Mononegavirales) is one of the main causes of respiratory tract illness in children. To help elucidate the assembly mechanism of the nucleocapsid  we present crystallographic structures of HMPV N in its assembled RNA-bound state and in a monomeric state, bound to the polymerase cofactor P. Our structures reveal molecular details of how P inhibits the self-assembly of N and how N transitions between the RNA-free and RNA-bound conformational state. Notably, we observe a role for the C-terminal extension of N in directly preventing premature uptake of RNA by folding into the RNA-binding cleft. Our structures suggest a common mechanism of how the growth of the nucleocapsid is orchestrated, and highlight an interaction site representing an important target for antivirals.Non-segmented, (-)RNA viruses cause serious human diseases. Human metapneumovirus (HMPV), an emerging pathogen of this order of viruses (DOI:http://dx.doi.org/10.7554/eLife.12627.001 Human metapneumovirus (HMPV for short) is a major cause of infections of the airways and lungs, particularly in children, elderly individuals and people with weakened immune systems. As for all viruses, HMPV cannot survive on its own. Instead, it must invade and hijack cells in order to replicate its own genetic material and form new viruses. In HMPV, this genetic information is in the form of a strand of RNA, and is protected by a shell-like structure called a nucleocapsid. Drugs that disrupt the nucleocapsid may therefore help to kill the viruses and treat the illnesses that they cause.Nucleocapsids are built out of many copies of a protein called nucleoprotein, which binds to a strand of RNA. However, viral nucleocapsids can only be built from nucleoproteins that are bound to viral RNA. Potentially, nucleoproteins could instead bind to RNA belonging to the cells that HMPV infects and they would then be trapped in a dead-end state. To prevent this type of unproductive binding, before the nucleocapsid is formed the nucleoprotein is kept unassembled with the help of another protein called the polymerase cofactor. However, it was not clear exactly how the polymerase cofactor helps to maintain this unassembled state.Using techniques called cryo-electron microscopy and X-ray crystallography, Renner et al. studied the structures formed when nucleoproteins are either bound to RNA or are unassembled and bind to the polymerase cofactor. Comparing these structures revealed that RNA normally binds to a specific cleft in the nucleoprotein. However, when nucleoprotein is bound to the polymerase cofactor a portion of the nucleoprotein folds into this cleft instead, blocking the insertion of RNA. This prevents the nucleoprotein from associating with the wrong RNA, allowing the nucleoprotein to remain in an unassembled state until it is needed for the virus.Renner et al. also found that the interactions between the nucleoprotein and the polymerase cofactor of HMPV occur at sites that are also found in several other related viruses, such as Ebola. Targeting this common region could therefore be a good strategy for developing new antiviral drugs.DOI:http://dx.doi.org/10.7554/eLife.12627.002 Mononegavirales) include measles, rabies, Ebola, respiratory syncytial virus (RSV) and Human metapneumovirus (HMPV). HMPV  is a leading cause of serious respiratory tract infections in children, the elderly, and immunocompromised individuals  and RNA  from vesicular stomatitis virus (VSV), Ebola virus, Nipah virus, and measles virus have highlighted its role in preventing assembly of N by blocking the C-terminal and N-terminal extensions of N (CTD-arm and NTD-arm) which facilitate N oligomerization . The CTD-arm in turn latches onto the top of the CTD of the next protomer (i-1 latches onto CTD of Ni). We observed that the binding site of the P peptide overlaps with the binding sites of the NTD- and CTD-arms  and a closed RNA-bound (N-RNA) conformation  it packs down against the core of N  the CTD-arm flips upwards and latches onto the next protomer, whilst in the monomeric state  to the filament necessitates that the CTD-arm of the terminal Ni unbinds and flips upwards  agarose . The column was washed and the protein was eluted in 25 mM Tris, pH 8, 1 M NaCl, 400 mM imidazole. The eluate was further purified by size exclusion chromatography using a Superose6 10/300 column  equilibrated in 25 mM Tris, pH 8, 1 M NaCl. The protein was buffer exchanged into 25 mM Tris, pH 8, 150 mM NaCl, 500 mM NDSB201, 50 mM Arginine using a PD10 column  and then concentrated to ~4 mg/mL for crystallization.The full-length N gene from human metapneumovirus  was cloned into the pOPINE expression vector, which includes a C-terminal His-tag, using the In-Fusion system  following standard procedures. The construct was verified by sequencing. Rosetta2 E.coli cells harboring the expression plasmid were grown at 37°C in terrific broth containing appropriate antibiotics and expression was induced at an OD600 of 0.8 by adding isopropyl β-D-1-thiogalactopyranoside to 1 mM. The temperature was then lowered to 18°C and after further 18 hrs the cells were harvested by centrifugation . Cell pellets were resuspended in 40 mL of 25 mM Tris, pH 8, 1 M NaCl per L of culture and lysed by sonication. The lysate was centrifuged  and the supernatant was filtered and loaded on a column containing pre-equilibrated Ni0-P hybrid gene was generated by fusing the sequence corresponding to the first 40 residues of HMPV P  to the 3’ end of the full-length N gene using overlapping primer PCR. The resulting hybrid construct was cloned into POPINE as described above and verified by sequencing. Protein expression was carried out as described for N, above. Cell pellets were resuspended in 20 mM Tris, pH 7, 1M NaCl, lysed by sonication and the lysate was subsequently centrifuged . The supernatant was purified using a column containing pre-equilibrated Ni2+-NTA agarose and elution was carried out using 20 mM Tris, pH 7, 1M NaCl, 300 mM imidazole. The protein was then buffer exchanged into 20 mM Tris, pH 7, 100 mM NaCl and loaded onto a HiTrap Heparin HP column  for further purification using a stepwise NaCl gradient. Finally, the N0-P hybrid was gel-filtrated using a Superdex 75 column  equilibrated with 20 mM Tris, pH 7, 100 mM NaCl, and concentrated to ~7 mg/mL for crystallization.The N3, Na2HPO4, (NH4)2SO4), 37.5% methyl-2 4-pentanediol, polyethylene glycol 1000 and polyethylene glycol 3350 of the MORPHEUS crystal screen. The crystal was frozen in liquid nitrogen and diffraction data up to 4.2 Å were recorded at 100 K on the I04-1 beamline at Diamond Light Source, Didcot, UK.Sitting drop, vapor diffusion crystallization trials were set up in 96-well Greiner plates using a Cartesian Technologies robot . A diamo0-P preparation just before setting up the crystallization trials. Initial crystals formed in mother liquor containing 100 mM PCB System, pH 7, 25% polyethylene glycol 1500 and improved crystals could be grown with additives of the Hampton Silver Bullet screen . The crystals were cryoprotected in 25% glycerol and frozen in liquid nitrogen. Diffraction data up to 1.9 Å were recorded at 100 K on the I04 beamline at Diamond Light Source, Didcot, UK. All data were processed and scaled with XIA2  and N-RNA (at 4.2 Å), respectively. Refinement and geometry statistics are given in The structure of the RNA-bound subnucleocapsid ring was solved with PHASER  using a lProbity  resultinMultiple sequence alignments (MSA) were carried out with PROMALS3D  and figuN-RNA rings were analysed via electron cryomicroscopy (cryo-EM). Aliquots (3 µl) of N-RNA preparations were pipetted onto glow-discharged Cflat holey carbon grids  and excess liquid was blotted with filter paper for 3 s. Grids were then plunge-frozen in an ethane-propane mixture at liquid nitrogen temperature using a CP3 plunging device (Gatan). Cryo-EM data were acquired using a 300-kV Polara transmission electron microscope (FEI) equipped with a K2 Summit direct electron detector (Gatan) and using defocus values ranging from -2.0 to -6.0 μm at a calibrated magnification of 37,000x, resulting in a pixel size of 1.35 Å. The contrast transfer function (CTF) parameters were determined using CTFFIND3  and 2D-c In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and John Kuriyan as the Senior Editor. Our decision has been reached after consultation between the reviewers and the Reviewing Editor. Based on these discussions, we regret to inform you that your work will not be considered further for publication in eLife. The decision was made not because of the quality of the work, which both reviewers judged to be fine, but rather because of the criteria for novelty and breadth of interest that the journal and its Board of Reviewing Editors have set for the journal.Thank you for submitting your work entitled "Nucleocapsid assembly in pneumoviruses is regulated by conformational switching of the N protein" for consideration by Renner and colleagues provide evidence that nucleocapsid assembly in pneumoviruses involves a conformational switch of the N protein. That conclusion is based on the structures of an N-protein RNA complex and a soluble form of N, referred to as No, both for human metapneumovirus (HMPV) N. The No structure was obtained in complex with a small N-terminal fragment of P. The structures are of interest and suggest that a conformational change occurs during the process of encapsidation of the nascent RNA. The authors propose, based on the structures, that the C-terminal arm of the last N protein molecule in the growing filament flips up to allow engagement of the new incoming No-P, displacing P concomitant with the insertion of the nascent RNA chain into the newly added N. The process of encapsidation of the nascent RNA lacks a detailed molecular model, and this manuscript may provide some important new clues as to how the process occurs not just for HMPV, but perhaps other viruses in the order mononegavirales (MNV). There are, however, some concerns regarding the No-P structure, and several statements of novelty and precedent in the manuscript that do not seem justified. Moreover, other structures of RNA free, soluble No and N-RNA complexes have been determined for other viruses in the MNV, diminishing the novelty of present work.1) The No-P structure was solved using an artificial construct in which the N-terminal 40 residues of P were fused to the C-terminal arm of full-length N, and crystals obtained by in situ proteolysis with trypsin. It is uncertain to what extent the structure obtained represents a physiologically relevant form of soluble N or is in fact a form that is simply obtained using the in situ proteolytic approach used here.2) The conformational switch model proposed suggests contacts that can be tested experimentally using HMPV reverse genetics – this would go a long way to alleviate the concern of point 1.3) Throughout the manuscript the authors make broad claims of precedent, stating that this is the first virus in the mononegavirales for which structures of both No and N-RNA have been determined. This is not the case – the authors show several previously published structures in  Renner and colleagues provide evidence that nucleocapsid assembly in pneumoviruses involves a conformational switch of the N protein. That conclusion is based on the structures of an N-protein RNA complex and a soluble form of N, referred to as No, both for human metapneumovirus (HMPV) N. The No structure was obtained in complex with a small N-terminal fragment of P. The structures are of interest and suggest that a conformational change occurs during the process of encapsidation of the nascent RNA. The authors propose, based on the structures, that the C-terminal arm of the last N protein molecule in the growing filament flips up to allow engagement of the new incoming No-P, displacing P concomitant with the insertion of the nascent RNA chain into the newly added N. The process of encapsidation of the nascent RNA lacks a detailed molecular model, and this manuscript may provide some important new clues as to how the process occurs not just for HMPV, but perhaps other viruses in the order mononegavirales (MNV). There are, however, some concerns regarding the No-P structure, and several statements of novelty and precedent in the manuscript that do not seem justified. Moreover, other structures of RNA free, soluble No and N-RNA complexes have been determined for other viruses in the MNV, diminishing the novelty of present work.I would like to thank you and the referees for the comments regarding our manuscript “Nucleocapsid assembly in pneumoviruses is regulated by conformational switching of the N protein”. Two of the three main points brought forward by the referees are either unjustified or easy to address.1) The No-P structure was solved using an artificial construct in which the N-terminal 40 residues of P were fused to the C-terminal arm of full-length N, and crystals obtained by in situ proteolysis with trypsin. It is uncertain to what extent the structure obtained represents a physiologically relevant form of soluble N or is in fact a form that is simply obtained using the in situ proteolytic approach used here.Linking of binding partners as we have performed in this manuscript is a powerful technique to circumvent problems of unstable binding and increase naturally occurring interaction. The method  is well-established and has been successfully used on a variety of systems, including MHC-receptors and, indeed, viral nucleoproteins . Due to the inherent propensity to form oligomeric assemblies such tricks are absolutely necessary for the study of N. The referees categorically denote the method as being artificial but offer no specific criticisms to the structure at hand. Similarly, the use of limited proteolytic digestion is a very common technique in crystallography to facilitate the pruning of flexible loops and thus promote crystallization . It seems that the major criticism of the referees was that we utilized two common and established techniques which happen to be unfamiliar to them. Indeed, we can bring forward several points that confirm our N0-P structure is correctly folded, even though this should not be necessary: ( (1)) the binding site of P is in line with previous functional studies , as mentioned in the manuscript, ( (2)) the P-site is also in line with structures of related viruses, ( (3)) the linker between N and P is cleaved by the in-situ proteolysis yielding non-covalently bound P which should completely negate this point of contention, ( (4)) the overall fold of N follows other N proteins. We therefore cannot understand why our structure is deemed artificial and would like to challenge the referees to bring forward specific criticisms. We addressed the criticisms in our manuscript and explain the rationale of using a chimeric protein and proteolytic digestion. We have also addressed the criticism of data/model quality for our N-RNA structure by including CC*, CCwork and CCfree statistics by resolution shell .2) The conformational switch model proposed suggests contacts that can be tested experimentally using HMPV reverse genetics – this would go a long way to alleviate the concern of point 1.We fully agree that testing the proposed interactions using a reverse genetics system is key in further dissecting the mechanism of nucleocapsid assembly. Unfortunately, we lack the resources or necessary collaborations to perform these experiments. This is why our manuscript was written as a short report focused on biophysical and structural analysis of this system and we feel that such experiments would be beyond its scope. However, we could conceive of mutagenesis experiments that could test the interactions in-vitro, and these may provide valuable additional validation3) Throughout the manuscript the authors make broad claims of precedent, stating that this is the first virus in the mononegavirales for which structures of both No and N-RNA have been determined. This is not the case – the authors show several previously published structures in Our study aims at structurally characterising assembly of N. The transition between assembled N-RNA and monomeric N0 -P was analysed in-depth in our manuscript. At the point of review, to our best knowledge, there was no instance where an assembled N-RNA and monomeric N0 -P structure was available for the same virus. The only instance where this might be contended is for vesicular stomatitis virus (VSV). However, in this case both structures form oligomeric, assembled rings that are conformationally identical . We could thus, for the first time, analyse the structural transition without having to resort to qualitative comparisons with phylogenetically distant homologues. Similar to major comment #1 the referees unfairly dismissed our claims without specific criticism. We would like to challenge them to show us for which virus they think monomeric N0 -P and assembled N-RNA was available during review. However, during to the lengthy review process, the measles virus N0 -P was published , meaning that we cannot make the contended claim anymore. To quote the manuscript by Guryanov et al: "Here, for the first time in Paramyxovirus research, our data allow direct comparison of the structure of the nucleoprotein from the same virus in two functional states: a P-bound naive state, and an RNA-bound helical assembly." Consequently, although they were true during the review stage, we have now removed these claims of precedence.


Metastasis to the pituitary gland is extremely rare and is often detected incidentally by symptoms associated with endocrine dysfunction. Breast and lung cancer are the most common primary metastasizing to pituitary gland. Metastasis from hepatocellular carcinoma to the pituitary gland is extremely rare, with only 10 cases having been previously reported. We present here the first case of pituitary metastasis of hepatocellular carcinoma presenting with panhypopituitarism diagnosed by magnetic resonance imaging.We report the case of an 80-year-old Japanese woman who presented with the sudden onset of hypotension and bradycardia after having previously been diagnosed with hepatocellular carcinoma. Based on low levels of pituitary hormones, she was diagnosed with panhypopituitarism caused by metastasis of the hepatocellular carcinoma to the pituitary gland. Magnetic resonance imaging with arterial spin-labeling was effective in the differential diagnosis of the intrasellar tumor. The patient died despite hormone replacement therapy because of hypovolemic shock.Metastasis to the pituitary gland causes various non-specific symptoms, so it is difficult to diagnose. The present case emphasizes the importance of diagnostic imaging in identifying these metastases. Clinicians should consider the possibility of pituitary metastasis in patients with malignant tumors who demonstrate hypopituitarism. Intracranial metastasis to the pituitary gland is rare. The reported incidence of pituitary metastasis is 0.87 % and 1.9 % of all intracranial metastases and autopsied cancer patients, respectively. Breast and lung cancer are the most common primary carcinomas causing pituitary metastasis [In November 2011, an 80-year-old woman exhibited sudden-onset anorexia accompanied by hypotension and bradycardia. She had previously been treated for hepatitis C virus-related cirrhosis and HCC in August 2002. The patient suffered multiple relapses of HCC and had undergone four radiofrequency ablation cycles and two transcatheter arterial chemoembolization cycles. Routine laboratory tests detected a mild elevation of serum aspartate aminotransferase to 81 IU/L . An additional endocrinological work-up detected thyroid hormone, cortisol, and adrenocorticotropic hormone insufficiencies, with normal levels of thyroid stimulation hormone  developed five days into the replacement therapy, and the patient’s daily output of urine was more than 2.5 L. Urine output decreased only temporarily following treatment with desmopressin, but increased to 6.0 L/day one week into treatment. Two weeks after the initial diagnosis of pituitary metastasis, MRI revealed enlargement and subsequent hemorrhaging of the metastatic pituitary tumor.In January 2012, the patient died of hypovolemic shock. Postmortem examination of the pituitary tumor revealed tumoral hepatocytes in a thick trabecular pattern, the typical appearance of well differentiated HCC Fig. . The tumAs noted above, metastasis to the pituitary gland is extremely rare. The incidence of pituitary metastasis is less than 1 % among all patients with intracranial metastases [The differential diagnosis of intrasellar tumors requires the exclusion of such diseases as hypophyseal adenoma, craniopharyngioma, meningioma, Rathke’s cleft cyst, and aneurysm. Fassett et al. reported that differentiation of a pituitary metastasis from other pituitary tumors based on neuroimaging alone can be difficult, although certain features, such as thickening of the pituitary stalk, invasion of the cavernous sinus, and sclerosis of the surrounding sella turcica, can indicate metastasis . On the ASL is a unique perfusion imaging method, especially for the brain, that can generate images without injection of contrast material. Its clinical utility has been established in several cerebral conditions such as brain tumors, infarction, and vascular lesions –14. In oThe patient exhibited a sudden onset of hypotension with bradycardia, symptoms which were suspected to be the result of panhypopituitarism. This was later confirmed by a blood test. On the other hand, the most common symptom of pituitary metastasis is DI –8, 15, bDistinguishing metastasis to the pituitary gland from metastasis to the skull base  is very difficult because of the close proximity of these regions . To estaFor the treatment of metastatic pituitary tumors, Morita et al. reported that there were no significant differences in survival times between surgical and nonsurgical treatments . The vasWe reported a case of HCC metastasis to the pituitary gland presenting with panhypopituitarism. The present case emphasizes the importance of diagnostic imaging in identifying these metastases. Metastasis to the pituitary gland causes various non-specific symptoms. It is important to consider a metastasis to the pituitary gland when an HCC patient exhibits nonspecific symptoms associated with panhypopituitarism.Written informed consent was obtained from the guardians of the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.


Escherichia coli K-12 substrain MG1655 and have compared it to its intracellular RNA complement. Our results demonstrate that a large part of the extracellular RNA complement is in the size range between 15 and 40 nucleotides and is derived from specific intracellular RNAs. Furthermore, RNA is associated with OMVs and the relative abundances of RNA biotypes in the intracellular, OMV and OMV-free fractions are distinct. Apart from rRNA fragments, a significant portion of the extracellular RNA complement is composed of specific cleavage products of functionally important structural noncoding RNAs, including tRNAs, 4.5S RNA, 6S RNA, and tmRNA. In addition, the extracellular RNA pool includes RNA biotypes from cryptic prophages, intergenic, and coding regions, of which some are so far uncharacterised, for example, transcripts mapping to the fimA-fimL and ves-spy intergenic regions. Our study provides the first detailed characterization of the extracellular RNA complement of the enteric model bacterium E. coli. Analogous to findings in eukaryotes, our results suggest the selective export of specific RNA biotypes by E. coli, which in turn indicates a potential role for extracellular bacterial RNAs in intercellular communication.The secretion of biomolecules into the extracellular milieu is a common and well-conserved phenomenon in biology. In bacteria, secreted biomolecules are not only involved in intra-species communication but they also play roles in inter-kingdom exchanges and pathogenicity. To date, released products, such as small molecules, DNA, peptides, and proteins, have been well studied in bacteria. However, the bacterial extracellular RNA complement has so far not been comprehensively characterized. Here, we have analyzed, using a combination of physical characterization and high-throughput sequencing, the extracellular RNA complement of both outer membrane vesicle (OMV)-associated and OMV-free RNA of the enteric Gram-negative model bacterium  Helicobacter pylori, which are taken up by gastric epithelial cells  of the enteric Gram-negative bacterium E. coli K-12 substrain MG1655. Bacteria were grown in Luria-Bertani (LB) broth or M9 minimal medium at 37°C. OMVs were isolated as previously described  with a 100 kDa hollow fiber membrane . The concentrated supernatants were then further passed through a 0.8/0.2 μm filter . As a control, filtered supernatants were checked for bacterial contamination by plating 1 mL of supernatant on a LB-agar plate followed by overnight incubation at 37°C. The filtered supernatants were then ultracentrifuged at 150,000g for 3 h at 4°C in order to separate the bacteria-free media into an OMV containing fraction (pellet) and OMV-free fraction (supernatant). Thus, OMVs were contained in the pellet fractions, which were then washed with Phospate buffered saline (PBS) (1X), and finally resuspended in PBS and stored at −80°C. The OMV-depleted supernatants were also stored at −80°C for extraction of RNAs. Unless otherwise stated, all experiments were performed with samples prepared by the method described above.OMVs were derived from the enteric Gram-negative bacterium The resulting OMV fractions were then further separated into low- and high-density fractions (HDFs) by gradient ultracentrifugation as previously described  and washed with PBS. Samples were placed on Formvar-coated nickel grids, air dried, and examined at 80 kV with a Morgagni 268D transmission electron microscope. Images were captured digitally by a Mega View III camera .4 was added to the sample and incubated on ice for 30 min. Samples were centrifuged at a speed of 14,000g for 30 min at 4°C. The supernatant was then separated from the samples and used for protein analyses.Protein samples were dissolved in lysis buffer (25 mmol/L of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.4, 5 mmol/L of Ethylenediaminetetraacetic acid (EDTA), 1 mmol/L of Dithiothreitol (DTT), 1 mg/mL of Lysozyme) and passed through three cycles of freezing and thawing. Finally, MgSOProtein concentrations were determined using a Qubit Protein Assay Kit  and separated on a 4–15% of polyacrylamide gel . Gels were stained with either Imperial Protein Stain  or the SilverQuest™ Silver Staining Kit .E. coli cells, OMVs, OMV-free supernatant, low-density OMV fractions (fractions 2–6), high-density OMV fractions (fractions 7–12), and culture media using the All-in-One Purification Kit . Samples were precipitated with 20% (w/v) trichloroacetic acid (TCA) followed by 80% (v/v) acetone washing before the extraction of RNA . A primer contamination table was generated by analyzing the sequence libraries. Contaminant adapters (RNA 3′ adapter and RNA PCR primer) were subsequently trimmed using the FastX toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). This step was repeated three times in order to achieve a high accuracy for adapter trimming. Following adapter trimming, the sequence reads were subjected to quality trimming to remove noise using a Phred quality scores threshold of 25, which corresponds to an error probability of 0.003 per base. In addition, sequence reads failing to retain all bases with Phred quality score of 25 per base throughout the read were removed. Reads shorter than 14 nucleotides were removed. Identical read sequences after adapter trimming and quality filtering were then collapsed while maintaining the count of the identical reads in the library. Finally, for further analyses of the sequence libraries, identical reads present in the libraries obtained from cell-free growth media and cell-free growth media incubated overnight were subtracted from the reads in all libraries.Raw small RNA sequence libraries of different samples were subjected to preprocessing for quality control and adapter removal using the FastX toolkit (E. coli strain K-12 substrain MG1655 reference genome (accession number: NC_000913.2) from the RefSeq database (http://www.ncbi.nlm.nih.gov/refseq/) using novoalign (http://www.novocraft.com) with zero mismatches allowed. Unmapped reads from the library were filtered using SAMtools  and 5 μmol/L of red fluorescent Lipophilic Tracer DiD (Life Technologies) for an hour at 37°C. Samples were washed once in PBS (1X) followed by centrifugation at a speed of 150,000g for 1 h at 4°C and mounted onto a glass slide with mounting medium, Fluoroshield™ (Sigma).OMVs were stained as described previously  and processed using the ZEN software package . The background was adjusted with dye and unstained control samples. For each sample, images were taken separately in rapid succession. The excitation and emission spectra were set at a wavelengths of μL of reaction mixture where primers were added at a concentration of 0.2 μmol/L, 5 μL of PerfeCTa SYBR Green SuperMix (Quanta BioSciences) and the remaining volume was adjusted for cDNA amount and water. Primer efficiency was calculated using a 10-fold dilution series of the template cDNA, and for all primer sets efficiency values were close to 100%. Real-time PCR was run on a LightCycler® 480 Real-Time PCR System  using the thermal cycles of 95°C for 2 min and 40 cycles of 95°C for 10 sec, 57–62°C for 5 sec, and 72°C for 10 sec. Ct values were obtained using automatic baseline and threshold settings provided by the LightCycler® 480 Software, Version 1.5. Data were analyzed manually using the comparative CT method (ΔΔCT). Individual targets were analyzed in three biological replicates and represented as a mean.Equal amounts of DNase I-treated RNA were polyadenylated and converted to cDNA using the qScript microRNA cDNA Synthesis Kit . About 1 ng of cDNA was amplified in combination with sequence-specific forward primers such as: tRNA-Asn: GTATGTCACTGGTTCGAGTC, 23S rRNA: GTTAAGCGACTAAGCGTACAC, tRNA-met: TGCTCTAACCAACTGAGCTAC, tRNA-Thr: CTCAGTAGGTAGAGCAACTG, tRNA-Ile: CTTGTAGCTCAGGTGGTTAGAG, tRNA-His: CTATAGCTCAGTTGGTAGAG, tRNA-Val: TCGAGTCCACTCGGACGCAC, tmRNA: GCTGATTCTGGATTCGACG, 4.5S RNA: CTCTGTTGGTTCTCCCGCAAC or 6S RNA: TCTCTGAGATGTTCGCAAGC (obtained from Eurogentec) and PerfeCTa Universal PCR Primer (Quanta BioSciences) in a total volume of 10 E. coli K-12 substrain MG1655 using standard isolation procedures and transmission electron microscopy  of acts Fig.. These cE. coli contains RNA, by applying a fluorescent labeling method which was adapted for staining of the OMV fraction , extracellular OMV-free fraction (RNAexOMV-f), and from whole cells . To minimize potential confounding effects of the presence of RNA in the RNAexOMV-f fraction due to the lysis of cells and/or OMVs, RNA was always isolated from the respective fractions from early stationary growth phase. The presence of distinct RNAs in the RNAexOMV-f fraction compared to RNAint  suggests the involvement of unknown mechanisms in the secretion of bacterial intracellular RNA into the extracellular milieu in addition to their release through OMVs. Each RNA fraction was subdivided into a <200 nt long RNA fraction  and >200 nt long RNA fraction (“large RNA fraction”) and analyzed using an Agilent Bioanalyzer . The majority of the RNA obtained from the RNAexOMV and RNAexOMV-f has a length less than 60 nt with an enrichment in the size range between 15 and 40 nucleotides . Reads which mapped to both the media RNA libraries and the E. coli genome were termed “cross-mapping reads.” The majority of the cross-mapping reads from RNAint map to tRNA and protein coding genes in E. coli, whereas protein coding genes dominate the cross-mapping reads in RNAexOMV, and tRNAs in RNAexOMV-f, respectively . In order to compare the extracellular RNA profiles with the intracellular RNA profiles, the extracted <200 nt long intracellular RNA fraction was also sequenced. To identify and remove possible contaminant RNAs present in the LB growth medium . Reads mapped to E. coli but not to known RNA biotypes, were designated as uncharacterized  and RNAs encoded by mobile genetic elements (including cryptic prophage) comprise 0.54% and 0.19% of the total mapped sequence reads in RNAexOMV, compared to 6.5% and 4.5% in RNAexOMV-f, and 0.19% and 0.05% in RNAint, respectively. Furthermore, the mapping of reads corresponding to individual RNA biotypes reinforces the intracellular origin of extracellular RNAs while discrete differences in coverage may indicate discrete processing of these RNAs according to their localization . On the one hand, the observation of numerous biotypes unique to RNAint underpins the notion of selective export of RNA biotypes, analogous to observations in eukaryotes . These map mostly to the 5′ and 3′ ends of mature tRNAs ] and two 17 nt long fragments of the 3′ end of the 5S rRNA [GTGGGGTCTCCCCATGC (+st) and AGGGAACTGCCAGGCAT (+st)], which seem to be exclusively exported as suggested by the mapping patterns of read sequences  nucleotides: “CGGAAAGACC.” For the tRF, thymine is substituted by adenine, whereas cytosine is substituted by guanine and vice versa. This sequence is present on a 15 nt long tRF, which was found in RNAexOMV , RNAexOMV-f (coverage of 270), and RNAint  . One of the functions of SRP involves the targeting of membrane and secretory proteins to the plasma membrane of bacteria following the recognition of a signal peptide sequence on the nascent polypeptide , 6S RNA, and others are also selectively enriched in the RNAex Table. Furtherex and RNAint mapped to mobile genetic elements of the E. coli MG1655 genome, primarily to the cryptic prophages CP4-6, DLP12, and e14, with the majority of those exported reads in the RNAexOMV-f fraction mapping to cryptic prophage CP4-6. Cryptic prophages provide selective advantages to their hosts in different conditions . Although, according to these results, OMVs contain RNA, our results also indicate that RNA may be associated with the OMV surface as previously described for DNA in Moraxella catarrhalis, where OMV-associated DNA is recognized by toll-like receptor 9 (TLR9) inside immune cells and thereby stimulating an immune response ] encoded by this region which was present in both RNAexOMV-f and RNAexOMV  How are non-OMV-associated bacterial RNAs transported across bacterial membranes and subsequently released into the extracellular space? (2) How are the RNAs selected for export? (3) Is there a difference in the extracellular RNA profile between pathogenic and non-pathogenic bacteria? Apart from seeking answers to these questions, extracellular bacterial RNAs should be investigated for their potential protein binding partners, host target cells and their potential as biomarkers. Understanding the role of bacteria-derived exogenous RNA in host–microbe interactions, in pathogenesis as well as in mutualism, will elucidate new mechanisms and perhaps allow the identification of new drug targets and/or the development of RNA-based vaccines. Further investigations in the field of extracellular bacterial RNAs are clearly needed to shed light on their potential role as mediators of microbe–microbe and host–microbe intercellular communication.


RNA silencing has become a major focus of molecular and biomedical research in the last decade. This mechanism, which is conserved in most eukaryotes, has been extensively studied and is associated to various pathways implicated in the regulation of development, in the control of transposition events, heterochromatin maintenance and also playing a role in defense against viruses. Despite of its importance, the regulation of the RNA silencing machinery itself remains still poorly explored. Recently several reports in both plants and metazoans revealed that key components of RNA silencing, such as RNA-induced silencing complex component ARGONAUTE proteins, but also the endonuclease Dicer are subjected to proteasomal and autophagic pathways. Here we will review these post-translational proteolytic regulations with a special emphasis on plant research and also discuss their functional relevance. RNA silencing involves processing of double stranded (dsRNA) by the enzyme Dicer, into small RNAs, 21–25 nucleotides in length called small interfering RNAs , with which FBW2 does not share any significant sequence similarity beside an F-box motif.Because viruses usually hijack host cell machineries, it was conceivable that AGO1 protein turnover by autophagy may also occur in a P0-independent context. Hence, this prediction was confirmed when it was shown that mutations affecting miRNA biogenesis and/or accumulation and thus disturbing RISC assembly, also result in AGO1 degradation by autophagy . This fiproteins . One gooein FBW2 . FBW2 waein FBW2 , FBW2 ovein FBW2 . InteresArabidopdsis AGO so tightly regulated at the post-translational level. Thus, at least in transient expression assays in tobacco leaves, P0 is also able to mediate the degradation of AGO2, AGO4-6, and AGO9  is both hydroxylated and phosphorylated . In partin vitro and in vivo through its RING and B-Box domains, all located in the Trim domain. Moreover, the ectopic overexpression of mLin41 reduced the level of endogenous Ago2 in embryonic carcinoma cells and this effect was attenuated by inhibition of the proteasome with MG132. However, more recent studies put into question the control of AGO2 stability by mLIN41 . Hence it was shown that both DICER and AGO2 levels increased in HeLa cells treated with chemical inhibitors known to block autophagy and in siRNA-depleted cells for different component of the autophagy pathway, such as ATG5, ATG6, ATG7 or NDP52  triggers AGO1 destabilization by the proteasome , a master ubiquitin protein ligase that usually targets cell cycle regulatory proteins  of these proteolytic pathways? The current model indicates that the stability of AGO proteins depends on miRNA biogenesis and thus unloaded AGOs are unstable . If AGO 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.


RNA metabolism and thus gene expression. Here, we attempt such decryption in an eukaryotic model organism, the fission yeast S. pombe. We first derive an improved genome annotation that redefines borders of 36% of expressed mRNAs and adds 487 non‐coding RNAs (ncRNAs). We then combine RNA labeling in vivo with mathematical modeling to obtain rates of RNA synthesis and degradation for 5,484 expressed RNAs and splicing rates for 4,958 introns. We identify functional sequence elements in DNA and RNA that control RNA metabolic rates and quantify the contributions of individual nucleotides to RNA synthesis, splicing, and degradation. Our approach reveals distinct kinetics of mRNA and ncRNA metabolism, separates antisense regulation by transcription interference from RNA interference, and provides a general tool for studying the regulatory code of genomes.To decrypt the regulatory code of the genome, sequence elements must be defined that determine the kinetics of  The ratio between the rates of RNA synthesis and degradation determines steady‐state levels of mature RNA, thereby controlling the amount of messenger RNA (mRNA) and the cellular concentration of non‐coding RNA (ncRNA). The rates of both RNA degradation and splicing contribute to the time required for reaching mature RNA steady‐state levels following transcriptional responses  is an attractive model organism to study eukaryotic RNA metabolism. S. pombe shares important gene expression mechanisms with higher eukaryotes that are not prominent or even absent in the budding yeast S. cerevisiae. These include splicing, which occurs for ~50% of the genes and is achieved with conserved spliceosomal components  from fission yeast grown in rich media  previously annotated ncRNAs and could not recover 1,011 (66%) of the previously annotated ncRNAs  of ORF‐TUs were generally much shorter than previously annotated (mean difference 91 nt), consistent with a previously curated set of ORF transcript boundaries . The data contained many reads that stemmed from intronic sequences and reads comprising exon–intron junctions, showing that 4tU‐Seq captured short‐lived precursor RNA transcripts. These reads from unspliced RNA gradually ceased during the time course  and used the obtained data for kinetic modeling Fig , step 1.urse Fig A and B, et alTo globally estimate rates of RNA synthesis, splicing, and degradation, we used a first‐order kinetic model with constant rates that describes the amount of labeled RNA as a function of time Fig C. We modet al, Our method yields absolute splicing and degradation rates, but provides synthesis rates up to a scaling factor common to all TUs. Absolute synthesis rates were obtained by scaling all values so that the median steady‐state level of ORF‐TUs matches the known median of 2.4 mRNAs per cell  and were degraded slightly faster . Thus, the differences in steady‐state levels of mRNAs and ncRNAs are achieved both by longer synthesis times and shorter half‐lives for ncRNAs, although the differences in synthesis times dominate. Moreover, splicing time did differ significantly between the two transcript classes . Transcription is known to be the major determinant of gene expression. However, among genes expressed above background level as investigated here, the dynamic ranges across the bulk of all TUs  showed similar amplitudes for all three rates  and about an order of magnitude slower than splicing , a fission yeast core promoter element, and the Homol E‐box (ACCCTACCCT), providing positive controls. In agreement with literature  is located near the TU 5′end Fig C, and aset al, et al, et al, S. pombe to mammals.Three motifs were detected in 3′ UTRs of ORF‐TUs that all were associated with decreased RNA half‐lives. One of these (TATTTAT) corresponds to the known AU‐rich element (ARE) that destabilizes RNAs  consistent with the predicted 1.35‐fold increased synthesis time  whereas the (7.G > A) in the gene mug65 led to a significantly decreased expression level . Among the novel motifs, the TTAATGA could be validated (3 out of 4 genes with a significant change in expression in the predicted direction P < 0.05) as well as the AACCAC motif (2 out of 2 genes with a significant change in expression in the predicted direction P < 0.05). The other motifs generally did not yield significant changes, possibly because the predicted and the observed effects were of small amplitude. In the 20 variants, the observed and predicted fold changes did not only agree in direction but also in amplitude  and at the branch site (BS). We complemented this set with the 3′‐splice site (3′SS) and extended motifs in each direction as far as significant single‐nucleotide effects were found  and correlated negatively with TU length  and depleted within exons (P = 0.001) of the opposite strand , consistent with repression of mRNA synthesis by antisense transcription. This effect was higher when the antisense ncTU covered a larger area of the ORF‐TU , which in fission yeast acts co‐transcriptionally.Although transcripts are generally not antisense to each other, we found 520 ncTUs antisense of ORF‐TUs , this approach recovered known regulatory motifs de novo, such as core promoter elements and the 3′ UTR AU‐rich element, but also provided two novel 3′ UTR motifs, and AC‐rich sequences in promoters and in 5′ UTRs. Our approach has several advantages. Conservation analysis is difficult in genomic regions that align poorly, as is often the case for regulatory regions, and give confounding results because selection can have regulatory and non‐regulatory origins. By analyzing the relation between sequence and individual RNA metabolic rates, we uncouple the contribution of sequence elements to each step of RNA metabolism. Whereas standard motif enrichment analysis discriminates between two classes of data , we used quantitative regression and therefore could exploit the full range of the data without applying any cutoff. Regression furthermore has the benefit to provide quantitative predictions regarding genetic perturbations that could be directly compared to expression fold changes for functional validations. Our method is general and can be applied to other organisms. Here, we analyzed steady state of a single growth condition, which might explain why we found regulatory elements such as the Homol D and the Homol E‐box or the AU‐rich element that are presumably constitutive. Application of the approach in a comparative fashion between different conditions could help in identifying a wider range of regulatory elements. Moreover, the approach could be extended to study other layers of gene regulation such as ribosome recruitment, or translation.We further introduced an approach to discover regulatory elements in the genome that combines S. cerivisiae, fly, mouse, A. thaliana, and human. Hence, our approach could help interpret the transcription profiling in human individuals. In the future, the application of our model may help to understand the consequences of regulatory variation in the human genome, with important implications for understanding gene regulation and interpreting the many disease‐risk variants that fall outside of protein‐coding regions .All experiments were done with the strain ED666 (BIONEER) . Labeled RNA was purified as published  was inoculated from glycerol stock. An overnight culture was inoculated (YES medium: 0.5% w/v yeast extract (Difco), 225 mg/l each of adenine, histidine, leucine, uracil, and lysine hydrochloride, 3.0% glucose) from a single colony and grown at 30°C. In the morning, a 120 ml culture (YEA medium: 0.5% w/v yeast extract (Difco), 75 mg/l adenine, 3.0% glucose) was started at OD™ Gold Kit  according to the manufacturer's recommendation with 1.5 μg labeled RNA and with 2.5 μg total RNA as input. Sequencing libraries for the time series samples were prepared according to the manufacturer's recommendations using the ScriptSeq™ v2 RNA‐seq Library Preparation Kit (Epicentre). Libraries were sequenced on Genome Analyzer IIx (Illumina). Raw files can be downloaded from Array Express accession E‐MTAB‐3653. Annotation and basic statistics of the libraries are given in rRNA was depleted using the Ribo‐ZeroSingle‐ and paired‐end RNA‐seq reads were mapped to the reference genome (ASM294v2.26) with GSNAP  were identified using a min‐length max‐gap algorithm on binarized RNA‐seq coverage track  resultinet al, –stranded = yes and ‐m intersection‐strict. To count reads that map to splice sites we used HTseq with one different parameter (‐m union) to allow counting of reads that spanned the junctions. For each intron, we defined the 5′SS as the 2‐nt region that contains the last position of the upstream exon and the first position of the intron. Accordingly, we defined the 3′SS as the 2‐nt region that contains the last position of the intron and the first position of the downstream exon. To distinguish spliced and unspliced junction mapping reads, a custom python script checked the cigar string of each alignment for occurrences of skipped reference bases (“N”). Alignments containing “N” and overlapped with a splice site were counted as spliced junction reads.Counts of reads aligning completely within exons or introns were obtained with the software HTseq‐counts  plus 1 μl bacterial alkaline phosphatase (Invitrogen). RNA was precipitated at −80°C for 5 min with 3 M sodium acetate and ice‐cold 100% ethanol. After centrifugation at 13,000 rpm for 5 min at RT, the supernatant was transferred to a new tube and an additional 100 μl of ice‐cold 100% ethanol was added. After 5 min at −80°C and centrifugation at 13,000 rpm for 5 min at RT, the supernatant was transferred to a new tube and the sample was dried with a SpeedVac at 45°C for about 25 min. For the HPLC, samples were run on a Kinetex 5 μm C18 100 250 × 4.6 mm (Phenomenex) column with buffer A acetonitrile and buffer B 0.05 M triethylammonium bicarbonate, pH 8.0 in a gradient 1% A/ 99% B to 15% A / 85% B in 20 min at 1.2 ml/min. The retention times of ribouridine (rU) and 4‐thiouridine (4sU) were calculated from running standards (cells metabolize 4tU into 4sU prior to incorporation into RNA). Percent of incorporation was calculated by taking the value for the area under the curve for the rU and the 4sU peaks with the software Chromeleon Chromatography Management System, version 6.80 (Thermo Scientific) and multiplying them with the extinction coefficients for 260 nm for rU or for 330 nm for 4sU and then dividing the resulting value for 4sU by the value for rU.PC and JG designed and supervised the research. PC conceived and designed the experiments. KCM performed the experiments. PE, LW, MB, JG, CD, and SI analyzed the data. PE, JG, PC, LW, and MB wrote the manuscript.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Dataset EV1Click here for additional data file.Dataset EV2Click here for additional data file.Dataset EV3Click here for additional data file.Dataset EV4Click here for additional data file.Dataset EV5Click here for additional data file.Dataset EV6Click here for additional data file.Review Process FileClick here for additional data file.


Lucilia sericata, is a species with importance in medicine, agriculture and forensics. Improved understanding of this species’ biology is of great potential benefit to many research communities. MicroRNAs (miRNA) are a short non-protein coding regulatory RNA, which directly regulate a host of protein coding genes at the translational level. They have been shown to have developmental and tissue specific distributions where they impact directly on gene regulation. In order to improve understanding of the biology of L. sericata maggots we have performed small RNA-sequencing of their secretions and tissue at different developmental stages.The green bottle fly maggot, L. sericata maggots. Illumina small RNA-sequencing of these secretions and the three tissues  revealed that the most common small RNA fragments were derived from ribosomal RNA and transfer RNAs of both insect and bacterial origins. These RNA fragments were highly specific, with the most common tRNAs, such as GlyGCC, predominantly represented by reads derived from the 5’ end of the mature maggot tRNA. Each library also had a unique profile of miRNAs with a high abundance of miR-10-5p in the maggot secretions and gut and miR-8 in the food storage organ the crop and salivary glands. The pattern of small RNAs in the bioactive maggot secretions suggests they originate from a combination of saliva, foregut and hindgut tissues. Droplet digital RT-PCR validation of the RNA-sequencing data shows that not only are there differences in the tissue profiles for miRNAs and small RNA fragments but that these are also modulated through developmental stages of the insect.We have successfully isolated RNA from the secretions of L. sericata and shown that there are distinct subsets of miRNAs expressed in specific tissues that also alter during the development of the insect. Furthermore there are very specific RNA fragments derived from other non-coding RNAs present in tissues and in the secretions. This new knowledge has applicability in diverse research fields including wound healing, agriculture and forensics.We have identified the small-RNAome of the medicinal maggots  Lucilia sericata larvae are commonly known as green-bottle blowfly maggots and are an important species in forensics, agriculture and biomedicine [Lucilia have also proven useful in forensics for estimation of post-mortem intervals [Lucilia, both L. sericata and to a greater extent L. cuprina, are parasites of sheep causing blow-fly strike which has detrimental economic effects worldwide [medicine ,2. Theirmedicine . Luciliantervals . Conversorldwide ,6.L. sericata maggots are believed to have a multifactoral influence on wound healing. Initially believed to be due to the mechanical eating of dead tissue, (debridement), they are now thought to mostly function through their biochemically active excretions and secretions (ES) [L. sericata ES have focused on the identification of molecules such as amino acids and fatty acids which may play a role in the wound healing [L. cuprina have also been reported to have anti-microbial activity, suggesting that this may be a common feature of fly larvae [Medicinal ons (ES) . The ES ons (ES) , proteasons (ES) , and eveons (ES) . Studies healing ,12. Prot healing ,14. A nu healing . The secy larvae .L. sericata is not publically available, only the mitochondrial genome is published [L. cuprina [Lucilia has already shown great value as it is accurate in developmental stage estimation for use in forensics [The full genome for ublished . Some shublished ,18. Due ublished . An expr cuprina . Gene exorensics .Drosophila [Apis Mellifera) [Anopheles; Aedes; Culex) [L. Sericata we have performed small RNA-sequencing on their larvae tissues and ES. When the data was matched to known small RNA databases we identified both common and tissue specific RNAs, derived from various families of annotated small RNAs. The abundant small RNAs were then were assayed across developmental stages of L. sericata and validated in the dissected tissues by droplet digital RT-PCR.The small RNA profiles in multiple flying insects such as osophila , honey bllifera)  and mosq; Culex) ,25 have Eggs and Instar 2/3 larvae were provided for sterilisation and dissection by Consultant Entomologist Dr Dallas Bishop, Upper Hutt, New Zealand. She also provided the developmental stages of the insect, as determined by visual inspection of morphology, snap frozen on dry-ice. The eggs were laid onto liver, removed and sent by overnight courier to our laboratory with an ice-pack to delay hatching. A colony was also established in Auckland, using the same egg supply and rearing techniques, by Mr Vernon Tintinger. All eggs and larvae were maintained at ambient temperature.All equipment and reagents were autoclaved for sterility prior to use. Insect handling was performed in a laminar flow hood and maggots were stored in a sealed box on plates at room temperature.2O. Eggs were transferred to sterile 10% sheep blood, BHI agar, Colombia blood agar base plates (CBB) . Larvae were hatched in a sealed sterile box. After 48 hours the instar 2 maggots were re-bleached briefly in 0.5% sodium hypocholorite and rinsed in 70% EtOH as for the eggs and plated overnight on soy agar plates  to ensure the blood agar was removed from their systems along with potential food-derived animal RNAs.Eggs were surface sterilised upon arrival approximately 16 hrs after oviposition to mimic the human clinical application state by immersion in 1% sodium hypochlorite for 5min followed by 5min in 70% EtOH with a final rinse in dHAt each stage of growth, sample sterilised eggs/maggots were crushed and streaked on CBB plates and grown at 37°C overnight to confirm there was no surface bacterial contamination.2O and blotted dry.On day three after eggs bleaching, at instar 2/3, maggots were used for collection of excretions and secretions (ES) and dissections. Prior to use, maggots were again washed in 70% EtOH for 5 min, rinsed in dHTo isolate the salivary glands, crop and gut we used a dissecting microscope. Maggots were anaesthetised on ice and quickly placed in a sterile petri dish containing sterile water. Forceps were used at the proximal and distal ends to hold the maggot and pull laterally, ensuring a good hold on the mouthparts. Whole salivary glands, then the diverticulated foregut crop and lastly gut were dissected out and placed into 0.5mL of Trizol LS .2O at room temperature in the dark for 1 hour. Maggots were removed and ES spun 5min 2,500g to pellet debris and filtered through a 0.2μM syringe filter. Trizol LS was added at a volume of 3:1 as per manufacturers recommendations.ES was collected in by placing batches of 50 larvae in 250μL dHRepresentative insects from each of the developmental stages , as deteRNA was extracted from the Trizol samples with a modified Purelink RNA mini kit (Life Technologies) protocol which ensures retention of the small RNA fraction by adding an equal volume of 100% EtOH to the aqueous phase prior to addition onto the filter column. RNA was DNase I treated prior to sequencing using Turbo DNase-free (Ambion). RNA quantity and quality were assessed by Nanodrop and Experion bioanalyser chip (BioRad).et. al. from Drosophila [Drosophila.Modified from the method of Seitz osophila , a compl20 pmol oligo synthesised by IDT was coupled to 0.8mg of M279 streptabeads beads  washed prior in 0.5xSSC, by mixing on ice for 30 min. Uncoupled oligos were removed by placing the beads on a magnet and washing once with 0.5x SSC, resuspending in a volume of 80μL and then incubating at 65°C for 5 min then quenching on ice.To the coupled beads we added 5μg total RNA denatured previously at 85°C for 5min, then standing for one hour at 50°C. After depletion the rRNA bound beads were removed using a magnet and the supernatant retained. RNA was extracted from the depleted RNA using an Ambion miRvana column  to enrich for the <200bp fraction as per manufacturers instructions.Sample inputs were 9.2ng enriched depleted SG, 25.4ng depleted gut, 58.0ng depleted crop, 246.8ng whole ES RNA, all collected from sterilised Instar 3 animals. Small RNA libraries were prepared using a TruSeq small RNA sample preparation kit (Illumina) as standard and run on a MiSeq with two samples per lane, 2x25bp paired end reads. Sequencing was performed as a service by New Zealand Genomics Ltd. The data sets supporting the results in this article are available from the Sequence Read Archive, project number SRP028914.MiSeq Reads were linker sequence trimmed, quality trimmed then self-aligned into unique reads. The reads were BLAST against miRBase V19 , the FunWe used ddPCR to validate the tissue specificity and developmental stage expression of selected miRNAs. Briefly, RNA was isolated from whole samples at seven developmental stages after homogenisation in Trizol with a Purelink Mini kit (Invitrogen) following the protocol for total RNA or from dissected tissues as previously detailed. Developmental stages were RNA extracted in triplicates from individuals (pupae/adult) or pools of insects. The dissected tissues were tested in singleplex due to the lack of excess source material and six replicate ES samples, collected from Instar 1 animals, were assayed. RNA was DNase treated using Zymo DNA-free RNA kit (Zymo Research) and RNA yield was assessed by a Qubit 2.0 fluorometer (Life Technologies) for accuracy.L. sericata to assess conservation of standard small RNAs such as U6.cDNA was synthesised from 100ng RNA using the Qiagen miScript cDNA kit and diluted 1:10 to 1:4000 with distilled water before use. PCR was performed with custom small RNA primers and predesigned miScript miRNA primer assays (Qiagen) using ddPCR EvaGreen Supermix (Bio-Rad). Reactions were carried out with 4μl of the diluted cDNA in a 20μl final volume, then mixed with 70μl Evagreen droplet generation oil for droplet synthesis using the QX200 system. From this, 40μl of PCR-oil mix was cycled as per manufacturers recommended conditions and read on a QX200 droplet reader. Positive droplet counts as determined by Quantasoft software with samples with less than 10,000 droplets discarded and repeated. The quantification is presented as copies per μl of PCR mixture adjusted for the cDNA dilution factor. The use of ddPCR which does not require a reference gene is ideal for this application due to a lack of genome for L. sericata salivary gland (SG) library was found to contain a single unique read at a very high abundance where of 2.71M total reads, 91% of these matched to this single RNA. This read aligned to the 30nt 2S rRNA which is reported to be present in all Diptera such as Drosophila [Drosophila [Small RNA-sequencing was performed on tissues from instar 3 larvae and also from their bioactive ES . A pilotosophila . This prL. sericata larvae for ES, salivary gland (SG), GUT and CROP  library which was found to contain an unexpectedly large amount of bacterial RNA. Medicinal maggots are used in the clinic after a process designed to generate “sterile” larvae, which we undertook to replicate by bleaching of eggs and then bleaching 3-day old larvae before collection of ‘sterilised’ tissue and ES samples. This bleaching is essentially a surface sterilisation process. Four tissue specific libraries were prepared from the instar 2/3 and CROP . The latand CROP . Most reReads were BLAST searched against three small RNA databases  to identify known annotated small RNAs and their species of origin . Only 2.When we focused on the database matched insect-derived small RNAs, each sample had a slightly different profile of RNA types . The mosProteus mirabilis. Another 23S rRNA fragment derived from closely related genus Providencia was also detected at high abundance in the SG and CROP samples.The top ten most common URs in each of the four samples were further BLAST searched against RefSeq databases . FirstlyDrosophila mature tRNA sequences they are predominantly at the 5’ end, particularly when the tRNA is highly abundant. The fragmentation patterns for tRNAs were generally consistent across samples with the only differences seen for those with lower read counts suggesting that they are random degradation products.We next looked at the patterns of insect tRNA fragments that were found in each sample . A tRNA Drosophila miRNAs were also found in L. sericata larval tissues, such as miR-8, miR-31a, bantam and miR-275 [Sarcophilus harrisii (Tasmanian devil) also matched to fRNAdb as a fragment of insect large subunit rRNA. To note, we have also identified some low read count mammalian miRNAs in our tissue libraries, such as miR-486-5p, a red blood cell abundant miRNA, that we hypothesise to be derived from the sheep blood agar which was provided as a food source for the maggots. Unfortunately, due to the current lack of a genome for L. sericata and the limited depth of our RNA-seq data we are unable to identify novel miRNAs in the species.Finally we focused on the identification of known miRNA homologues by BLAST searching against miRBase V19 . The miR miR-275 . In our L. sericata from whole eggs through larval stages to pupae and eventually to newly emerged adult fly . We assessed the highly common 2S rRNA fragment, tRNA GlyGCC 5’ fragment, and 7 miRNAs in these samples   . This meWe also detected differences in many of the small RNAs assayed through developmental stages. For the most part, their abundance is lowest in eggs with an increase after hatching into instar 1 larvae. The miRNAs have specific patterns of expression throughout the developmental process with miR-10-5p gradually declining in abundance and miR-956-3p having the opposite pattern of expression.The aim of this study was to use small RNA-sequencing technology to identify the profiles in a range of medicinal maggot tissue samples and in its ES. The standard for analysis of such data is to initially map the reads to the genome of interest before further analysis, discarding any which are unmapped. In our case this was not possible due to lack of a complete published genome. We note therefore that this dataset is limited to common subcategories of small RNAs from orthologous insects. However because of our non-genome-mapping approach to the analysis we were able to identify small RNAs in an unbiased manner, including both fly and bacterial sequences. The RNA component present in ES appears to be derived from all its potential tissue sources . In terms of the overall RNA profiles, tRNA and miRNA patterns, the SG and CROP source libraries are most similar to one another, possible due to the close proximity of these tissues in the maggot foregut.L. sericata tissues and ES highlighted the presence of a varied profile of commonly annotated small RNAs, derived from insect, bacterial and food sources. The surface bleaching of eggs and larvae into medicinal-grade maggots, did as expected and removed a large proportion of bacterial burden; however the database read matches still identified bacterial RNAs in the tissues and ES. The bacterial content was highest in the crop, a food storage organ [Proteus mirabilis 23s rRNA, a known symbiotic bacterium in flies [Providencia, identified as an insect pathogen in Drosophila [Mycobacterium tuberculosis [Small RNA-seq of ge organ , which hge organ . Thus thin flies  and alsoosophila . The bacosophila  or even rculosis .L. Sericata has shown that each has a distinct pattern of small RNA expression. The most common reads in all samples were fragments from 2S rRNA and tRNA GlyGCC. The 2S rRNA is found only in Diptera, processed as a 30nt fragment from the 5S rRNA [L. sericata samples we assayed by RNA-seq and across the developmental stages of the insect by ddPCR. Its function, other than as a ribosomal component is unknown. The ddPCR validation showed that the 2S rRNA was present in all tissues and the ES, at levels 13 to 22-fold higher than the most abundant miRNA assayed.Here we have shown for the first time that the bioactive maggot ES contains RNA. Furthermore the RNA profiling of the three tissues from the gastrointestinal tract of  5S rRNA . It is c 5S rRNA ,39, its Drosophila, tRFs have been found complexed with the piRNA-binding protein PIWI and have a role in RNA silencing [Haloferax volcanii these tRFs bind to the ribosome to interfere with peptidyl transferase activity and reduce translation [Proteus and Providencia. Other fragments identified in addition to these 5’ tRNA fragments were at very low counts and were likely to be non-specific degradation products.The second most common single read in the RNA-seq libraries was a very specific fragment (tRF) from the 5’ end of the GlyGCC tRNA. This tRF was in particular highly abundant in the GUT and ES libraries and ddPCR showed it was 37 to 60-fold more abundant in these than any of the other miRNAs assayed. Other 5’ tRFs that had high counts in all tissue libraries were AspGTC, LysCTT and HisGTG. Indeed, all of the tRFs with high count numbers were derived from the 5’ end of the mature tRNAs. Furthermore the dominant fragment for each tRNA was found to be consistent across the different tissues. tRFs have been explored in response to stress in mammals where they can function as novel global translation inhibitors ,41. In Dilencing . In the nslation . In the nslation . These pnslation . It was nslation . It is tDrosophila to mosquitos and ticks, which are commonly responsible for the transmission of disease in mammals [L. sericata. These sequence matches were predominantly to miRNAs from flying insects such as Drosophila , suggesting that they’re directly derived from the maggot rather than from the bacterial and food source contamination. Some low count mammalian miRNAs were found but these were rare as we ensured that the maggots were starved overnight of their sterile blood food source before sampling.MicroRNA profiling in insects has been performed on many different species from the model organism  mammals ,25,46. MDrosophila miR-263a and miR-8 [Each of the tissue libraries had a different profile of miRNAs which in the most part was validated by ddPCR. For example, two anti-apopototic miRNAs in nd miR-8  were theL. sericata miRNAs are compared to published insect profiles, we see many overlap in terms of their overall abundance or their tissue expression profiles. For example miR-8, miR-184, and bantam are found in all Drosophila tissues [Haemaphysalis longicornis [When the  tissues  and miR-gicornis .L. sericata eggs and then rapidly increased in instar 1 larvae (3.5 to 55-fold increase). It appears therefore that miRNAs are switched on during larval development and are likely important in the regulation of this process. Furthermore, most miRNAs slightly declined in levels between instar 1 and early instar 3 before either continuing this decline (e.g. miR-10-5p) or increasing again from late instar 3 to adults . This increase in expression at late instar 3 could be associated with feeding and rapid growth. The increase in later larval stages, pupae and adults of these miRNAs was also reported for the silkworm moth Bombyx mori [Drosophila [All of the miRNAs assessed by ddPCR were expressed at relatively low levels in byx mori . Conversosophila .L. sericata. It is possible that novel siRNA-based therapeutics, aimed at the interference of these insect-specific miRNAs might be useful in the treatment of blow-fly infections in agriculture to hinder larval development.The variation in miRNA abundance between the developmental stages proposes the potential for them to be used in aging of maggots. Gene expression analysis has proven valuable in forensics for accurate determination of developmental stages. A set of miRNAs, such as miR-10-5p which decreases through instars and miR-956-3p which increases could therefore be useful in forensic staging. Furthermore miRNAs are more stable than messenger RNAs which are currently assayed . In addiet al discussed the potential for miRNAs to act as antimicrobials by downregulating bacterial gene expression [Apis) which produces both honey and royal jelly. The components of the royal jelly secretions have epigenetic effects on gene expression such that a worker bee fed on it will develop into a queen bee. Like the maggot ES, royal jelly has recently been shown to contain specific miRNAs, including miR-184, -276a, -10, -8, -31a and bantam [The multimodal effects seen when maggot ES is used in the clinical setting is probably due to their diverse molecular composition, with proteins, fatty acids and peptides all found to be functional in promoting wound healing ,12–15,52pression . In the d bantam , all of d bantam  and has d bantam .L. sericata may hold great interest for many research fields from forensics to agriculture. We have identified common subcategories of small RNA in the tissues of L. sericata and shown that these also vary by developmental stage. Furthermore, these tissues commonly contain specific fragments of ribosomal and transfer RNAs which appear to be more than simply degradation products. Finally, we have shown for the first time that the secretions of L. sericata maggots contain RNA, which we propose may be directly involved in its potency as an antimicrobial and wound healing agent.The small RNA profile and in particular the miRNA profile in S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Altered RNA metabolism is a key pathophysiological component causing several neurodegenerative diseases. Genetic mutations causing neurodegeneration occur in coding and noncoding regions of seemingly unrelated genes whose products do not always contribute to the gene expression process. Several pathogenic mechanisms may coexist within a single neuronal cell, including RNA/protein toxic gain-of-function and/or protein loss-of-function. Genetic mutations that cause neurodegenerative disorders disrupt healthy gene expression at diverse levels, from chromatin remodelling, transcription, splicing, through to axonal transport and repeat-associated non-ATG (RAN) translation. We address neurodegeneration in repeat expansion disorders  and in diseases caused by deletions or point mutations . Some neurodegenerative disorders exhibit broad dysregulation of gene expression with the synthesis of hundreds to thousands of abnormal messenger RNA (mRNA) molecules. However, the number and identity of aberrant mRNAs that are translated into proteins – and how these lead to neurodegeneration – remain unknown. The field of RNA biology research faces the challenge of identifying pathophysiological events of dysregulated gene expression. In conclusion, we discuss current research limitations and future directions to improve our characterization of pathological mechanisms that trigger disease onset and progression. RNA-mediated neurodegeneration is implicated in the causes of Huntington's disease (HD), spinocerebellar ataxias (SCAs), spinal muscular atrophy (SMA) and major subtypes of amyotrophic lateral sclerosis (ALS). In contrast, the current neuropathological classification of Parkinson's disease (PD), Alzheimer's disease (AD) and prion disease relates to the abnormal accumulation of misfolded and aggregated proteins in the brain .post mortem HD brains may have lost up to 25% of their weight. HD usually develops in patients aged 35–45 years, although onset can occur from childhood to old age. Caucasian populations have a high and likely underestimated prevalence . The disease progressively affects movement , behaviour and cognitive functions. Neuropsychiatric problems worsen over time and ultimately lead to dementia HTT). Unaffected individuals normally carry 11–34 CAG repeats while HD patients have >36–250 CAG repeats HD is a fatal disease initiated by selective death of neurons in the striatum before neurodegeneration spreads to other cerebral regions. Startlingly, some The SCAs form a large heterogeneous group of autosomal-dominant diseases with typical onset at 30–50 years old. They are caused by repeat expansions in both coding and noncoding regions of multiple genes. Repeat expansions are formed by CAG repeat sequences in most cases; however, some subtypes are characterized by the presence of trinucleotide (CTG), pentanucleotide  or hexanucleotide (GGCCTG) repeats. SCAs are slowly progressive diseases involving neurodegeneration of the cerebellum and spinal cord. They are associated with dysarthria, poor coordination of gait and fine movements, but with retention of cognitive function SMN1) gene SMA is the second most common genetic cause of infant mortality after cystic fibrosis (approximately 1 in 10 000 newborns). Lower motor neurons in the anterior horns of the spinal cord progressively degenerate, leading to muscle atrophy, paralysis and often fatal respiratory failure ALS is characterized by relentless degeneration of upper and lower motor neurons, which leads to progressive paralysis and death usually within 3–5 years from symptom onset. ALS is generally an adult disease with age of onset peaking at around 55–60 years of age. It is the most common form of motor neurodegenerative disease and has a prevalence of 6–10 cases per 100 000 individuals, with a lifetime risk of 1 in 400 Here, we review the molecular mechanisms by which genetic mutations can alter normal gene expression in neurodegenerative diseases. We also discuss research limitations and future strategies to better understand the functional cellular consequences of widespread RNA dysregulation.Eukaryotic gene expression is tightly regulated and integrates activating and repressive mechanisms Biogenesis/processing of mRNA is orchestrated in separate, but extensively coupled steps within the nucleus HTT gene encodes a very large protein huntingtin (HTT) of over 3000 amino acids (approximately 350 kDa) which is essential for embryonic development and brain function The HTT in HD Poly-glutamine (poly-Q) amino-terminal truncations of HTT, generated through aberrant splicing of Loss of HTT protein function plays a critical role in HD. HTT is involved in various cellular functions On the other hand, the poly-Q expansions in HTT also trigger several toxic gain-of-functions described below. HTT has the ability to interact with over 100 proteins involved in various cellular functions Poly-Q HTT aberrantly interacts and may sequester several transcription factors (TFs) which disrupt transcription and widely affect gene expression. Several TFs have been found in nuclear HTT inclusions, including the general transcription factor TATA box binding protein (TBP) and co-transcriptional activators such as CREB-binding protein (CBP) and specificity protein 1 (SP1) PPARGC1A), as well as its protein product PGC-1α, a transcriptional master co-regulator that regulates energy homeostasis, mitochondrial biogenesis and antioxidant defences. Reduced PPARGC1A transcription and disrupted PGC-1αfunction are likely to contribute to the mitochondrial dysfunction observed in HD HTTPoly-Q HTT directly binds to the promoter of peroxisome proliferator-activated receptor gamma coactivator 1-alpha  CBP and P/CAF. Post-translational modifications of histones control the accessibility of the chromatin to the transcriptional machineries. Alteration of chromatin remodelling in HD leads to widespread dysregulation of gene transcription in large genomic regions The expanded HTT RNA Finally, CBP dysregulation also results in reduced acetylation of the rRNA-specific upstream binding factor 1 (UBF1) which dysregulates rRNA transcription and leads to nucleolar stress There are 36 SCA subtypes identified to date, with a combined prevalence of 5–7 cases per 100 000 people 70–72. Most SCA subtypes are caused by repeat expansion mutations that occur in over 20 known genes. SCAs can be sporadic, or have autosomal recessive or autosomal dominant inheritance dependent on the subtype. The most widely characterized genetic subset are the autosomal dominant cerebellar ataxias (ADCAs), which are subdivided into three broad types based on their clinical presentation Many of the ADCAs  are caused by the presence of expanded CAG repeats within exonic regions of genes. These repeats are reminiscent of those found in HD and have led to these SCAs being classified as poly-Q diseases A–C). An ATTCT pentanucleotide repeat expansion in intron 9 of ATXN10 causes SCA10 ATXN10 pre-mRNA, but the expanded AUUCU RNA is resistant to degradation and aggregates in nuclear and cytoplasmic foci of SCA10 cells and transgenic mouse brain. The expanded AUUCU RNA binds the splicing factor heterogeneous nuclear ribonucleoprotein K (hnRNPK), resulting in hnRNPK sequestration and loss of function. As a result, protein kinase C δ (PKCδ) accumulates in the mitochondria of SCA10 cells, leading to caspase-3 mediated apoptosis BEAN) and thymidine kinase 2 (TK2), genes that are transcribed in the opposite direction. Affected individuals have over 250 repeats. The expanded UGGAA RNA binds and sequesters the serine arginine-rich splicing factor (SRSF) SRSF1 and SRSF9 in vitroin vivo, pre-mRNA processing and stability are likely to be disrupted. SCA36 is intriguing because it exhibits similarities with C9ORF72-related ALS. SCA36 patients initially develop cerebellar ataxia and frontal lobe atrophy. However, symptoms typical of ALS follow, with upper and lower motor neuron involvement, including tongue atrophy, skeletal muscular atrophy and fasciculation NOP56) C9ORF72miR-1292 gene Other SCA subtypes such as SCA10, SCA31 and SCA36 are caused by a variety of different noncoding repeat expansions, which have the potential to cause RNA toxicity important in disease pathogenesis 3ATXN) ATXN8OS and a CAG repeat expansion in ATXN8, genes which are transcribed in opposite directions ATXN8OS is transcribed from the sense strand, and the transcript contains an untranslated CUG repeat, while the CAG repeat expansion within ATXN8 on the antisense strand is translated into an almost pure poly-Q protein. The CUG repeat-containing RNA forms nuclear foci which sequesters the RNA processing factor MBNL1 post mortem human central nervous system (CNS) tissue In some other SCAs, there are contributions from both protein gain-of-function and RNA toxicity. The expanded CAG repeat in SCA3 not only produces poly-Q proteins, but also produces expanded CAG repeat RNA that potentially binds and sequesters RNA-binding proteins. The dysregulated transcription and splicing could contribute to neurodegeneration SMN genes that encode the same 294-residue protein. The majority of SMA cases show homozygous deletions, rearrangements or large truncations of the telomeric SMN1 gene copy. However, other cases are caused by short deletions/mutations in the splice sites of SMN1 introns 6 and 7. The SMN1 gene differs from the centromeric SMN2 copy by a few silent base changes SMN2 transcripts, leaving the vast majority of SMN2 mRNAs as defective transcripts lacking exon-7 SMN2 alterations are not associated with clinical pathology. Healthy motor neurons naturally express lower amounts of fully spliced SMN2 mRNA which may account for the higher vulnerability of motor neurons to SMN1 mRNA loss SMN2 exon-7 for the development of therapeutic approaches. As described below, the intricate regulation of exon-7 splicing involves binding of multiple RNA recognition motif (RRM) containing proteins such as SRSFs and hnRNPs that act directly on binding exon-7 and flanking introns as well as through direct protein : protein interactions.SMA is caused by a drastic reduction of SMN protein levels. The chromosomal 5q13 SMA locus is a duplicated region carrying two inverted copies of almost identical SMN1, while hnRNPM further stimulates the recruitment of the splicing factor U2AF65 to the flanking intron-7 4A). In contrast, several base changes in SMN2 exon-7 and flanking introns inhibit exon-7 inclusion. They alter the +6 ESE sequence leading to reduced interaction with SRSF1 SMN2 exon-7 is stimulated by ESE-binding of hnRNPQ1 SMN2 exon-7 splicing is provided in Figure 4B. Notably, SMA patients develop respiratory impairment in the late stages of disease. Hypoxia was reported to induce both hnRNPA1 and Sam68 protein levels, promoting SMN2 exon-7 skipping and further reduction of SMN protein levels. SMA SMNΔ7 mice housed in hyperoxia conditions, in contrast, displayed increased splicing of exon-7 and improved motor function SRSF1 CG8408) Nrxn2a) β-actin mRNA cpg15) mRNA in neuronal processes SMN proteins form part of a large oligomeric SMN complex of nine core-proteins composed of SMN (Gemin1), Gemins 2–8 and Unrip in mammals. This complex is found in the nucleus in Gemini of coiled (GEMs) bodies often associated with Cajal bodies where it is thought to play a role in RNA polymerase II dependent transcription and/or pre-mRNA splicing. The SMN complex is required in the cytoplasm for the assembly of uridine-rich small nuclear RNPs (U snRNPs) that compose the spliceosome, providing a binding-platform for the Sm core-domain and the targeted recruitment of snRNA SMN2 exon-7 skipping 2A) that results in broad splicing and axonal mRNP trafficking defects.In SMA, reduction in SMN levels also induces down-regulation of Gemins 2/3, hnRNPQ, hTra2-β1 SOD1 mutations in experimental models and human biosamples. In the presence of mutant superoxide dismutase 1 (SOD1) and in sporadic cases of ALS, multiple interacting factors contribute to the neurodegenerative process, including oxidative stress, excitotoxicity, mitochondrial dysfunction, disruption of the cytoskeleton/axonal transport, protein aggregation and altered glial-motor neuron cross-talk SOD1 mutations, highlighting alteration of mRNA-processing ALS is a multifactorial disease caused by mutations in one of over 20 genes encoding proteins with a variety of functions C9ORF72 (uncharacterized) in 40–50% cases SOD1TARDBP (encoding the TDP-43 protein) FUS (fused in sarcoma) The most common genetic causes of familial ALS involve autosomal-dominant mutations in the following four genes: C9ORF72 encodes a protein of uncharacterized function which might belong to the family of DENN-containing proteins, GDP-GTP exchange factors (GEFs) for Rab GTPases domain involved in the regulation of membrane trafficking C9ORF72 gene. The C9ORF72 RNA repeat expansions form stable parallel uni and multimeric G-quadruplex structures 150–152 which are well known for avidly interacting with RNA-processing factors. Both sense 89,91,153–155 and anti-sense 152,155–157 intraneuronal RNA foci have been observed in association with the C9ORF72 hexanucleotide expansion and form a pathological hallmark of C9ORF72-related ALS.In contrast to TDP-43 and FUS, the C9ORF72 protein does not display homology to RNA-binding proteins and is not thought to play a direct role in mRNA metabolism. C9ORF72 allele(s) 89,158–162 ; (ii) RNA mediated gain-of-function toxicity by the GGGGCC-expanded intron that prevents aberrantly bound/sequestered RNA-processing factors from functioning normally in the nucleus 91,92,153–155,163,164; and (iii) protein gain-of-function toxicity by dipeptide repeat proteins (DPRs) aberrantly generated from RAN translation of the expanded intron-containing mRNA 3A–C).Three potential molecular mechanisms of gene expression alteration and neuronal injury have been proposed and may coexist simultaneously: (i) haploinsufficiency due to decreased expression from the altered C9ORF72 coding sequence, suggesting that pathogenicity due to loss-of-function is less likely C9ORF72-mediated RNA toxicity in vitro alter RNA biogenesis and kill cells Haploinsufficiency was reported by several groups SOD1 encodes a ubiquitously expressed free radical scavenging enzyme. ALS SOD1 mutations Sod1 mice. The transcriptomes of dissected spinal cord motor neurons from SOD1-related ALS models SOD1-related ALS, anti-apoptotic phosphatidylinositol 3-kinase and protein kinase B (AKT3) were up-regulated with a concomitant reduction in the level of phosphatase and tensin homologue (PTEN) which inhibits the pro-survival AKT pathway, suggesting a mechanism for how these intact motor neurons survived the neurodegenerative process. Therapeutic modulation of this pathway is now being developed as a potential neuroprotective approach.SOD1 have been reported in ALS cases . They are distributed over the full length of the 154 amino acids of the human SOD1 protein, suggesting that loss-of-function mutations are unlikely. Furthermore, knock down of Sod1 does not trigger ALS in mice, and several SOD1 mutants, including A4V and G93A, retain virtually normal superoxide dismutase activity. The pathophysiological mechanisms of SOD1 mutations are thought to involve protein toxic gain-of-function  due to protein misfolding and formation of ubiquitinated intracytoplasmic neuronal and astrocytic inclusions in the CNS of SOD1-related ALS patients and mice More than 170 missense mutations of n Figure B due to TARDBP and FUS encode ubiquitously expressed DNA/RNA-binding proteins implicated in multiple aspects of gene expression regulation, including transcriptional control, alternative splicing of mRNA A,B). However, toxicity does not seem to depend on nuclear localization of mutant TDP-43 or on the formation of intracytoplasmic inclusions, but rather on the increased cytoplasmic mislocalization of soluble ALS-linked TDP-43 mutant proteins FUS and TDP-43 are hnRNP proteins which shuttle between the nucleus and the cytoplasm. They are composed, respectively, of one and two RRM domains flanked by carboxyl-terminal arginine/glycine (RG)-rich regions, also called the prion-domain because these unstructured regions have a propensity for aggregation. FUS also exhibits an RG-rich region in its amino-terminus. The vast majority of ALS mutations cluster in the RG-rich regions in exon 6 of TDP-43 and in exons 3–6 or 12–15 of FUS. These mutations disrupt protein : protein interactions, including those of TDP-43 with the splicing hnRNPs A1, A2/B1, C1/C2 and A3 2SETX) is predicted to play roles in several steps of gene expression, including functions associated with RNA processing and the maintenance of genome integrity. Angiogenin (ANG), the expression of which is increased during hypoxia to promote angiogenesis, is a tRNA-specific RNase that also regulates the transcription of ribosomal RNA. ALS mutations of ANG are likely to act through a loss of function, as overexpression extends the lifespan of ALS SOD1 mutant mice. The mechanism(s) by which mutations in SETX and ANG cause ALS remains to be determined.Further evidence of dysfunctional RNA metabolism has been provided by autosomal-dominant mutations in genes encoding senataxin ELP3) TAF15EWSR1) ATXN2), a gene product involved in the translation control of the circadian rhythm Saccharomyces cerevisiae and Drosophila melanogaster, and TDP-43 interacts with the polyA-binding protein (PABP) Additional genetic mutations were more recently identified in elongator protein 3  and disruption of multiple RNA-processing steps. Accordingly, transcriptome studies have revealed dysregulation of hundreds to thousands of RNA molecules in the presence of disease-causing mutations post mortem tissues enriched for neurons that survived the neurodegenerative process) C9ORF72 can be translated into toxic proteins Transcriptomics as a research methodology has contributed greatly to our understanding of the mechanisms of neuronal cell death As yet, the functional consequences of RNA dysregulation for the processes that trigger age-related and selective progression of neuronal cell death remain very poorly understood. The levels, frequency and identity of aberrantly spliced mRNA isoforms that undergo nuclear export and are subsequently translated into aberrant proteins are still largely uncharacterized. These aberrant proteins, many of which are likely to have essential enzymatic or structural activities that impinge upon a multitude of cellular pathways, may have either lost their wild-type biological function or will have acquired new deleterious functions post mortem CNS tissue. Single-cell next-generation RNA sequencing Reliable subcellular fractionation of cytoplasmic RNA, as well as purification of ribosomes for the extraction and next-generation sequencing of actively translating mRNA (i.e. the ‘translatome’) will become essential methodologies that will enable dissection of the molecular events that contribute to neurodegeneration. We anticipate that emerging methodologies investigating gene expression dysregulation at the level of active protein synthesis rather than at RNA/protein steady-state levels will yield biological results of higher relevance to the understanding of disease pathophysiology. Also, the specific sequences of mRNAs undergoing translation would provide rich information on the nature of mutations and/or aberrant splicing variations that are acquired during mRNA processing, and then subsequently translated into aberrant protein expression. Recent advances in molecular biology systems have also allowed the engineering of stable inducible neuronal cell models of neurodegeneration. In these models, cellular insults, such as disease-mutated protein or expanded nucleotide repeat expression, can be turned on as required. Such systems will allow investigators to observe early events in gene expression dysregulation that are more likely to be an upstream cause rather than a consequence of disease. Furthermore, these systems allow for the tracking of the progression of this dysregulation at multiple time points, meaning that it may be possible to define the upstream pathways in the pathophysiology of neuronal injury. Significantly, these types of analysis are prohibitively difficult to perform in animal models and impossible in human Widespread dysregulation of RNA metabolism is now clearly recognized as a pathophysiological component triggering neurodegeneration in the neurodegenerative diseases that are the focus of this review. It is very likely that other neurodegenerative diseases involve widespread dysregulation of gene expression. For example, excessive phosphorylation of the ribosomal RPS15 subunit by the PD mutated LRRK2 G2019S kinase was recently shown to trigger a toxic burst in global protein synthesis