Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that affects the upper and lower motor neurons. It has a focal onset but gradually spreads, leading to disability and, eventually, death. About 5–10% of ALS is inherited, usually following a dominant pattern. Pathological analysis revealed that motor neuron degeneration and death in the familial and sporadic forms is closely connected to protein aggregation and deposition, abnormal level and function of RNA molecules, abnormal neuronal cytoarchitecture, and non-neuronal cell death (4C2 HRE) in chromosome 9 open reading frame 72 (C9orf72) gene is found in 40% of the familial ALS cases. It is also found in other neurodegenerative disorders, including frontotemporal dementias (FTDs) (.The GGGGCC hexanucleotide expansion G4C HRE in cs (FTDs) .The molecular mechanisms of C9orf72 ALS neurodegeneration are currently a subject of controversy. Two main pathophysiological models have been hypothesized with experimental evidence supporting both . The fir4C2 HRE (4C2 HRE-mediated neurodegeneration by using (G4C2)30Drosophila models. Different phenotypes were obtained by expressing the abnormal gene at different stages of fly development. If expressed in 1-day-old flies, the HRE sequence caused progressive defects in the photoreceptor organization, suggesting age-dependent neurodegeneration. Locomotor defects were noted in 15-day-old (G4C2)30 flies pointing toward motor neuron pathology. Both of these phenotypes were rescued either by RanGAP overexpression or by using a heterozygous RanGAP gain-of-function mutation. Conversely, photoreceptor degeneration was accelerated by RanGAP knockdown using RNA interference. When the (G4C2)30 HRE was expressed in larval motor neurons, it caused severe neuromuscular junction defects that were not rescued by RanGAP overexpression. These results suggested that RanGAP overactivity suppressed HRE-mediated neurodegeneration in adult Drosophila, but HRE-mediated neurotoxicity in the larval stage is RanGAP independent.Zhang et al. have recently investigated the complex mechanisms that lead to impairment of the normal trafficking through the nuclear pore complex (NPC) associated with G4C2 HRE . They de4C2 RNA interaction in induced pluripotent stem cell (iPSC) neurons, derived from multiple C9orf72 ALS patients and noted that RanGAP G4C2 RNA can colocalize in these cells. This interaction seems to lead to RanGAP loss-of-function, which was shown by a disrupted Ran nucleocytoplasmic gradient in cells expressing G4C2 HRE. HRE toxicity also appeared to be modulated by nucleocytoplasmic transport, and the data suggested that genetically enhancing nuclear import and/or inhibiting nuclear export can suppress G4C2-repeat-mediated degeneration in both Drosophila and human cells.The authors also studied the RanGAP/G4C2 HRE RNA is involved in the neurodegenerative pathophysiological pathway, the investigators treated C9 ALS iPSC neurons with antisense oligonucleotides targeting these molecules. The treatment rescued the disrupted Ran gradient, suggesting that the abnormal RNA may be responsible for the nucleocytoplasmic transport deficits. Neurodegenerative phenotype suppression also occurred using a nuclear export inhibitor, suggesting that nuclear export inhibition may compensate for disrupted nuclear import.In order to prove that abnormal G4C2 HRE disrupts nucleocytoplasmic transport in Drosophila models and in human cells. RanGAP seems to be a key link in the pathophysiological chain, but other nuclear pore proteins may also be involved. Nuclear pore transport was previously implicated in both ALS and FTD pathophysiology, and data from this study suggest that the RNA fragments cause nucleocytoplasmic trafficking defects by direct interaction with NPC proteins. However, other authors argued that DPRs can cause a reversible dysfunction of the NPC in the absence of abnormal C9orf72 RNA (In summary, GTM – summarized the original article, selecting important points for presentation, and wrote the manuscript. TL – manuscript review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The “RNA world” hypothesis is seen as one of the main contenders for a viable theory on the origin of life. Relatively small RNAs have catalytic power, RNA is everywhere in present-day life, the ribosome is seen as a ribozyme, and rRNA and tRNA are crucial for modern protein synthesis. However, this view is incomplete at best. The modern protein-RNA ribosome most probably is not a distorted form of a “pure RNA ribosome” evolution started out with. Though the oldest center of the ribosome seems “RNA only”, we cannot conclude from this that it ever functioned in an environment without amino acids and/or peptides. Very small RNAs (versatile and stable due to basepairing) and amino acids, as well as dipeptides, coevolved. Remember, it is the amino group of aminoacylated tRNA that attacks peptidyl-tRNA, destroying the bond between peptide and tRNA. This activity of the amino acid part of aminoacyl-tRNA illustrates the centrality of amino acids in life. With the rise of the “RNA world” view of early life, the pendulum seems to have swung too much towards the ribozymatic part of early biochemistry. The necessary presence and activity of amino acids and peptides is in need of highlighting. In this article, we try to bring the role of the peptide component of early life back into focus. We argue that an RNA world completely independent of amino acids never existed. We are well aware that some parts of the reconstruction are more speculative than others: When trying to piece together a possible scenario for the origin of life this cannot be otherwise. Before introducing such a coevolutionary theory, however, we have to clarify the history of the term and what we mean by it.The idea of an independent RNA world without oligopeptides or amino acids stabilizing structures and helping in catalysis does not seem a viable concept. On the other hand, the idea of catalytic protein existing without RNA storing the polypeptide sequences, which have catalytic activity, and organizing the production of these sequences, also does not seem a viable concept. Here we argue for a “coevolutionary” theory in which amino acids and  peptides as well as small RNAs existed together and where their separate abilities not only reinforced each other’s survival, but allowed life to more quickly climb the ladder of complexity. Essential for our approach is the following: Starting with small molecules (easily) derived from prebiotic chemistry, we will try to reconstruct a possible history in which every stage of increased complexity arises from the previous more simple stage because specific nucleotide/amino acid (RNA/peptide) interactions allowed it do so. These interactions occurring during pre-biological evolution would have been selected for molecular functionality, as exemplified by “4, NH3, H2O and H2S 2+ ions [2+ ions , however2+ ions , the min2+ ions  wetting-an form , taking into account the catalytic effects of GlyGly [Of course, such an interaction is a far cry from f GlyGly   can be envisaged more easily. We will discuss some remarkable issues concerning this early evolution of the RNA world next.Thus, what we mean with “coevolutionary” is that evolutionary lengthening of peptides and nucleic acids happened simultaneously. Such “mutual assistance” is also already mentioned in an article by Carter and Kraut , howeverAmino acids are all around. They are produced in outer space and rain upon earth with meteorites . They arAbundant presence of amino acids on earth is one thing, peptide formation quite another. In this context, especially the work of Rode and colleagues seems to be enlightening. Some dipeptide formation can occur by comet impact . The cat+ ions essential for the SIPF reaction) could have functioned as a conservation pocket for RNA molecules washed into montmorillonite-bottomed pools. We touch upon a well-known general problem here: The fact that specific parts of scenarios invoked to explain the origin of life can only function in radically different milieus, with completely different chemical parameters. However, prebiotic earth probably was very rich in quite different microenvironments with many points of contact. Such frontiers must have been essential during the development of life. For a more general discussion of these aspects, see [Above we mentioned the possible role of (aspartate-containing) peptides in protecting RNA from breakdown catalyzed by magnesium ions . This obcts, see .selection functioning at the level of individual molecules at this stage. Presumably “collective evolution” [communities of molecules. A possible example of influence “in trans”: We start out with the GlyGly producing RNA benefiting from the protection by Asp-containing oligopeptides that are able to sequester Mg2+ ions. These Asp-containing oligopeptides are in turn produced by GlyGly. How other RNAs and (di)peptides would influence further evolution we can only speculate about. A possible scenario will have slowly developing communities of ribozymes, amino acids and small peptides in which more complex mutual dependencies could evolve. Thus we would get much more variation in molecular communities which would enhance the speed of evolution.We now come at a crucial and, we have to admit, somewhat theoretical juncture: GlyGly production from glycine should have been a pretty simple function for RNA to come up with, if it improved survival of the RNA, and thus would have been selected for. To put it succinctly: RNA produces GlyGly; GlyGly produces Aspartic acid-containing oligopeptides; Aspartic acid-containing oligopeptides protect RNA. In this way, a positive feedback loop creating RNA could have arisen. Thus, coevolution is illustrated by the presumption that RNAs could not persist without peptide protection, that very short (very early) peptides were made more abundant by RNA producing them, and that they co-evolve forming longer RNAs and peptides. This would constitute an RNA/peptide world of ribozymes and short oligopeptides. These oligopeptides had RNA protection functions  of the ribosome is orienting this amino group such that it can efficiently attack the peptide-tRNA connection of the peptidyl-tRNA, during protein synthesis. In principle one would expect that montmorillonite also has the potential to align two loaded RNAs in the correct orientation such that peptide bond formation could occur. The conformational change of the tRNA upon release of the amino acid could be crucial in making the process directional. Such a scenario would couple the dynamic nature of the tRNA , with tnslation . Possiblspecifically loaded by Glycine, at least in environments containing ValAsp and activated Glycine. ValAsp could thus be seen as the appearance of the first “protein” aminoacyl tRNA synthetase (aaRS) with the first anticodon of the genetic code (GCC). The “synthetase” is of course still non-specific: The “tRNA” itself is performing the specific selection of amino acid. If one of the short RNA stem-loops fitted into the combined montmorillonite phosphotransferase system, the amount of Glycine entering oligomerization could be boosted. This would further increase concentrations of, e.g., GlyGly and AspGlyAsp.Twenty years ago, Shimizu reported that an RNA stem-loop (the “C4N structure”), with two dangling ends, a 5' GCC terminus and a 3' UCCA terminus, was loaded specifically with Glycine, in the presence of ValAsp and activated amino acids . FurtherIt is strange to notice that follow-up experiments  seem not to have been performed, as far as we know. This becomes even more puzzling when we consider the momentous step the Shimuzu experiments seem to illuminate: The possible origin of coded synthesis.Nanoarchaeum equitans, which he thinks reflects the primordial configuration [Cyanidioschyzon merolae, as ancestral [In a string of publications, Di Giulio defended his remarkable suggestion that tRNAs originated from the duplication of a “hairpin gene” ,47,48,49guration . He alsoncestral . Howeverncestral .We could thus envisage two relatively small hairpin sequences basepair, forming a primitive tRNA precursor. If we begin with two “Shimizu” RNA stem loop molecules (C4N structures), each with a 5' anticodon-stem loop stem-discriminator nucleotide-CCA 3' structure see  and allo(1)Chlamydomonas reinhardtii mitochondrial DNA [Much less extensive RNA molecules are necessary than was thought previously to produce rather large RNA structures. Base pairing could lead to something resembling a primitive tRNA starting out with relatively small RNA molecules. The same general point  of course holds true for rRNA. That this arrangement can work is nicely illustrated by the mitochondrial ribosomal RNA, which is now encoded in scrambled gene pieces in rial DNA . This co(2)both ends had an anticodon! The anticodon close to the acceptor side chooses Glycine, Alanine, or Valine. The anticodon in the middle becomes available for mRNA.The scenario described could explain the perplexing fact that originally the anticodon should be close to the CCA acceptor stem to allow acylation with the correct amino acid while it is now only found at the other side of the tRNA: In the beginning (3)not coincide with the two original regions of the “Di Giulio dimer”, as can be clearly seen in This scenario should be distinguished from another popular model regarding tRNA evolution: The one in which “tRNA” started out as a small CCA acceptor only, which would be aminoacylated according to the second genetic code, with the current genetic code appearing at a later stage upon extension of the acceptor minihelix with an anticodon stem-loop . It shoet al. [et al. showed that 120–130 residue fragments of aaRSs of both Class I and Class II  already catalyse tRNA acylation at a high rate (both urzymes function at ~60% of present-day efficiencies) [Up till now we have envisaged coordination occurring on montmorillonite, with a hypothetical community of four different tRNAs, several mRNAs and some very short peptides made up of Glycine, Alanine, Aspartic acid and/or Valine collaborating to achieve a highly primitive and presumably very inefficient, but coded (!) protein synthesis. GlyGly is most likely produced rather unambiguously. ValAsp, however, is produced in a less straightforward way, because the tRNA with a middle-U anticodon is loaded with different kinds of amino acid depending on their relative amino acid concentrations. Two ways are available to achieve a higher abundance of Aspartic acid with the middle-U loading: (1) enhancing metabolism transforming Glycine, Alanine and Valine into Aspartic acid ; (2) producing the first different aaRSs (variations of the peptides present in our scenario). Such “variations of Glycine, Alanine, Valine and/or Aspartic acid peptide synthetases” would first catalyze  unselective tRNA loading, but then selective middle-U tRNA loading with acidic amino acids would have to develop. In this respect it is telling that Rodin and Ohno suggested that aaRSs originated as couples of core synthetase proteins, coded sense and anti-sense on the same nucleic acid double strand . Ribas det al.  showed hiencies) . Thus suet al. suggest [Fox  suggest  that witde novo”) and translocation capacities seem to have been progressively built up, while also extending the exit tunnel. The core, representing an early time point, seems almost devoid of secondary structural elements such as protein beta-sheets and alpha-helices and RNA basepair helices. Ribosomal protein components near the PTC are indicative of peptide ancestors of ribosomal proteins. Length restrictions did not allow them protein secondary structure, which is almost absent from the PTC-origin. However, such structures steadily increase over time. The evolution of the early PTC also made use of Mg2+ ions (but see below) for coordination of partially single-stranded RNA species, which are replaced by proteins in periphery. The fact that we do not find “paleontological traces” of amino acids or tiny peptides in the center does not mean they were not involved in protection against breakdown (see previous paragraphs) or assisting folding of the “precursor” RNA. However, it does strongly support the notion that catalysis of peptide formation is achieved by RNA only. The role of amino acids and di- or tripeptides would thus have been restricted to protection of this primordial RNA against breakdown. This is still essential, however, as the presence of Mg2+ ions is a mixed blessing. These ions cannot only coordinate RNA, but can also catalyze hydrolysis [First of all, we seem to need quite large rRNA-like molecules to build such a cavity, while peptide catalysts still seem to be limited to relatively small oligopeptides. Secondly (see below), when looking at reconstructions of the ancient PTC, we seem to find absolutely no traces of the involvement of small peptides or amino acids here ,64,65. Sdrolysis , unless drolysis  or shortdrolysis ,67. Thisdrolysis  these “a2+ to protect the RNAs from breakdown. Sequence comparison shows it to be the sequence, which evolved (upon extension in the “PTC-tunnel core”) to replicate RNA (probably in cooperation with RNA and Mg2+). AspGlyAsp evolves into AspAlaAspValAspGlyAsp, (DADVDGD), being the obvious ancestor sequence of the universal RNA polymerase active site sequence NADFDGD. Of this sequence, the three aspartic acids interspaced by a bulky residue and a glycine seem to be a universal RNA polymerase signature, e.g., of both the enzyme that produces mRNA with DNA as the template as well as of the enzyme that makes microRNAs with RNA as template. The pentapeptide DVDGD or its heptapeptide extension on their own are without known catalytic polymerase activity [2+. It is highly likely that this sequence formed a crucial part of the oldest RNA replicases, whether they were more based on RNA or on protein for their remainder. Such interactions, where “acid amino acid rich” peptides or even simpler di- and tri-carboxylic acids would coordinate Mg2+ to protect RNA, minimize wobble mismatch formation, and in general to enhance chances of some kind of RNA replication to evolve, have most likely been crucial in early evolution [We have arrived at a juncture where  coded limited peptide synthesis seems up and running. If these small peptides could boost RNA synthesis, the precursors for present-day mRNA molecules could be generated. The last of the RNA trinity  could thus come about. The ancient, crucial, tripeptide AspGlyAsp most likely chelates Mgactivity . In crysvolution .2+. The ribozyme world evolves in this context: Reigning peptides are made up of Glycine, Alanine, Aspartic acid and Valine. Despite being very small, they  already do have rudimentary enzymatic functions [We are now in a position to describe a possible scenario for the first stages in genetic code development. We start out with stem-loop tRNA predecessors that can be charged by whatever amino acid is present, most likely mainly Glycine, Alanine, Aspartic acid and Valine. In this situation, tRNA-tRNA interactions can produce dipeptides, among which GlyGly would predominate. The presence of “Shimizu-like” C4N structures would allow even further increases in the amount of GlyGly dipeptides. Their presence would increase the chances of the development of small ribozymes such as the precursor of the large rRNA. Variations in C4N structures would give the possibility to expand the number of specifically loaded tRNAs to three next: Alanine and Valine would join Glycine. The fourth variant tRNA precursor is still incorporating activated amino acids non-selectively. Among these, Aspartic acid will be of major importance. During this development small RNA stem-loop molecules interact with each other to produce cloverleaf tRNA precursors (according to Di Giulio’s major insight). This produces an anticodon at the “right side” , and expunctions ,18,36. Irelease factors emerged. Originally the tRNA  could catalyze “termination” itself . Such termination of the peptidyl-tRNA by hydrolysis of the ester bond would occur in the precursor of the P-site. This mechanism has been mentioned by Noller [every unloaded tRNA  had the ability to terminate further peptide bond formation. In this way, peptide synthesis was aborted in case of amino acid shortage. Later a specialized tRNA would be able to catalyze termination, the one with anticodon CUA being the obvious candidate. This, however, is in a rather advanced stage, when more amino acids are in the repertoire than just Glycine, Alanine, Aspartic acid and Valine. We find it quite amazing that the possible RNA activity in termination is not getting more attention in the research community, as illustrated by the paucity of citations of articles reporting termination activity of A-site unloaded tRNAs.At the stage just described, water, the availability of loaded tRNA precursors and “rRNA” were the only factors involved in termination, giving very limited options for improvements in both speed and specificity of the process. At a later stage y Noller  citing [y Noller ,70,71,72We have suggested an intricate relation between the world of small RNAs and tiny “preproteins” , the emerging ribosome, and the earliest synthetases that replaced ValAsp and “ribozyme synthetases” in the emerging translational system. For Glycine, Alanine, Aspartic acid and Valine, ribozymatic mechanisms existed to load the tRNAs. However, one has to think in terms of “Shimizu interactions” as described above (and of which the model studies should be urgently revisited and extended), and feedback mechanisms, such as “soak-it in Aspartic acid” strategies. Long ribozymes doing complex things were not a part of the early living world. Among the first functions possibly taken up by preproteins were peptide production (GlyGly), aminoacylation , RNA replication (AspGlyAsp), ribosome formation (r-preproteins), and more specific aminoacylation (Class 1 and Class 2 synthetase couples). During the era of coupled synthetases, three of such couples emerged: The couple distinguishing aliphatic, hydrophobic  from small ; the couple doing the same for Glutamic acid and its counterpart Aspartic acid; and the couple handling Tyrosine and Phenylalanine. These couples of course coincide with the synthetase subclasses. The last couple, however, emerges after the “Glycine, Alanine, Aspartic acid and Valine world” has been replaced by a more diverse protein world. These later stages of the history of life do not fall within the scope of this article. The original coupled function of synthetases has left its mark on the structure of the genetic code . One of our conclusions regarding the early RNA world must be: Long complicated ribozymes performing highly complex tasks were probably not part of the early living world .is found in all domains of life. It is indirectly involved in translation. tmRNA, which rescues stalled ribosomes and thus is directly involved in translation, is only found in eubacteria and some mitochondria [is found in all three domains. Its universality in living forms reminds us of the fact that translation probably began to evolve in the presence of membranes. Lipid (like) membranes already segregated the early biochemistry of life. Membranes were instrumental in maintaining and protecting high concentrations of precursors and products. Again, this function could be a mixed blessing, as dead end products or interfering molecules could also be trapped. As such, membranes remind us of the microenvironments at the interphase of different earth systems discussed above, which have to concentrate and combine while retaining flexibility to allow further processes. The “flexibility” of membranes in this era had to come from non-protein channels, possibly consisting of polyhydroxybutyrates and/or inorganic polyphosphates [In our article we did not discuss some very important other aspects of early evolution. In support of our considerations we find that of all the RNA species found in living organisms today, only the ones directly involved in translation (the process intimately linking amino acids and RNA from early on in evolution) are consistently found highly conserved in all three domains of life: tRNA, rRNA, and mRNA. Practically all other RNA molecules, e.g., involved in posttranscriptional processing, regulation and DNA replication are not found in all domains ,74. Therchondria ,76. SRP osphates . Here we


Constant efforts have been made to develop new method to realize sequence-specific RNA degradation, which could cause inhibition of the expression of targeted gene. Herein, by using an unmodified short DNA oligonucleotide for sequence recognition and endogenic small molecue, vitamin B2 (riboflavin) as photosensitizer, we report a simple strategy to realize the sequence-specific photocleavage of targeted RNA. The DNA strand is complimentary to the target sequence to form DNA/RNA duplex containing a G•U wobble in the middle. The cleavage reaction goes through oxidative elimination mechanism at the nucleoside downstream of U of the G•U wobble in duplex to obtain unnatural RNA terminal, and the whole process is under tight control by using light as switch, which means the cleavage could be carried out according to specific spatial and temporal requirements. The biocompatibility of this method makes the DNA strand in combination with riboflavin a promising molecular tool for RNA manipulation. Previous research focused on gene suppression strategies through RNA degradation involving antisense oligonucleotides and artificial ribonucleases12349Dilara Crate and Charles Wilson reported a site-specific inactivation of RNA transcripts through Laser-induced hydrolysis of the malachite green (MG)-binding RNA, which extend the RNA cleavage into a new scenario, namely under tight spatial and temporal controlin vitro selection1819Riboflavin, as the most efficient natural photoshensitizer, has been shown to induce photooxidative DNA damage via the generation of an 8-hydroxydeoxyguanosine intermediate14151632P-labeled RNA target with 26 nt in length was incubated with the DNA sequence in presence 200 μM of riboflavin at 25 °C. After 4 h of irradiation with 45 W household light, a clear cleaving fragment with 17 nt in length was observed based on the PAGE gel analysis. Meanwhile, no cleavage could be observed when the DNA strand, riboflavin or light irradiation was absent in the cleaving reaction . However, the catalytic activity of the DNA strand strongly depended on the concentration of riboflavin (oC (data no shown). Under optimal condition, the DNA strand in combination with riboflavin catalyzed the photocleavage of target RNA with a kobs as high as 0.25 h−1, the half life being around 6 hours, which is much faster than most of reported artificial ribonucleasesUYX for this strategy has been carefully studied (X located at the 5′ of the uracil of the G•U base pair could affect the cleavage efficiency of RNA substrates (G ≈ A > C > U). The nucleoside Y RNA in substrate that will be removed from 3′ of U revealed the tread of C > U > A > G in terms of cleavage efficiency (The boflavin . The phoboflavin . Moreove studied . The nucficiency . The RNAficiency .32P-labeled RNA marker (17 nt), revealing the presence of one more phosphate group at the 3′-end  that encodes the same sequence of RNA target at 5′end was applied to self-cleavage under the same conditions. Both the intramolecular and intermolecular reaction provided the cleaved products with the same length  in the middle of mircoRNA21 was selected as the cleaving site, and the DNA strand was designed to complement to the target sequence except for the formation of a G•U wobble . As showTo further confirm the DNA stand in combination with riboflavin as tool to hinder the RNA function through cleavage, RNA mimic of GFP (Green Fluorescent Protein) was chosen as the object of study. Recently, Jaffrey group has described a complex of RNA aptamer (24–2) and a derivative of the GFP fluorophore, 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), termed as “Spinach” that emits fluorescence upon transformation in specific conformationin vivo. (4) Unlike antisense oligonucleotide, the DNA strand in our method could interfere with the function of RNA through the irreversible photocleavge, forming cleaved fragments with unnatural 3′ -phosphate RNA terminal.The established sequence-specific photocleavge of RNA described here appears to have several promising features to become a new molecular tool for research and application as follows: (1) the designation of DNA strand to realize the seqence specific cleavage of RNA target is very easy to fulfill only if there are uracil nucleotides in the RNA sequence and the DNA strand is complementary to the target sequence expect to form a G•U wobble in between. (2) Though the minimal size of DNA strand is 13 nt in length, high sequence specificity could be achieved by increase the length of DNA stand to form the longer DNA/RNA duplex with target. The efficiency of the cleavage reaction based on the DNA strand in combination with riboflavin is very high. And the cleaving specificity and efficiency could be further improved by introducing more strands to cleave the same RNA at multiplex cites. (3) The cleavage of RNA by DNA strand is under tight control by using light as switch, which means the cleavage could be carried out according to specific spatial and temporal requirements. Moreover, the photosensitizer, riboflavin (vitamin B2), is endogenic coenzyme existing in the cell of most living creature, which makes this method possible to apply in vitro, which was proved to proceed through the cleavage reaction other than the hybridization with target. The biocompatibility of this method makes the DNA strand in combination with riboflavin a promising manipulable molecular tool in vivo. The direct conjunction the DNA strand with riboflavin and the application our method in the cell experiment is underway in our lab.In summary, we developed a new RNA-cleaving DNA strand that catalyzes the sequence-specific photo-induced cleavage of RNA at the 3′ of the uracil of G•U wobble base pair in the presence of rioflavin as cofactors. Unlike most artificial ribonuclease that need conjugate functional chemical group to DNA sequence to fulfill the cleavage of RNA molecules specifically, the DNA strand described here is unmodified DNA sequence, cooperating with riboflavin to realize the photocleavage of RNA target through oxidative elimination mechanism. This method has broad substrate scope if the targeted RNA contains required uracil nucleotide that has high frequency of one repeat in every 4 bp. The function of RNA molecules were successfully hinder by photocleavage based on our method How to cite this article: Zhao, Y. et al. Sequence-specific RNA Photocleavage by Single-stranded DNA in Presence of Riboflavin. Sci. Rep.5, 15039; doi: 10.1038/srep15039 (2015).


Conventional RNA polymerase II transcription assays are based on radioactive labelling of the newly synthesized RNA. Due to the inefficient in vitro transcription, the detection of the RNA involving purification and gel electrophoresis is laborious and not always quantitative.Many studies of the eukaryotic transcription mechanism and its regulation rely on in vitro transcription assay that has been established in our laboratory. Upon transcription, the newly synthesized RNA is directly detected and quantified using the QuantiGene assay. Alternatively, the RNA can be purified and a primer extension followed by PCR detection or qPCR quantification can be performed. When applied to assess the activity of RNA polymerase II inhibitors, this new method allowed an accurate estimation of their relative potency.Herein, we describe a new, non-radioactive, robust and reproducible eukaryotic in vitro transcription assay that allows for sensitive detection and precise quantification of the newly transcribed, unlabelled RNA and is particularly useful for quantification of strong transcriptional inhibitors like α-amanitin. Moreover, the method can be easily adapted to quantify the reaction yield and the transcription efficiency of other eukaryotic in vitro systems, thus providing a complementary tool for the field of transcriptional research.Our novel assay provides a non-radioactive alternative to a standard  The control of transcription plays herein a key role, and research is still ongoing to decipher its mechanisms. Many of these studies rely on n assays -3.in vitro polymerase II transcription run-off assay [Effective transcription is especially critical for transformed cells . Since iff assay ,12.32P-CTP to the reaction mixture. After the transcription reaction, the RNA has to be detected and/or quantified. Most commonly, the RNA is purified by phenol-chloroform extraction and ethanol precipitation. An RNA gel electrophoresis is performed and the labeled RNA detected by autoradiography [For a typical polymerase II run-off reaction, a well-defined nuclear extract providing the RNA polymerase II and a basal set of transcription factors are used together with a linear DNA template containing the desired promoter. The newly synthesized RNA is radioactively labeled by adding a iography ,2. Alteriography . After rHowever, these methods are difficult to use for an unexperienced laboratory. Since the synthesized amounts are minute, reproducible and quantitative RNA recovery during purification is challenging and strongly depends on the experience of the laboratory staff. Quantification of the newly synthesized RNA requires a phosphor-imaging device. Moreover, because of the laborious and time-consuming procedure, studies involving high numbers of probes are difficult.in vitro transcription assay that relies on a commercially available eukaryotic transcription kit and quantitative PCR RNA detection. Moreover, the method was further optimized by using a novel hybridization method for RNA detection and quantification. With this optimized detection the purification step can be avoided so that the method can be used for the concomitant analysis of a considerable number of samples including replicates. We applied both new methods to quantify the activity of the strong RNA polymerase II direct inhibitor α-amanitin vs. natural, synthetic and semisynthetic amanitin-derivatives  was directly used as a template. The reactions were set up using the HeLa Scribe kit without adding radioactive nucleotides. To assess transcription inhibition, α-amanitin or other compounds were added in various concentrations to the reaction mix. A transcription reaction mixture that did not include NTPs served as a negative control, since it contained the same amount of template DNA and would thus monitor DNA interference. After transcription, the newly synthesized RNA was purified and DNase digested. For RNA detection, a primer pair was designed and used in a reverse transcription plus PCR approach to amplify a 295 bp long DNA stretch within the first 400 bp of the EGFP transcript  values , HDP30.841  and HDP30.890 , sharing the α-amanitin structure but with the natural amino acid 3 (aa3) di-hydroxy-isoleucine replaced by different synthetic phenyl-glycine derivatives, showed 150-1000-fold weaker inhibitory activity than α-amanitin (IC50 = 2.1 μM). The α-amanitin-analog HDP30.0931 .When applied to assess the inhibitory activity of several α-amanitin analogs vs. α-amanitin, this optimized method allowed for a quantitative estimation of the inhibitory potency using the inhibition concentration 50  A clear RNA concentration dependency of the signal was seen for both the purified RNA and the unpurified reaction mixture dilutions Figure A. Moreovin vitro transcribed RNA from a set of reactions containing serial α-amanitin dilutions. For qPCR quantification, half of the reaction mix was subject to RNA purification. Three different series were used to the QuantiGene detection: an aliquot of the purified RNA, a dilution prepared directly from the reaction mix and the same reaction mix dilution that was digested by DNase prior QuantiGene assessment. As expected, almost identical inhibition curves were obtained when the purified RNA was assessed by the QuantiGene or qPCR methods. Most importantly, the inhibition curves derived from the reaction mix samples also showed a clear concentration dependency and did not differ significantly from the curves achieved using the purified RNA, regardless of the DNase digestion step , HDP30.0378 (IC50 = 180 μM) and HDP30.0516 (IC50 = 629 μM), were 30-100-fold lower than of α-amanitin (IC50 = 5.7 μM), demonstrating that all these compounds could still bind to the RNA polymerase II despite methylation and substitution by linker, and that an optimum of activity could be achieved when using a linker with a length of 6C atoms.This method was thus applied to compare the inhibitory activity of α-amanitin and α-amanitin-derivatives in a series of s Figure C. In this Figure F, respecTo assess for transcription efficiency in the absence of TBP activity, a template lacking the TATA box was used, in which the TATATA sequence had been substituted by the TAGCTA sequence .50 inhibition values thus depend on the concentration of RNA polymerase core subunit. On the other side, by binding to P-TEFb, flavopiridol inhibits the phosphorylation of the RNA polymerase core subunit. Slower transcription elongation at 20–50 nucleotides per min, however, occurs even in the absence of CTD phosphorylation and the resulting transcripts are therefore shorter [In another set of experiments, the activity of flavopiridol was assessed, an alternative inhibitor of transcription elongation. Flavopiridol acts on transcription by stoichiometric binding to P-TEFb, a protein kinase composed of the cyclin-dependent kinase 9 (cdk9) and a cyclin subunit, which regulates transcription elongation by phosphorylating the C-terminal domain (CTD) of the large subunit of the RNA polymerase II ,23. As e shorter . The quain vitro transcription reactions in comparison to the wild-type template. Using the QuantiGene quantification method a transcription yield of ca. 75% of the wilde-type was observed for the TATA mutant template. An intact TATA box determines the transcription start position by providing the binding site for TBP but is not strictly required for human transcription to occur. The mutation can cause a shift of the start of transcription site to one or multiple weaker ones, as well as influence the transcription efficacy [But apart from a high-throughput assessment of transcription inhibitors, the new methods can be employed for a large variety of transcription-related applications, since the transcript sequence can be combined with any desirable promoter. As an example, a DNA template containing a mutated TATA box was used for efficacy . Our resin vitro transcription experiments, since the ultrasensitive RNA detection compensates for the low initiation efficacy of such in vitro transcription assays. However, determining the composition of the transcripts would be required before determining potential HTS set-up.Moreover, provided that the composition of the transcripts has been previously analyzed to ensure an accurate interpretation of the results, the QuantiGene detection can be easily adapted for the detection and quantification of any specific transcript. The transcription reaction composition can be changed so that the effect of specific factors can be independently and quantitatively assessed. By using immobilized DNA templates, pre-initiation complexes can be assembled, isolated and their translational activity further investigated. The immobilized linear DNA can be processed to reconstitute a more physiological nucleosomal template for in vitro transcription. We showed that a simple qualitative PCR detection of the RNA transcript can be used after a run-off assay to avoid radioactive labeling. Moreover, the optimized qPCR detection method allows for unprecedented precise quantification of the transcription efficacy. When using the QuantiGene detection, the laborious RNA purification can be avoided and a large number of samples processed simultaneously.In conclusion, on the basis of a commercially available kit we have established a relatively simple, robust and reproducible non-radioactive method for RNA quantification after eukaryotic CMV-EGFP-frw and CMV-EGFP-rev primers. The positive control DNA  was used as a template for the PCR-amplification of the 1182 bp linear DNA fragment HS-DNA using the HELA NUCLEAR FW and HELA NUCLEAR RV primers , the respective 5′-biotinilated forward primers were used. Bead immobilization was performed as recommended by the manufacturer at 0.5 μg DNA per 100 μg beads and stored at 4°C in the binding buffer. The binding efficacy of about 80% resulted in a DNA concentration of 0.4 ng/100 g beads. Prior to transcription reaction setup, the required amount of loaded beads was transferred to a new tube and washed there times with in vitro transcription buffer.The pEGFP-N1 plasmid  was used as a template for the amplification of the 1136 bp linear DNA fragment CMV-EGFP using the EGFPrev1 for CMV-EGFP and HNqPCRrev1 for HS-DNA. For Quantigene detection, the customized oligonucleotide mix designed by the manufacturer based on the sequence of the transcribed region of the HS-DNA template.Two primer pairs were designed for PCR and qPCR detection on the CMV-EGFP – derived RNA/cDNA, six for the detection of the HS-DNA transcription product, of which only the two sets showing satisfactory results are shown in Table Flavopiridol was obtained from Sigma-Aldrich. α-Amanitin Figure A was obtThe semisynthetic linker derivatives HDP30.0378, HDP 30.0516 and HDP 30.0592  corresponding to 22 μl minus the nuclear extract volume, MgCl2 to a final concentration of 3 mM, 40 Units RNase inhibitor, DNA template  and the nuclear extract following the manufacturer’s recipe. Aliquots of the master mix were transferred into reaction tubes containing different α-amanitin concentrations or water and incubated for 20 min at room temperature. Standard transcription reactions containing no α-amanitin were referred as ‘positive’, reactions containing α-amanitin or other inhibitors as ’inhibited’. A standard reaction containing no α-amanitin/inhibitors but also no NTPs was used as a ‘negative control transcription’. The reactions were started by the addition of the NTP mix to a final concentration of 400 μM each, except for the negative control where water was added. After incubating for 30 min at 30°C, the reactions were stopped by diluting with 400 μl of the RLT buffer from the Qiagen RNeasy Micro kit and stored at −80°C until proceeding with RNeasy cleanup. This was performed as recommended by the manufacturer, including on-column DNase treatment but without the addition of carrier RNA. The RNA was eluted using 15 μl water and stored at −80°C.Transcription reactions were set up as using the Promega HeLaScribe Nuclear Extract EGFPrev1 primer . For reverse transcription with the Qiagen Sensiscript RT kit, 2 μl of the digestion reaction and 1 μM EGFP-LNA1 at 250 nM, same primer set as well as an alternative primer set  or HNrev1 (Bead-HS-DNA template) primers were used. After reverse transcription, 15 μl qPCR master mix aliquots were added directly to the reverse transcription mixtures and the amplification was performed as described. For the Bead-CMV-EGFP - derived RNA the primer-probe-mix EGFP1  was used, for the Bead-HS-DNA - derived RNA the primer-probe-mix H1 . A qPCR standard curve was prepared from the respective template DNA and used to calculate the RNA copy number in the samples. By relating the RNA copy numbers in the inhibited reactions to the copy number in the positive transcription reactions, a percent transcription inhibition was calculated for each compound and concentration and an inhibition curve plotted. A sigmoidal dose–response curve fit was used to calculate the compound concentrations that inhibited RNA synthesis by 50% .After reaction, the beads were spun down and 20 μl of the supernatants were diluted into the RLT buffer of the Qiagen RNeasy Mini kit and stored at −80°C until proceeding to the RNA cleanup. The RNA was eluted from the RNeasy mini-columns with 2×30 μl water and subsequently dried using a speed-vac. DNase digestion and reverse transcription steps were performed as described above with some modifications. The dried RNA was directly taken up into 10 μl DNase digestion mix. For reverse transcription in a final volume of 10 μl using 3 μl of the DNase-digested RNA, the 2+ that would interfere with the QuantiGene capture step. Alternatively, a dilution series was prepared from the purified RNA, starting at 1:100 unto 1:100,000. Both dilution series were assessed using the QuantiGene 2.0 System (Affimetrix) following the instructions provided by the manufacturer. The negative control transcription reaction products were only assessed as a 1:2 dilution for the unpurified negative reaction mix or 1: 10 dilution for the DNase-digested RNA purified from the negative control transcription reaction.Positive and negative control transcription reactions using the bead-immobilized Bead-HS-DNA template were set up as duplicates as described above in a final volume of 100 μl per reaction. After transcription, 50 μl of the reaction mixture were used for RNA cleanup. The copy number of synthesized RNA was quantitated using the established primer extension and qPCR method. From this number, the RNA copy number in the reaction mixture was estimated. Using the rest of the reaction mixture of the positive reactions, a dilution series was prepared starting at 1:10 unto 1:10,000. 0.3 mM EDTA was added to the first dilution to bind MgFor the capture step, the working reagent was prepared according to the recipe provided in the ‘Capturing Target RNA from Fresh, Frozen, or FFPE Tissue Homogenates’ section of the QuantiGene Handbook. 60 μl of working reagent were distributed into the QuantiGene plate and 40 μl of reaction mix or RNA dilution were added. The subsequent hybridization, signal amplification and detection steps were performed as described in the manual. Finally, the chemoluminescence signals were measured using a FLUOstar Optima chemoluminometer (BMG LABtech) and were plotted against the estimated RNA copy number.50 values of the tested compounds.For further experiments, positive, inhibited and negative control transcription reactions were set up in 25 μl as duplicates covering a complete range of α-amanitin or compound concentrations. After transcription, 20 μl of the bead supernatant was used for RNA cleanup followed by the optimized primer extension and qPCR quantification method. 3 μl of the reaction mix were diluted with 97 μl water. 4 μl of this dilution were once more diluted with 45 μl 0.3 mM EDTA containing RNase-inhibitor (2U/μl). Alternatively, 36 μl DNase Zero master mix containing buffer, RNase inhibitor and enzyme were added to 4 μl of the reaction mix dilution and DNase digestion was performed for 15 min at 37°C. The digestion was stopped by the addition of 5 μl stop solution (EDTA) and 10 min heating at 65°C. A blank DNase digestion with water was used for the Qiagen background sample. For QuantiGene quantification, 40 μl of the purified RNA, the twice diluted reaction mix or the digested diluted reaction mix were added to 60 μl working reagent in the QuantiGene capture plate. Upon chemoluminescence detection, the background signals were subtracted from the signals of the positive or inhibited or negative control transcription samples. For each sample type, reaction mixture, digested reaction mixture and purified RNA, the negative control sample signals were averaged and subtracted from the signals of the respective positive and inhibited samples. A percent inhibition was calculated by relating the signals of the inhibited reactions to the signal derived from the positive transcription reactions, which were set to 100%. A sigmoidal dose–response curve fit of the inhibition curves was used to calculate the ICCV and JA developed the concept and co-wrote the manuscript; CV and BS performed most experiments; SW-S, CL and WS performed the chemical synthesis of the derivatives; JA supervised the project. All authors read and approved the final manuscript.HeLaScribe Positive Control DNA Sequence. DNA sequence information for the DNA template ‘HeLa Nuclear Extract Positive Control DNA’.Click here for fileTemplate comparison. qPCR amplification curves of the data presented in Table Click here for fileDetailed Protocol. Detailed protocol for non-radioactive eukaryotic in vitro transcription and RNA quantification.Click here for fileAlignment of the HS-DNA, HS-DNA_mut and HS-DNA_long sequences. HS-DNA_mut is a mutant of the standard HS-DNA template containing a mutated TATA box. HS-DNA_long is a template similar to the standard HS-DNA template but including additional 98 bp within the run-off transcript (and primer extension/qPCR product) sequence. The sequence alignment highlights the differences between the three templates.Click here for file


GSE48084).Standard RNA expression profiling methods rely on enrichment steps for specific RNA classes, thereby not detecting all RNA species. For example, small and large RNAs from the same sample cannot be sequenced in a single sequence run. We designed RNAome sequencing, which is a strand-specific method to determine the expression of small and large RNAs from ribosomal RNA-depleted total RNA in a single sequence run. RNAome sequencing quantitatively preserves all RNA classes. This characteristic allows comparisons between RNA classes, thereby facilitating relationships between different RNA classes. Here, we describe in detail the experimental procedure associated with RNAome sequencing published by Derks and colleagues in RNA Biology (2015) [1]. We also provide the R code for the developed Total Rna Analysis Pipeline (TRAP), an algorithm to analyze RNAome sequencing datasets (deposited at the Gene Expression Omnibus data repository, accession number  Concentrated rRNA-depleted samples were fragmented by sonication , resulting in fragments smaller than 500 nucleotides. Agilent 2100 Bioanalyzer analysis shows that rRNA depletion and fragmentation of the rRNA-depleted RNA were highly reproducible . The excGSE48084.Pooled cDNA libraries consisted of equal concentration bar-coded samples, i.e., 3 mock- and 3 cisplatin-treated samples and were sequenced in 2 lanes, 36 bp single read on the Illumina HiSeq2000. This yielded on average 52 million sequence reads per sample. The dataset has been deposited at the Gene Expression Omnibus database, The analysis of the sequencing dataset was performed with TRAP, which refers to Total RNA Analysis Pipeline . The anahttp://rna-ome.erasmusmc.nl/). Note that TRAP can also be used for standard poly(A)+-sequencing, standard small RNA sequencing.Next, TRAP can be used to analyze reads smaller than 36 nucleotides B. In modThe Netherlands Toxicogenomics Center (NTC), and National Institute of Health (NIH)/National Institute of Aging (NIA) (1PO1 AG-17242-02), NIEHS (1UO1 ES011044).This work was supported by


Diabetic patients’ lifestyle adaptations to improve glycaemic control are not always followed by improvements in self-rated general health (SRH). The perceived impact of diabetes on patients’ daily lives may influence changes in their SRH. This paper examines the association of illness severity, treatment, behavioural, and coping-related factors with changes in SRH from diagnosis of type 2 diabetes until one year later, in a population-based sample of 599 patients aged 40 years or over who were treated in general practice.Change in SRH was estimated by a cumulative probit model with the inclusion of covariates related to SRH .At diagnosis, 11.6% of patients reported very good, 35.1% good, 44.6% fair and 8.5% poor SRH. Physical inactivity, many diabetes-related symptoms, and cardiovascular disease were related to lower SRH ratings. On average SRH improved by 0.46  during the first year after diagnosis without inclusion of covariates. Mental and practical illness burden was the only factor associated with change in SRH, independent of patients’ diabetes severity and medical treatment . Compared to otherwise similar patients without illness burden, increase in SRH was marginally smaller among patients who expressed minor illness burden, but much smaller among patients with more pronounced illness burden.Much as one would expect, many patients increased their SRH during the first year after diabetes diagnosis. This increase in SRH was not associated with indicators of illness severity or factors reflecting socio-demographic circumstances, but patients experiencing illness burden had a smaller increase than those who reported no illness burden. We suggest that during the diabetes consultation, general practitioners explore further how patients manage their illness burden. We further suggest that diabetes guidelines extend their current focus on clinical and social aspects of diabetes to include questions on patient’s perceived illness burden and SRH.The online version of this article (doi:10.1186/s12889-015-1790-6) contains supplementary material, which is available to authorized users. General practitioners’ (GPs) and patients’ evaluation of the patient’s health may differ -4. The pMajor decisions about lifestyle changes and treatments are made based on characteristics measured shortly after diabetes diagnosis. It is suggested that the first year after diagnosis of T2DM is both emotionally and practically turbulent for patients -11 who hFew studies have examined change in SRH and its association with health conditions -17,35-39The aim of this article therefore is: a) to describe the change in SRH from diagnosis until one year later in patients with T2DM seen in general practice, and b) to investigate the association between changes in SRH and coping-related factors, health behaviour, social support, and treatment.This follow-up study includes the intervention group of patients who participated in the Danish randomised trial “Diabetes Care in General Practice” , and it From 1989–1992 the 243 GPs who were part of the intervention group included all newly diagnosed patients with T2DM on their patient list  , genital itching, balanitis, recurrent urinary tract infections, stomatitis, recurrent skin infections, foot ulcer, gangrene, angina pectoris, intermittent claudication) and one open category. The questionnaire was based on a literature search and interviews with experienced diabetologists. In questionnaires, patients gave information about angina pectoris, intermittent claudication, cancer disease, lifestyle, socio-demographic factors and SRH . SRH wasAt the 1-year follow-up GPs handed out a patient questionnaire called “A life with Diabetes” (no reminder was issued). The questionnaire was constructed by the study coordinator, Niels Olivarius, and an experienced sociologist, Dorte Gannik. It was based on: a) a literature review of previous qualitative and quantitative research about patients’ views on living with chronic illness; b) theories about living with a chronic illness drawing their main inspiration from the symbolic interactionist theory; c) a review of existing instruments; d) in-depth qualitative interviews with three newly diagnosed patients with T2DM (performed by Dorte Gannik), and e) discussions with health professionals. A group of people familiar with the construction of questionnaires (GPs and sociologists) reviewed the questionnaire several times. Questions were rephrased and/or removed in cases of unclear wording or nonexclusive response categories. After the questionnaire review, 15 T2DM patients tested the questionnaire, and this led to a further revision. Before the questionnaire was sent to the GPs participating in our study, it was shown again to GPs and sociologists familiar with the construction of questionnaires. They had no further comments on the wording and response categories. The questionnaire contained questions on SRH and health behaviour (the patients’ dietary habits and their indication of change in their way of living after diagnosis). In multiple response questions the patients reported whether they received the necessary support and understanding from family and significant others  and how they were coping . The responses concerning social support and coping were summarised into new categories  was defined as lack of a sense of pin prick and/or touch of cotton wool on at least one foot and/or absent patellar reflex on at least one knee. Urinary albumin concentration was used to define microalbuminuria (≥15 - <200 mg/ml) and proteinuria (≥200 mg/l). CVD was defined as history of myocardial infarction and/or history of stroke and/or angina pectoris and/or intermittent claudication and/or absent arterial pulses on both feet and/or amputation of the lower extremities. Diabetic retinopathy was defined as presence of at least one microaneurysm.A symptom score was constructed by adding together positive answers to the symptom questions. The open category was coded as “other symptoms”. Body mass index (BMI) was calculated as (weight in kg)/(height in m)First, the relation between SRH  and socio-demographic status, diagnostic HbA1c, and other factors indicating the illness severity at diagnosis Table . IllnessVariables indicating the patients’ coping strategies and social support are regarded as latent variables. To ensure that a co-variation between SRH and these variables does not reflect a common underlying latent variable, we performed a graphical Rasch analysis  conditioWith a multivariate analysis we estimated how the covariates influenced change in SRH from diagnosis until one year later. Illness severity at diagnosis may influence the variation of the covariates: patients with diabetic complications may have health behaviours which are different from those without complications, and an analysis accounted for the distribution of these factors.The expected level of the two SRH measurements was modelled using a cumulative probit model for ordinal data. One interpretation of this model is that the ordinal measure of SRH is the realization of a continuous, latent SRH, assumed to be normally distributed, with a mean that may be allowed to depend on the covariates, and a variance of one. The larger this latent SRH, the better the SRH. The expected differences in the person’s level of SRH at diagnosis were estimated using baseline information. To avoid a scenario where an effect of a covariate on the level of SRH at diagnosis was misinterpreted as an effect on the estimated change between the two time points, the covariates were also used for modelling the mean SRH at diagnosis. The expected change in SRH, defined as latent SRH one year after diagnosis minus latent SRH at diagnosis, was allowed to depend on the patients’ indication of the impact of diabetes on daily life and their evaluation of social support, indication of change in their way of living, and other variables shown to be significant for the physical condition of diabetes and life in general. . The test of whether a variable relates to change in SRH corresponds to a test of interaction with time in the cumulative probit model.A patient’s SRH at two different time points may be correlated. The analysis therefore took account of a possible correlation of measurements within one person to avoid possible incorrect conventional confidence intervals for the different parameters by using generalized estimating equation methods . The hypotheses were tested using a generalized Wald test. Hypotheses regarding the effect of ordinal, categorical variables on change in latent SRH or at the level of latent SRH at diagnosis were tested as a trend test using a model where the ordinal variable was included as a continuous variable. In the multivariate analyses we performed backwards elimination of variables based on p ≤ 0.25. The nominal statistical significance level was <0.05.At diagnosis, 11.6% of patients reported very good, 35.1% good, 44.6% fair, and 8.5% poor SRH. Only a few variables were related to SRH, but the number of symptoms increased with decreasing SRH  showed that patients on average experienced an SRH increase of 0.46 ) during the first year after diabetes diagnosis. After inclusion of covariates, only illness burden was associated with a change in SRH after backward elimination of non-significant associations  a low level of physical activity ,24,39,50Our cross-sectional analysis one year after diabetes diagnosis tallies with the results obtained from the American Centers for Disease Control and Prevention  and JacoOnly the patients’ perceived illness burden was associated with SRH change in our study. The lack of association between change in SRH and any indicator of objective health status, health practices, or utilization of health services confirms the results from an American study. This study included middle-aged people where health indicators were related to actual SRH but not to change in SRH over a 1-year period . For exaIn some studies medical treatment and lifestyle changes to accommodate to a disease have been shown to have less impact on people’s perceived health than would be expected from an evaluation of other health indicators ,58,59. TJylhä also recommends that GPs use SRH as a kind of screening tool for patients’ health status which could be interpreted together with other measurements . Good SRDuring the first year after diagnosis of T2DM SRH improves for many patients, and their perceived illness burden seems to be associated with the change in SRH. The causal direction is unknown, but the increase in SRH was lower among patients with a greater illness burden. However, change in SRH was not associated with indicators of illness severity or factors reflecting socio-demographic circumstances.The diabetes consultation gives the GP and other health professionals an opportunity to gain knowledge about how the individual patient tolerates and manages the illness burden and it can bring to light those personal health resources that may potentially minimize this burden. Current guidelines recommend that clinical and social aspects of diabetes are considered by the GP in an effort to optimize treatment. Our results indicate that it may also be relevant to extend these guidelines with simple key questions about the patient’s SRH and personal perception of the illness burden.


D that is only 5.6-fold weaker than for RNA:DNA duplexes. Most IP protocols do not pre-clear the genomic nucleic acid with RNase A to remove free RNA. Fold back of ssRNA can readily generate RNA:RNA duplexes that may bind the S9.6 antibody, and adventitious binding of RNA may also create short RNA:DNA regions. Here we investigate whether RNase A is needed to obtain reliable IP with S9.6.Long genomic R-loops in eukaryotes were first described at the immunoglobulin heavy chain locus switch regions using bisulfite sequencing and functional studies. A mouse monoclonal antibody called S9.6 has been used for immunoprecipitation (IP) to identify R-loops, based on the assumption that it is specific for RNA:DNA over other nucleic acid duplexes. However, recent work has demonstrated that a variable domain of S9.6 binds AU-rich RNA:RNA duplexes with a KAs our test locus, we chose the most well-documented site for kilobase-long mammalian genomic R-loops, the immunoglobulin heavy chain locus (IgH) class switch regions. The R-loops at this locus can be induced by using cytokines to stimulate transcription from germline transcript promoters. We tested IP using S9.6 with and without various RNase treatments. The RNase treatments included RNase H to destroy the RNA in an RNA:DNA duplex and RNase A to destroy single-stranded (ss) RNA to prevent it from binding S9.6 directly (as duplex RNA) and to prevent the ssRNA from annealing to the genome, resulting in adventitious RNA:DNA hybrids. We find that optimal detection of RNA:DNA duplexes requires removal of ssRNA using RNase A. Without RNase A treatment, known regions of R-loop formation containing RNA:DNA duplexes can not be reliably detected. With RNase A treatment, a signal can be detected over background, but only within a limited 2 or 3-fold range, even with a stable kilobase-long genomic R-loop.Any use of the S9.6 antibody must be preceded by RNase A treatment to remove free ssRNA that may compete for the S9.6 binding by forming RNA:RNA regions or short, transient RNA:DNA duplexes. Caution should be used when interpreting S9.6 data, and confirmation by independent structural and functional methods is essential.The online version of this article (doi:10.1186/s13104-015-1092-1) contains supplementary material, which is available to authorized users. Recent work has shown that a single-chain variable domain of the S9.6 antibody can bind RNA:RNA duplexes with an affinity that is only 5.6-fold weaker than to RNA:DNA duplexes, raising the serious concern that S9.6 can indeed cross react with RNA species [While S9.6 antibody can recognize RNA:DNA duplexes -28, comp species . Most of species -28,30,31 species , which i species -35.6 cells/ml were supplemented with anti-CD40 (eBioscience Cat. No. 16-0404-86), IL-4 (R&D Cat. No. 404-ML-010) and TGF- β1 (R&D Cat. No. 240-B-002) for 24 hours.CH12F3.2a and its derivative cells were cultured in RPMI medium supplemented with 10 % FCS and 50 μM β-mercaptoethanol . For expATCC HB-8730 hybridoma line (generously provided by Bradley Cairns) was cultured in a CELLine 1000 bioreactor  according to manufacturer’s instructions. Harvested antibody (culture supernatant) was purified on a column packed with Protein G Sepharose 4 Fast Flow  equilibrated with 1x phosphate buffered saline (PBS).Genomic DNA from CH12F3.2a cells with and without cytokine stimulation was prepared by overnight proteinase K digestion, phenol-chloroform extraction and ethanol precipitation. Genomic DNA was digested with EcoRI; importantly, RNase A was added at this step to prevent S9.6 antibody binding to RNA species in subsequent steps in the experiments with RNase A treatment . Five mWe stimulated CH12F3.2a cells (which are capable of robust cytokine-dependent CSR in culture) for 24 hr before genomic DNA was harvested and digested in preparation for immunoprecipitation (IP) as described previously . The traIn these IP experiments , we findIt is surprising that we detected signals clearly above the background within the fragments far upstream and downstream of the alpha locus even though these signals do not change with cytokine stimulation. We have previously documented that the region upstream of the germline transcript promoter for the IgH alpha locus and the downstream region do not harbor any R-loops, regardless of cytokine treatment ,3,4,33. We also examined the beta-actin gene in the DNA samples from the same IP experiments. The beta-actin gene is at another location in the genome, and it has not been documented to have any extended R-loop by physical methods. The signal at the beta-actin gene is similar to all regions other than IgH Sα Figure , Group 4Taken together, the S9.6 antibody appears to have good specificity for binding to RNA:DNA hybrids, but its ability to distinguish extended R-loops versus small RNA:DNA hybrids arising during DNA replication (RNA primer sites) or from other causes is highly uncertain. With the potential high background due to the antibody binding to non-R-loop RNA:DNA hybrids, the dynamic range of the R-loop signal is greatly affected.In all the IP experiments of DNA samples without RNase A treatment regardless of cytokine stimulation, qPCR did not detect any signal above background in any regions examined (Figure The IgH class switch region R-loops can be up to kilobases in length, and thus provide the largest possible target upon which to test the S9.6 antibody for detection of RNA:DNA duplex in the mammalian genome ,2. DespiWe will immediately provide anyone with our primary data.


Mycobacterium bovis BCG to illustrate the method. Based on a combination of mechanical disruption and liquid and solid-phase technologies, the method produces all major species of ncRNA in high yield and with high integrity, enabling direct chemical and sequence analysis of the ncRNA species. The reproducibility of the method with BCG was evident in bioanalyzer electrophoretic analysis of isolated RNA, which revealed quantitatively significant differences in the ncRNA profiles of exponentially growing and non-replicating hypoxic bacilli. The method also overcame an historical inconsistency in 5S rRNA isolation, with direct sequencing revealing a novel post-transcriptional processing of 5S rRNA to its functional form and with chemical analysis revealing seven post-transcriptional ribonucleoside modifications in the 5S rRNA. This optimized RNA isolation procedure thus provides a means to more rigorously explore the biology of ncRNA species in mycobacteria.A major challenge in the study of mycobacterial RNA biology is the lack of a comprehensive RNA isolation method that overcomes the unusual cell wall to faithfully yield the full spectrum of non-coding RNA (ncRNA) species. Here, we describe a simple and robust procedure optimized for the isolation of total ncRNA, including 5S, 16S and 23S ribosomal RNA (rRNA) and tRNA, from mycobacteria, using  Mycobacterium tuberculosis (Mtb) represents one of the most widespread microbial diseases, with nearly one-third of the world's population showing signs of exposure, more than 20 million people actively infected and almost 80% of the population of some countries testing positive in tuberculin tests  as the model mycobacterial species, the method represents a combination of bead-beating with phenol-chloroform and solid-phase extraction steps optimized for both yield and quality of tRNA, 5S, 16S and 23S rRNA, as well as mRNA for seven genes. Application of the method to BCG revealed hypoxia-induced alterations of the relative quantities of 16S and 23S rRNA, a novel post-transcriptional processing of 5S rRNA, and the first complete analysis of the full set of modified ribonucleosides in mycobacterial 5S rRNA.Infection with in tests ,2. This in tests –6 and inin tests ,8, both in tests ,10. One in tests ,12, whicin tests ,14, whilin tests –18. In ain tests –22. Howein tests , these min tests . To addrM. bovis BCG  was grown at 37°C in a shaking incubator in Middlebrook 7H9 broth  to an OD600 of 0.6–0.8. BCG cells in a hypoxia-induced non-replicative state were obtained using the Wayne model adapted from Low et al.  1-l glass flask  with stirring at 170 rpm and air headspace volume of 450 ml. With a plateau in cell growth at 7 days (as determined by OD600), induction of a non-replicative state was confirmed by the absence of change in OD600 for the next 14 days, at which point the cells were harvested .For exponentially growing mycobacteria, w et al. . BrieflyThe optimized RNA isolation protocol was developed around a simple framework consisting of lysis by bead-beating in a denaturing extraction solvent, with subsequent RNA retrieval from the solvent. Using this framework, we systematically tested a variety of conditions and parameters, including extraction reagent, bead beating conditions and RNA retrieval method, to define an optimal protocol to extract all types of ncRNA from BCG. Two nucleic acid extraction reagents were considered: TRIzol  and phenol:chloroform:isoamyl alcohol . While harshly denaturing, these reagents are insufficient to lyse mycobacterial cells. To assist in lysing cells, mechanical disruption by bead beating on a reciprocal shaker was chosen, with optimization of bead size, extraction solvent volumes and duration. For retrieval of RNA from the extraction solvent following mechanical disruption, we compared simple isopropanol precipitation of RNA to solid-phase isolation of RNA using the Purelink miRNA Isolation Kit . A summary of all conditions tested and their respective nucleic acid yields before DNase I treatment as assessed by UV spectroscopy is detailed in Supplementary Table S1; the analyses were performed in three biological replicates.9 cells were washed in phosphate-buffered saline (PBS) containing 0.5% Tween 80 to remove media debris that could interfere with RNA isolation and resuspended in 1 ml of ice-cold phenol:chloroform:isoamyl alcohol  and 400 μl of 2-M sodium acetate (pH 5.2). The suspension was combined with 100 μl of 0.1-mm zirconia-silica beads  in a 2-ml screw-capped tube and vigorously shaken on a Qiagen Tissuelyser II  with chambers pre-chilled to −20°C to account for heating to ambient temperature by the end of the 30-min run at a frequency of 30 Hz. After centrifugation , the aqueous phase (∼400 μl) was transferred into a new tube to which 215 μl of 100% ethanol was added with mixing and the solution loaded onto a Purelink miRNA Isolation Kit  column (column #1) to retain large RNA species. Following centrifugation of the column , the flow-through containing small RNAs was collected, placed on ice, combined with 700 μl of 100% ethanol and loaded onto a second Purelink miRNA Isolation column (column #2) to retain the small RNA species. The column was again centrifuged  and the flow-through discarded. Both columns (#1 and #2) were washed twice with washing buffer provided with the kit. RNase-free water (50 μl) was added to the columns, which were incubated at ambient temperature for 2 min followed by centrifugation . The eluates from columns #1 and #2 contained mainly large and small RNA, respectively, and these were combined to recreate a total RNA preparation. Aliquots of each RNA sample were adjusted to a concentration of 1 ng/μl and thee integrity and relative quantities of the RNA species in the sample were assessed using an Agilent 2100 Bioanalyzer . Column eluates were stored at −80°C.Supplementary Figure S2 illustrates the final optimized ncRNA isolation method, which was applied to BCG cultures growing exponentially or in a hypoxia-induced non-replicative state. Cells are harvested from culture medium by centrifugation at 4000 x g for 5 min at 4°C. Pellets containing 10Individual RNA species were purified by size-exclusion (SEC) high-performance liquid chromatography (HPLC), as described in a recent publication . FollowiTo confirm the identity of the purified 5S rRNA, the 3′ end of 25 pmol of the RNA was ligated to 50 pmol of a pre-adenylated DNA adaptor  using 1.5 μg of RNA ligase 2  with incubation at 22°C for 16 h. The ligated product was purified by SEC3 HPLC and then reverse transcribed with 200 U of Superscript III RT . The resulting cDNA was ligated to 50 pmol of another pre-adenylated 3′ adaptor  using 10 U of T4 RNA ligase 1  with incubation at 4°C for 16 h. Following SEC3 HPLC purification, the ligated cDNA was PCR amplified, TA cloned , and sequenced to confirm its identity . Sequence analysis was performed with Nucleotide BLAST (NCBI) against the annotated full genome sequence of the same BCG strain (NC_008769.1). The cDNA sequence from mature 5S rRNA is available on GenBank (Accession number KC203333).To compare the mRNA yield from the optimized RNA isolation approach to that of the conventional TRIzol method, RNA was isolated from exponentially growing BCG cultures using TRIzol or the optimized RNA isolation method and DNA was removed using the TURBO DNA-free Kit . RNA (50 ng/μl) was reverse transcribed using the iScript cDNA synthesis kit  according to the manufacturer's instructions. The reverse transcription program was run as follows: 25°C for 5 min, 42°C for 30 min and 85°C for 5 min, followed by a cooling step at 4°C. Two-step real-time quantitative polymerase chain reaction (qPCR) was then performed using the SsoAdvanced universal SYBR Green supermix . Primer sequences and melting temperatures can be found in Supplementary Table S3. The qPCR program was run as follows: 95°C for 30 s followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 30 s. A melting curve analysis consisting of 0.5°C increments from 65 to 95°C was performed for all reactions to ascertain the specificity of the primers.The full spectrum of modified ribonucleosides in the purified 5S rRNA was characterized by analysis of ribonucleoside hydrolysates of the RNA by chromatography-coupled quadrupole time-of-flight and triple quadrupole mass spectrometry, using methods described previously ,26,27. BCT values for the RNA isolation methods and quantities of RNA species in exponentially growing and hypoxic, non-replicative (Wayne day 21) cultures was performed using Student's t-test with significance set at P ≤ 0.05.Statistical analysis for comparison of qPCR in vivo. The workflow shown in Supplementary Figure S2 represents the culmination of a series of optimization studies in which different procedures were assessed for the yield of RNA, the relative quantities of the various ncRNA species and the quality of the RNA in terms of degradation. Subsequent analysis of the isolated ncRNA species from BCG revealed several novel features of RNA biology in mycobacteria.Given the challenges of working with mycobacteria, we sought to develop a method for purification of ncRNA that optimized the cell lysis, RNA extraction and RNA purification steps to yield the full spectrum of intact ncRNA species in quantities that reflect their abundances in vivo, with contaminating DNA subsequently removed by DNAse I digestion or HPLC purification, without sacrificing longer RNA species .With all of the optimization data shown in Supplementary Table S1, optimization of the ncRNA isolation method was initiated with a comparison of two standard extraction reagents, TRIzol and the 25:25:1 mixture of phenol, chloroform and isoamyl alcohol. An extraction reagent was added to the cells prior to bead beating to quench adventitious enzymatic reactions during cell lysis, including nucleases and RNA modification enzymes. Given the evidence for biased isolation of miRNA with TRIzol , we compNext, we optimized for cell lysis, comparing the RNA yield obtained using 0.5- and 1.0-mm glass beads and 0.1-mm zirconia-silica beads. While 0.5-mm glass beads offered a 61% increase in nucleic acid yield against 1.0-mm glass beads, we observed an additional 37% increase in RNA yield with 0.1-mm zirconia-silica beads (Supplementary Table S1). Using 0.1-mm zirconia-silica beads, we optimized for the duration of bead beating. A duration of 10 min was sufficient for lysis of mycobacterial cells using the TissueLyser II, while a duration of 30 min was required for the TissueLyser LT. Use of a French press was ruled out due to the time required for the lysis process (>15 min) in the absence of denaturants, which allows enzymatic reactions to affect the spectrum of tRNA and rRNA modifications or cause RNA degradation.Finally, to recover RNA from the extraction solvents, we chose the Purelink miRNA Isolation Kit system over isopropanol-based RNA precipitation. While the Purelink columns were optimized for use with TRIzol, we modified the manufacturer's protocol to use phenol:chloroform:isoamyl alcohol instead, which allowed for the maximal recovery of both large and small RNA. Using 35% ethanol, larger RNA species were recovered on column #1 while smaller RNA species were recovered on column #2 using 70% ethanol.To assess the performance of the optimized RNA isolation method and the quality of the isolated ncRNA, the method was applied to BCG cells growing exponentially and to cells arrested in a hypoxia-induced non-replicative state that mimics the granuloma of pulmonary mycobacterial infections. The quality and relative quantities of the various ncRNA species in total RNA extracted from BCG were assessed using a Bioanalyzer with either the Agilent Pico Chip for large RNA 16S, 23S) and the Agilent Small RNA Chip for small RNA species . As shown in Figure 6S, 23S aTo assess the performance of the Purelink miRNA Isolation columns, the proportions of ncRNA species in column eluates were calculated by manual integration of electropherogram peaks with Bioanalyzer 2100 Expert software. A comparison of the TRIzol and phenol:chloroform:isoamyl alcohol methods is summarized in Supplementary Table S2. Using this approach, large ncRNAs  were almost entirely recovered from column #1 along with a significant portion of the small ncRNAs (<200 nt), while small ncRNAs represented >95% of the RNA eluted from column #2. The small ncRNAs, whose identities were assigned based on size, consisted of three major species: miRNA-like species, tRNA and 5S rRNA. This retention of small ncRNAs on column #1 points to potential bias in the small ncRNAs recovered from column #2 as ‘small RNA’. However, by combining the eluates from both columns, the yield of all ncRNA species was maximized Figure  and F, wConsidering both the RIN and the recovery of all ncRNAs from the columns using the phenol:chloroform:isoamyl alcohol approach, the results with exponentially growing BCG RNA suggest that the degradation apparent in hypoxic, non-replicative BCG is not the result of the RNA isolation procedure and instead reflects physiological processing of the RNA. These differences were next assessed more quantitatively.CT values against five gene transcripts as illustrated in Table CT value (i.e. higher concentration of mRNA) compared to the TRIzol approach.While optimized for the isolation of ncRNA, we investigated the possibility of utilizing our approach for mRNA isolation. Here, we performed qPCR against seven gene transcripts on samples derived from RNA isolation with our approach and samples derived from the conventional TRIzol RNA isolation approach. Analysis revealed that there were no significant differences in R = 3.9 ± 0.02) of 16S and 23S rRNAs using an Agilent size-exclusion HPLC column (SEC5 1000 Å), while tRNA and 5S rRNA were well resolved (R = 4.1 ± 0.04) using a solid phase with a smaller pore size (SEC3 300Å). The absolute quantities of individual ncRNA species were calculated by interpolating the UV absorbance peak areas for HPLC-resolved ncRNA species with a standard curve prepared from purified 28S rRNA. These quantities were then combined with cell counts from colony forming assays for cultures used in the RNA isolation to calculate the quantity of each rRNA species on a per cell basis. As shown in Figure P ≤ 0.05), yet there was no difference in the quantities of 5S rRNA and tRNA for the two growth conditions. These data for cellular quantities of ncRNA are consistent with theoretical estimates of mycobacterial molecular composition  of 4 x e−49 and a maximal identity of 100% for several Mycobacterium species, including BCG, M. tuberculosis, Mycobacterium canettii, and Mycobacterium africanum. Comparison of the annotated BCG 5S rRNA sequence  and the cDNA sequence of our purified 5S rRNA revealed several differences in the 3′- and 5′-ends, as shown in Figure Using the optimized RNA isolation method with BCG, we were able to reproducibly detect an RNA species with a size consistent with 5S rRNA. Though relatively abundant when compared to other bacterial small RNAs, the observation of 5S rRNA is notable in light of the fact that this species has never been isolated for direct sequencing. In other studies of mycobacterial ncRNA, small RNAs were identified by genomic analysis, by microarray detection of non-coding transcripts, by shotgun cloning of small RNAs or by co-purification with RNA-binding proteins –34. Here1-methyladenosine (m1A), N6,N6-dimethyladenosine , N7-methylguanosine (m7G), N1-methylguaonsine (m1G), 2′-O-methylguanosine (Gm) and inosine (I), each with distinct mass-to-charge transition ratios and retention times as shown in Supplementary Figure S6. The structures of six of the modified ribonucleosides were confirmed with synthetic standards, while the structure of the seventh is tentatively assigned as N4-methyl-2′-O-methylcytidine (m4Cm) based on CID fragmentation patterns.To take advantage of the ability to isolate 5S rRNA using the optimized method, we further explored 5S rRNA maturation by analyzing post-transcriptional modifications. Here, we used a highly sensitive chromatography-coupled mass spectrometric platform to identify modified ribonucleosides in RNA ,26,27. AM. tuberculosis based on genomic analyses  provided versatility and ease in retrieving RNA from a wide variety of sources . Howeverficiency . We also . This amount of cellular RNA is consistent with recent studies on the molecular composition of mycobacteria that gave a theoretical total content of RNA in a single BCG bacillus at 2.2–10 fg with dependence on the growth rate , we sought to determine if our method was suitable for mRNA isolation or gene expression studies. We thus probed for the presence of seven mRNA transcripts on samples derived from our RNA isolation approach and samples derived from the conventional TRIzol RNA isolation approach. Analysis showed that the optimized phenol:chloroform:isoamyl alcohol approach resulted in a similar yield of mRNA when compared to TRIzol-based RNA isolation, while the RIN data attest to the quality of the total RNA preparation. We therefore believe that total RNA derived from our approach can be used for gene expression studies.The results presented here demonstrate the utility of a mycobacterial RNA isolation method that maximally preserves the quantitative and qualitative fidelity of ncRNA species. While optimized for ncRNA, the method also provides high fidelity isolation of mRNA from mycobacteria.GenBank KC203333.Supplementary Data are available at NAR Online.


Although the complex phenotype of Dazl deficiency in both sexes, with defects at multiple stages of germ cell development and during meiosis, demonstrates its obligate significance in fertility in animal models, its involvement in human fertility is less clear. As an RNA binding protein, identification of the in vivo mRNA targets of DAZL is necessary to understand its influence. Thus far, only a small number of Dazl targets have been identified, which typically have pivotal roles in germ cell development and meiotic progression. However, it is likely that there are a number of additional germ cell and meiosis-relevant transcripts whose translation is affected in the absence of Dazl. Efforts to identify these RNA targets have mainly been focused on spermatogenesis, and restricted to mouse. In women, prophase I occurs in fetal life and it is during this period that the ovarian follicle pool is established, thus factors that have a role in determining the quality and quantity of the ovarian reserve may have significant impact on reproductive outcomes later in adult life. Here, we suggest that DAZL may be one such factor, and there is a need for greater understanding of the role of DAZL in human oogenesis and its contribution to lifelong female fertility.The RNA binding protein  Female reproductive potential is governed by two key factors: oocyte quality and quantity, with the loss of quality underpinning the exponential increase in age-related aneuploidy, and the loss of quantity underlying the age at menopause  belong to the DAZ family of genes encoding RNA binding proteins, and have essential roles in gametogenesis. It is likely that BOLL is the ancestral gene, first identified in Drosophila (where it is known as bol), and DAZL is considered to have arisen from BOLL through a gene duplication event  11.5 in post-migratory germ cells  and poly(G) RNA in vitro  tail length, recruitment to polysomes and translational activation  , also known as premature ovarian failure, in infertile men and women, respectively , there has been no examination as to whether the quality of oocytes is affected too. Given that DAZL was shown to influence total sperm count and sperm motility, quantity and quality parameters respectively, it seems possible that the same should occur in women; a key significant difference being the developmental period at which the effect happens. The generation of sperm via spermatogenesis, and thus meiosis, is a continuous process beginning at the onset of puberty and continuing throughout male adult life. However, in females, the process of oogenesis is initiated in the fetus, with the formation of stored oocytes that are used periodically over a defined reproductive lifespan. It is not clear if the genetic associations between DAZL polymorphisms and age at menopause and POI reflect a role for DAZL in fetal oocyte development or maintenance of oocytes during their prolonged post-natal arrest, but these associations are consistent with the fetal oocyte death reported in Dazl null mice . Funding to pay the Open Access publication charges for this article was provided by the Medical Research Council.The authors' work in this field is supported by grants from the None declared.