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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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Characterization of DXZ4 small RNAs. (A) In vitro Drosha assay with extracts from HEK293T cells either mock transfected or cotransfected with expression plasmids for Drosha and DGCR8. Radiolabeled in vitro transcripts from two DXZ4 regions characterized by specific histone modifications and the KSHV precursor-miRNA miR-K5 served as substrates for Drosha/DGCR8 processing and were separated by polyacrylamide gel electrophoresis. (B) Effect of Dicer depletion on DXZ4 small RNA expression in HEK293T cells. Relative expression of two small RNAs was determined by quantitative reverse-transcription polymerase chain reaction (n = 5). The difference with DXZ4-2355as is not statistically significant (P-value = 0.089, t-test). (C) Sequence logos for DXZ4 small RNAs grouped according to their lengths.
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fig2: Characterization of DXZ4 small RNAs. (A) In vitro Drosha assay with extracts from HEK293T cells either mock transfected or cotransfected with expression plasmids for Drosha and DGCR8. Radiolabeled in vitro transcripts from two DXZ4 regions characterized by specific histone modifications and the KSHV precursor-miRNA miR-K5 served as substrates for Drosha/DGCR8 processing and were separated by polyacrylamide gel electrophoresis. (B) Effect of Dicer depletion on DXZ4 small RNA expression in HEK293T cells. Relative expression of two small RNAs was determined by quantitative reverse-transcription polymerase chain reaction (n = 5). The difference with DXZ4-2355as is not statistically significant (P-value = 0.089, t-test). (C) Sequence logos for DXZ4 small RNAs grouped according to their lengths.

Mentions: The size range of DXZ4 small RNAs corresponds to several classes of small RNAs, including siRNAs, miRNAs, and piRNAs (Kim et al. 2009). To characterize these RNAs, we tested processing of potential precursor-miRNAs by the Drosha/DGCR8 complex (Lee et al. 2003; Denli et al. 2004; Gregory et al. 2004). Because processing requires folding of the primary miRNA into a characteristic hairpin stem loop structure (Zeng et al. 2005), approximately 65-nucleotide long DXZ4 RNA fragments were tested with a secondary structure algorithm (Zuker 2003). For several fragments, one or several thermodynamically stable hairpin structures were identified, implying DXZ4 transcripts could resemble precursor-miRNAs. Two of those potential precursors were analyzed in a Drosha cleavage assay (Zeng and Cullen 2006). The Kaposi’s sarcoma-associated herpesvirus miR-K5 precursor that previously had been identified as a substrate (Gottwein et al. 2006) yielded the expected ~65 nucleotide cleavage product (Figure 2A). In contrast, both DXZ4 transcripts were not cleaved, suggesting they are not proper Drosha substrates. It has been shown that subtle differences in the stem-loop structure can severely impair its ability to be processed (Gottwein et al. 2006). Although those DXZ4 fragments could potentially fold into stem loops, we conclude that the limited number of DXZ4 RNA fragments tested is not regular precursor-miRNAs. It is known that some miRNAs are generated by Drosha-independent noncanonical pathways (Okamura et al. 2007; Ruby et al. 2007; Bogerd et al. 2010; Valen et al. 2011).


Small RNA expression from the human macrosatellite DXZ4.

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

Characterization of DXZ4 small RNAs. (A) In vitro Drosha assay with extracts from HEK293T cells either mock transfected or cotransfected with expression plasmids for Drosha and DGCR8. Radiolabeled in vitro transcripts from two DXZ4 regions characterized by specific histone modifications and the KSHV precursor-miRNA miR-K5 served as substrates for Drosha/DGCR8 processing and were separated by polyacrylamide gel electrophoresis. (B) Effect of Dicer depletion on DXZ4 small RNA expression in HEK293T cells. Relative expression of two small RNAs was determined by quantitative reverse-transcription polymerase chain reaction (n = 5). The difference with DXZ4-2355as is not statistically significant (P-value = 0.089, t-test). (C) Sequence logos for DXZ4 small RNAs grouped according to their lengths.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Characterization of DXZ4 small RNAs. (A) In vitro Drosha assay with extracts from HEK293T cells either mock transfected or cotransfected with expression plasmids for Drosha and DGCR8. Radiolabeled in vitro transcripts from two DXZ4 regions characterized by specific histone modifications and the KSHV precursor-miRNA miR-K5 served as substrates for Drosha/DGCR8 processing and were separated by polyacrylamide gel electrophoresis. (B) Effect of Dicer depletion on DXZ4 small RNA expression in HEK293T cells. Relative expression of two small RNAs was determined by quantitative reverse-transcription polymerase chain reaction (n = 5). The difference with DXZ4-2355as is not statistically significant (P-value = 0.089, t-test). (C) Sequence logos for DXZ4 small RNAs grouped according to their lengths.
Mentions: The size range of DXZ4 small RNAs corresponds to several classes of small RNAs, including siRNAs, miRNAs, and piRNAs (Kim et al. 2009). To characterize these RNAs, we tested processing of potential precursor-miRNAs by the Drosha/DGCR8 complex (Lee et al. 2003; Denli et al. 2004; Gregory et al. 2004). Because processing requires folding of the primary miRNA into a characteristic hairpin stem loop structure (Zeng et al. 2005), approximately 65-nucleotide long DXZ4 RNA fragments were tested with a secondary structure algorithm (Zuker 2003). For several fragments, one or several thermodynamically stable hairpin structures were identified, implying DXZ4 transcripts could resemble precursor-miRNAs. Two of those potential precursors were analyzed in a Drosha cleavage assay (Zeng and Cullen 2006). The Kaposi’s sarcoma-associated herpesvirus miR-K5 precursor that previously had been identified as a substrate (Gottwein et al. 2006) yielded the expected ~65 nucleotide cleavage product (Figure 2A). In contrast, both DXZ4 transcripts were not cleaved, suggesting they are not proper Drosha substrates. It has been shown that subtle differences in the stem-loop structure can severely impair its ability to be processed (Gottwein et al. 2006). Although those DXZ4 fragments could potentially fold into stem loops, we conclude that the limited number of DXZ4 RNA fragments tested is not regular precursor-miRNAs. It is known that some miRNAs are generated by Drosha-independent noncanonical pathways (Okamura et al. 2007; Ruby et al. 2007; Bogerd et al. 2010; Valen et al. 2011).

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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