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Non-coding RNA derived from a conservative subtelomeric tandem repeat in chicken and Japanese quail somatic cells.

Trofimova I, Popova D, Vasilevskaya E, Krasikova A - Mol Cytogenet (2014)

Bottom Line: We focused on tissue-specific differences of subtelomeric repeats transcription, structure of the resulting transcripts and the behavior of subtelomere-derived RNA during mitosis.We revealed one or two major foci of PO41 repeat transcripts associated with RNA polymerase II, representing nascent RNA, and dispersed PO41 repeat transcripts localized in euchromatin or interchromatin space, representing released RNA.Potential regulatory role of the PO41 repeat transcripts in RNA-dependent process of subtelomere heterochromatin maintenance is discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Cytology and Histology, Saint-Petersburg State University, Oranienbaumskoie sch. 2, Stary Peterhof, 198504 Saint-Petersburg, Russia.

ABSTRACT

Background: Subtelomeres are located close to the ends of chromosomes and organized by tandemly repetitive sequences, duplicated copies of genes, pseudogenes and retrotransposons. Transcriptional activity of tandemly organized DNA at terminal chromosomal regions and the distribution of subtelomere-derived non-coding RNAs are poorly investigated. Here we aimed to analyze transcriptional activity of subtelomeric tandem repeat in somatic tissues and cultured cells of birds. We focused on tissue-specific differences of subtelomeric repeats transcription, structure of the resulting transcripts and the behavior of subtelomere-derived RNA during mitosis.

Results: Transcriptional activity of short subtelomeric PO41 ("pattern of 41 bp") tandem repeat in the somatic and cultured cells of chicken (Gallus gallus domesticus) and Japanese quail (Coturnix coturnix japonica) was examined using RNA fluorescence in situ hybridization approach. We discovered transcripts from both strands of the PO41 repeat in chicken MDCC-MSB1 cells as well as in chicken and Japanese quail somatic tissues, such as tissues of cerebellum, telencephalon, muscles, oviduct, small and large intestine. Normal somatic and transformed cells demonstrate similar distribution of PO41 repeat transcripts in interphase nuclei. We revealed one or two major foci of PO41 repeat transcripts associated with RNA polymerase II, representing nascent RNA, and dispersed PO41 repeat transcripts localized in euchromatin or interchromatin space, representing released RNA. During mitosis PO41 non-coding RNA distribute between condensed chromosomes till anaphase, when they concentrate at the cleavage plane. At telophase, clusters of PO41 RNA surround terminal regions of chromosomes. Treatments with RNases of different substrate specificity indicate that PO41 repeat transcripts are single-stranded RNAs with short double-stranded regions due to appearance of inverted repeats.

Conclusion: Transcription of a subtelomeric tandem repeat in avian somatic cells is reported here for the first time. PO41 repeat transcription is conserved among Galliformes and has similar pattern in somatic tissues. We demonstrated redistribution of non-coding PO41 RNA occurring during the cell cycle. Potential regulatory role of the PO41 repeat transcripts in RNA-dependent process of subtelomere heterochromatin maintenance is discussed.

No MeSH data available.


Related in: MedlinePlus

PO41 tandem repeat is transcribed in chicken lymphoblastoid MDCC-MSB1 cells. FISH with PO41pos (green) and PO41neg (red) probes on chicken MDCC-MSB1 cells. (a, a’) DNA/RNA hybridization revealed transcripts from both strands of PO41 repeat in interphase nuclei. (b, b’) DNA/DNA hybridization (positive control) revealed clusters of PO41 repeat in interphase nuclei. (c, c’) RiboShredder RNase cocktail treatment before DNA/RNA hybridization (negative control) removed all hybridization signals. (d) 3D reconstruction of interphase nucleus with isosurfaces around G-rich transcripts of PO41 repeat (red) and chromatin (blue). DNA was counterstained with DAPI. Scale bars: 5 μm (a-c, a’-c’); 3 μm (d).
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Fig1: PO41 tandem repeat is transcribed in chicken lymphoblastoid MDCC-MSB1 cells. FISH with PO41pos (green) and PO41neg (red) probes on chicken MDCC-MSB1 cells. (a, a’) DNA/RNA hybridization revealed transcripts from both strands of PO41 repeat in interphase nuclei. (b, b’) DNA/DNA hybridization (positive control) revealed clusters of PO41 repeat in interphase nuclei. (c, c’) RiboShredder RNase cocktail treatment before DNA/RNA hybridization (negative control) removed all hybridization signals. (d) 3D reconstruction of interphase nucleus with isosurfaces around G-rich transcripts of PO41 repeat (red) and chromatin (blue). DNA was counterstained with DAPI. Scale bars: 5 μm (a-c, a’-c’); 3 μm (d).

Mentions: To study the transcriptional activity of subtelomeric tandem PO41 repeat, we performed FISH according to DNA/RNA hybridization protocol with single stranded oligonucleotide probes (PO41pos and PO41neg) to each strand of the repeat. RNA FISH was used as a reliable approach to reveal specific transcripts in either cultured cells or tissues [29]. At first, we analyzed the transcription of PO41 repeat in chicken MDCC-MSB1 cell line as in one of the few widespread permanent cell lines of birds with a high level of proliferative activity [30]. In MDCC-MSB1 cells, we detected transcripts from both strands of PO41 repeat in interphase nuclei, but not cytoplasm (Figure 1a, a’). The pattern of intranuclear distribution was identical for probes to each of the strands of tandem repeat: transcripts localized predominantly in one, rarely two foci typically observed in euchromatin (~0.59 μm and 0.54 μm for C- and G-rich transcripts respectively) (Figure 1d). It indicates that transcription of PO41 repeat is probably initiated at one locus in a genome. The transcription from multiple sites could not be excluded but remained undetected due to bright signals from the major foci. Since bright foci bearing PO41 repeat transcripts are present in nuclei of all cells, we suggest that PO41 repeat transcription begins in early G1 phase. Treatment of MDCC-MSB1 cells with a cocktail of RNases (RiboShredder RNase Blend) before DNA/RNA FISH effectively eliminated all fluorescent signals (Figure 1c, c’) which confirms probes hybridization to RNA-transcripts.Figure 1


Non-coding RNA derived from a conservative subtelomeric tandem repeat in chicken and Japanese quail somatic cells.

Trofimova I, Popova D, Vasilevskaya E, Krasikova A - Mol Cytogenet (2014)

PO41 tandem repeat is transcribed in chicken lymphoblastoid MDCC-MSB1 cells. FISH with PO41pos (green) and PO41neg (red) probes on chicken MDCC-MSB1 cells. (a, a’) DNA/RNA hybridization revealed transcripts from both strands of PO41 repeat in interphase nuclei. (b, b’) DNA/DNA hybridization (positive control) revealed clusters of PO41 repeat in interphase nuclei. (c, c’) RiboShredder RNase cocktail treatment before DNA/RNA hybridization (negative control) removed all hybridization signals. (d) 3D reconstruction of interphase nucleus with isosurfaces around G-rich transcripts of PO41 repeat (red) and chromatin (blue). DNA was counterstained with DAPI. Scale bars: 5 μm (a-c, a’-c’); 3 μm (d).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4301066&req=5

Fig1: PO41 tandem repeat is transcribed in chicken lymphoblastoid MDCC-MSB1 cells. FISH with PO41pos (green) and PO41neg (red) probes on chicken MDCC-MSB1 cells. (a, a’) DNA/RNA hybridization revealed transcripts from both strands of PO41 repeat in interphase nuclei. (b, b’) DNA/DNA hybridization (positive control) revealed clusters of PO41 repeat in interphase nuclei. (c, c’) RiboShredder RNase cocktail treatment before DNA/RNA hybridization (negative control) removed all hybridization signals. (d) 3D reconstruction of interphase nucleus with isosurfaces around G-rich transcripts of PO41 repeat (red) and chromatin (blue). DNA was counterstained with DAPI. Scale bars: 5 μm (a-c, a’-c’); 3 μm (d).
Mentions: To study the transcriptional activity of subtelomeric tandem PO41 repeat, we performed FISH according to DNA/RNA hybridization protocol with single stranded oligonucleotide probes (PO41pos and PO41neg) to each strand of the repeat. RNA FISH was used as a reliable approach to reveal specific transcripts in either cultured cells or tissues [29]. At first, we analyzed the transcription of PO41 repeat in chicken MDCC-MSB1 cell line as in one of the few widespread permanent cell lines of birds with a high level of proliferative activity [30]. In MDCC-MSB1 cells, we detected transcripts from both strands of PO41 repeat in interphase nuclei, but not cytoplasm (Figure 1a, a’). The pattern of intranuclear distribution was identical for probes to each of the strands of tandem repeat: transcripts localized predominantly in one, rarely two foci typically observed in euchromatin (~0.59 μm and 0.54 μm for C- and G-rich transcripts respectively) (Figure 1d). It indicates that transcription of PO41 repeat is probably initiated at one locus in a genome. The transcription from multiple sites could not be excluded but remained undetected due to bright signals from the major foci. Since bright foci bearing PO41 repeat transcripts are present in nuclei of all cells, we suggest that PO41 repeat transcription begins in early G1 phase. Treatment of MDCC-MSB1 cells with a cocktail of RNases (RiboShredder RNase Blend) before DNA/RNA FISH effectively eliminated all fluorescent signals (Figure 1c, c’) which confirms probes hybridization to RNA-transcripts.Figure 1

Bottom Line: We focused on tissue-specific differences of subtelomeric repeats transcription, structure of the resulting transcripts and the behavior of subtelomere-derived RNA during mitosis.We revealed one or two major foci of PO41 repeat transcripts associated with RNA polymerase II, representing nascent RNA, and dispersed PO41 repeat transcripts localized in euchromatin or interchromatin space, representing released RNA.Potential regulatory role of the PO41 repeat transcripts in RNA-dependent process of subtelomere heterochromatin maintenance is discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Cytology and Histology, Saint-Petersburg State University, Oranienbaumskoie sch. 2, Stary Peterhof, 198504 Saint-Petersburg, Russia.

ABSTRACT

Background: Subtelomeres are located close to the ends of chromosomes and organized by tandemly repetitive sequences, duplicated copies of genes, pseudogenes and retrotransposons. Transcriptional activity of tandemly organized DNA at terminal chromosomal regions and the distribution of subtelomere-derived non-coding RNAs are poorly investigated. Here we aimed to analyze transcriptional activity of subtelomeric tandem repeat in somatic tissues and cultured cells of birds. We focused on tissue-specific differences of subtelomeric repeats transcription, structure of the resulting transcripts and the behavior of subtelomere-derived RNA during mitosis.

Results: Transcriptional activity of short subtelomeric PO41 ("pattern of 41 bp") tandem repeat in the somatic and cultured cells of chicken (Gallus gallus domesticus) and Japanese quail (Coturnix coturnix japonica) was examined using RNA fluorescence in situ hybridization approach. We discovered transcripts from both strands of the PO41 repeat in chicken MDCC-MSB1 cells as well as in chicken and Japanese quail somatic tissues, such as tissues of cerebellum, telencephalon, muscles, oviduct, small and large intestine. Normal somatic and transformed cells demonstrate similar distribution of PO41 repeat transcripts in interphase nuclei. We revealed one or two major foci of PO41 repeat transcripts associated with RNA polymerase II, representing nascent RNA, and dispersed PO41 repeat transcripts localized in euchromatin or interchromatin space, representing released RNA. During mitosis PO41 non-coding RNA distribute between condensed chromosomes till anaphase, when they concentrate at the cleavage plane. At telophase, clusters of PO41 RNA surround terminal regions of chromosomes. Treatments with RNases of different substrate specificity indicate that PO41 repeat transcripts are single-stranded RNAs with short double-stranded regions due to appearance of inverted repeats.

Conclusion: Transcription of a subtelomeric tandem repeat in avian somatic cells is reported here for the first time. PO41 repeat transcription is conserved among Galliformes and has similar pattern in somatic tissues. We demonstrated redistribution of non-coding PO41 RNA occurring during the cell cycle. Potential regulatory role of the PO41 repeat transcripts in RNA-dependent process of subtelomere heterochromatin maintenance is discussed.

No MeSH data available.


Related in: MedlinePlus