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Small RNA expression from the human macrosatellite DXZ4.

Pohlers M, Calabrese JM, Magnuson T - G3 (Bethesda) (2014)

Bottom Line: DXZ4 was found to express a wide range of small RNAs potentially representing several classes of small RNAs.A subpopulation of these RNAs is bound by Argonaute.We hypothesize that the RNAs are involved in Argonaute-dependent methylation of DXZ4 DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, the Carolina Center for Genome Sciences, University of North Carolina, Chapel Hill, North Carolina 27599 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599.

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Small RNA expression from DXZ4. (A) Northern hybridization detecting small RNAs expressed from DXZ4 in the cell lines HEK293T and human umbilical vein endothelial cell (HUVEC). The numbers below correlate with the start position of the Northern probes in DXZ4, which colocalize with DXZ4 regions associated with characteristic histone modifications. (B) Distribution of the lengths of DXZ4-matching unique small RNA reads obtained from merged high-throughput sequencing data from K562, HEK293, MCF10a, H1, and IMR-90 cell lines (n = 97). Coloring corresponds to size range of known small RNA classes. (C) Alignment of DXZ4 small RNAs from (B) and from the K562 chromatin fraction only (n = 47), respectively. Refer to the text for details. The colors of the dots reflect the colors used in (B) indicating small RNA size. (D) Relative expression of DXZ4 small RNAs in HEK293T detected by quantitative reverse-transcription polymerase chain reaction. cDNA synthesis was performed in the presence or absence of reverse transcriptase. Each reaction was performed n ≥ 6. (E) Alignment of sequenced DXZ4 small RNAs coprecipitated in AGO immunoprecipitations in HeLa cells. Alignment includes multiple RNA copies.
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fig1: Small RNA expression from DXZ4. (A) Northern hybridization detecting small RNAs expressed from DXZ4 in the cell lines HEK293T and human umbilical vein endothelial cell (HUVEC). The numbers below correlate with the start position of the Northern probes in DXZ4, which colocalize with DXZ4 regions associated with characteristic histone modifications. (B) Distribution of the lengths of DXZ4-matching unique small RNA reads obtained from merged high-throughput sequencing data from K562, HEK293, MCF10a, H1, and IMR-90 cell lines (n = 97). Coloring corresponds to size range of known small RNA classes. (C) Alignment of DXZ4 small RNAs from (B) and from the K562 chromatin fraction only (n = 47), respectively. Refer to the text for details. The colors of the dots reflect the colors used in (B) indicating small RNA size. (D) Relative expression of DXZ4 small RNAs in HEK293T detected by quantitative reverse-transcription polymerase chain reaction. cDNA synthesis was performed in the presence or absence of reverse transcriptase. Each reaction was performed n ≥ 6. (E) Alignment of sequenced DXZ4 small RNAs coprecipitated in AGO immunoprecipitations in HeLa cells. Alignment includes multiple RNA copies.

Mentions: Northern blots with RNA from the cell lines HEK293T and human umbilical vein endothelial cell were probed for regions that express 85-nucleotide DXZ4 RNAs detecting multiple transcripts including small RNAs between 20 and 40 nucleotides long (Figure 1A). This size range corresponds to several classes of small RNAs known to mediate epigenetic modifications (Kim et al. 2009). In addition to these small RNAs, the probes detected several longer transcripts. Because of their design, the probes are predicted to detect the 85-nucleotide transcripts as well as any larger transcripts (Chadwick 2008), providing a putative explanation for the detection of multiple signals per probe. However, although we used stringent hybridization and washing conditions, we cannot exclude the presence of unintended cross hybridization signals.


Small RNA expression from the human macrosatellite DXZ4.

Pohlers M, Calabrese JM, Magnuson T - G3 (Bethesda) (2014)

Small RNA expression from DXZ4. (A) Northern hybridization detecting small RNAs expressed from DXZ4 in the cell lines HEK293T and human umbilical vein endothelial cell (HUVEC). The numbers below correlate with the start position of the Northern probes in DXZ4, which colocalize with DXZ4 regions associated with characteristic histone modifications. (B) Distribution of the lengths of DXZ4-matching unique small RNA reads obtained from merged high-throughput sequencing data from K562, HEK293, MCF10a, H1, and IMR-90 cell lines (n = 97). Coloring corresponds to size range of known small RNA classes. (C) Alignment of DXZ4 small RNAs from (B) and from the K562 chromatin fraction only (n = 47), respectively. Refer to the text for details. The colors of the dots reflect the colors used in (B) indicating small RNA size. (D) Relative expression of DXZ4 small RNAs in HEK293T detected by quantitative reverse-transcription polymerase chain reaction. cDNA synthesis was performed in the presence or absence of reverse transcriptase. Each reaction was performed n ≥ 6. (E) Alignment of sequenced DXZ4 small RNAs coprecipitated in AGO immunoprecipitations in HeLa cells. Alignment includes multiple RNA copies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199704&req=5

fig1: Small RNA expression from DXZ4. (A) Northern hybridization detecting small RNAs expressed from DXZ4 in the cell lines HEK293T and human umbilical vein endothelial cell (HUVEC). The numbers below correlate with the start position of the Northern probes in DXZ4, which colocalize with DXZ4 regions associated with characteristic histone modifications. (B) Distribution of the lengths of DXZ4-matching unique small RNA reads obtained from merged high-throughput sequencing data from K562, HEK293, MCF10a, H1, and IMR-90 cell lines (n = 97). Coloring corresponds to size range of known small RNA classes. (C) Alignment of DXZ4 small RNAs from (B) and from the K562 chromatin fraction only (n = 47), respectively. Refer to the text for details. The colors of the dots reflect the colors used in (B) indicating small RNA size. (D) Relative expression of DXZ4 small RNAs in HEK293T detected by quantitative reverse-transcription polymerase chain reaction. cDNA synthesis was performed in the presence or absence of reverse transcriptase. Each reaction was performed n ≥ 6. (E) Alignment of sequenced DXZ4 small RNAs coprecipitated in AGO immunoprecipitations in HeLa cells. Alignment includes multiple RNA copies.
Mentions: Northern blots with RNA from the cell lines HEK293T and human umbilical vein endothelial cell were probed for regions that express 85-nucleotide DXZ4 RNAs detecting multiple transcripts including small RNAs between 20 and 40 nucleotides long (Figure 1A). This size range corresponds to several classes of small RNAs known to mediate epigenetic modifications (Kim et al. 2009). In addition to these small RNAs, the probes detected several longer transcripts. Because of their design, the probes are predicted to detect the 85-nucleotide transcripts as well as any larger transcripts (Chadwick 2008), providing a putative explanation for the detection of multiple signals per probe. However, although we used stringent hybridization and washing conditions, we cannot exclude the presence of unintended cross hybridization signals.

Bottom Line: DXZ4 was found to express a wide range of small RNAs potentially representing several classes of small RNAs.A subpopulation of these RNAs is bound by Argonaute.We hypothesize that the RNAs are involved in Argonaute-dependent methylation of DXZ4 DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, the Carolina Center for Genome Sciences, University of North Carolina, Chapel Hill, North Carolina 27599 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599.

Show MeSH
Related in: MedlinePlus