The canonical siRNA-processing pathway is not involved in the regulation of SNORD44 sdRNAs.

<p><i>A–D</i> HeLa cells were transfected with control or Drosha-targeting siRNA and incubated for 48 hours. Cells were fixed and stained for Drosha (<i>A</i>) or DGCR8 (<i>B</i>), and counterstained for DNA. Merged images are shown. Scale bar, 10 µm. (<i>C, D</i>) Image quantifications for (<i>C</i>) Drosha and (<i>D</i>) DCGR8. Mean normalized fold intensity is shown. Error bars, SD. <i>E</i> Nuclear (containing nucleoli) and cytoplasmic fractions were prepared of the cells described in A, and RNA was isolated. Northern hybridization was conducted using the SNORD44 5′ probe. Signal intensities for 120 nt and sdRNAs were quantified and normalized against the mature SNORD44. <i>F</i> HCT116 and HCT116 Dicer −/− cells were subjected to subcellular fractionation, RNA was isolated and Northern hybridization was conducted using the SNORD44 5′ probe. Signal intensities for sdRNAs normalized to mature SNORD44 are provided below. hY1 probe was used as control.</p

Size-distribution of the annotated small RNA sequence reads in the subcellular compartments.

<p><i>A</i> Cellular RNA reads. <i>B</i> Cytoplasmic RNA reads. <i>C</i> Nuclear RNA reads. <i>D</i> Nucleolar RNA reads.</p

sRNA-seq strategy and preparation of small RNA libraries.

<p><i>A</i> Subcellular fractionation and RNA purification scheme. Cyto, cytoplasmic; Nu, nuclear; No, nucleolar. <i>B</i> RNA-PAGE analysis by 16% denaturing PAGE before (<i>left</i>) and after (<i>right</i>) purification of the <200 nt RNA fraction. Subcellular fractions are indicated at the top. <i>C</i> RNA profiles of the <40 nt (<i>left</i>) and >40 nt fractions (<i>right</i>) as analyzed by BioAnalyzer. Subcellular RNA fractions are indicated on <i>top</i>, RNA size markers to the <i>left</i>.</p

RNA-Seq of the Nucleolus Reveals Abundant SNORD44-Derived Small RNAs

<div><p>Small non-coding RNAs represent RNA species that are not translated to proteins, but which have diverse and broad functional activities in physiological and pathophysiological states. The knowledge of these small RNAs is rapidly expanding in part through the use of massive parallel (deep) sequencing efforts. We present here the first deep sequencing of small RNomes in subcellular compartments with particular emphasis on small RNAs (sRNA) associated with the nucleolus. The vast majority of the cellular, cytoplasmic and nuclear sRNAs were identified as miRNAs. In contrast, the nucleolar sRNAs had a unique size distribution consisting of 19–20 and 25 nt RNAs, which were predominantly composed of small snoRNA-derived box C/D RNAs (termed as sdRNA). Sequences from 47 sdRNAs were identified, which mapped to both 5′ and 3′ ends of the snoRNAs, and retained conserved box C or D motifs. SdRNA reads mapping to SNORD44 comprised 74% of all nucleolar sdRNAs, and were confirmed by Northern blotting as comprising both 20 and 25 nt RNAs. A novel 120 nt SNORD44 form was also identified. The expression of the SNORD44 sdRNA and 120 nt form was independent of Dicer/Drosha–mediated processing pathways but was dependent on the box C/D snoRNP proteins/sno-ribonucleoproteins fibrillarin and NOP58. The 120 nt SNORD44-derived RNA bound to fibrillarin suggesting that C/D sno-ribonucleoproteins are involved in regulating the stability or processing of SNORD44. This study reveals sRNA cell-compartment specific expression and the distinctive unique composition of the nucleolar sRNAs.</p></div

Small RNA read distribution.

<p>sRNAs: miRNAs, snoRNAs, snRNAs.</p><p>misc RNAs: Y RNAs, vault RNAs, lincRNAs.</p><p>Small RNA read distribution.</p

Distribution of sdRNAs in the cellular subfractions.

<p><i>A–C</i> Scatter plots of sdRNA reads in the different cellular compartments. Note the differential scaling of the axes. Pearson correlation coefficiences are indicated (<i>r</i>). <i>A</i> Nucleolar (No) <i>vs</i>. nuclear (Nu) sdRNA reads. <i>B</i> Nucleolar (No) <i>vs</i>. whole cell (Ce) sdRNA reads. <i>C</i> Nuclear (Nu) <i>vs.</i> whole cell (Ce) sdRNA reads. <i>D</i> Nucleolar sdRNA reads (reads ≥50 are shown). <i>E</i> Scatter plot of frequencies of nucleolar and nuclear 5′ reads present at ≥10 reads in both libraries. Selected SNORDs are identified by their numbering. <i>F</i> C/C' and D/D' box frequencies of nucleolar sdRNA reads.</p

RNA-seq read alignment.

<p>sRNA: miRNA, snoRNA, snRNA, Y RNA, Vault RNA, LincRNA.</p><p>RNA-seq read alignment.</p

Baculovirus-mediated expression of C-terminal His-tagged human MBD2-MBD in Sf9 insect cells and purification by Ni-NTA beads

<p><b>Copyright information:</b></p><p>Taken from "Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation"</p><p>Nucleic Acids Research 2006;34(3):e19-e19.</p><p>Published online 9 Feb 2006</p><p>PMCID:PMC1363782.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Unbound lysate, washes and eluted purified protein were run on an SDS–PAGE gel and stained with Coomassie Blue R-250. The eluate contained the purified ∼9.8 kDa MBD2-MBD-His

5hmC levels are reduced in the germinal regions of the human fetal forebrain.

<p>Fetal brain sections were stained with hematoxylin and eosin (A-C) and evaluated for 5hmC or TET2 levels using immunohistochemistry. Sections of the forebrain germinal region stained with antibodies for 5hmC (D) showed markedly reduced levels. In contrast, periventriclular regions outside the forebrain germinal matrix (E) and in the fetal cortex (F) showed high levels of 5hmC staining. TET2 immunohistochemistry (G-I) showed a similar distribution to 5hmC staining. Original magnification was 200X for all photomicrographs, except panels A, D, and G which were 100X.</p

Summary of 5hmC immunohistochemistry.

<p>For (a) and (b) values are based on a single IDH1 negative tumor. For (c) the value is based on a single IDH1-positive tumor. N = number of tumors of the designated histologic type. IDH R132H mutants = tumors that were immunopositive for antibodies against IDH1 mutant R132H. IDH1neg = tumors immunonegative for IDH1 mutant R132H. ND = not determined due to the absence of IDH1 mutant pilocytic astrocytomas.</p