring the net rate of diversification and how it varies through time has become an important metric in the integrated study of macroevolution and macroecology . MethodsThis radiation and subsequent slowdown is also termed “density-dependent” diversification and can resemble a “niche-filling” process. Such a process was recently uncovered in the trophic diversification of several tropical reef fish families  where a We have yet to definitively identify within a complete phylogenetic framework how rates of net diversification on tropical reefs have been altered by ecological or biogeographical processes. If such processes have underpinned the radiation of fishes on coral reefs it may change our understanding of the origins of biodiversity and what factors are important in maintaining diversity in the present.Across several taxonomic groups there is consistent evidence of a link between the rate of molecular evolution and the observed biodiversity of clades . This paWhen exploring the link between molecular rate and diversity there are three main explanations that have been discussed . First, If speciation drives the rate of molecular evolution through population subdivision, higher diversity and molecular rates in reef associated fish lineages could be driven by the fragmentation of habitat and peripheral isolation, both process that have been reported in evolutionary studies of tropical reef fishes . If corrIf mutation rate drives speciation rate, would this mean that coral reefs provide the molecular fuel for speciation? It has already been demonstrated that coral reefs promote both the diversification of lineages  and morpIn reviewing the current state of phylogenies and historical biogeography of tropical reef fishes I have summarized a series of historical events that have underpinned the origins and proliferation of reef fish biodiversity in the tropics. This review also highlights several groups that require increased sampling and further analysis. While some focal groups are almost completely sampled, an additional push is needed to obtain complete species level sampling. Although the traditional nine coral reef fish families have been important models in the exploration of marine speciation and evolution on coral reefs, there are other fish families and lineages that may provided as much, if not more insight into the origins of tropical biodiversity. It is in this respect that a robust and well-resolved Fish Tree of Life will be beneficial to both the examination and comparison of evolutionary rates among discrete tropical clades found on and off reefs, and the investigation of overarching patterns of tropical diversification. I suggest that future research concerning the macroevolutionary patterns of fishes found on coral reefs examine the historical variation in rates of speciation, extinction, and dispersal among biogeographic regions and across multiple lineages. Further discussion is needed to evaluate how hypotheses concerning the origin and maintenance of biodiversity are modeled to account for the interaction between macroecology, macroevolution and molecular processes.There are several questions that offer exciting pathways for future research:• How has temporal and spatial variation in rates of speciation, extinction and dispersal lead to present day patterns of tropic fish biodiversity?• Do present day biogeographic delineations reflect the evolutionary history of the tropical belt, and which scheme is best?• Are there limits to the biodiversity of tropical regions and if so, how are these limits linked to the diversity of clades and regional assemblages?• Do tropical clades experience increase rates of molecular change on coral reefs and how does this link to patterns of biodiversity across the tropical belt?With increasing access to genomic methods, there is a unique opportunity to reconstruct the evolutionary history of all fishes to the level of resolution that is available in other vertebrate clades. Within this framework we can move beyond categorizing patterns and predictors of extant biodiversity, and statistically examine the evolutionary history under hypotheses driven models of diversification.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Caenorhabditis elegans is characterized by a large number of acetylcholine receptor subunit genes, a feature shared across the nematodes. This dynamic family is characterized by both gene duplication and loss between species. The pentameric levamisole-sensitive acetylcholine receptor has been characterized from C. elegans, comprised of five different subunits. More recently, cognate receptors have been reconstituted from multiple parasitic nematodes that are found to vary in subunit composition. In order to understand the implications of receptor composition change and the origins of potentially novel drug targets, we investigated a specific example of subunit duplication based on analysis of genome data for 25 species from the 50 helminth genome initiative. We found multiple independent duplications of the unc-29, acetylcholine receptor subunit, where codon substitution rate analysis identified positive, directional selection acting on amino acid positions associated with subunit assembly. Characterization of four gene copies from a model parasitic nematode, Haemonchus contortus, demonstrated that each copy has acquired unique functional characteristics based on phenotype rescue of transgenic C. elegans and electrophysiology of receptors reconstituted in Xenopus oocytes. We found evidence that a specific incompatibility has evolved for two subunits co-expressed in muscle. We demonstrated that functional divergence of acetylcholine receptors, driven by directional selection, can occur more rapidly than previously thought and may be mediated by alteration of receptor assembly. This phenomenon is common among the clade V parasitic nematodes and this work provides a foundation for understanding the broader context of changing anthelmintic drug targets across the parasitic nematodes.Helminth parasites rely on fast-synaptic transmission in their neuromusculature to experience the outside world and respond to it. Acetylcholine plays a pivotal role in this and its receptors are targeted by a wide variety of both natural and synthetic compounds used in human health and for the control of parasitic disease. The model,  Caenorhabditis elegans has allowed major advances in our understanding of neurotransmitter receptors, including acetylcholine receptors targeted by the drug levamisole. Characterization of equivalent receptors in parasitic nematodes has revealed changes in receptor composition, the consequences of which are not yet clear. The aim of this study was to examine the mechanisms that produce new receptor subunits and characterize changes in receptor function that arise. We identified multiple duplications of unc-29, a subunit of the levamisole-sensitive acetylcholine receptor in parasitic nematodes closely related to C. elegans. We show the parasite, Haemonchus contortus, meets the criteria for a good model of this process and demonstrate that new copies of unc-29 experience selective pressure to rapidly acquire new functional properties, leading to the possibility of new drug targets. An evolved change in compatibility between receptor subunits appears to play a major role in determining the changes in receptor composition.Parasitic nematodes pose a global threat to human health and severely impact livestock animals and crops. Most anthelmintic drugs paralyze worms, targeting pentameric neurotransmitter receptors in the neuromuscular synapse. A detailed understanding of this signaling allows the most effective use of existing drugs and the best opportunities for developing new treatments. The model nematode,  The ability to control movement based on a nervous system is unique to the animal kingdom and is a major target for anthelmintic drugs. The fundamental importance of neuronal signalling is revealed by the fact that members of all branches of the tree of life including archaea, bacteria, fungi, plants and animals produce toxins that specifically inhibit signalling causing pain, paralysis or death. A large pharmaceutical industry has risen around drugs targeting the nervous system to control pain, mood and behaviour in humans as well as to control insect pests that transmit disease and parasitic nematodes that damage crops and cause infection in livestock. A detailed understanding of how the nervous system is controlled is therefore essential for understanding animal behaviour and the search for new drugs to combat disease.Examination of the evolutionary history of genes associated with neuronal signaling suggests that extensive gene duplication, interspersed with periods of gene loss are a common feature . UnderstCaenorhabditis elegans has been examined in detail. The pentameric ligand-gated ion-channel family of neurotransmitter receptors mediate fast, synaptic signaling in C. elegans and in the great majority of animals. Five related, or identical, subunits combine into a pentamer with a large extracellular domain where the activating neurotransmitter binds at the interface between two subunits. The receptor is embedded in the post-synaptic membrane by four transmembrane (TM) regions in each subunit. The second TM regions combine to produce a barrier, gating the flow of either anions or cations. Three classes of pLGIC play a predominant role in the neuromuscular synapse of C. elegans: two stimulatory acetylcholine (ACh) receptors, one sensitive to nicotine (N-AChR), the other sensitive to levamisole (L-AChR), and one inhibitory GABA receptor (UNC-49) [C. elegans [C. elegans, requiring five different genes contributing subunits [Control of body muscle contraction in the nematode UNC-49) . The fun elegans  and orth9 . The fThe nematodes, along with insects, are members of an ancient group of organisms, the ecdysozoa, that are characterized by larval stages proceeding through a series of moults . AnalysiC. elegans led to a renewed interest in cognate receptors from the parasitic nematodes [Ascaris suum, Oesophagostomum dentatum and the sheep parasite, Haemonchus contortus. Whereas the L-AChR of C. elegans has a fixed composition, the parasitic nematodes have lost specific subunits and functional receptors, with different pharmacology, have been produced with different subunit combinations with each species [Cel-unc-29 L-AChR subunit gene are present as four copies in the trichostrongylid nematodes, Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis [unc-29 paralogs have appeared since divergence from the Rhabditid nematodes within clade V. Sequence divergence among the unc-29 paralogs reported in these parasitic species varies from 20–25% and is similar to divergence from C. elegans. This recent duplication event therefore represents an attractive system in which to investigate the basis for such gene duplication and functional divergence.Diversification of L-AChR subunits predates the common, ecdysozoan, ancestor, but duplication, divergence and loss of subunits has continued throughout the evolution of nematodes , 11. Recematodes . Functio species –15. Of sriformis . Importaunc-29 subunits within the clade V nematodes as a model system that will reveal the physiological conditions that increase the likelihood of gene duplication and functional divergence and provide insight into the mechanisms by which such functional adaptation occurs. We show that this system meets three necessary criteria for an appropriate model, that duplication of unc-29 is a common feature within the clade V nematodes, that the different copies of unc-29 have diverged in function and that their sequences are sufficiently similar for individual sites under selection during functional divergence to be identified.We propose recent diversification of unc-29 is a common, recurrent feature of the clade V parasitic nematodes and must therefore be taken into account to understand the action of levamisole (LEV) more generally. Functional divergence to produce receptors with novel pharmacology is rapid and driven by directional selection. A major feature of this process includes changes in subunit compatibility that may help to explain the differences reported for the L-AChRs from different parasitic nematodes.Using this system we have shown that duplication of C. elegans unc-29 gene in different nematode species representing clades III and V [unc-29 with almost complete coding sequence were identified in each clade III nematode species, Ascaris suum, Thelazia callipaeda, Brugia malayi, Loa loa, Dirofilaria immitis, Onchocerca volvulus, Litomosoides sigmodontis and Acanthocheilonema viteae and each clade V Rhabditoidea, Caenorhabditis elegans, C. japonica, C. briggsae, C. remanei and C. tropicalis, examined. In contrast, multiple copies of unc-29 were identified in every member of the Strongylida and Diplogasterida, including, Haemonchus contortus, Teladorsagia colubriformis, Trichostrongylus colubriformis, Dictyocaulus viviparus, Angiostrongylus costaricensis, Ancylostoma caninum, Ancylostoma ceylanicum, Nippostrongylus brasiliensis, Pristionchus pacificus and Pristionchus exspectatus. unc-29 sequences. The genomic organization and inferred events leading to the duplicate genes are shown in Diplogasterida and nine within the Strongylida  down to 76% (unc-29.2 and unc-29.3), in comparison to 74% comparing C. elegans unc-29 with unc-29.2.The clade III nematodes and the clade V achieved , 15 and Comparison of substitution rates using PAML 4.8 found that branch substitution rates were well below the level at which sites became saturated . A modelComparison of different nested substitution rate class models M0 (single rate), M3k2 (two rate), M3k3 (three rate) and MD (three rate branch-site) for each branch labelled (A-J) in Strongyloidea. Eight of these branches  consistent with relaxed, neutral evolution. For unc-29.2, a significant change was observed in the intermediate rate class, rising from ω = 0.030 to 0.227  to determine specific amino acid sites under positive selection for branches corresponding to the different duplication events in unc-29 from H. contortus as representatives of the wider phenomenon of unc-29 subunit duplication in the clade V nematodes. To evaluate functional divergence of Hco-unc-29 copies from C. elegans, we evaluated the ability of each to functionally replace the defective Cel-unc-29 and rescue sensitivity to LEV in the defining unc-29 mutant strain, CB1072 [myo-3 promoter that allows expression in body muscles where the native C. elegans L-AChR is present [We chose the four copies of unc-29(e1072) animals were barely affected by the drug over two hours of exposure  [Xenopus oocytes, sequentially replacing UNC-29.1 with each paralog in turn. For convenience we refer to these as L-AChR1.1, L-AChR1.2, L-AChR1.3 and L-AChR1.4, respectively. Reconstitution of the L-AChR1.1 as a reference, produced large inward currents in the μA range for both ACh and LEV at 100 μM, with LEV acting as a superagonist    in Xenopesponse) . The relectively , Table 2unc-29.2 cRNA leading to measurable current in combination with C. elegans subunits.Surprisingly, replacing UNC-29.1 by UNC-29.2 failed to produce any detectable current in response to either 100 μM perfusion of ACh or LEV . This wa50 of approximately 3.2 μM  and anthelmintics . In each case, the rank order for these drugs, normalized to the response to 100 μM ACh, was LEV > DMPP >>> Bephenium ~ Pirantel ~ Nicotine.  Table 3.We also tested activity of the cholinergic antagonists d-tubocurarine (dTC), dihydro-β-erythroidine (DHβE) and mecamylamine (MECA) on the reconstituted L-AChR1.1, L-AChR1.3 and L-AChR1.4. The application of 100 μM of the canonical antagonist dTC efficiently blocked the ACh responses , Table 3Xenopus oocytes in addition to the previously identified L-AChR1.1 Overall, each of the four paralogs of unc-29 displayed a unique functional characteristic, either in their (in)ability to reconstitute an ACh responsive channel, or in the agonist characteristics of LEV and response to the antagonist Meca.In summary, two novel receptors, L-AChR1.3 and L-AChR1.4, with high affinity to ACh and LEV, were reconstituted in in vitro and in vivo with C. elegans subunits, they may have the potential to co-assemble, or interfere with each other, if co-expressed in the same tissue in vivo in H. contortus. We raised antibodies against peptides, specific for either UNC-29.1 or UNC-29.2, in the highly variable intracellular loop between TM3 and TM4. Specificity and cross-reactivity of the antibodies was verified by an ELISA assay for binding to the antigen peptides and to native protein expressed in Xenopus oocytes. Antibodies against both UNC-29.1 and UNC-29.2 were specific and showed fluorescence at the oocyte surface only with their cognate receptor  concentration of cRNA for UNC-29.2 relative to UNC-29.1. The presence of unc-29.2 cRNA had no effect on the pharmacological response to either ACh or LEV nor in the observed current  that mediate fast synaptic neurotransmitter signalling [C. elegans model has proved particularly effective in the identification of specific anthelmintic drug targets in the parasitic nematodes, including the pLGICs mediating sensitivity to ivermectin, nicotine, levamisole and monepantel [C. elegans [The free living nematode dination . One feagnalling , 36. Expgnalling , 37–39. nepantel , 40–42. nepantel , 43–46.  elegans . UnderstC. elegans, the stimulatory N-AChR and L-AChR, specifically activated by nicotine and levamisole respectively, and the inhibitory GABA gated chloride channel, UNC-49 [unc-29 gene occurring since the divergence of the trichostrongylid parasitic nematodes from the common ancestor with C. elegans [unc-29 gene within the Trichostrongyloidea meets these three criteria.Three major classes of pLGIC are found within the same neuromuscular synapse of , UNC-49 . These t, UNC-49 , 31, 48  elegans . This suStrongylida from the Caenorhabditis group. It is not yet possible to place a date on this event with any confidence, but Cutter estimates certainly more than 30 MYr based on analysis of neutral substitution rates [Strongylida and the most recent occurring within N. brasiliensis after divergence from its neighbouring species. The four copies of unc-29 within the Trichostrongylidae can therefore be considered as representative of the phenomenon in general.The phylogeny in on rates . The firunc-29 and within the clade V nematodes found up to 26% of amino acid positions were not conserved, with most substitutions occurring within the intracellular loop between TM3 and TM4 of the receptor subunit. The level of nucleotide substitution among copies of unc-29 is well below the level required to reach saturation, suggesting that it will be possible to identify specific sequence changes that correlate with any change in function [Overall protein identity among copies of function . RelativCaenorhabditis group unc-29 and the duplicate copies found within the Strongylida share a common origin within the clade V nematodes. A first step in determining if there has been any functional divergence among unc-29 copies is to determine if these are functionally equivalent. Decreased sensitivity to LEV in C. elegans carrying the definitive unc-29 mutant (e1072) can be rescued by transgenic reintroduction of the Cel-unc-29 gene [unc-29 from H. contortus , T. callipaeda (Tzc), B. malayi (Bma), L. loa (Llo), D. immitis (Dim), O. volvulus (Ovo), L. sigmodontis (Lsi), A. viteae (Avi) and P. pacificus (Ppa), P. exspectatus (Pex), C. tropicalis (Ctr), C. japonica (Cjp), C. remanei (Cre), C. brigsgae (Cbr), C. elegans (Cel), H. contortus (Hco), T. colubriformis (Tco), T. circumcincta (Tci), A. costaricensis (Acs), D. viviparus (Dvi), O. dentatum (Ode), A. caninum (Aca), A. ceylanicum (Ace) and N. brasiliensis (Nbr). Sequences were aligned as codons using the MAFFT plugin (v1.3.3) of Geneious  [Coding sequence orthologous to ematodes , 8. Thesers Ltd) . Regionsers Ltd) .Relative substitutions rates, branch lengths and transition/transversion ratios were estimated using codeml (PAML v4.8) based on the unrooted tree shown in The remaining substitution rate analysis was carried out using HyPhy v2.2.6, with the same alignment and phylogenetic tree as above . The besXenopus laevis) and sheep were maintained under Animal Use Protocols #AUP5284 and #AUP3845, respectively, approved by the Macdonald Campus Facility Animal Care Committee of McGill University. These protocols adhered to the standards set out by the Canadian Council on Animal Care in Science. Xenopus laevis oocytes were surgically removed from adult female frogs by certified personnel following guidelines laid out in the AUP. Sheep were infected with approximately 5,000 infective L3 larvae by oral gavage and adult parasites collected at necropsy.Frogs , Cel-lev-8 (NP_509932), Cel-unc-29 (NP_492399), Cel-unc-38 (NP_491472) and Cel-unc-63 (NP_491533); Hco-unc-29.1 (GU060980), Hco-unc-29.2 (GU060981), Hco-unc-29.3 (GU060982), Hco-unc-29.4 (GU060983), Hco-unc-38 (GU060984), Hco-unc-63a (GU060985), Hco-acr-8 (EU006785), Hco-unc-50 (HQ116822), Hco-unc-74 (HQ116821), Hco-ric-3.1 (HQ116823).cDNA clones used in this study correspond to sequences, Acetylcholine chloride (ACh), BAPTA-AM, bephenium hydroxynaphtoate, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), mecamylamine hydrochloride, morantel citrate, (-)-tetramisole hydrochloride (levamisole), (-)-nicotine hydrogen tartrate, pyrantel citrate, (+)-tubocurarine chloride hydrate (dTC) were purchased from Sigma-Aldrich. Dihydro-berythroidine hydrobromide (DHβE) was obtained from Tocris Bioscience.H. contortus cDNAs encoding AChR subunits and ancillary proteins were available as expression clones in the pTB207 vector and C. elegans expression clones were a gift from T. Boulin. [NheI-linearized templates and was performed with the mMessage mMachine T7 Kit (Ambion) according to the manufacturer’s instructions. cRNAs were precipitated with lithium chloride and redissolved in RNAse-free water and stored at -80°C. cRNA concentration was determined using a Nanodrop spectrophotometer (Thermo Scientific) and cRNA purity and integrity was assessed by electrophoresis through a 1% gel stained with GreenGlo nucleic acid dye (Denville Scientific Inc.).The  Boulin. , 14. cRNXenopus females according to standard procedures [2 1.8 mM, MgCl2 1 mM, and HEPES 5 mM, pH 7.3 supplemented with sodium pyruvate 2.5 mM) [Oocytes were collected from mature ocedures  and incu 2.5 mM) . Defolli 2.5 mM) .2, 5 mM HEPES, pH 7.3). All measurements were performed using a Geneclamp 500B amplifier and Digidata 1322M (Axon Instruments), oocytes were voltage-clamped at -60 mV. Drug solutions were applied to oocytes by gravity flow using perfusion syringes directly connected to the chamber. Data were collected and analyzed using Clampex 9.2 and Clampfit 9.2 . Traces were filtered at 30 Hz and analyzed using GraphPad Prism 5.0 software . Final results are presented as means ± SE.Two-electrode voltage-clamp experiments were carried out using a perfusion system connected to a flat RC-1Z chamber (Harvard Apparatus). Glass electrodes were filled with 3M KCl and checked for resistance between 1 and 3 MΩ. The oocyte bath was connected to the ground through a 3 M KCl agar bridge. Agonist and antagonists were dissolved or diluted in recording solution  as previously described .Cel-unc-29, unc-29.1, unc-29.2, unc-29.3, unc-29.4 cDNA sequences were amplified by PCR using primers containing restriction site inserted with restriction enzymes in the plasmid backbone (Addgene L2534) containing the myo-3 promoter. Strains used in these study were the wild-type Bristol isolate N2 and the LEV resistant isolate CB1072: unc-29 (e1072). Worms were grown on Escherichia coli OP50 seeded NGM (Nematode Growth Medium) plates at 20°C and maintained using standard culture methods [C. elegans were transformed by microinjection of plasmids into the gonads [Hco-unc-29 cDNA sequences driven by the myo-3 promoter and the ttx-3::GFP-expressing plasmid [The e gonads . Transfo plasmid  at identunc-29 (e1072) mutant strain and the transformed lines were done using two-way ANOVA with Bonferroni’s multiple comparison post test. A level of p < 0.05 was considered significant.Three day old adult fluorescent worms were transferred onto unseeded NGM plates, equilibrated at 20°C, containing 200 μM LEV. Drug induced paralysis was determined by visual inspection every 15 min. Worms were defined as paralyzed if no movement was observed within five seconds after prodding the tail with a hair tip . StatistPeptide-derived polyclonal antibodies were generated in rabbits against subunits UNC-29.1 and UNC-29.2, in rats . Animals were immunized with a mixture of two specific peptides per subunit corresponding to parts of the intracellular loop located between the third and the fourth TM domains. For UNC-29.1, the synthetic peptides were "EKTASPKTLNCAMELTTRDPQL" and "PIPEPAPPEITQLP" The UNC-29.2 peptides corresponded to "VSIKVLNCGDDIPKDP" and "MTKKKRGATVAKLP" sequences. Purified antibodies were tested for specificity and titrated against both peptides by ELISA.Oocytes were collected, treated and injected as previously described for receptor reconstitution. Approximately 12 oocytes were injected per subunit mix. After 5 days, TEVC was performed to verify AChR expression with exposure to 100 μM ACh. Oocytes were then incubated overnight in 1X PBS with 4% paraformaldehyde (PFA) at 4°C. After being washed 3 times, 5 min each in 1X PBS, oocytes were then blocked overnight in an antibody diluent (AbD) containing 1X PBS, 0.2% gelatin from cold water fish skin, 2.5% Triton X-100 and 0.1% sodium azide. Primary antibodies were diluted at 1:100 in AbD and applied on oocytes overnight at 4°C. The eggs were washed 3 to 4 times in AbD and incubated with the corresponding 1:500 secondary antibodies conjugated to Alexa Fluor 488  overnight at 4°C. Final washes were performed 2 times with AbD and 2 times with 1X PBS/0.1% (v/v) Triton X-100. The membrane of the oocytes was observed using a Zeiss LSM710 confocal microscope  equipped with the Zeiss Zen 2010 software. Negative controls were either water-injected oocytes or AChR-expressing oocytes but omitting the primary antibodies.H. contortus were extracted and fixed overnight in PBS 1X supplemented with 4% PFA at 4°C. The worms were washed 3 times in PBS 1X and incubated overnight at 4°C in PBS 1X with 30% sucrose under gentle rocking. The nematodes were rapidly frozen in dry ice after embedding in an optimal cutting temperature compound (OCT) . Cryosections were performed transversally using a Thermo Shandon cryotome . 10 μm slices were placed on microscope slides and blocked overnight in AbD. Worms sections were then independently incubated overnight at 4°C with a 1/100 dilution of each specific anti-Hco-UNC-29 antibody. After washing a minimum of five times with AbD at 4°C, the sections were incubated overnight with the corresponding 1:500 secondary antibodies conjugated to Alexa Fluor 488 . Final washes were performed two times in AbD and 2 times in 1X PBS/0.1% (v/v). Slides were then mounted using a Mounting medium (Sigma) and examined with the same confocal microscope.Male and female PF23 The omission of the primary antibodies as well as the use of peptide adsorbed primary antibodies were used as controls. In the latter case, primary antibody were pre-adsorbed with 0.25 mg/ml of the corresponding peptide antigen.S1 FigXenopus oocytes injected with Hco-unc-63, Hco-unc-38, Hco-acr-8, Hco-unc-29.2 and Hco-ric-3.1, Hco-unc-74, Hco-unc-50 cRNAs. Representative recording trace from a single oocyte perfused with 100μM of the following cholinergic agonists: acetylcholine (ACh), Dimethylpiperazinium (DMPP), Pyrantel (PYR), Nicotine (NIC), Bephenium (BEPH) and Levamisole (LEV).TEVC experiments were performed in (TIF)Click here for additional data file.S2 FigCel-unc-63, Cel-unc-38, Cel-lev-8, Cel-lev-1 cRNAs and co-expressed with H. contortus accessory proteins, ric-3, unc-50 and unc-74. The addition of cRNAs encoding Cel-unc-29, unc-29.1, unc-29.2, unc-29.3 and unc-29.4 were tested independently. Current (nA) evoked with 100 μM ACh recorded from the different subunit combinations is indicated above each column. Error bars indicate SD.TEVC experiments were performed on oocytes injected with (TIF)Click here for additional data file.S3 FigAll drugs were applied at a concentration of 100μM. All values are normalized to the current evoked by perfusion of 100μM ACh. Statistics. Experimental data are shown as mean ± SE. Statistical comparisons were done using one-way ANOVA with Bonferroni’s multiple comparison post test. Asterisks, ***, indicate a significant difference between receptors (p<0.001).(TIF)Click here for additional data file.S4 FigXenopus oocytes injected with Hco-unc-63, Hco-unc-38, Hco-acr-8, Hco-unc-29.1 and Hco-ric-3.1, Hco-unc-74, Hco-unc-50 cRNAs. Hco-unc-29.2 cRNA was co-injected at 0.2, 1 or 5 times the concentration of unc-29.1. Dose-response curves are shown for each ratio for ACh (red) and LEV (blue). All responses are normalized to 100μM ACh, which corresponds to the saturating dose. The ACh and LEV 50% effective concentration (EC50) values as well as Hill coefficients are indicated in TEVC experiments were performed on (TIF)Click here for additional data file.S5 FigXenopus oocytes were injected with the L-AChR1.1 and L-AChR1.2 corresponding cRNA mixtures. After 5 days, oocytes were checked for expression using the TEVC technique (when applicable), fixed and incubated with affinity-purified antibodies raised against Hco-UNC-29.1 and Hco-UNC-29.2 specific peptides. Localization of subunits was performed using Alexa 488-labeled secondary antibodies (green). Controls were un-injected oocytes incubated in anti-UNC-29.1 antibodies and L-AChR1-expressing oocytes incubated with the secondary antibodies only. Confocal microscopy was performed on the whole oocytes. All slides were observed under 20x magnification. Scale bars correspond to 100 μm.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Fileunc-29 sequences.gb:Zip archive containing:  Nucleotide sequences of unc-29 used to prepare paml unc-29 phylogeny.newick: Newick format file used for the substitution rate analysis. paml unc-29 alignment.phy: Phylip format file of the sequence alignment used for the substitution rate analysis.(ZIP)Click here for additional data file.


Myc)-mediated hypermetabolic analytical model using cultured cardiomyocytes. Nascent RNA labeling combined with single cell imaging demonstrated that Myc protein significantly increased the amount of global RNA production per cardiomyocyte. Chromatin immunoprecipitation with high-throughput sequencing clarified that overexpressed Myc bound to a specific set of genes and recruits RNA polymerase II. Among these genes, we identified Btg2 as a novel target of Myc. Btg2 overexpression significantly reduced cardiomyocyte surface area. Conversely, shRNA-mediated knockdown of Btg2 accelerated adrenergic stimulus-induced hypertrophy. Using mass spectrometry analysis, we determined that Btg2 binds a series of proteins that comprise mRNA deadenylation complexes. Intriguingly, Btg2 specifically suppresses cytosolic, but not nuclear, RNA levels. Btg2 knockdown further enhances cytosolic RNA accumulation in cardiomyocytes under adrenergic stimulation, suggesting that Btg2 negatively regulates reactive hypertrophy by negatively regulating RNA accumulation. Our findings provide insight into the functional significance of the mechanisms regulating RNA levels in cardiomyocytes.Under hypertrophic stimulation, cardiomyocytes enter a hypermetabolic state and accelerate biomass accumulation. Although the molecular pathways that regulate protein levels are well-studied, the functional implications of RNA accumulation and its regulatory mechanisms in cardiomyocytes remain elusive. Here, we have elucidated the quantitative kinetics of RNA in cardiomyocytes through single cell imaging and c-Myc ( Cardiac hypertrophy is initially an adaptive response to exogenous stimuli caused by either high blood pressure or neurohumoral factors, and is recognized as the causative mechanism during the progression of heart failure. To adapt to the increased metabolic demand when they are subject to exogenous hypertrophic stimuli, cardiomyocytes accelerate RNA synthesis to promote translation and increase cellular protein synthesis1235Myc) is a major oncogene and is also a well-defined pro-hypertrophic transcription factor in cardiomyocytes789912131112131517c-Myc , we determined Myc-regulated genes in cardiomyocytes and identified Btg2 as a novel Myc target gene. Based on biochemical and functional analysis using single cell imaging, we found that Btg2 negatively regulates cardiomyocyte hypertrophy and decreases cytosolic RNA levels by interacting with the Ccr4-Not deadenylation complex.A chemical method to detect RNA synthesis based on the biosynthetic incorporation of the uridine analog into newly transcribed RNA enables the quantitative evaluation of transcription rates in cultured cellsMyc (Ad-Myc) into a primary culture of rat neonatal cardiomyocytes using adenovirus to generate cellular conditions with increased amounts of Myc. We used a rabbit monoclonal antibody against Myc that specifically detected both endogenous and overexpressed Myc protein , indicating that the amplification of active gene expression by Myc15Gata4, Gata6, Tbx20, Nkx2-5), structural genes , or heart failure related genes , but did not induce Pol II recruitment  in cardiomyocytes transduced with Myc both under serum-supplemented conditions and serum-free conditions. As shown in Nppa, Nppb, or Acta1 mRNA, suggesting that Myc alone is not sufficient to upregulate expression of these genes. These data are consistent with a previous finding that overexpressed Myc invades accessible chromatin in lymphocytes27The transcribed RNA labeled by EU includes both protein-coding mRNAs transcribed by RNA polymerase II (Pol II) and non-coding RNAs transcribed by either RNA polymerase I or III control . For oth control . To evalruitment . We furtBtg2, also known as Tis21 and Pc3) as ranking second in Pol II enrichment in both experimental replicates. Myc and Pol II bindings at the Btg2 locus were confirmed by quantitative PCR using specific primer sets  that induced significant cardiac hypertrophy  and transduced it in cardiomyocytes. Overexpressed Btg2 was diffusely distributed mainly in the cytosol and partially in the nucleus in cardiomyocyte  that are transcriptionally upregulated under adrenergic stimulation or pressure-overloaded heart failure. These data suggest that Myc binding is not sufficient to promote transcription of hypertrophy-related genes, although Myc is required to activate these genes15In this study, we used single cell quantitative imaging combined with nascent RNA labeling to elucidate the functional significance of the mechanisms that regulate RNA levels in cardiomyocytes. In our analytical model, Myc promoted nuclear RNA synthesis in cardiomyocytes in a dose-dependent fashion, thereby amplifying transcription. From the genome-wide binding profiles for Myc and Pol II using ChIP-seq, we determined that Myc binds preferentially to and recruits Pol II to a specific set of genes that includes genes involved in ribosomal and RNA biogenesis. This finding suggests that even in primary cardiomyocytes, Myc targets genes involved in biomass accumulation, which has been reported as a primordial function of Myc in tumor cellsBtg2 as a novel Myc target gene. Immediate early genes with extremely short half-lives like Btg2 may be difficult to detect by expression profiling because mRNA expression levels depend on the amount of mRNA at the time analyzed. In contrast, Pol II accumulation at a specific gene locus might indicate the transcriptional signature including the past time. Therefore, molecular screening using quantified Pol II signatures is useful to identify downstream targets with very short half-lives regulated by specific transcription factors. These data are consistent with previous findings that Pol II occupancy detected by ChIP-seq provides a precise readout for transcription and is useful for detecting genes that have high mRNA turnover ratesBtg2 is rapidly induced by pro-hypertrophic stimuli in cardiomyocytes, its physiological role appears to be that of a negative regulator that opposes anabolism. It has not been shown conclusively that mRNA deadenylation is the only downstream functional pathway of Btg2 in cardiomyocytes since the previously determined molecular function of Btg2 and interacting effectors are wide-ranging, and EU-mediated RNA labeling could not distinguish mRNA from other non-coding RNAs. Nevertheless, the results of MS spectrometry analysis, regional quantitation of RNA amount in cardiomyocytes, and the specific suppression of cytosolic RNA abundance by Btg2 strongly support the functional relationship between Btg2 and the Ccr4–Not complex–mediated deadenylation system in cardiomyocytes.RNA pol II dynamics, distribution and regulation in cardiac myocyte hypertrophy have been reported5354Drosophila and in mouseBtg2 must be strictly regulated as an immediate early and transiently expressed gene because of its strong anti-anabolic effect. Although mRNA deadenylation is a global mechanism, searching for the cardiomyocyte-specific target of Btg2 is an attractive goal considering the role of RNA metabolism during the progression of pathological hypertrophy under clinical settings.Movement towards the anabolic state is an adaptive mechanism under stress conditions accompanied by increased levels of proteins and nucleic acids. The regulatory mechanisms that maintain the appropriate amount of these biomasses are critical components for cardiomyocyte function. Depleting the Ccr4–Not complex leads to heart failure with reduced systolic function in Phenylephrine (SIGMA), MG132 (SIGMA). Sarcomeric Alpha Actinin , Cardiac Troponin I (Abcam), Gapdh , Myc , RNA polymerase II , Btg2 , Cnot7 , α-Tubulin (MBL), anti-FLAG , and Alexa 488-and Alexa 568-labeled secondary antibodies (Invitrogen).Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health  and were approved by the Osaka University Committee for Laboratory Animal Use.Neonatal rat cardiomyocytes were prepared as previously describedhttp://david.abcc.ncifcrf.gov/)The gene lists with significant changes were subjected to gene ontology term analysis and functional enrichment clustering using the DAVID software  and converted to cDNA using high capacity RNA-to cDNA RT kit (Life technologies). Quantitative real-time PCR was performed with SYBR® green reagent or TaqMan® probe using StepOnePlus Real-Time PCR Systems (Applied Biosystems). All of the samples were processed in duplicate. The level of each transcript was quantified by the threshold cycle (Ct) method using Gapdh as an internal control.C57BL/6 mice aged 8 weeks and weighing 20–25 g were subjected to pressure overload, as described previously® Protein G (Life technology) were washed with PBS and incubated with 150 mM 1xRIPA buffer  including 2 μg of the monoclonal anti-Myc antibody or monoclonal anti-RNA pol II antibody at 4 °C for 1 h. The crosslinked cell pellet was lysed with NP-40 buffer  and centrifuged. The cell pellet as nuclear fraction was lysed with SDS Lysis buffer , and sonicated by Bioruptor TOS-UCW-310-EX . After centrifuge, the supernatant was collected as soluble chromatin fraction. The chromatin fraction was diluted two fold in ChIP dilution buffer  and the 1.7% of the solution was stored as input control. The chromatin fraction was incubated with the antibody-conjugated beads at 4 °C with rotation overnight. The incubated beads were wash once with 150 mM 1xRIPA buffer, 500 mM 1xRIPA buffer, then two times with 1xTE buffer. The bound fraction and input were incubated with ChIP elution buffer  and at 65 °C for 2 h. The eluted samples were incubated with RNase at 30 °C for 30 min, then incubated with Proteinase K at 55 °C for 1 h. The ChIP DNA and input were purified using QIAquick PCR purification Kit (QIAGEN) according to the manufacturer’s instructions and the DNA amount was quantified using Qubit® 2.0 fluorometer with Qubit® dsDNA HS Assay Kit (Life Technology). Each ChIP DNA and input samples were sheared by sonication, end-repaired, ligated to the sequencing adapters and amplified. The size distribution of library DNA was checked by Agilent 2100 Bioanalyzer, and ChIP efficiency was analyzed by quantitative PCR in each purification step. Sequencing of ChIP-Seq libraries were performed on a SOLiD instrument with 50-bp reads according to manufacturer’s instructions (Life Technologies). All experiments were performed on full sequencing slides with barcodes. Libraries were quantified by SYBR green quantitative-PCR (qPCR) to determine appropriate mixing ratios, which also depended on the desired sequencing depth for each of the libraries in the mixture. We repeated the two independent experiments for reproducibility.In ChIP experiments, isolated neonatal cardiomyocytes were cultured in medium containing 10 μM BrdU to inhibit proliferation of cardiac fibroblasts. Twenty-four hours after transduction of Ad-Myc or Ad-LacZ, cultured rat neonatal cardiomyocytes were washed by PBS, then cross-linked for 10 min at room temperature by addition of 16% paraformaldehyde to PBS at a final concentration of 0.5%. The crosslinking reaction was stopped by adding glycine to a final concentration of 175 mM. The fixed cells were washed with cold PBS on ice two times, harvested with cold PBS, then centrifuged. The cell pellet was flash frozen in liquid nitrogen and stored at −80 °C until following experiments. Forty μl DynabeadsUnfiltered sequence reads were aligned to the rat reference genome (rn4) using Bowtie6 cells in 10 mm culture dishes and transduced with Ad-Btg2 or Ad-LacZ as a control. Forty-eight h after transduction, the cells were lysed with the lysis buffer (50 mM Tris–HCl (pH 7.4), 100 mM NaCl, 0.1 mM EDTA, 1% CHAPS, and protease inhibitor mixture ). The lysates were immunoprecipitated with anti-FLAG M2-agarose (Sigma-Aldrich) at 4 °C for 1 h. After extensive washing, the proteins were eluted with 250 μg/mL FLAG peptide at 4 °C for 30 min. The eluate was subjected to shotgun mass spectrometry analysis. The same eluate was electrophoresed on 4–12% NuPAGE Bis-Tris gel and silver-stained. Gel pieces, including specific bands from the silver-stained gels, were excised, digested with trypsin, and analyzed by mass spectrometry.Cardiomyocytes were seeded at 1.5 × 104 cells/well) were seeded on Greiner CELLSTAR® 96 well plates which were previously coated with collagen (Celmatrix Type I-C (KURABO)), and incubated in DMEM supplemented with 10% FBS. 1 mM EU was added to the complete culture medium from a 100 mM stock in DMSO. After EU labeling for the indicated time, cells were washed with PBS and fixed. The Labeled EU of the cells were visualized by Click-iT RNA imaging kits (Life Technology) according to the manufacturer’s instructions. The samples were incubated with the working solution of Click-iT reaction cocktail, containing the Alexa Fluor 594 azide and CuSO4, for 30 min at room temperature. We choose 594 probe to completely distinguish the fluorescent images of the labeled RNA from nuclear fluorescence stained by Hoechst. After removal of the reaction cocktail, cells were washed with Click-iT reaction rinse buffer and PBS, then transferred to immunostaining procedures. After incubation with blocking buffer (1% BSA in PBS), the cells were immunostained with anti-cardiac Troponin I polyclonal antibody for 1 h at room temperature. After washing with PBS, the cells were incubated with a goat anti–rabbit IgG secondary antibody, conjugated with Alexa Fluor 488 (Invitrogen) diluted by blocking buffer containing Hoechst. The immunofluorescent images were obtained using IN Cell Analyzer 6000. A total of 64 nonoverlap images (16 images per well) were obtained from each sample in one experiment using a 20 ×/0.45NA Nikon lens. The average data were obtained from at least three biological replicates. All the images are shown in figures after color conversion between green (488) and red (594). The obtained images were analyzed using IN Cell Developer toolbox  with the algorism described in Cardiomyocytes .