The starting material for RNA sequencing (RNA-seq) studies is usually total RNA or polyA+ RNA. Both forms of RNA represent heterogeneous pools of RNA molecules at different levels of maturation and processing. Such heterogeneity, in addition to the biases associated with polyA+ purification steps, may influence the analysis, sensitivity and the interpretation of RNA-seq data. We hypothesize that subcellular fractions of RNA may provide a more accurate picture of gene expression.de novo assembly of RNA-seq data. Conversely, the nuclear fraction shows an enrichment of unprocessed RNA compared with total RNA-seq, making it suitable for analysis of nascent transcripts and RNA processing dynamics.We present results for sequencing of cytoplasmic and nuclear RNA after cellular fractionation of tissue samples. In comparison with conventional polyA+ RNA, the cytoplasmic RNA contains a significantly higher fraction of exonic sequence, providing increased sensitivity in expression analysis and splice junction detection, and in improved Our results show that cellular fractionation is a more rapid and cost effective approach than conventional polyA+ enrichment when studying mature RNAs. Thus, RNA-seq of separated cytosolic and nuclear RNA can significantly improve the analysis of complex transcriptomes from mammalian tissues. The transcriptome is the complete catalogue of transcripts in the human cell. At any given time, a wide range of different RNA molecules are present at different levels of maturation and processing. The rates and the dynamics of RNA transcription and processing are unique for each cell. Previous studies have highlighted the importance of creating a complete map of transcripts, and the importance of understanding how different physiological conditions, developmental stages and disease can affect expression and regulation ,2.In the recent years, RNA-sequencing (RNA-seq) has emerged as a standard procedure to study transcriptomes and measure levels of gene expression -6. StudiThe starting material for RNA-seq is typically total RNA or polyadenylated (polyA+) RNA, which both represent heterogeneous pools of RNA molecules at different stages of maturation and processing . SeveralAlthough total RNA-seq has been shown to provide insight into ongoing transcription and co-transcriptional splicing in the nucleus ,20, the Recent improvements in RNA extraction protocols now make it possible to study specific pools of RNA molecules, either by fractionation of subcellular compartments or by molecular capture of specific RNA-associated targets. Several kits for RNA extraction from different cellular fractions are commercially available . However, these kits are associated with significant amounts of cross contamination between the fractions. To overcome the effects of cross contamination, recent studies used the selection of polyA+ from the cytoplasmic fraction and chromatin-associated transcripts from the nuclear fraction to obtain more homogenous pools of mature and nascent transcripts respectively ,23. AlthIn this study, we investigate the benefits of analyzing RNA sequencing data from separated nuclear and cytosolic RNA. We have made improvements to an existing protocol for cell fractionation in order to more efficiently fractionate cytoplasmic and nuclear RNAs from tissue samples. We find that extraction of RNA using our modified protocol results in pure subcellular RNA populations with minimal levels of cross contamination. RNA-seq results from nuclear and cytosolic fractions are compared to polyA+ and total RNA-seq from the same tissue samples. Our results highlight significant advantages of performing RNA-seq on cytosol and nuclear RNAs, as compared to standard RNA-seq protocols. Sequencing of nuclear RNA provides insight into nascent transcript formation and processing, and cytosolic RNA-seq leads to improved de novo assembly and splice junction detection.To improve the efficiency of RNA extraction from different subcellular fractions, the cytoplasmic and nuclear RNA purification kit (Norgen) was modified with the addition of a sucrose gradient and extra washing step (see Methods for more details). The method for cellular fractionation of RNA is outlined in Figure To investigate the separation and detection of mature transcripts from the cytoplasm and nascent transcripts from the nucleus, we purified cytoplasmic and nuclear RNA from two human fetal frontal cortex tissue samples, denoted Sample 1 and Sample 2. We then sequenced (SOLiD5500) the cytoplasmic and nuclear RNA from both samples, along with total and polyA+ RNA from the same tissues. While many genes have very clean peaks corresponding to the exons . The EI-ratio is a number ranging from 1  to 0 . In the cytoplasmic RNA, the EI-ratios were 0.74 and 0.72 in the two tissues. For polyA+ RNA the EI-ratio ranged from 0.27-0.45 and even lower values were seen in total RNA (0.20-0.42) and nuclear RNA (0.12-0.31)  as a measure to quantify the expression levels of all exons in the human genome. As expected, cytoplasmic RNA-seq gives the highest dcpm levels for exons , type of RNA or association with certain subcellular compartments or structures. Examples include polyA+ selection, extraction of chromatin associated RNA and extraction of cytosolic RNA ,23,31 anIn addition to the improved efficiency of this method to enrich for mature RNA transcripts, cytoplasmic RNA purification provides several technical advantages over conventional polyA+ enrichment approaches. The enrichment of polyA+ RNA directly from tissues and cell lines is a complicated and time-consuming procedure, requiring significant amounts of starting material. Cytoplasmic RNA purification represents a more affordable and rapid protocol (1.5 hours). Moreover, it requires only 15 mg of tissue to provide sufficient amounts of RNA from both cytosol and nuclear fractions for RNA-seq and subsequent validation experiments. Our approach has lower resolution for specific sub-classes of RNA compared to more specialized molecular approaches such as GRO-seq and NET-seq, but has the benefit of providing a global picture of all RNA in the cell, with increased resolution compared with conventional extraction protocols.There are several potential biases when using polyA+ RNA, which may be avoided using the cytosolic fraction. We find a surprisingly high fraction of intronic sequence reads in the polyA+ RNA-Seq data. Although the results were better for the polyA+ RNA purchased from a commercial vendor, where a more stringent selection protocol was applied, we still find that > 50% of the intragenic reads map to introns. These findings indicate that there may be significant unspecific capture of RNA in the polyA+ selection step, irrespective of the protocol used. Another explanation for the high intronic background in polyA+ data is that some fraction of introns (or transcripts) is spliced only after the addition of the polyA-tail . Such a Compared to cytosolic RNA, the nuclear fraction is less well suited for measuring mRNA expression levels. Instead, the enrichment of nascent RNA from the nuclear fraction is suitable for studying transcription dynamics without any further rRNA depletion steps. We show that signatures of co-transcriptional splicing are more distinct in the nuclear fraction than in total RNA. The level of intron coverage is also a good indicator of the rate of nascent transcript production, which does not always correlate with the level of processed mRNA. Additionally, nuclear extraction is advantageous for studies of RNA molecules that are primarily present in the nucleus, such as pri-microRNA, rRNA precursors and some long non-coding RNAs -36.By extracting both cytosolic and nuclear RNA fractions from the same tissue, it is also possible to study the relative abundance of the same transcripts in each fraction. Such analysis may provide insight into transcript processing, turnover and degradation. For example, a high level of nascent transcription for a transcript found at relatively lower levels in the cytosol might be an indication of rapid cytosol turnover. Conversely, high levels a transcript in the cytosolic RNA, combined with low nuclear levels for the same transcript may be an indication of high stability and long half-life for the mRNA of that transcript.de novo transcriptome assembly and tissue specific expression profiling.In this paper, we report on the advantages of using RNA-seq on separated cytosolic and nuclear RNA, extracted using a modified and improved protocol. Analysis on separated nuclear and cytoplasmic fractions is valuable to study RNA degradation patterns (degradome) and transport dynamics, intron retention patterns and mRNA turnover. Our results show that extraction of nuclear RNA is better than total RNA for measuring of nascent transcript levels and for studies of mechanisms of splicing. Furthermore, we show that RNA-sequencing of the cytoplasmic fraction shows an increased exonic coverage and minimal levels of intronic reads. This results in significantly higher number of transcripts that can be assembled from this fraction when compared to total or polyA+ preparations. Our data shows that sequencing of cytosolic RNA yields substantially lower background from immature transcripts, and we propose that cytosolic RNA-seq should be the method of choice for Tissue and total RNA samples from two fetal frontal cortex tissues, 23/24 weeks female (Sample 1) and 38 weeks male (Sample 2) were purchased from Capital Biosciences. The commercial adult frontal lobe polyA+ RNA sample with two rounds of polyA+ selection was acquired from Clontech .Cytoplasmic and nuclear RNA was purified from two fetal frontal cortex using Cytoplasmic and nuclear RNA purification kit (Norgen) with modifications as illustrated in Figure The supernatant containing the cytoplasmic fraction and the pellet containing the nuclei were mixed with 400 μl 1.6 M sucrose solution and carefully layered on the top of two 500 μl sucrose solution in two separate tubes. Both fractions were the centrifuged on 13,000-RPM for 15 minutes (4C°). The cytoplasmic fraction was collected form the top of the sucrose cushion and the cytoplasmic RNA was then further purified according to Norgen kit recommendations. The nuclear pellet was collected from the bottom of the tube and washed with 200 μl 1× PBS. The nuclei were collected after another centrifugation at 13,000 RPM for 3 minutes. The nuclear RNA was purified from the nuclear fraction according to the Norgen kit recommendations.polyA+ RNA from sample 1 and 2 was purified from 1 μg total RNA using MicroPoly(A)Purist kit (Ambion) according to the manufacturer’s instructions.Starting with 1 μg of cytoplasmic or nuclear RNA, cDNA was synthesized using the Maxima first strand cDNA synthesis kit (Fermentas) according to the manufacturer’s recommendations. 1 μl of the resulting cDNA was used for qrtPCR to measure the relative intronic and exonic expression in each fraction. The qrtPCR was performed with Stratagene Mx3000P in 96-well plates. The reactions were carried out with an initial denaturation at 95°C for 10 min followed by 40 cycles of denaturation at 95°C for 15 s, primer annealing at 60°C for 30 s and extension at 72°C for 30 s.The qrtPCR contained 12.5 ng single stranded cDNA, 0.4 μM for each primer and 12.5 μl Maxima SYBR Green/ROX qPCR Master Mix (Fermentas) in 25 μl reactions. All samples were amplified in triplicate and the mean values were used to calculate the expression level of each target. The intronic/exonic expression level ratios were determined by calculating the differences between the CT values (ΔCT) for exonic and intronic expression for each gene in each fraction. Raw data were analyzed using MxPro Mx3000P software (Stratagene).The quality of the input RNA was controlled using a RNA 6000 Pico chip on a Bioanalyzer (Agilent Technologies) and only RIN-values above 7 were accepted. Removal of rRNA was performed using the RiboMinus Eukaryote Kit (Life Technologies) according to manufacturer’s protocols. The samples were then fragmented using RNaseIII for 7 min. RNA libraries were constructed using the AB Library Builder Whole Transcriptome Core Kit (Life Technologies) and amplified (12 cycles). Emulsion PCR was performed using the EZ Bead System (Life Technologies).polyA+ and nuclear RNA samples were checked for rRNA contamination using a RNA 6000 Pico chip on a Bioanalyzer (Agilent Technologies). The samples were then fragmented using RNaseIII for 5 min. RNA libraries were constructed using the AB Library Builder Whole Transcriptome Core Kit (Life Technologies) and amplified 15 cycles. Emulsion PCR was performed using the EZ Bead System (Life Technologies).de novo transcriptome assembly analysis of RNA fractions from Sample 2. Reads for all samples were aligned to the human reference genome (hg19 assembly) using v2.5 of the LifeScope software.The RNA-seq libraries were sequenced on the SOLiD5500xl system, generating reads of length 75 bp. All four RNA fractions from Sample 2 were sequenced using the Exact Call Chemistry (ECC), which enables high accuracy conversion of reads from ‘color space’ to normal nucleotide sequences. This was done to facilitate the comparative The expression level for each exon was quantified from the RNA-seq data using the average depth of coverage per million mapped reads (average dcpm) as proposed by Hillier et al. . The dcpTopHat 2.0.8b  and BowtEI-ratios), the BEDTools software [E) and introns (I). Only reads mapping to the same strand as the gene were considered for this analysis. Having extracted the exonic and intronic reads, the EI-ratio was defined as E/(E + I).To calculate the Exon-Intron ratios . We note that down-sampling might introduce bias, but if so, then in favor of the polyA+ selected sample, which had the lowest read counts. We then ran sed blat  requirinhttp://www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-1898.RNA-Seq reads are available in the ArrayExpress database  differences between cytoplasmic and polyA+ selected RNA populations. Additional data file 4 is a table listing the cut-off values and number of expressed exons out of refSeq exons. Additional data file 5 is a table listing primer sequences for the quantification of intronic and exonic expression in NRXN1, CELF4 and GRID2.Click here for file


Here we use iCLIP to globally analyze and compare the RNA binding properties of two SR proteins, SRSF3 and SRSF4, in murine cells.The SR proteins comprise a family of essential, structurally related RNA binding proteins. The complexity of their RNA targets and specificity of RNA recognition in vivo consensus binding motifs were distinct. Interactions with intronless and intron-containing mRNAs as well as non-coding RNAs were detected. Surprisingly, both SR proteins bound to the 3' ends of the majority of intronless histone transcripts, implicating SRSF3 and SRSF4 in histone mRNA metabolism. In contrast, SRSF3 but not SRSF4 specifically bound transcripts encoding numerous RNA binding proteins. Remarkably, SRSF3 was shown to modulate alternative splicing of its own as well as three other transcripts encoding SR proteins. These SRSF3-mediated splicing events led to downregulation of heterologous SR proteins via nonsense-mediated decay.SRSF3 and SRSF4 binding sites mapped to largely non-overlapping target genes, and SRSF3 and SRSF4 display unique RNA binding properties underlying diverse cellular regulatory mechanisms, with shared as well as unique coding and non-coding targets. Importantly, CLIP analysis led to the discovery that SRSF3 cross-regulates the expression of other SR protein family members. While investigation of transcriptional regulation by transcription factors has led to a deep understanding of how DNA binding proteins direct RNA polymerases to genes, regulation of RNA processing by RNA-binding proteins is still enigmatic. Hundreds of proteins encoded by metazoan genomes have RNA-binding capacity conferred by specific protein structural domains, such as RNA recognition motifs (RRMs), KH domains and zinc fingers . RNA-binIn vitro binding specificities have been determined for some SR protein family members [in vivo crosslinking was used to define mRNA targets of SRSF1 ; this study identified thousands of SRSF1 target sites, which resembled the sequences derived in vitro [SR proteins are a family of seven RNA-binding proteins with a functional repertoire that has expanded to many aspects of RNA metabolism ,3. They  members ,11, whicin vitro . Mature in vitro . Howeverin vivo binding sites of RNA-binding proteins in a genome-wide manner [in vivo binding sites of SRSF3 and SRSF4. Our analysis shows that SRSF3 and SRSF4 bind to distinct sequences and target RNAs, including non-coding RNAs (ncRNAs). The subsequent analysis showed that SRSF3 or SRSF4 binding to these sites conferred regulatory functions in several steps of RNA metabolism in cells, supporting the widespread contribution of SR proteins in gene expression regulation.To understand the widespread functions of SR protein family members, the identification of endogenous RNA target sites is required. The development of ultraviolet (UV) crosslinking and immunoprecipitation (CLIP) followed by high-throughput sequencing has made possible the identification of e manner . Here wee manner , which ae manner . Using tin vivo UV crosslinking mRNAs, whereas SRSF4 only immunoprecipitated significant levels of its own (pre-)mRNA . Intriguingly, the SRSF3 CLIP-tag clusters were also found in the NMD-associated exons or introns of three heterologous SR protein-encoding genes, 7 Figure . In contA Figure . These dSRSF2, SRSF3, SRSF5 and SRSF7  [SRSF5 and SRSF7 mRNA levels decrease significantly in cells over-expressing SRSF3  . The use3 Figure . Upon CH3 Figure . Taken t3 Figure . This cr3 Figure  raises tHere we used iCLIP to investigate the RNA-binding landscape of two SR proteins, SRSF3 and SRSF4, in mouse cells. The value of this study is enhanced by the global comparison of RNA targets and binding sites for two members of this prominent family of RNA binding proteins with a variety of known roles in gene expression. Through detailed analysis of the transcripts and transcript regions bound by SRSF3 and SRSF4, we provide evidence for previously unknown functions of these highly conserved RNA binding proteins. Here we discuss our findings in the context of five major conclusions.in vivo [in vivo binding sequences. We show that SRSF4 binds a consensus GA-rich sequence. The CU-rich SRSF3 in vivo consensus binding sequence is similar to that derived in vitro by SELEX [in vivo RNA targets because the shortness and degeneracy of consensus sequences leads to over-representation within the transcriptome [in vivo targets, accomplished here by CLIP, allowed us to further investigate the identified RNA classes and RNA regions bound.First, SRSF3 and SRSF4 exhibit largely non-overlapping binding sites and RNA targets, indicating that SRSF3 and SRSF4 regulate specific sets of genes through their interaction with different RNA sequences. This finding is consistent with the previous finding that SRSF3 and SRSF4 are present in non-overlapping mRNPs in vivo , althougby SELEX ,19, indicriptome . The ideMALAT1, which is enriched in nuclear speckles and interacts with a subset of SR proteins, including SRSF3 [MALAT1 [MALAT1 interaction is common among RNA-binding proteins in nuclear speckles. Furthermore, SRSF3 and SRSF4 interacted with 7SK, another ncRNA localized to speckles [NEAT1 [Second, an important class of RNAs bound by both SRSF3 and SRSF4 were ncRNAs. SRSF3 and SRSF4 crosslinked to multiple sites along the lincRNA ng SRSF3 . Other s [MALAT1 ,21; it aspeckles  but not s [NEAT1 . One esps [NEAT1 . It remas [NEAT1 . BecauseXenopus oocytes [Third, we provide evidence that SRSF3 and SRSF4 bind many intronless genes, further supporting their role as regulators of gene expression independent of pre-mRNA splicing . Surpris oocytes . UV-RIP  oocytes , and are oocytes . It was  oocytes . However oocytes ,36. Thus oocytes ,28. It wIn vitro studies suggested that SRSF1 is first recruited to an exonic splicing enhancer and the RS domain subsequently contacts the branch point [Fourth, intron-containing protein-coding transcripts were a major class of SRSF3 and SRSF4 targets. SR proteins are thought to bind primarily to exonic splicing enhancers, where they influence recognition of adjacent 5' and 3' splice sites . The patch point ,40. The ch point .SRSF2, SRSF3, SRSF5, and SRSF7). This reveals an unexpected role of SRSF3 in cross-regulating expression of other SR protein family members . For branch point RNA maps, we used computationally predicted branch points from Corvelo et al. . Only thSRSF3 or SRSF4, esiRNA sequences described previously were used [SRSF3 and SRSF4 cDNA were cloned into pYFP-N2 and pEGFP-N3 vectors (Clontech), respectively. To construct the minigenes, the region of interest was PCR amplified from P19 genomic DNA and cloned into pcDNA3 vector (Invitrogen). The plasmids carrying the minigenes were co-transfected together with either SRSF3 or SRSF4 esiRNA or overexpression construct into P19 cells using Lipofectamine 2000 (Invitrogen). Empty vectors or esiRNA targeting EGFP were used as controls. Twenty-four hours post-transfection, total RNA was isolated using acid phenol-chloroform extraction (Ambion). After DNaseI treatment the RNA was reverse transcribed with Superscript III (Invitrogen). The splicing patterns of minigenes were analyzed using vector-specific PCR primers. To abrogate the nonsense-mediated decay pathway, P19 wt cells were treated with 300 μg/ml cycloheximide for 3 hours. Untreated samples were processed in parallel. Total RNA was extracted and the samples treated as described above. Gene-specific primers spanning the exons flanking the PTC-containing cassette exon were used. Total expression of SRSF2, SRSF5 and SRSF7 was determined by RT-qPCR using primers spanning an exon-exon junction. ACTB was used as a reference gene to normalize for cDNA synthesis efficiency. Primer sequences are available upon request.For the knockdown of ere used . For the2; 0.3% (v/v) Nonidet P-40, RNaseOUT; complete protein inhibitor cocktail), the nuclear pellet was collected by centrifugation, and the supernatant was collected as the cytoplasmic faction. The cytoplasmic fraction was used as such or RNA was extracted as above. Input, mock and immunoprecipitation samples were independently reverse primed with either oligo-dT or hexamer primers. Primers specific for individual histone mRNAs were used for qPCR amplification; POFUT1 was used as a reference gene for SRSF3 and DTMYK for SRSF4 to normalize for cDNA synthesis efficiency. Oligo-dT and hexamer samples were normalized independently of each other to their respective input. The primer sequences are available upon request.UV-crosslinked cells (see above) were harvested and the cytoplasmic fraction was separated. The total cell pellet was suspended in NET-2 buffer  Nonidet P-40) for western blot analysis or Trizol (Invitrogen) for RNA isolation. For the fractionation, the cell pellet was suspended in hypotonic buffer PCR: reverse transcription (quantitative) polymerase chain reaction; scaRNA: small Cajal body-specific RNA; SELEX: systemic evolution of ligands by exponential enrichment; SLBP: stem loop binding protein; snoRNA: small nucleolar RNA; UTR: untranslated region; UV: ultraviolet.The authors declare that they have no competing interests.M-LÄ, MMM and KMN designed the experiments; M-LÄ and MMM performed the experiments; M-LÄ, HB, IH, JU, TC and CG analyzed the data; M-LÄ, MMM and KMN wrote the manuscript. The final manuscript has been read and approved by all the authors.Supplementary Information. Supplementary Materials and methods, References, Figures S1 to S7 and Tables S1 and S3 to S5.Click here for fileSupplementary Table S2. Genes with significant SRSF3 or SRSF4 crosslink sites.Click here for file


Integrated CMOS Hall sensors have been widely used to measure magnetic fields. However, they are difficult to work with in a low magnetic field environment due to their low sensitivity and large offset. This paper describes a highly sensitive digital Hall sensor fabricated in 0.18 μm high voltage CMOS technology for low field applications. The sensor consists of a switched cross-shaped Hall plate and a novel signal conditioner. It effectively eliminates offset and low frequency 1/f noise by applying a dynamic quadrature offset cancellation technique. The measured results show the optimal Hall plate achieves a high current related sensitivity of about 310 V/AT. The whole sensor has a remarkable ability to measure a minimum ±2 mT magnetic field and output a digital Hall signal in a wide temperature range from −40 °C to 120 °C. They are used not only for direct measurement of magnetic fields, but also for non-direct measurements, like speed or position, etc. Hall devices can be realized in standard integrated circuit processes such as the bipolar or CMOS technologies. Compared to bipolar Hall devices, CMOS Hall devices exhibit the following advantages: high reliability, small size, low cost and compatibility with other CMOS technologies [Presently, Hall magnetic field sensors are widely established for their great applications in industrial control systems, intelligent instruments, and consumer electronic products, nologies –4. Unfornologies –4. Firstnologies .i.e., the so-called spinning current technique, has been widely employed in Hall sensors [Therefore, the low magnetic sensitivity and the large offset of Hall sensors limit both the minimum value of the magnetic field that can be measured as well as the accuracy of the measurements. So far the techniques used to reduce the offset and 1/f noise from the electronic circuits, can be mainly divided into auto-zero (AZ), correlated double sampling (CDS) and chopper stabilization (CHS) techniques . Compare sensors ,10.This paper deals with a highly sensitive digital Hall sensor fabricated in 0.18 μm high voltage (HV) CMOS technology for low magnetic field applications. A new chopper stabilized instrumental chain is employed to perform the dynamic offset cancellation, which mainly consists of an optimal switched Hall plate, and a novel and simple signal conditioner. In particular, the proposed signal conditioner features a switched hysteresis comparator to replace the sample-hold circuit and Schmitt trigger of conventional signal conditioner, which further reduces the size and cost of the proposed Hall sensor.This paper is arranged as follows: first, the design and optimization of the CMOS horizontal Hall plate is briefly introduced. Further, the structure of an analog front-end with the dynamic offset cancellation is described in detail. Then, the simulation and experimental results are presented and discussed. Finally, conclusions are drawn.2.V or current I bias is supplied via one pair of terminals and a perpendicular magnetic field ZB is applied to the device surface, the Hall voltage HV appears on the other pair of terminals due to the Hall effect. Considering the geometry of a real Hall plate, HV can be expressed with the current related sensitivity IS [IS is determined by the geometrical correction factor G, the Hall mobility Hμ or the Hall factor Hr, the doping concentration NWn of the N-well, and the effective depth of the N-well NWt.The cross-shaped Hall plate as a horizontal Hall device has been broadly used due to its relatively high sensitivity and low offset. The structure of the CMOS cross-shaped Hall plate is schematically shown in ivity SI :(1)VH=SVS:L and W are the finger length and finger width of the cross-shaped Hall plate, respectively.nθ is the Hall angle, equal to tan−1(HBμ).For a cross-shaped Hall plate, the geometrical correction factor can be calculated by :(3)G=1−IS is inversely proportional to the carrier concentration of the N-well. Therefore, a Hall device fabricated by a standard CMOS process has a low sensitivity due to high N-well doping concentration. In order to improve the current related sensitivity, we select a HV CMOS process to fabricate the Hall plate as it can provide an obviously lower N-well doping level than the standard CMOS process, despite a relatively deep N-well depth. On the other hand, the geometrical correction factor should also be enhanced, which is determined by the ratio of finger length L to finger width W in terms of W/L = 2) has been reasonably selected in the layout design.It is well known that CMOS Hall devices seriously suffer from a large offset. One of the main origins of offset comes from the mask-misalignment, which can be minimized by designing a Hall device with an appropriate and symmetric layout. In fact, the masks defining the terminals and N-well implant active layer of the Hall plate could be shifted or rotated relative to each other during photolithography. Any misalignment between terminals mask and the N-well mask will result in an offset, even in the absence of magnetic field. However, the smaller terminals designed within the N-well could lead to a larger masks misalignment. In the layout design, an optimized cross-shaped Hall plate structure is developed. Compared to the conventional Hall plate, the length of terminals reaches a maximum allowable value in the N-well for a given technology. Thus, the effect of the masks misalignment on the offset can be greatly reduced.3.The block diagram of the new chopper stabilized instrumental chain is illustrated in 3.1.a and terminal c of the Hall plate are connected to power and ground. Then current flows from terminal a to terminal c, and Hall signal appears between terminal b and terminal d. When NCLK is high level, M1, M4, M6 and M7 turn on, so there is a current flowing from terminal b to terminal d. Accordingly, a Hall signal is present between terminal c and terminal a. Thus, if the change of the magnetic field is much slower than the clock frequency, the differential output Hall voltage HV periodically changes its polarities with the same magnitude in the course of current spinning. On the contrary, the differential output offset voltage OPV always keeps the same magnitude and a constant polarity, as the same imbalance occurs in adjacent branches of the equivalent Wheatstone bridge network. It is important to note that the offset OAV of the instrumentation amplifier becomes indistinguishable from OPV. Consequently, a demodulation should be performed to extract the Hall signal and eliminate the Hall offset and the instrumentation amplifier’s offset simultaneously by the following signal conditioner at no extra cost.3.2.The traditional signal conditioners execute sample-and-hold (S/H) and adding functions to remove offset without using low-pass filters ,8. FirstIn this work, we propose a signal conditioner based on a high-pass filtering—demodulation configuration, as shown in During the 90° state, the differential input voltage of the instrumentation amplifier changes to:1RAB or 1/C2RAB, where RAB is the equivalent resistance between the points A and B. In order to obtain low 1/f noise performance, C1 and C2 are set to 10 pF.After it is amplified by the instrumentation amplifier, the Hall signal can pass through the high-pass filters, but the offset and 1/f noise are blocked. It is important to notice that a simple passive first order high-pass filter is sufficient to perfectly cancel the offset and 1/f noise. The cut-off frequency of the high-pass filter has to be higher than the 1/f noise frequency, which is typically between 500 Hz and 1 kHz, so a first order high-pass filter with 2 kHz cut-off frequency can achieve this requirement. The cut-off frequency is determined by 1/CthV is a trigger threshold level and its polarity is controlled by the feedback Hall output signal. When the Hall output signal is high level, switch M3 turns on and thV is equal to R1V (the current of 10 μA flowing across resistor 1R). Otherwise, switch M4 turns on, then thV becomes −R2V (the current of 10 μA flowing across resistor 2R). We selected 1R = 2R = 3R = 4R = 2 K to make thV = ±20 mV. At the moment, the cut-off frequency of the high-pass filters is much higher than the chopping frequency of 100 KHz, hence both Hall signal and offset and 1/f noise are blocked.When NCLK is high level, switches M1 and M2 turn on, then, the differential input voltage of the comparator B is clamped to:uA is the voltage gain of the instrumentation amplifier A, and the residual offset voltage is neglected.When NCLK is low level, switches M1 and M2 turn off. At this time, the cut-off frequency of the high-pass filters becomes less than 100 kHz but higher than 2 kHz. Thus, only the Hall signal can pass through the high-pass filters. Since the amount of electric charge on the capacitances C1 and C2 remains unchanged, the differential input voltage of the comparator B changes to:AV > BV, so the output voltage of comparator B is a high level. At this time, thV is equal to R1V. When 2uVHA reversely increases more than |R1V|, the comparator B outputs a low level and thV becomes −R2V. Only when the value of 2uVHA forward increases more than |R2V|, the output becomes a high level. In order to output a standard CMOS level, a D flip-flop (DFF) is used.First assume that the initial state is Therefore, the proposed signal conditioner not only effectively eliminates the offset and 1/f noise, but also realizes the hysteresis characteristics of a digital Hall sensor without a Schmitt trigger. Meanwhile, the whole signal conditioner only needs two capacitances, which further make the chip cost-effective.4.A SPICE simulation of the front-end chopper stabilized instrumental chain was performed with 100 kHz chopping clock frequency using a Cadence spectre simulator. The simulation model parameters of the devices were derived from the X-FAB 0.18 μm HV CMOS technology. The Hall plate is modeled by an equivalent simulation model written in Verilog-A language . The Hal5.2 is situated in the central region of the chip. The spinning current circuit and the signal conditioner are located close to the Hall device. In addition, the single-chip motor driver also integrates other important functional blocks such as control logic, H-bridge driver, oscillator, bandgap voltage reference, thermal shutdown (TSD) and under voltage lockout (UVLO) protection, etc.A highly sensitive digital Hall sensor using the dynamic offset cancellation technique was implemented in a monolithic brushless DC motor driver chip based on the X-FAB 0.18 μm HV CMOS process for detecting magnetic pole position. A microphotograph of the driver chip is shown in Firstly, the performances of the single Hall device were tested by using a special Hall effect measuring instrument NDWH-648. The instrument can achieve a Hall voltage resolution of 1 μV, providing a sufficiently high measuring accuracy over the measuring temperature range from 77 K to 400 K. When the Hall plate is biased with 100 μA DC current, the current related sensitivity was measured at room temperature. A comparison of the current related sensitivity has been made between the proposed Hall device and the reported CMOS Hall devices, as shown in It is obvious that a higher sensitivity of about 310 V/AT is achieved in this work. The temperature first order coefficient of sensitivity is estimated to about 800 ppm/K. The variation of the current related sensitivity with the DC bias current is shown in Next, the functions of the whole Hall sensor were measured. The digital output of the Hall sensor was observed by using an Agilent 3032A oscilloscope, as illustrated in 6.A highly sensitive digital Hall magnetic sensor using the X-FAB 0.18 μm HV CMOS technology is introduced. The cross-shaped structure of Hall device is optimized to reduce Hall offset and improve sensitivity. In order to eliminate the relatively large offset, including Hall offset, amplifier’s offset and 1/f noise, the dynamic offset cancellation technique through Hall current spinning is applied. A novel signal conditioner with a simple structure is proposed for saving chip area and improving the performance of the sensor. The recovery of digital Hall output and offset cancellation are achieved with only two high-pass filters and a switch-controlled comparator. The whole signal conditioner only requires a pair of complementary clocks. Additionally, it is convenient to change the hysteresis characteristics by adjusting resistances, without needing an actual Schmitt trigger. The experimental results show that the sensor has a remarkable ability to measure a minimum ±2 mT magnetic field and output a digital Hall signal over a wide temperature range from −40 °C to 120 °C. Therefore, this Hall sensor is well suited for low magnetic field applications, such as integrated brushless DC motor drivers which require small chip size and high sensitivity.


Adenosine-to-inosine (A-to-I) editing is a site-selective post-transcriptional alteration of double-stranded RNA by ADAR deaminases that is crucial for homeostasis and development. Recently the Mouse Genomes Project generated genome sequences for 17 laboratory mouse strains and rich catalogues of variants. We also generated RNA-seq data from whole brain RNA from 15 of the sequenced strains.Cacna1d gene that fell into the IQ domain portion of the Cav1.2 voltage-gated calcium channel, indicating a potential role for editing in the generation of transcript diversity.Here we present a computational approach that takes an initial set of transcriptome/genome mismatch sites and filters these calls taking into account systematic biases in alignment, single nucleotide variant calling, and sequencing depth to identify RNA editing sites with high accuracy. We applied this approach to our panel of mouse strain transcriptomes identifying 7,389 editing sites with an estimated false-discovery rate of between 2.9 and 10.5%. The overwhelming majority of these edits were of the A-to-I type, with less than 2.4% not of this class, and only three of these edits could not be explained as alignment artifacts. We validated 24 novel RNA editing sites in coding sequence, including two non-synonymous edits in the Cds2 gene we find evidence for RNA editing acting to preserve the ancestral transcript sequence despite genomic sequence divergence.We show that despite over two million years of evolutionary divergence, the sites edited and the level of editing at each site is remarkably consistent across the 15 strains. In the  HTR2C gene transcripts, which are edited at five sites in close proximity to each other , and the resulting reads (paired end 76 bp reads) were aligned to the mouse reference sequence (mm9) with BWA .ADAR: adenosine deaminase acting on RNA; bp: base pair; FDR: false discovery rate; GAT: Genomic Association Tester; GO: Gene Ontology; SINE: short interspersed element; SNV: single nucleotide variant; UTR: untranslated region.The authors declare that they have no competing interests.PD, RD, JF, TMK and DJA conceived the ideas for the study. PD and TMK carried out the computational analysis. RM, JB, and SB carried out the wet lab validation experiments. CN and CP carried out the functional classes and GO categories analysis of the calls. PD, TMK, CN, and DJA wrote the manuscript, which was approved by all of the authors.Release-v1.vcf.gz is a VCF file listing the RNA-editing sites and their relative strain distribution patterns.Click here for fileSupplementary figures.Click here for fileSupplementary Table s1 with the sites validated on the Sequenom platform.Click here for file


Ampelopsis japonica (Thunb.) Makino(Vitaceae), is a herbal medicine which has been widely used in East Asia. The present study wasdone to explore whether the standardized extract of Ampelopsis Radix (AJW) protects dopaminergicneurons via antioxidant mechanisms in Parkinson's disease (PD) models. The effects of AJWon primary mesencephalic cultures stressed with 1-methyl-4-phenylpyridinium were investigated usingtyrosine hydroxylase (TH) immunohistochemistry and reactive oxygen species measurement.The eliminative effects of AJW on the 2,2-diphenyl-1-picrylhydrazyl and2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) radicals wereexplored using colorimetric methods. The effects of AJW on the mice treated with1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were determined by pole test as wellas TH and 8-hydroxydeoxyguanosine immunohistochemistry. AJW protected dopaminergic neuronsby inhibiting reactive oxygen species generation in vitro. Moreover, AJW showedpotent radical scavenging activities in vitro. In the mouse PD model, AJW protected the dopaminergicneurons in the brain, leading to motor improvements. AJW inhibited the MPTP-evoked accumulation of8-hydroxydeoxyguanosine in the brain. These data suggest that AJW has neuroprotective effects with antioxidant mechanisms in PD models.Ampelopsis Radix, the root of  Oxidative stress occurs as a result of imbalance of the free radical generation and antioxidant defense system . Even un+), by an oxidizing enzyme in astrocytes [+ is transferred to dopaminergic neurons through the dopamine transporter, where it inhibits the respiratory chain by disrupting mitochondrial complex I, and leads to oxidative stress via mitochondria-dependent or -independent pathways [+ subsequently results in the peroxidation of macromolecules, ultimately resulting in cell death [As a very common neurotoxin for PD models, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes clinical and chemical alterations in humans and rodents similar to those that occur in PD , 8. Aftetrocytes . MPP+ ispathways . The oxill death .Ampelopsis japonica (Thunb.) Makino, belongs to the grape family (Vitaceae) and has been widely used as a traditional medicine to treat pain and fever in East Asia [+-induced toxicities in various cellular systems [+ and C57BL/6 mice stressed with MPTP. We also examined the possible antioxidant mechanisms of this protection.Ampelopsis Radix, the root of ast Asia . It contast Asia , 12. Resast Asia , 14. (+) systems . In addi systems . Neverth+, 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA), phosphate-buffered saline (PBS), paraformaldehyde (PFA), 3,3-diaminobenzidine (DAB), sucrose, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich . Rabbit anti-tyrosine hydroxylase (TH) antibody was purchased from Chemicon International Inc. . Mouse anti-8-hydroxydeoxyguanosine (8-OHdG) antibody was purchased from JaICA . Biotinylated anti-rabbit and anti-mouse antibodies, normal goat serum, and avidin-biotin-peroxidase complex (ABC) standard kit were purchased from Vector Lab . Zoletil was purchased from Virbac .Minimal essential medium was purchased from Gibco . Fetal bovine serum (FBS) was purchased from Hyclone Lab Inc. . (+)-Catechin, (−)-epicatechin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), potassium persulfate, poly-L-lysine (PLL), glucose, glutamine, MPTP, MPPA. japonica was obtained from Jung Do Hrbal Drug Co. . The voucher specimen (KHUOPS-MH023) was deposited in the herbarium of the College of Pharmacy, Kyung Hee University . It was extracted with distilled water at 100°C for 2 h. The powder (AJW) was made from the extracts by filtering and lyophilization. The yield of AJW was 16.10% and it was kept at 4°C. AJW was standardized with (+)-catechin and (−)-epicatechin known as constituents in A. japonica [μm; Waters, Miliford, MA, USA) at 25°C. Mobile phases included aqueous solution of 0.2% acetonitrile (eluent M1) and methanol (eluent M2). The gradient elution was carried in the way that the eluent M2 was linearly increased from 0 to 50% for 0 to 24 min and from 50 to 100% for 24 to 40 min at a flow rate of 1.2 mL/min. The injection volume was 10 μL, and the detector wavelength was set at 280 nm. AJW and reference to the calibration curve obtained with (+)-catechin and (−)-epicatechin were analyzed in triplicates. (+)-Catechin and (−)-epicatechin were found in AJW at a mean level of 1.81 ± 0.16 mg/g and 1.40 ± 0.04 mg/g, respectively.A dried root of japonica , using r5 cells/well. Cultures were maintained in a humidified incubator of 5% CO2 at 37°C in minimal essential medium with 6 g/L glucose, 2 mM glutamine, and 10% FBS. On the 6th day in vitro, cells were treated with AJW for 1 h and stressed with 12 μM MPP+ for a further 23 h. An equal volume of vehicles was given to the control and toxin groups. Then, cells were fixed with 4% PFA at room temperature for 30 min. The cells were stored in PBS at 4°C for immunohistochemistry.Mesencephalic cell cultures were derived from 14-day embryos of Sprague-Dawley rat . Mesencephalons were dissected, collected, dissociated, and seeded in PLL precoated 24-well plates at a density of 1.0 × 102DCF-DA. Intracellular H2O2 or low-molecular weight peroxides oxidize H2DCF-DA to the highly fluorescent compound dichlorofluorescein (DCF). Cells were seeded in PLL precoated 24-well plates at a density of 1.0 × 105 cells/well, and on the 6th day in vitro, cells were treated with AJW for 1 h and stressed with 12 μM MPP+ for a further 23 h. An equal volume of vehicles was given to the control and toxin groups. Cells were incubated with 20 μM H2DCF-DA at 37°C for 30 min, the cells on cover slips were mounted on gelatin-coated glass slides; they were photographed with a fluorescence microscope . The fluorescence intensity of DCF was measured by a fluorescence microscope, and was expressed as a percentage of the value in the vehicle-treated control group.Intracellular ROS was measured using a fluorescent probe, Hμg/mL was mixed with 0.2 mM DPPH ethanol solution (1 : 1). After incubation at room temperature in the dark for 30 min; the absorbance of the mixture was determined at 517 nm using spectrophotometer . Also, the activity of AJW was expressed as half maximal inhibiting concentration (IC50) which is defined as the concentration of AJW required to scavenge 50% of DPPH radicals. IC50 values were estimated by a nonlinear regression using the GraphPad Prism software .AJW at various concentrations of 1–1000 μg/mL was mixed with 7.4 mM ABTS solution with 2.6 mM potassium persulfate. After incubation at room temperature for 5 min, the absorbance of the mixture was determined at 732 nm using spectrophotometer. Also, the activity of AJW was expressed as IC50 which is defined as the concentration of AJW required to scavenge 50% of ABTS radical cations. IC50 values were estimated by a nonlinear regression using the GraphPad Prism software.ABTS solution of 7.4 mM was added to 2.6 mM potassium persulfate for 1 day before starting the experiment in the dark. AJW at various concentrations of 1–1000 Animal maintenance and treatment were carried out in accordance with the Principles of Laboratory Animal Care  and the Animal Care and Use guidelines of Kyung Hee University, Seoul, Republic of Korea. Male C57BL/6 mice (8 weeks old) were purchased from Daehan Biolink. Animals were assigned to four groups: (1) Group 1 (vehicle-treated group), (2) Group 2 (MPTP-only treated group), (3) Group 3 (MPTP + AJW 10 mg/kg/day treated group), and (4) Group 4 (MPTP + AJW 100 mg/kg/day treated group). The mice were housed at an ambient temperature of 23 ± 1°C and a relative humidity of 60 ± 10% under a 12-h light/dark cycle and were allowed free access to water and food. AJW dissolved in saline was administered orally at 10 or 100 mg/kg/day for 7 days. MPTP  in saline was injected intraperitoneally four times at 2 h intervals on the last day of AJW treatment.μm coronal sections using a cryostat microtome . Then, the tissues were stored in storing solution containing glycerin, ethylene glycol, and PB at 4°C for immunohistochemistry.We performed the pole test, on the seventh day after the last MPTP injection. The mice were placed head upward near the top of a vertical rough-surfaced pole . The time taken for the mice to turn completely downward  and the time taken to reach the floor  were recorded, with a cut-off limit of 30 sec. After the pole test, the mice were anesthetized with 50 mg/kg Zoletil (intramuscularly) and were rapidly perfused transcardially with PBS, followed by 4% PFA in 0.1 M phosphate buffer (PB). Then, brains were rapidly taken out, postfixed in 4% PFA, and processed for cryoprotection in 30% sucrose at 4°C. Frozen brains were cut into 30 2O2 in PBS for 15 min. For dopaminergic neuron detection, they were incubated with a rabbit anti-TH antibody (1 : 2000 dilution) overnight at 4°C. For detection of oxidative DNA damage, the treated brain sections were heated in citric acid  for 5 min for antigen retrieval, incubated in blocking solution (10% goat serum + 1% BSA in PBS) for 1 h, and incubated with mouse anti-8-OHdG antibody (1 : 50 dilution in blocking solution) overnight at 4°C. They were then incubated with a biotinylated anti-mouse and anti-rabbit IgG, respectively, for 1 h followed by incubation in ABC solution for 1 h at room temperature. The activity was visualized with DAB for 3 min. After every incubation step, the cells and tissues were washed three times with PBS. Finally, the mesencephalic cells on cover slips were mounted on gelatin-coated glass slides and air-dried. The free-floating brain tissues were mounted on gelatin-coated slides, dehydrated, cleared with xylene, and cover-slipped using histomount medium. All of them were photographed with an optical light microscope . For quantification of the effect of AJW in the mesencephalic dopaminergic cells, TH-positive cells were counted on at least four cover slips from independent experiments for each condition. Quantification of the effect of AJW in brain tissues was performed by counting the TH-positive cell numbers and the 8-OHdG-positive cell numbers in the SNc at ×100 magnification under a microscope and by measuring the optical density of TH-positive fibers in the striatum at ×40 magnification using an ImageJ software . Data were expressed as a percentage of the value in the vehicle-treated control group.Fixed mesencephalic cells on cover slips and free-floating brain sections were rinsed in PBS at room temperature before immunostaining. They were pretreated with 1% HP < 0.05 was deemed to indicate statistical significance.The data were expressed as mean ± standard error of the mean (SEM). Statistical significance was determined by one-way analysis of variance, followed by Tukey's multiple comparison test, using GraphPad Prism software. In all analyses, + toxicity in primary dopaminergic neurons, we counted TH-positive cell numbers. Exposure to 12 μM MPP+ exhibited remarkably reduced numbers of TH-positive cells as 42.50 ± 2.45% relative to the control group and the treated cells had shrunken bodies and shortened neurites. Treatment of AJW at 1 μg/mL protected dopaminergic cells, showing increased survival rate by 61.96 ± 3.70% relative to the control group and better neuronal morphology of  TH-positive cells , used as a positive control. AJW at 1000  in DPPH  and ABTS in DPPH  assays, in vivo PD model, we treated mice with AJW and/or MPTP and carried out the pole test. As a result, T-turn and T-LA of MPTP-only treated mice were markedly prolonged as 25.15 ± 4.17 sec and 26.74 ± 3.26 sec, compared with the control group  (To confirm the effect of AJW on dopaminergic neurons in an .88 sec) . However.88 sec) .In the mouse brain, MPTP induced severe dopaminergic cell death in the SNc and the striatum. In the MPTP-only treated mice, the number of TH-positive cells in the SNc and the optical density in the striatum were decreased by 37.46 ± 3.46 and 39.53 ± 1.38% relative to the control group, respectively . HoweverTo evaluate the inhibitory effects of AJW on 8-OHdG accumulation by MPTP in mice, we used anti-8-OHdG antibody. In the SNc of MPTP-only treated mice, large accumulation of 8-OHdG occurred, whereas in the control group, there is nothing shown . This ac+ toxicities in a primary culture system and against MPTP toxicities in mice through antioxidant mechanisms.In this study, we demonstrated that AJW protects dopaminergic neurons against MPP+ toxicity, we performed an immunohistochemistry of TH, the crucial enzyme in dopamine biosynthesis [+, the active metabolite of MPTP, is known to result in selective dopaminergic neuronal degeneration via inhibiting mitochondrial complex I and increasing calcium permeability of the mitochondrial membrane generating ROS in cellular PD models [+ toxicity, leading to an increase in the TH-positive cell numbers and preservation of TH-positive cell morphologies.First, to investigate whether AJW protects dopaminergic neurons from MPPynthesis , in primD models . In this+-induced neurotoxicity, we measured intracellular ROS level in primary cultured mesencephalic cells treated with MPP+ and/or AJW. ROS, generated mostly during the incomplete metabolic reduction of oxygen to water in aerobes, include superoxide, nitric oxide, hydroxyl radical, H2O2, and peroxynitrite [+, the overproduction of ROS readily damages dopaminergic neurons, which are especially vulnerable to oxidative stress [+ in primary mesencephalic cells. In addition, AJW showed potent radical scavenging activity comparable to SBE, a positive control that has strong antioxidant and neuroprotective effects [+.Next, to explore the protective mechanisms of AJW from MPPynitrite . All of ynitrite . Moreovee stress , 19, 20. effects , in DPPHin vivo, we performed behavioral tests and brain tissue stereology after treating C57BL/6 mice with MPTP and/or AJW. The MPTP-treated mouse has been widely used as an in vivo PD model because MPTP induces PD-like motor deficits such as bradykinesia and rigidity and selective dopaminergic neuronal loss in the SNc [in vitro and in vivo.Next, to evaluate the effects of AJW  the SNc . In the  the SNc , AJW-tre the SNc . TherefoCassia obtusifolia L. seed extract showed protective effects in experimental models of memory impairment, hippocampal cell death, ischemia, and PD [Polygala tenuifolia Willd was reported to confer neuroprotection in various experimental models, such as memory impairment, cerebral ischemia, chronic stress, anxiety, and PD [Scutellaria baicalensis Georgi root extract, another herbal medicine, protected neurons against ischemia, PD, Alzheimer's disease, and spinal cord injury in various experimental models [β toxicities in vitro [Most neurodegenerative diseases are heterogeneous and genetically complex. In PD, more than 90% of the cases are not linked to a single mutation . Due to , and PD . Similar, and PD . In addil models . Chemical models  and the l models  and to hl models . In addil models , 15, 30,l models , 32. Furin vitro . Taken t+ toxicities in primary mesencephalic cultures and against MPTP toxicities in C57BL/6 mice. The neuroprotective effects of AJW are due to the antioxidant activities including inhibition of ROS, free radicals, and 8-OHdGs. These results indicated that AJW is a potential candidate as a neuroprotective agent in PD, although the major active ingredients in AJW and the possible protective effects of AJW in other PD models need to be further studied.AJW protected dopaminergic cells from MPP


RNA editing is vital to provide the RNA and protein complexity to regulate the gene expression. Correct RNA editing maintains the cell function and organism development. Imbalance of the RNA editing machinery may lead to diseases and cancers. Recently, RNA editing has been recognized as a target for drug discovery although few studies targeting RNA editing for disease and cancer therapy were reported in the field of natural products. Therefore, RNA editing may be a potential target for therapeutic natural products. In this review, we provide a literature overview of the biological functions of RNA editing on gene expression, diseases, cancers, and drugs. The bioinformatics resources of RNA editing were also summarized. RNA editing is the change of nucleotide sequence of RNA transcripts relative to that of the encoding DNA . RNA ediThe A-to-I editing is an enzymatic process mediated by proteins of the family of adenosine deaminase acting on RNA (ADAR). Two types of A-to-I RNA editing such as site selective and hyper-editing have been summarized . The sitRNA editing is essential in many organisms. Correct RNA editing is important in organism's development . For exaSince the RNA editing is essential in regulating gene expression of organisms, imbalance of RNA editing may lead to dysfunction of some proteins involved in normal physiology such as neural and immune functions. A large number of nervous system targets such as neurotransmitter receptors and ion channels , 19 undeADARs can regulate the innate immune system by editing the RNA transcripts of immune-related genes . ADAR  and glioma-associated oncogene 1 (GLI1). A-to-I RNA editing of AZIN1 is increased in hepatocellular carcinoma . RNA edimRNA transcript diversity such as RNA editing has profound impact on drug discovery , 54. In RNA editing has been suggested to be a therapeutic target for CNS disorders . For exa Trypanosoma brucei RNA editing ligase 1 were reported to be potential therapeutic drugs [Recently, some drugs for inhibiting RNA editing enzymes were discovered. For example, novel inhibitors ofic drugs , 59. Although there is a high chance of finding natural products that could target the RNA editing enzymes or lead to the RNA editing of some target genes, such investigations are still rare. It is possible that certain natural products have the potential to be the inhibitors or modulators for RNA editing and may have impacts on the disease and cancer therapy. Therefore, we collected the bioinformatics resources of RNA editing  to help  In brief, dbRES  containsIn the future, we expect that RNA editing studies related to natural products may be accumulating. We hope that this concept can inspire the scientific idea to connect the fields of RNA editing research and natural products for drug discovery in cancer therapy.


Dictyostelium discoideum, an organism widely established as a model for studying eukaryotic cell biology. Using in bacterio functional assays, we demonstrate that the mitochondrial transcription factor B homolog not only functions as a mitochondrial transcription factor, but that it also has a role in rRNA methylation. Additionally, we show that the transcriptional activation properties of the D. discoideum protein are located in its extended C-terminal tail, a feature not seen before in the mitochondrial transcription factor B family, but reminiscent of the human mitochondrial transcription factor A. This report contributes to our current understanding of the complexities of mitochondrial transcription, and its evolution in eukaryotes.Unlike their bacteriophage homologs, mitochondrial RNA polymerases require the assistance of transcription factors in order to transcribe mitochondrial DNA efficiently. The transcription factor A family has been shown to be important for transcription of the human mitochondrial DNA, with some of its regulatory activity located in its extended C-terminal tail. The mitochondrial transcription factor B family often has functions not only in transcription, but also in mitochondrial rRNA modification, a hallmark of its α-proteobacterial origin. We have identified and characterised a mitochondrial transcription factor B homolog in the soil dwelling cellular slime mould  Mitochondria are vital to the eukaryotic cell as they generate the bulk of the energy required for cellular processes. The double membrane organelles have many characteristics in common with bacteria as a result of their evolution from a primitive α-proteobacterium. Interestingly, mitochondria have maintained their own genome, which can vary in size significantly. Most of the genes in the mitochondrial genome encode proteins, which play a role either in oxidative phosphorylation, or are components of the mitochondrial translation machinery In most eukaryotes, mitochondrial DNA (mtDNA) is transcribed by a T-odd numbered bacteriophage-like RNA polymerase rns). In metazoans, the mtTFB gene has undergone a duplication event, with an evolutionary trend towards separating the two functions into individual proteins. This has been demonstrated with the human mtTFB homologs, h-mtTFB1 and h-mtTFB2 , which display both functions, but have varied degrees of methyltransferase activity and opposing efficiencies as transcriptional activators Saccharomyces cerevisiae, mtf1, on the other hand, functions solely as a mitochondrial transcription factor and exhibits no methyltransferase activity rns transcripts are not methylated in this manner Members of the mitochondrial transcription factor B (mtTFB) family have also been found to be important positive regulators of mitochondrial transcription. Originally believed to be homologous to bacterial sigma factors, most mtTFB proteins are now known to contain an rRNA adenosine dimethyltransferase domain, a hallmark of their α-proteobacterial origin Drosophila melanogaster also has no function in the transcription of mtDNA D. melanogaster that is the primary activator of transcription and transcription-primed mtDNA replication, while mtTFB1 plays a role in mitochondrial translation Dictyostelium discoideum.Little is known about how mtTFB proteins contribute to mitochondrial transcription, as they often seem to lack obvious DNA binding and activation domains. In human mitochondria, it is believed that h-mtTFB1/h-mtTFB2 interact directly with the mtRNAP POLRMT, which can then recognise the DNA associated mtTFA, TFAM, allowing mitochondrial transcription to be initiated D. discoideum is an amoeba which has been well utilised to study many different aspects of eukaryotic cell biology. This is due to its unique life cycle which provides multiple developmental stages to study differentiation and development, while encompassing many features of the humble microbe that make it rather easy to cultivate and manipulate in a laboratory setting D. discoideum, whose entire genome has been sequenced, has also more recently become an attractive model for studying mitochondrial genetics and disease D. discoideum, rpmA, is the only trans component of the mitochondrial transcription machinery to have been characterised thus far. This phage-like RNA polymerase was found to specifically bind and initiate transcription in the NCR of the D. discoideum mitochondrial genome, but does so rather inefficiently D. discoideum which not only functions as a rRNA adenosine dimethyltransferase, but also upregulates mitochondrial transcription in association with rpmA. Additional features of this transcription factor contribute to its transcriptional activation properties, which seem to be lacking in other mtTFB homologs characterised to date, providing new insights into the process of mitochondrial transcription initiation.This study characterises a mtTFB homolog in mtTFB gene sequence in D. discoideum had been identified by Shutt and Gray, 2006 D. discoideum genome in a search for any mitochondrial transcription factor gene sequences leading to the identification of only one candidate (Genbank accession no. XM_632683), which was the same as the one identified previously. This gene, which we have named tfb1m, is 1458 bp in size and consists of a continuous open reading frame. The corresponding protein sequence is 485 amino acids long, has a predicted molecular weight of ∼56 kDa, and contains a putative N-terminal mitochondrial targeting signal , encoding the putative mitochondrial targeting signal, fused to the gene encoding the green fluorescent protein (GFP) in the D. discoideum shuttle vector pA15GFP D. discoideum cells, the encoded fusion protein was found to co-localise with mitochondrial markers   markers , indicatEscherichia coli have demonstrated that the knockout of its gene results in a lack of methylated 16S rRNA and, as a consequence of this, resistance to kasugamycin, an aminoglycoside that functions by interacting with 16S rRNA, the substrate for KsgA E. coli, where introduction of a mtTFB encoding gene into a ksgA mutant restored methylation of 16S rRNA and therefore sensitivity to kasugamycin D. discoideum Tfb1m was expressed in E. coli ksgA mutants. The ability of Tfb1m-expressing E. coli ksgA mutant cells to grow in the presence of kasugamycin was reduced significantly compared to their highly resistant phenotype in the absence of Tfb1m  of Tfb1m (Tfb1mΔC) remained resistant to kasugamycin, despite the fact the methyltransferase domain remained intact. Thus, although the C-terminus does not carry any enzymatic activity, its deletion abolishes the methyltransferase activity of Tfb1m, suggesting that the C-terminus is still required for methylation.The mtTFB family is hypothesised to have evolved from an α-proteobacterial rRNA adenosine dimethyltransferase belonging to the KsgA family of Tfb1m . The resin bacterio transcription system for analysing mitochondrial transcription. This system has been developed as an alternative to studying D. discoideum mitochondrial transcription in vitro, which proved to be problematic for several reasons as described previously in bacterio transcription system involves the use of an E. coli BL21 DE3 strain harbouring two vectors  vector (Novagen), in which the selectable marker has been changed to make it suitable for this system rpmA) in the presence or absence of Tfb1m (expressed from vector pET-tfb1m) in E. coli cells harbouring pZ-NCRrnl. The bacterial RNA was extracted, separated on agarose gels, and blotted onto a nylon membrane to detect any rnl transcript using an rnl specific probe. E. coli cells carrying only pZ-NCRrnl acted as a negative control and demonstrated that any transcription products derived from this construct were not due to endogenous RNA polymerase activity. Bacterial cells harbouring only pZ-NCRrnl and pQ-rpmA did not express any RNA that hybridised to the rnl specific probe. Transcription of the reporter gene from pZ-NCRrnl was only detected in cells expressing both rpmA and Tfb1m  to efficiently initiate transcription at a mitochondrial-specific transcription initiation site, resulting in the expression of a reporter gene , which, as our results demonstrated, contributes to the activator function of the transcription factor . Approxi E. coli , it seem E. coli . The coiD. melanogaster also possesses a shorter, but nonetheless extended C-terminal tail  , the probability scores for coiled-coil formation were not as significant as for Tfb1m. In addition, the significance of the C-terminal tail in the D. melanogaster homolog in regulating mitochondrial transcription remains to be elucidated. The excessive length of the C-terminal tail and the presence of coiled-coil structures in Tfb1m clearly distinguishes it from other mtTFB homologs, suggesting that D. discoideum uses a unique mechanism for regulating mitochondrial transcription.To our knowledge, this is the first report of a mtTFB homolog containing an extended and coiled-coil forming C-terminal tail with a role in transcription. While it is known that the C-terminus of the yeast mtTFB homolog also has a function in mitochondrial transcription, the overall length of the protein (341 amino acids compared to 485 amino acids for Tfb1m) is comparable to that of other mtTFB homologs and the C-terminus is not extended roteins) . AlthougD. discoideum yielded no candidates (data not shown), indicating that D. discoideum may not have a mtTFA homolog. Thus, the lack of a mtTFA homolog, and the presence of an extended coiled-coil forming C-terminal tail in Tfb1m, may mean that D. discoideum has developed an alternative mechanism for mitochondrial transcription involving only two proteins, an RNA polymerase and a single transcription factor that combines the features of both mitochondrial transcription factor families  and kasugamycin (800 µg/mL). The diluted cultures were then aliquoted into a 96 well microtiter plate (BD Biosciences) in triplicate and incubated at 37°C for 15 h. Growth was analysed by measuring the OD570 on a Lucy 2 microtiter plate reader (Quantum Scientific).Kasugamycin resistance was assayed to ascertain the methyltransferase activity of Tfb1m using a protocol adapted from Seidel-Rogol et al., 2003 E. coli cultures of interest, 0.02 vol of overnight cultures were inoculated into LB supplemented with the relevant combination of antibiotics  to an OD600 of 0.4–0.6. Expression of heterologous proteins was then induced via the addition of 1 mM IPTG and cultures were incubated at 21°C for 5 h. In parallel, another set of the same cultures were set up in the same manner, but without the addition of IPTG to serve as un-induced controls. Following in bacterio transcription, cultures were pelleted in preparation for RNA extraction. Transcription of the rnl reporter was detected via northern blot hybridisation using an in vitro transcribed radiolabelled RNA probe for the mitochondrial rnl.Analysis of mitochondrial transcription was carried out using the method modified from Le et al., 2009 2O.Extraction of total RNA from bacterial cultures was performed using TRIzol® reagent (Life Technologies). Cell pellets were treated with 200 µg/mL of lysozyme (Sigma) for 30 min to degrade the cell wall, enhancing cell lysis and RNA yield. Following lysozyme treatment, samples were resuspended in TRIzol® reagent, 0.2 vol of chloroform was added and the samples were mixed vigorously. Samples were then centrifuged to separate the RNA from DNA and proteins. The aqueous phase was collected, and the RNA was precipitated in an equal vol of isopropanol, and centrifuged as previously. RNA pellets were washed with 75% ethanol, air dried and resuspended in DEPC treated sdHin vitro transcription of RNA using a pBS-rnl vector to create an antisense rnl RNA probe. The reaction consisted of; 5× transcription buffer (Promega): 4 µL, 100 mM DTT: 2 µL, RNasin (Promega) (40 U): 0.5 µL, 2.5 mM rNTPs : 3 µL, 100 µM rCTP: 2.4 µL, [α-32P] rCTP (10 mCi/mL): 5 µL, linearised pBS-rnl vector: 1–2 µg, T3 RNA polymerase (Promega): 1 µL and DEPC sdH2O (to 20 µL). The reaction was incubated at 37°C for 1 h and the transcribed RNA was precipitated with an equal volume of isopropanol and used for hybridisation.Synthesis of gene-specific radiolabelled RNA probes was performed via E. coli RNA samples were fractionated on a 2% Tris-acetate (TAE) agarose gel and subsequently transferred to a nylon membrane (Hybond). Following transfer, the membrane was probed with a radiolabelled RNA probe and incubated at 55°C overnight. The blot was exposed to a PhosphorImager screen, and radioactivity was detected using the Amersham Storm 860 Imaging system.Northern blot hybridisation was performed similarly to that described previously


Mixodiaptomus laciniatus (intra- and interstage variability) was comparable to the interspecific variability across other zooplankton species. Further, according to the metabolic theory of ecology, temperature should enhance growth rate and hence RNA demands. RNA content in zooplankton was correlated with temperature, but the relationships were nutrient-dependent, with a positive correlation in nutrient-rich ecosystems and a negative one in those with scarce nutrients. Overall our results illustrate the mechanistic connections among organismal NA content, growth rate, nutrients and temperature, contributing to the conceptual unification of metabolic and stoichiometric theories.Metabolic and stoichiometric theories of ecology have provided broad complementary principles to understand ecosystem processes across different levels of biological organization. We tested several of their cornerstone hypotheses by measuring the nucleic acid (NA) and phosphorus (P) content of crustacean zooplankton species in 22 high mountain lakes . The P-allocation hypothesis (PAH) proposes that the genome size is smaller in cladocerans than in copepods as a result of selection for fast growth towards P-allocation from DNA to RNA under P limitation. Consistent with the PAH, the RNA:DNA ratio was >8-fold higher in cladocerans than in copepods, although ‘fast-growth’ cladocerans did not always exhibit higher RNA and lower DNA contents in comparison to ‘slow-growth’ copepods. We also showed strong associations among growth rate, RNA, and total P content supporting the growth rate hypothesis, which predicts that fast-growing organisms have high P content because of the preferential allocation to P-rich ribosomal RNA. In addition, we found that ontogenetic variability in NA content of the copepod  The metabolic theory of ecology (MTE) and biological stoichiometry (BS) have greatly advanced our understanding of the factors that control ecological processes Both theories place special emphasis on unraveling the mechanistic basis of individual metabolism and growth as it affects the energy flux, and the storage and turnover rates of elements in ecosystems It has been suggested that natural selection operating on GRs drives differences in the body P content of copepods and cladocerans Daphnia species. To explain this paradox, the P-allocation hypothesis (PAH) proposes that small genomes in cladocerans are the consequence of P allocation from DNA (mainly non-coding DNA) to RNA under sustained selection for rapid growth in P-limited environments Considerable interest in the genetic basis of GRH has emerged since the development of genomics earlier this century. A genetic approach offers an opportunity to study the way natural selection simultaneously operates on genome size and GR, which are traditionally studied separately in evolutionary biology Mixodiaptomus laciniatus. Finally, we studied the NA content in all zooplankton species in order to test the MTE and BS predictions for the combined effect of temperature and nutrients on the GR. Extreme low-nutrient alpine environments are ideal model systems for testing these hypotheses. First, their oligotrophic to ultra-oligotrophic status implies a strong food limitation for zooplankton, which may play a major ecological role in determining species composition We tested the PAH by determining the P and NA content and RNA:DNA ratio of several crustacean zooplankton species in 22 high mountain lakes . The GRH was tested by examining the relationship between RNA and P content in the studied zooplankton taxa and among RNA, P, and GR across the life cycle of the copepod The study was carried out in 22 high mountain lakes (1600–3100 m above sea level) in the National Park of Sierra Nevada and the Pyrenees (Spain). These lakes are small, shallow, and highly transparent, with absent or very scarce littoral vegetation  profiles were measured along the water column using a Biospherical Instrument Compact 4-Channel Radiometer . Each lake was characterized by the mean temperature of the water column and the diffuse attenuation coefficient (dK) at each wavelength, calculated from the slope of the linear regression of the natural logarithm of downwelling irradiance vs. depth. An integrated extinction coefficient of UVR (d UVRK) was calculated as the mean extinction coefficient of the three UVR wavelengths   .a (Chl a), seston carbon (C), nitrogen (N), P, and primary production measurements.Chemical and biological samples were taken with a 6-L Van Dorn sampler at the deepest point of the lake. When possible, water from up to four depths  was mixed in a 5-L bucket, and subsamples were taken in triplicate for total P (TP). After removing zooplankton by sieving water through a 40-µm mesh, another set of subsamples was taken in triplicate for P , chlorophyll Acanthocyclops vernalis . The crustacean zooplankton biomass was estimated by using length-weight regressions specifically developed for the copepods Cyclops abyssorum, 10–20 of Diaptomus cyaneus, 5–15 of Alona affinis, 20–25 of Daphnia longispina, and 5–15 of Daphnia pulicaria were isolated. We distinguished among ontogenetic stages and between adult genders in copepods and between non-ovigerous and ovigerous reproductive statuses in copepods and cladocerans. Three replicates per lake were collected for the most abundant species or copepod stages, whereas single or duplicate samples were collected when these were less abundant. Simultaneously, samples were taken and fixed in 4% formaldehyde for later individual body size measurements and, after biomass conversions, for estimation of total P content as % of dry weight (%P). GR and %P data for Mixodiaptomus laciniatus were obtained from Carrillo et al. later , and stored at −80°C until analysis Mixodiaptomus laciniatus ontogenetic stage and sampling day.Additional samples of zooplankton for P and NA determinations were collected by vertical hauls of a 40-µm mesh net and transported chilled in lake water to the laboratory. In the laboratory, zooplankton was concentrated by sieving through a 40-µm mesh and diluted to 1 L with 0.7-µm filtered lake water. For the analysis of P content, the species of live individuals was identified with the aid of an inverted microscope and sorted into precombusted (1 h at 550°C) 1.0-µm glass fiber filters (Whatman GF/B). When possible, samples containing 30–50 individuals of 14C method proposed by Steeman-Nielsen 14CO3  were incubated in situ for 4 h symmetrically distributed around noon at a depth where the UVR was 75% the surface value. All flasks were held horizontally during the incubations. Primary production was measured as total organic carbon (TOC) by acidifying a 4-mL subsample in a 20-mL scintillation vial with 100 µL of 1 N HCl and allowing the vial to stand open in a hood for 24 h (no bubbling), as recommended by Lignell 1) was determined by filtering an aliquot of 40 mL through 1.0 µm pore-size Nucleopore filters of 25-mm diameter, applying a low pressure (<100 mm of Hg) to minimize cell breakage. Filters were placed in scintillation vials and the dissolved inorganic 14C was removed by adding 100 µL of 1 N HCl. We added 16 mL of liquid scintillation cocktail  to the vials, and the radioactivity was counted at 12 h in a Beckman LS 6000 TA scintillation counter equipped with autocalibration . The total CO2 in the lake water was calculated from the alkalinity and pH measurements Primary production was measured with the a was measured fluorimetrically after filtration of 300 mL per replicate through 0.7-µm glass fiber filters (Whatman GF/F) at low pressure (<100 mm Hg) and 24-h pigment extraction in 90% acetone in the dark at 4°C. A Chl a standard  was used to transform the fluorescence data into Chl a concentrations.TP and TP′ were determined by analyzing 50-mL aliquots with the acid molybdate technique after digestion with a mixture of potassium persulfate, boric acid, and sodium hydroxide at 120°C for 30 min −1; Q-biogen, Weston, Massachussets, USA); N-lauroysarcosine ; Tris-EDTA buffer (Q-biogene). Fluorescence measurements were performed using a FLUOstar Optima fluorometer  and black solid flat-bottom microplates . The plate was scanned with a 0.2-s well measurement time, making 10 measurements per well, before and after RNase digestion (30 min under dark conditions at 37°C). Fluorescence measurements were converted into RNA and DNA concentrations by using standard curves for RNA  and DNA . RNA and DNA contents were expressed relative to dry weight (%RNA and %DNA) after biomass conversions using the above-mentioned length-weight regressions and also as RNA:DNA ratio. The %P allocated to RNA (%P-RNA) and DNA (%P-DNA) were calculated by multiplying the content of each NA by its P proportion  and the total P content of the organism (%P) as a measure of the relative P investment in NAs .NA analysis was carried out with a microplate fluorometric high-range assay with RiboGreen using length-measured individual zooplankters after extraction with N-laurylsarcosine followed by RNase digestion Mixodiaptomus laciniatus from lake Laguna de la Caldera. When significant effects were found, pairwise comparisons were made with Tukey’s HSD post-hoc tests. The low number of replicates for %P precluded the study of the effects of lake of origin, temperature, and TP′ for all species. Therefore, %P data collected from different lakes were pooled for analysis comparing groups and species. Given that all samples were collected in 1997  was performed to test for the effects of group (copepods vs. cladocerans), species and lake of origin, and analysis of covariance (ANCOVA) for the effects of group, species, lake temperature, TP′, and temperature×TP′ interaction. We also used ANCOVA to test for the effects of (i) group, species, and lake temperature below- and above-median TP′, and (ii) group, species, and lake TP′ below- and above-median temperature on %RNA. For these analyses, group and species were considered as fixed effects factors with species nested within group, lake as a random factor and temperature and TP′ as covariates. One-way ANOVA was used to analyze intraspecific differences due to ontogeny and gender  for copepods and due to female reproductive status (non-ovigerous vs. ovigerous) for copepods and cladocerans. The effect of sampling year was also included as a random factor in the main effects ANOVA to explore intraspecific differences due to ontogeny, gender and female reproductive status in the copepod 1997 see , the effariances .Mixodiaptomus laciniatus and between GR and %P and between GR and %RNA in stages of Mixodiaptomus laciniatus; (ii) the relationship between body size and DT; and (iii) the relationships of body size and DT (independent variables) with all RPIIs (dependent variables) in Mixodiaptomus laciniatus. A homogeneity of slopes model (ANCOVA) was used to test the effect of NA  across body sizes (continuous predictor variable) on RPII Partial regression plots were used to display the relationships of (i) temperature with %RNA at below- and above-median TP′, and (ii) TP′ with %RNA at below- and above-median temperature in all species, following recommendations by Moya-Laraño & Corobado a, seston C, TOC, POC1). Distributions of these variables were skewed. For instance, Chl a was in the range 0.25–11.85 µg L−1, with 82% of observations <5 µg L−1; or seston C was in the range of 126–1032 µg C L−1, with 86% of observations <500 µg C L−1). Food quality for consumers was high, with seston C:P and C:N ratios of 104–364 (<306 for 86% of lakes) and 6–12 (<9.6 for 95% of lakes), respectively , and algal standing stock variables , Chydorus sphaericus (Laguna Hondera), Cyclops abyssorum, Alona affinis and Daphnia longispina (Estany Baix de Montcasau and Estany de la Munyidera) were also present, although only sporadically and in extremely low abundance . While the %RNA and %DNA values were consistently higher in copepods than cladocerans , the RNAboth NAs ; Table 4A values ; Table 4A values ; Table 4Cyclops abyssorum and Diaptomus cyaneus, higher than in Mixodiaptomus laciniatus but not significantly different from that in cladocerans  collected over three years in lake Laguna de la Caldera . A strong effect of both ontogeny and sampling year was observed for all variables . However, RPIIRNA was always higher than RPIIDNA at each stage, especially at nauplius stages  was >4-fold higher  than the slope between body size and RPIIDNA . Because of the tight correlation between body size and DT . The adults evidenced gender differences in all variables, except for %P; neither the %P differed between ovigerous and non-ovigerous adult females  and among (interstage) and within (intrastage) developmental stages of ciniatus . Intrasphin them .Mixodiaptomus laciniatus  constraints on the NA content of zooplankton, illustrating how the integration of MTE and BS principles can successfully explain fundamental processes at the organism level.Alona affinis were more similar to the NA profile of Mixodiaptomus laciniatus or Eudiaptomus vulgaris than to that of the phylogenetically closer Daphnia species. Furthermore, the RNA and P content was markedly higher in Cyclops abyssorum or Diaptomus cyaneus than in Mixodiaptomus laciniatus or Daphnia species. Hence, we observed a pronounced taxa-specific variation in NA composition within each group (copepods and cladocerans).RNA:DNA ratios were higher in cladocerans than in copepods, as previously reported, consistent with the PAH premise that the allocation of P from DNA to RNA may drive elevated RNA:DNA ratios and therefore a reduced genome size in cladocerans Diaptomus cyaneus is often associated with non-permanent water bodies and completes its life cycle within 3–4 weeks Mixodiaptomus laciniatus inhabits more permanent water bodies and typically completes an univoltine cycle in alpine systems within 4–5 months Mixodiaptomus laciniatus feeds on scarce pelagic high quality seston Diaptomus cyaneus may be met by omnivorous feeding Cyclops abyssorum might be associated with the switch from an herbivorous diet in nauplii to a carnivorous diet based on high-P Daphnia in copepodites at metamorphosis Alona affinis to overcome P-limitation and maintain a large genome in comparison to Daphnia species We propose that much of the variation in NAs observed may also be attributed to life-history strategies. Thus, while Daphnia has also indicated that NA variability may be attributable to ontogenetic variation Mixodiaptomus laciniatus, especially for RNA, is comparable to or larger than the variation with other zooplankton species. The decrease in %RNA up to nauplius stage III may be explained by the depletion of inherited maternal RNA for early protein synthesis TNAs, which reflects a reduction in P allocation to NAs towards adulthood, which may be linked to an increase in other P-enriched biomolecules Previous research on Mixodiaptomus laciniatus stages (intrastage variability)  approach the ecdysis associated with a molt event  see . This covent see . AlternaMixodiaptomus laciniatus. Trends were all consistent with GRH predictions Mixodiaptomus laciniatus in this study  than those reported by Elser et al. Daphnia pulicaria  and Daphnia galeata . These variations likely reflect interspecific differences in life-history strategies between ‘fast-growth’ Daphnia species (like r-strategists) and ‘slow-growth’ copepod species (like K-strategists).Our results also showed strong relationships between the contents of RNA and P across studied species of mesozooplankton and between these contents and GR for the developmental stages of However, the finding that NA values in the species were influenced not only by phylo- and ontogenetic constraints but also by lake of origin, is consistent with the notion that other factors may have an important impact on the NA content of organisms. Many relevant parameters might vary among lakes. In particular, the opposed effects of temperature on RNA content under distinct nutrient scenarios contrast with the well-established positive effect of temperature on metabolic rate as predicted by MTE Taken together, our results are consistent with stoichiometric predictions (PAH and GRH) for NAs in crustacean zooplankton. However, RNA and DNA content did not evidence a consistent pattern of species-phylogenetic affiliation to cladocerans or copepods. Therefore, the PAH needs to take into account other important mechanisms, including life-history strategies, ontogenetic variations, and their temperature- and nutrient-dependence. We highlight the observation that the interaction between temperature and TP′ accounts for a substantial part of the variability of NAs in zooplankton, because it provides empirical evidence for the mechanistic MTE and BS-principles underpinning NAs composition and therefore advances our progress towards a more synthetic theory of Ecology.Figure S1Zooplankton biomass and taxonomic composition of the study lakes. (A) Biomass and taxonomic composition of the zooplankton, and (B) % of total zooplankton biomass for each taxonomic group of the study lakes: Acanthocyclops (A.) vernalis, Cyclops (C.) abyssorum, Diaptomus (D.) cyaneus, Eudiaptomus (E.) vulgaris, Mixodiaptomus (M.) laciniatus, Alona (A.) affinis, Alona sp., Chydorus (C.) sphaericus, Daphnia (D.) longispina, Daphnia (D.) pulicaria, Rotifera (rotifers) and Ciliata (ciliates). Lakes are: Caballo, Laguna del Caballo; Yeguas, Laguna de las Yeguas; Gr-Virgen, Lagunillo Grande de la Virgen; Ch-Virgen, Lagunillo Chico de la Virgen; A-Verdes, Laguna de Aguas Verdes; Al-Río Seco, Laguna Alta de Río Seco; Gr-Río Seco, Laguna Grande de Río Seco; Larga, Laguna Larga; Caldera, Laguna de la Caldera; Caldereta, Laguna de la Caldereta; Borreguil, Laguna del Borreguil; Llebreta, Estany de Llebreta; Al-Mont, Estany Alt de Montcasau; Llong, Estany Llong; Redó, Estany Redó; Barbs, Estany dels Barbs; Coveta, Estany de la Coveta; and Cabana, Estany de la Cabana. Lakes with zooplankton biomass values <1 µg dry weight L−1  were excluded.(PDF)Click here for additional data file.Figure S2Relationship between developmental time and mean body size for ontogenetic stages of the copepod Mixodiaptomus laciniatus. Stages are nauplii (NI-NVI) and copepodites (CI–CV). Solid line is the linear regression fit. See Results in the main text for regression parameters and statistics.(PDF)Click here for additional data file.Table S1dUVRK, mean extinction coefficient for ultraviolet radiation (UVR) of 305, 320, and 380 nm; dPARK, extinction coefficient for photosynthetic active radiation (PAR); temp., temperature. Units are given in brackets.Characterization of high-mountain lakes in Sierra Nevada and the Pyrenees during the study period. Variables: latitude; longitude; altitude; perimeter; area; maximum depth; (PDF)Click here for additional data file.Table S2Cyclops abyssorum, Diaptomus cyaneus, Mixodiaptomus laciniatus), and between female reproductive statuses (non-ovigerous vs. ovigerous) for copepods and cladocerans (Daphnia longispina). Significant results  are indicated in bold; n.s., not significant.Results of ANOVAs to analyze differences in reciprocal square root-transformed body size (µm), RNA and DNA contents , RNA:DNA ratio, and phosphorus allocated to total nucleic acids  among ontogenetic stages (nauplius vs. copepodite vs. adult) and between adult genders  for copepods ((PDF)Click here for additional data file.