Analysis of Surgically Treated Intraspinal Tumors in Southern Taiwan

The medical records of 117 patients with spinal tumors who underwent surgery with pathologic confirmation from January 1999 to April 2004 at Kaohsiung Medical University Hospital were reviewed. Data from this review were compared with those obtained from the same institution 10 years earlier (covering the period 1988-1995) and from other reported series. There were 69 male and 48 female patients aged from 13 to 87 years old (mean age, 51.9). The most common pathologic findings were metastasis in 45.3% (53/117), nerve sheath tumors in 28.2% (33/117), menin-giomas in 12% (14/117) and neuroepithelial tumors in 6% (7/117). The peak ages at diagnosis were 41-50 years and 61–70 years. A slight male predominance was noted for all tumors, except meningiomas. Motor weakness, even paralysis, was the major clinical presentation (64–86%), followed by sensory deficits (50%) and pain (42%). The location of tumors was most often in the thoracic (50.4%; 59/117), lumbosacral (27.4%; 32/117) and cervical spine (22.2%; 26/117) segments. Among the metastatic tumors, the lung (22.6%) and breast (15.1%) were the most common primary sites of origin, followed by unknown origin, the liver (hepatocellular carcinoma), the gastrointestinal tract and the nasopharynx (nasopharyngeal cancer)

Distinct and concurrent pathways of Pol II-and Pol IV- dependent siRNA biogenesis at a repetitive trans-silencer locus in Arabidopsis thaliana

SUMMARY Short interfering RNAs (siRNAs) homologous to transcriptional regulatory regions can induce RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS) of target genes. In our system, siRNAs are produced by transcribing an inverted DNA repeat (IR) of enhancer sequences, yielding a hairpin RNA that is processed by several Dicer activities into siRNAs of 21-24 nt. Primarily 24-nt siRNAs trigger RdDM of the target enhancer in trans and TGS of a downstream GFP reporter gene. We analyzed siRNA accumulation from two different structural forms of a trans-silencer locus in which tandem repeats are embedded in the enhancer IR and distinguished distinct RNA polymerase II (Pol II)-and Pol IV-dependent pathways of siRNA biogenesis. At the original silencer locus, Pol-II transcription of the IR from a 35S promoter produces a hairpin RNA that is diced into abundant siRNAs of 21-24 nt. A silencer variant lacking the 35S promoter revealed a normally masked Pol IV-dependent pathway that produces low levels of 24-nt siRNAs from the tandem repeats. Both pathways operate concurrently at the original silencer locus. siRNAs accrue only from specific regions of the enhancer and embedded tandem repeat. Analysis of these sequences and endogenous tandem repeats producing siRNAs revealed the preferential accumulation of siRNAs at GC-rich regions containing methylated CG dinucleotides. In addition to supporting a correlation between base composition, DNA methylation and siRNA accumulation, our results highlight the complexity of siRNA biogenesis at repetitive loci and show that Pol II and Pol IV use different promoters to transcribe the same template

Overlapping RdDM and non-RdDM mechanisms work together to maintain somatic repression of a paramutagenic epiallele of maize <i>pericarp color1</i>

<div><p>Allelic variation at the <i>Zea mays</i> (maize) <i>pericarp color1</i> (<i>p1</i>) gene has been attributed to epigenetic gene regulation. A <i>p1</i> distal enhancer, 5.2 kb upstream of the transcriptional start site, has demonstrated variation in DNA methylation in different <i>p1</i> alleles/epialleles. In addition, DNA methylation of sequences within the 3’ end of intron 2 also plays a role in tissue-specific expression of <i>p1</i> alleles. We show here a direct evidence for small RNAs’ involvement in regulating <i>p1</i> that has not been demonstrated previously. The role of <i>mediator of paramutation1</i> (<i>mop1</i>) was tested in the maintenance of somatic silencing at distinct <i>p1</i> alleles: the non-paramutagenic <i>P1-wr</i> allele and paramutagenic <i>P1-rr</i>’ epiallele. The <i>mop1-1</i> mutation gradually relieves the silenced phenotype after multiple generations of exposure; <i>P1-wr</i>;<i>mop1-1</i> plants display a loss of 24-nt small RNAs and DNA methylation in the 3’ end of the intron 2, a region close to a <i>Stowaway</i> transposon. In addition, a MULE sequence within the proximal promoter of <i>P1-wr</i> shows depletion of 24nt siRNAs in <i>mop1-1</i> plants. Release of silencing was not correlated with small RNAs at the distal enhancer region of the <i>P1-wr</i> allele. We found that the somatic silencing of the paramutagenic <i>P1-rr</i>’ is correlated with significantly reduced H3K9me2 in the distal enhancer of <i>P1-rr</i>’; <i>mop1-1</i> plants, while symmetric DNA methylation is not significantly different. This study highlights that the epigenetic regulation of <i>p1</i> alleles is controlled both via RdDM as well as non-RdDM mechanisms.</p></div

DNA methylation level of native <i>P1-rr</i> and paramutagenic <i>P1-rr</i>’ allele in Mop1 and <i>mop1-1</i> background.

<p>(A) Line diagram depicting <i>P1-rr</i> gene structure (Accession: AF427146). The exons and introns are shown as red filled and open boxes, respectively. The transcription start site is indicated by a bent arrow. The DNA methylation of the 503 bp DE region shown above the gene structure was analyzed by bisulfite sequencing. Double-headed arrow represents the region analyzed by ChIP-qPCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187157#pone.0187157.g005" target="_blank">Fig 5</a>). Hatched boxes indicate the P1.2 region. (B) Average DNA methylation in CG, CHG and CHH contexts at the DE and F8C regions of <i>P1-rr</i> and <i>P1-rr</i>’ in the <i>Mop1</i> or <i>mop1-1</i> plants. (C) Average DNA methylation of the Int2-1 and Int2-2 sub-regions of F8C. The percentage methylation is shown on the y-axis. Average methylation is calculated as the mean of two biological replicates with error bars indicating the SE of the mean. Comparisons of <i>Mop1</i> and <i>mop1-1</i> plants were not significant using the Student’s <i>t</i> test at a <i>P</i> of ≤ 0.05. A single <i>P1-rr</i> sample was analyzed so there is no error bar for <i>P1-rr</i> (the data is consistent with a previous study from Sekhon et al, 2012 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187157#pone.0187157.ref039" target="_blank">39</a>]).</p

H3K9me2 and transcript analysis at <i>P1-rr</i>’ in <i>Mop1</i> and <i>mop1-1</i>.

<p>(A) ChIP-qPCR of H3K9me2 of the DE region of <i>P1-rr</i> and <i>P1-rr</i>’ plants. (B) ChIP-qPCR of H3K9me2 of the DE region of <i>P1-rr</i>’ plants in <i>Mop1</i> or <i>mop1-1</i> plants and (C) their corresponding <i>p1</i> expression from qRT-PCR. Ears are shown for the corresponding phenotypes in which the pericarp samples were used for ChIP experiments. Data are presented as the mean of three biological replicates with error bars indicating SE of the mean. * P<0.05 using an unpaired t-test.</p

24-nt siRNA abundance at the <i>P1-wr</i> gene.

<p>(A) Line diagram of <i>P1-wr</i> showing gene structure and different regulatory elements (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187157#pone.0187157.g001" target="_blank">Fig 1</a> legend for details). Abundance of 24-nt siRNA was shown on sense and antisense strands from <i>P1-wr</i>;<i>Mop1</i>/<i>mop1-1</i> (3 plants) and <i>P1-wr</i>;<i>mop1-1</i>/<i>mop1-1</i> (2 plants) samples as reads per million (RPM) and normalized to the 22-nt size class (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187157#sec013" target="_blank">Methods</a>). Green, grey, and red shaded areas indicate regulatory regions, TEs, and exons, respectively. Proximal enhancer is shown as region between black dashed lines. (B) Repetitive elements and 24-nt siRNA abundance in the proximal enhancer region. (C) 24-nt siRNA abundance in the Int2-1, Int2-2, and downstream regions. siRNA abundances from <i>P1-wr</i>;<i>Mop1</i> samples (blue) and from <i>P1-wr</i>;<i>mop1-1</i> (red) are shown. Coordinates for each graph are shown below. Black arrows indicate inverted repeats.</p

RNA polymerase V-dependent small RNAs in Arabidopsis originate from small, intergenic loci including most SINE repeats

In plants, heterochromatin is maintained by a small RNA-based gene silencing mechanism known as RNA-directed DNA methylation (RdDM). RdDM requires the non-redundant functions of two plant-specific DNA-dependent RNA polymerases (RNAP), RNAP IV and RNAP V. RNAP IV plays a major role in siRNA biogenesis, while RNAP V may recruit DNA methylation machinery to target endogenous loci for silencing. Although small RNA-generating regions that are dependent on both RNAP IV and RNAP V have been identified previously, the genomic loci targeted by RNAP V for siRNA accumulation and silencing have not been described extensively. To characterize the RNAP V-dependent, heterochromatic siRNA-generating regions in the Arabidopsis genome, we deeply sequenced the small RNA populations of wild-type and RNAP V null mutant (nrpe1) plants. Our results showed that RNAP V-dependent siRNA-generating loci are associated predominately with short repetitive sequences in intergenic regions. Suppression of small RNA production from short repetitive sequences was also prominent in RdDM mutants including dms4, drd1, dms3 and rdm1, reflecting the known association of these RdDM effectors with RNAP V. The genomic regions targeted by RNAP V were small, with an estimated average length of 238 bp. Our results suggest that RNAP V affects siRNA production from genomic loci with features dissimilar to known RNAP IV-dependent loci. RNAP V, along with RNAP IV and DRM1/2, may target and silence a set of small, intergenic transposable elements located in dispersed genomic regions for silencing. Silencing at these loci may be actively reinforced by RdDM

Construction and characterisation of a knock-down RNA interference line of OsNRPD1 in rice (Oryza sativa ssp japonica cv Nipponbare)

In plants, RNA-directed DNA Methylation (RdDM) is a mechanism of silencing that relies on the production of 24-nt siRNAs by RNA POLYMERASE IV (Pol IV) to trigger methylation and thus inactivation of transposable elements (TEs). We present the construction and characterisation of osnrpd1, a knock-down RNA interference line of OsNRPD1 gene that encodes the largest subunit of Pol IV in rice (Oryza sativa ssp japonica cv Nipponbare). We show that osnrpd1 displays a lower accumulation of OsNRPD1 transcripts, associated to an overall reduction of 24-nt siRNAs and DNA methylation level in all three contexts, CG, CHG and CHH. We uncovered new insertions of known active TEs, the LTR retrotransposons Tos17 and Lullaby and the LINE-type retrotransposon Karma. However, we did not observe any clear developmental phenotype, contrary to what was expected for a mutant severily affected in RdDM. In addition, despite the presence of many putatively functional TEs in the rice genome, we found no evidence of in planta global reactivation of transposition. This knock-down of OsNRPD1 likely led to a weakly affected line, with no effect on development and a limited effect on transposition. We discuss the possibility that a knockout mutation of OsNRPD1 would cause sterility in rice