dataesr/scientific-paragraphs-categorization huggingface dataset:
DISCUSSION The biogenesis and impact of secondary siRNAs induced by exogenous dsRNA have most intensively been studied in plants and C. elegans. Here, we report RdRP-dependent secondary siRNA production and transitivity of the silencing response for the first time in a ciliate, and to our knowledge, in the SAR clade of eukaryotes (Stramenopiles, Alveolates, Rhizaria). Paramecium secondary siRNAs do   2 ). SiRNAs oriented sense/+ to mRNAs/non-coding transcripts are represented in gray, those oriented antisense/-are shown in black. SiRNA size distributions for each cluster are shown in Supplementary Figure  S9 , graphic representations of their distribution on genomic regions are shown in Supplementary Figure  S10. Overlapping poly(A)-containing transcripts were identified using directional RNA-Seq data sets (normalized values).
ACKNOWLEDGEMENTS We thank  Véronique Tanty  and all other lab members for continuous support and input, and  Marcel Schulz  ( Max Planck Institute for Informatics, Saarbr ücken ) for critical discussion of sequencing data. We are thankful to  Mireille Bétermier  ( Centre de Génétique Moléculaire, Gifsur-Yvette ) for providing unpublished RNA-Seq data sets. The sequencing of small RNAs benefited from the facilities and expertise of the high-throughput sequencing platform of  IMAGIF (Centre de Recherche de Gif --www. imagif. cnrs. fr). We thank  Jasmin Kirch  for technical assistance and  Karl Nordstr  öm for support in bioinformatics ( Department of Epigenetics, University of Saarbr ücken ).
acquisition software (Tokyo, Japan), Fiji (NIH, Baltimore, MD, USA) and Imaris (Bitplane, Zurich, Switzerland) software were used to acquire and reconstruct z-series images into standard deviation projections at the NRI Microscopy Facility. Live imaging of control and drugtreated colonies were carried out using a Leica MZ16FA epifluorescent stereomicroscope. Images from live animals were also used for morphometrical analysis by using ImageJ (NIH, Baltimore, MD, USA) plug-ins. Figure panels were mounted using FigureJ (Image J) and Photoshop (Adobe, San Jose, CA, USA).
Identification of the Wnt genes in B. schlosseri Using a de novo assembled transcriptome database (http://octopus. obs-vlfr. fr/public/botryllus/blast_botryllus. php), we identified several B. schlosseri wnt genes among which four sequences showed strong similarities (e-value <1e -40 ) with orthologs in other species (Additional file 1: Table  S1 ). The predicted amino acid coding sequences were aligned with other metazoans Wnts, and a multiple phylogenetic reconstruction showed orthologies for three out of the four selected sequences (Figure  1 ). Whereas B. schlosseri Wnt2B, Wnt5A, and Wnt9A showed a statistically significant orthology with wnt genes from other ascidians and vertebrates, we did not have enough resolution to establish the orthology of Botryllus Wnt9. Therefore, we did not include the latter on the analysis of the expression patterns.
Isolation of B. schlosseri Wnts and phylogenetic analyses B. schlosseri Wnts were identified by performing a Basic Local Alignment Search Tool (BLAST) search of our in-house reference transcriptome database (http://octopus. obs-vlfr. fr/public/botryllus/blast_botryllus. php). The whole open reading frame (ORFs) of the B. schlosseri othologues of Wnt2B, Wnt9A, and Wnt5A were amplified and cloned into a pGEM-T-EZ vector (Promega), sequenced and used to produce RNA probes for wholemount fluorescent in situ hybridization (FISH).
Acknowledgements We would like to thank  Dr. Delany Rodriguez ,  Dr. Adam Langenbacher , and  Mr. Daryl Taketa  for their suggestions and discussions, and to  Mr. Daniel Lewis  and  Miss. Rebecca Shin  for their help on PCRs and in situ hybridization experiments. We are grateful to  Mr. Mike Caun  for his help within the animal mariculture facility and to the  NRI microscopy facility  ( Dr. Mary Raven ) for the use of confocal and electron microscopes. We thank two anonymous reviewers for helping to improve the manuscript. Financial support was provided by the AFMTelethon grant (# 16611 ),  IRG Marie Curie grant  (# 276974 ) and Nikon Fellowship (2009) to ST, and the  NIH  ( R01-AG037966 ) to AWD.
Crystallization and structure solution Crystals were grown after 3-4 days at 19 • C from a mixture of YlTrm9N38-Trm112 complex (10 mg/ml) with an equal volume of the crystallization solution containing 100mM tri-sodium citrate, 20% polyethylene glycol 4000 (PEG4K), 20% 2-propanol. Crystals were cryoprotected by transfer into the crystallization solution supplemented with 15 then 30% v/v ethylene glycol and then flash-frozen in liquid nitrogen. The data sets were collected at 100 K on Proxima-1 beam line (SOLEIL, St Aubin, France). The structure was solved by Zn-MAD (Multiple Anomalous Dispersion) using data sets collected at three wavelengths corresponding to inflexion, peak and remote of Zn-edge (see Table  1  for statistics). Data were processed with XDS  (30)  and scaled using XSCALE. The space group was I432 (a = b = c = 176.
2 Å) with one heterodimer per asymmetric unit, corresponding to a solvent content of 56. 4%. As expected from the presence of one Trm112 protein in the asymmetric unit, one Zn atom could be located by the HYSS submodule of the PHENIX package in the 50-3 Å resolution range  (31). Refinement of the Zn atom coordinates, phasing and density modification were performed with SHARP program using the 50-3 Å resolution range  (32). The ScTrm112 crystal structure  (13)  was positioned into the experimental density maps by molecular replacement using the MOLREP program  (33)  and then modified to match with the YlTrm112 sequence. The Trm9 model was built into these maps using the COOT molecular modeling program  (34). Iterative cycles of manual model rebuilding using COOT followed by refinement with PHENIX led to an almost complete model, which was then completed and refined using a higher resolution data set (2.
(38). Steady-state kinetics analyses were performed by using various tRNA (from 0. 1 to 1. 5 M) or SAM (from 1 to 20 M) concentrations. The initial rate calculated for each tRNA or SAM concentration was plotted as a function of the concentration. The data were fit to the Michaelis-Menten equation using the ORIGIN software and the K m , k cat and k cat /K m values were calculated from the curve fitting.
Generation of YlTrm9N20-Trm112 model Residues 20-39 from YlTrm9, which are missing in the crystal structure of YlTrm9N38-Trm112 complex, were modeled by superimposing the crystal structure of RPA2492, the closest structural YlTrm9 homologue (PDB code: 3E23), onto our structure. The SAM substrate and backbone atoms from residues 8-27 (corresponding to residues 20-39 from YlTrm9) were retained from the crystal structure 3E23. Missing side chains were placed using the SCWRL4 software  (39). The cm 5 U residue was docked manually into the YlTrm9 active site using constraints issued from the functional analysis of the various Trm9 mutants and by positioning the carboxyl group of cm 5 U at a distance compatible with methyl transfer from SAM. Finally, as the loop containing R40 (corresponding to ScTrm9 R39) is one residue shorter in YlTrm9 than the corresponding region from RPA2492, which was used as a template to build this fragment, we applied a restraint with a force constant of 1000 kcal/mol/ Å2 between the carboxyl group of cm 5 U and guanidinium group of R40 to position the side chain of this latter group toward the binding pocket.
Active site mapping Despite extensive efforts, we could not obtain crystals of the YlTrm9-Trm112 or YlTrm9N38-Trm112 complexes bound to SAM or SAH. We have then modeled a SAM molecule by superimposing onto YlTrm9 structure our SAM-bound structure of the ScBud23-Trm112 complex (15) (rmsd of 2 Å over 134 C␣ atoms; 19% sequence identity). We have further observed that upon SAM binding, ScBud23 Nterminal region folds as an ␣-helix that lies onto SAM. In addition, a similar ␣-helix is present in the structure of RPA2492 from Rhodopseudomonas palustris, the protein sharing the highest structural similarity with Trm9 (Supplementary Figure  S3A ; rmsd of 1. 9 Å over 200 C␣ atoms; 17% sequence identity; PDB code: 3E23). Finally, secondary structure predictions for the N-terminal region from Trm9 proteins strongly suggest that this region has indeed a high propensity to fold as an ␣-helix.
Note that for the anti-myc probing, the lower bands observed in some of the input lanes result from Trm9-myc protein degradation whereas the band observed in the IP lanes comes from mouse primary antibody heavy chain cross-reacting with anti-mouse secondary antibody. IP were performed using 9E10 anti-myc monoclonal antibodies. (D) Enzymatic activity of Trm112-Trm9 mutants. The curves obtained after fitting of the experimental data with equation given in the Materials and Methods section are shown by lines using the same color code as for the symbols. (E) Detailed representation of the YlTrm9 active site. Side chains from residues which substitution by Ala strongly (brown) or only weakly (green) affect enzymatic activity are shown as sticks. For clarity, ScTrm9 numbering is used. A manually docked cm 5 U nucleotide is shown as beige sticks and potential hydrogen bonds that it could form with Trm9 active site residues are depicted by dashed black lines.
Soluble protein extracts (Input: 1/50 th of total proteins, i. e. 10 g) and immunoprecipitates (IP: 1/10 th of immunoprecipitated material) were subjected to 15% SDS-PAGE analysis and immunoblotted using mouse anti-Myc (Trm9-13Myc) or rabbit anti-Trm112 as primary antibodies and sheep anti-mouse or sheep anti-rabbit HRP-conjugated IgG as secondary antibodies, respectively. Note that for the anti-myc probing, the lower bands observed in some of the input lanes result from Trm9-myc protein degradation whereas the band observed in the IP lanes comes from mouse primary antibody heavy chain cross-reacting with anti-mouse secondary antibody. IP were performed using 9E10 anti-myc monoclonal antibodies. (D) Enzymatic activity of Trm112-Trm9 mutants. The curves obtained after fitting of the experimental data with equation given in the Materials and Methods section are shown by lines using the same color code as for the symbols.
ACKNOWLEDGEMENTS We are indebted to  M. Argentini  and  D. Cornu  for mass spectrometry analysis (SICaps,  IMAGIF Platform, Gif/Yvette, France ), to  Dr V. Heurgué-Hamard  for her help with MTase assay and for sharing with us plasmids and antibodies directed against S. cerevisiae Trm9-Trm112 complex. We thank  Dr G. Bourgeois  for helpful discussion and advices. We thank  Dr B. Séraphin ,  Dr K. Breunig  and  Dr R. Schaffrath  for sharing reagents with us. We acknowledge SOLEIL for provision of synchrotron radiation facilities.
Circular dichroism spectral analysis of recombinant wild-type and alanine-substituted Sco-CHH-Gly Circular dichroism (CD) spectra were obtained using an AVIV 202 spectropolarimeter (Aviv Associates). The HPLC-purified glycine extended wild-type Sco-CHH (Sco-CHH-Gly), and glycine extended and alanine-substituted Sco-CHHs (I2A, F3A, D4A, D12A, R13A, Q51A, E54A, D60A, I69A, and V72A Sco-CHH-Gly) were individually dissolved in 10 mM PBS (150 mM NaCl, pH 6. 8) at 20-30 μM; an aliquot (200 μl) of the samples was placed in a 1-mm cell and data collected from 260 nm to 200 nm in 0. 5 nm increments at 25°C. Raw data were baseline corrected, smoothed, and transformed to obtain spectra in units of mean residue ellipticity and the ellipticity at 222 nm was used to estimate the α-helical content of the peptides  [40].
Mass spectrometric analysis of recombinant wild-type and alaninesubstituted Sco-CHH Mass determination was performed for the amidated recombinant peptides through a commercially available service (Mass Solutions Technology Co. , Ltd. ) using an LC-MS/MS system with an Accela HPLC station coupled to a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermos Scientific). Full mass spectrometry scan was performed with the range of m/z 500-4000 and the isotopically resolved mass spectra further deconvoluted using Thermo Scientific Protein Deconvolution software v2. 0.
Acknowledgments The authors thank  Dr. P-C Lyu  of  National Tsing Hua University  for providing circular dichroism spectropolarimeter and related technical support. C-YL  and  J-YT  also thank  Terres Asutrales et Antarctiques Françaises (TAAF) ,  Institut Polaire Français Paul Emile Victor (IPEV) , and the staff of  Terre Adélie  (TA) 65 for providing a warm and supporting community for them at a  French Antarctic station (Dumont D'Urville)  where an earlier draft of the manuscript was completed. J-YT was supported by an  NCUE Visiting Professorship in April 2014  from  National Changhua University of Education  for which he wishes to express his thankfulness.
These four variants were validated using Sanger sequencing. Co-segregation analysis confirmed that the mutations were inherited from heterozygous carrier parents. These variants were not observed in over 6,500 individuals of European and African American ancestry in the NHLBI Exome Sequencing Project database, in the ExAc data set nor in our internal variant databases. Furthermore, these variants were not present in more than 300 Centre d'Etude du Polymorphisme Humain (CEPH) control chromosomes. In silico analysis predicted those to be disease-causing (Supplementary Table  2c ). These variants were found to affect highly conserved amino-acid residues (Fig. 1 ). To identify additional mutations, we sequenced all exons, exon-intron boundaries and 3 0 and 5 0 untranslated repeat of POLR1C (see Supplementary Table  3  for primers) in 16 other individuals selected on the basis of clinical (compatible neurological features with at least one non-neurological feature such as dental abnormalities or hypogonadotropic hypogonadism, together with hypomyelination on MRI) and/or radiological characteristics (typical MRI features of 4H leukodystrophy)  3, 10, 11  but negative for POLR3A and POLR3B mutations and uncovered five additional compound heterozygous or homozygous cases (Supplementary Table  2 , Fig.
Cases one and two were sequenced using PerkinElmer's sequencing service using the Agilent Sure Select Human All Exon Capture V4 Kit and exome sequencing for these two cases was performed (two paired-end 100-bp reads) with the Illumina HiSeq 2000 system. Reads were aligned to the reference human genome (UCSC Genome Browser hg19) with the Genome Analysis Toolkit (GATK)  32, 33  , SAMtools  32, 34  , Picard (see web resource) and CASAVA v1. 8 (ref. 35)  and annotated using the snpEff software tool (http://snpeff. sourceforge. net/), as well as visualization tools from PerkinElmer. Subsequent analyses were performed using the Ingenuity software package (Qiagen, Redwood City, USA). Exome enrichment for case three was performed using the Illumina Nextera Rapid Capture kit and sequenced on an Illumina HiSeq 2000 (2 Â 100 bp paired-end).
Reads were aligned to the reference human genome (GRCh37) and pedigreeinformed variant calling was performed using the Real Time Genomics (RTG) integrated analysis tool rtg Family v3. 2 (ref. 36). Variants were annotated using SnpEff v3. 4 (ref. 37). In all cases, filtering queries were created as specific presets that allowed in silico reduction of variant lists down to candidates with correlation to phenotype, transmission mode of inheritance and alteration classification of pathogenicity. Sanger sequencing and co-segregation analysis were performed on genomic DNA using primer pairs designed with the primer3 software package and the genomic sequence of POLR1C (NM_203290; GRCh37/hg19). PCR products were forward-and reverse-sequenced at the McGill University and Genome Quebec Innovation Centre using an ABI 3730xl DNA Analyzer (ABI; Applied Biosystems, Foster City, CA, USA).
For western blot analysis, anti-FLAG (dilution 1/3,000) was used to detect the FLAG-POLR1C variants and anti-GAPDH (dilution 1/2,000;) for loading control  18. Protein affinity purification coupled to mass spectrometry. Generation of cell lines stably expressing FLAG-tagged POLR1C subunits (WT and mutated) and affinity purification from the soluble fraction were performed using standard procedures  38, 39. The eluates were digested with trypsin and the resulting tryptic peptides were purified and identified with tandem mass spectrometry (LC-MS/MS) using a microcapillary reversed-phase high-pressure liquid chromatographycoupled LTQ-Orbitrap (ThermoElectron) quadrupole ion trap mass spectrometer with a nanospray interface, as recently described  40. Protein database searching and protein spectral count quantification were performed with Mascot (version 2.
3. 02)  41. The NCBI_Human protein sequence database was downloaded on 20 February 2014. Known protein contaminants such as keratins, which are not expressed in HeLa cells, were excluded from the data set. Undistinguishable protein isoforms were considered as a single protein. For each LC-MS/MS analysis, protein spectral counts were normalized by the spectral count of the FLAG-POLR1C in order to allow the comparison of different purifications. To simulate the background noise of the LC-MS/MS analysis of a given sample, spectral counts reported as 0 by Mascot were replaced by randomly generated spectral count values that are normally distributed with a mean and s. d. equal to those of the lowest 20% spectral count values from the LC-MS/MS analysis. Each replicate LC-MS/MS analysis of the affinity purifications of the FLAG-POLR1C mutants (MUT; N32I, N74S and R279Q) was paired with a LC-MS/MS analysis of the WT FLAG-POLR1C that was performed at the same time.
The resulting P values were adjusted for multiple hypothesis testing using the Benjamini-Hochberg procedure  42. To maximize the The rDNA gene encodes a 45S pre-rRNA precursor that will generate the 5. 8S, 18S and 28S rRNAs. There are B400 copies of the rDNA gene arranged in tandem repeats in the human genome. rDNA repeats are not present in the reference genome assemblies; therefore, unique reads were aligned directly to the human rDNA reference sequence (NCBI accession number: HSU13369)  56. No differences were observed in POLR1C occupancy between wild-type and mutant variants. A schematic of a rDNA repeat is included below the graph. specificity of our approach, a protein is deemed to show a level of differential interaction with POLR1C that is statistically significant when its adjusted P value is o0.
The beads were then washed and eluted, followed by phenol:chloroform-isoamyl (   48  and the tRNA Genes track of the UCSC Genome Browser  49, 50. Data visualization was performed with the Integrative Genomics Viewer 51. To compare chromatin binding between WT and mutated POLR1C variants over all POLR3transcribed genes divided into three classes according to their regulatory elements (see legend to Fig. 4 ), we used the annotation mode of the Versatile Aggregate Profiler with the transcription start site as the only reference point and 100 windows of 10 base pairs each on both sides of the reference point  52.
IGS, intergenic spacer. (c) IGV view of one ribosomal DNA (rDNA) repeat. The rDNA gene encodes a 45S pre-rRNA precursor that will generate the 5. 8S, 18S and 28S rRNAs. There are B400 copies of the rDNA gene arranged in tandem repeats in the human genome. rDNA repeats are not present in the reference genome assemblies; therefore, unique reads were aligned directly to the human rDNA reference sequence (NCBI accession number: HSU13369) 56. No differences were observed in POLR1C occupancy between wild-type and mutant variants. A schematic of a rDNA repeat is included below the graph.
Acknowledgements We are grateful for the support and cooperation of the subjects, as well as their families. G. B. has received a  Research Scholar Junior 1 award  from the  Fonds de Recherche du Quebec en Sante(FRQS). I. T. has received a scholarship from the  Reseau de medecine genetique appliquee du FRQS. N. I. W. and M. S. v. d. K. are supported by  ZonMw  ( TOP grant   91211005 ). B. C. holds the  Bell-Bombardier Research Chair of Excellence (IRCM). K. C. received a scholarship from the  FRQS. D. T. received support from the  German Research Council and European Union  ( Marie Curie ITN ). R. J. T. , C. S. and A. V. are supported by a  National Health and Medical Research Council, Australia  project grant ( APP1068278 ). The  Myelin Disorders Bioregistry Project  was supported by the  NIH National Center for Advancing Translational Sciences  (award number  UL1TR000075 ).
S. F. was supported by the  Ligue Regionale contre le Cancer  (comiteDordogne),  INSERM  and the  Conseil Regional d'Aquitaine. This work was supported by  Canadian Institute for Health Research  grant  MOP-G-287547  to G. B. , B. B. and B. C. We also thank the  McGill University and Genome Quebec Innovation Center , the  IRCM Molecular Biology and Functional Genomics Platform and Denis Faubert, Josee Champagne ,  Sylvain Tessier  and  Marguerite Boulos  of the  IRCM Proteomics Discovery Platform  for their services and  Dr Franc¸ois Robert  and  Alexis Blanchet-Cohen  for bioinformatics support. We thank  Dr Claudia Kleinman  for her advice regarding ChIP-Seq data analysis. J. R. Y. has been funded by  National Institutes of Health (NIH)  grants  P41 GM103533 ,  R01 MH067880 ,  R01 MH100175  and  UCLA/NHLBI Proteomics Centers  ( HHSN268201000035C ).
Results GMPS architecture. We purified and crystallized PfGMPS, PfGMPS_C89A and PfGATase, and solved their structures by molecular replacement to resolutions of 3. 6, 3. 15 and 2. 6 Å, respectively (Table  1 ). A 2. 72 Å resolution crystal structure of PfGMPS/XMP is available in the Protein Databank (PDB-ID 3UOW-unpublished). For the full-length enzymes (dimers as shown experimentally, see below), monomers are composed of two catalytic domains, an N-terminal independent GATase (1-236) and a C-terminal ATPPase domain (237-555) (Fig. 1a, b ). The GATase segment displays an a/b-structure of nine b-strands surrounded by five a-helixes. Compared with Hs-and EcGMPS, the PfGMPS GATase domain exhibits an extension (119-151) inserted between b5 and b6. This unique insertion lacks even in other Plasmodium GMPS and its role is yet to be determined.
25 to 20 mM and NH 4 Cl from 1 to 250 mM. The saturating concentration of ATP, XMP, Gln and NH 4 Cl were 2 mM, 150 mM, 5 mM and 100 mM, respectively. Initial velocity data were fitted to the Michaelis-Menten equation v ¼ (V max S)/(K m þ S) by nonlinear regression using GraphPad Prism version 5. 00 (GraphPad Software). Initial velocity measurements at varying substrate concentrations were performed in duplicate and a minimum of 14 substrate concentrations were used in all cases. Assay for glutaminase activity. Glutaminase activity was measured at 25 °C by estimating the concentration of glutamate formed using either glutamate dehydrogenase (Sigma-Aldrich, USA) or P. falciparum aspartate aminotransferase (PfAAT, in-house generated) as the coupling enzyme. In the GDH-coupled assay, the glutamate generated is converted to a-ketoglutarate along with the concomitant reduction of NAD þ to NADH.
The total flow rate was 8 ml s À 1 resulting in a dead time of 3. 8 ms. The instrument was controlled using the Biokine32 software, version 4. 62, allowing accumulation of 8,000 data points for each trace. Data were acquired every ms, and traces from three or more shots were averaged and A 290 was normalized (the offset was corrected to zero) and plotted against time. The resulting traces were fitted to equations  28. A 290 ¼ A 1 Â e Àk 0 t À 1 À k 00 t ð1Þ and A 290 ¼ k 00 t ð2Þ where k 0 is the observed rate constant of the exponential phase (first faster phase), k 00 is the observed rate constant of the steady-state rate phase and A 1 the amplitude of the exponential phase. Absorbance was monitored at 290 nm using an A ¨KTA Basic HPLC equipped with a UV-900 detector at a flow rate of 0.