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A novel collection of snRNA-like promoters with tissue-specific transcription properties.

Garritano S, Gigoni A, Costa D, Malatesta P, Florio T, Cancedda R, Pagano A - Int J Mol Sci (2012)

Bottom Line: The investigation of a subset of these elements showed that they play relevant roles in physiology and/or pathology.The bioinformatic analysis of a subset of these elements that map in introns of protein-coding genes in antisense configuration suggest their association with alternative splicing, similarly to other recently characterized small RNAs.Interestingly, the analysis of the transcriptional activity of these novel promoters shows that they are active in a cell-type specific manner, in accordance with the emerging body of evidence of a tissue/cell-specific activity of pol III.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Medicine (DiMES), University of Genoa, 16132 Genoa, Italy; E-Mails: sgarritano@biologia.unipi.it (S.G.); arianna.gigoni@hotmail.it (A.G.); delfinacosta@gmail.com (D.C.); paolo.malatesta@unige.it (P.M.); ranieri.cancedda@unige.it (R.C.).

ABSTRACT
We recently identified a novel dataset of snRNA-like trascriptional units in the human genome. The investigation of a subset of these elements showed that they play relevant roles in physiology and/or pathology. In this work we expand our collection of small RNAs taking advantage of a newly developed algorithm able to identify genome sequence stretches with RNA polymerase (pol) III type 3 promoter features thus constituting putative pol III binding sites. The bioinformatic analysis of a subset of these elements that map in introns of protein-coding genes in antisense configuration suggest their association with alternative splicing, similarly to other recently characterized small RNAs. Interestingly, the analysis of the transcriptional activity of these novel promoters shows that they are active in a cell-type specific manner, in accordance with the emerging body of evidence of a tissue/cell-specific activity of pol III.

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Promoter activity transfection assay of five novel pol III type 3 promoters in cells of different origin: (A) NCTC (Murine Liver); (B) SH-SY5Y (Human Neuroblastoma); (C) HeLa (Human Cervical Cancer) and; (D) U2OS (Osteosarcoma) cell lines. NP, pShag-No promoter negative control; U6, pShag-U6 positive control; NDUFS4, pShag-NDUFS4; NF1, pShag-NF1; Park2, pShag-Park2; Runx2, pShag-Runx2; Tnc, pShag-Tnc. Results are reported as the fraction of luciferase emission detected in cells transfected with pShag driven by different pol III type 3 promoters (U6; NDUFS4; NF1; Park2; Runx2; Tnc) with respect to the no promoter control (NP). Data are reported as mean values ± standard deviation resulting of three determinations.
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f1-ijms-13-11323: Promoter activity transfection assay of five novel pol III type 3 promoters in cells of different origin: (A) NCTC (Murine Liver); (B) SH-SY5Y (Human Neuroblastoma); (C) HeLa (Human Cervical Cancer) and; (D) U2OS (Osteosarcoma) cell lines. NP, pShag-No promoter negative control; U6, pShag-U6 positive control; NDUFS4, pShag-NDUFS4; NF1, pShag-NF1; Park2, pShag-Park2; Runx2, pShag-Runx2; Tnc, pShag-Tnc. Results are reported as the fraction of luciferase emission detected in cells transfected with pShag driven by different pol III type 3 promoters (U6; NDUFS4; NF1; Park2; Runx2; Tnc) with respect to the no promoter control (NP). Data are reported as mean values ± standard deviation resulting of three determinations.

Mentions: In order to assess whether the predicted novel putative transcription units are indeed transcribed, we selected five elements, mapped in five protein-coding genes of interest (NF1, Neurofibromin 1 NM_001042492; Park2, parkin NM_004562; TNC, Tenascin C NM_002160; Runx2, Runt-related transcription factor 2 NM_004348; NDUFS4, NADH dehydrogenase Fe-S protein 4 NM_002495) as experimental models to test the transcriptional activity of their pol III type 3 promoters in vitro, in different cell lines. To detect quantitatively the transcription rate of the novel elements we fused their pol III type 3 promoters to a luciferase silencer hairpin (pSHAG-NF1/-Park2/-TNC/-Runx2/- NDUFS4, hereafter referred to as pS-NF1/-Park2/-TNC/-Runx2/-NDUFS4). In this condition, if the promoter is active in a specific cell line, the transcription of the hairpin drives the post-transcriptional silencing of a co-transfected luciferase cDNA and, ultimately, leads to the decrease of luciferase signal; on the contrary, an unaltered luminescent signal would indicate that the luciferase is not silenced and thus the promoter of the specific ncRNA is not actively transcribed in the cell line tested. Although a different experimental approach such as in vitro transcription and/or primer extension is needed to unambiguously validate the transcriptional activity of these putative promoters, as shown in Figure 1, they affect luciferase activity in a cell type-specific manner, suggesting specific biological roles associated with their corresponding protein-coding genes. Accordingly, the Runx2-associated ncRNA is actively transcribed in Murine Liver NCTC and in osteosarcoma Osteosarcoma U2OS cell lines (respectively 80% and 72% decrease of luciferase emission in cells transfected with pShag-Runx2), in which the Runx2 protein-coding gene (that encodes a nuclear protein essential for osteoblastic differentiation and skeletal morphogenesis) is expressed at high levels. Similarly, in neuroblastoma SH-SY5Y cells we detected the highest expression level of the park2-associated ncRNA (77% decrease of luciferase emission in cells SH-SY5Y transfected with pShag-Park2) that maps in the Park2 protein (parkin, NM_004562) [20].


A novel collection of snRNA-like promoters with tissue-specific transcription properties.

Garritano S, Gigoni A, Costa D, Malatesta P, Florio T, Cancedda R, Pagano A - Int J Mol Sci (2012)

Promoter activity transfection assay of five novel pol III type 3 promoters in cells of different origin: (A) NCTC (Murine Liver); (B) SH-SY5Y (Human Neuroblastoma); (C) HeLa (Human Cervical Cancer) and; (D) U2OS (Osteosarcoma) cell lines. NP, pShag-No promoter negative control; U6, pShag-U6 positive control; NDUFS4, pShag-NDUFS4; NF1, pShag-NF1; Park2, pShag-Park2; Runx2, pShag-Runx2; Tnc, pShag-Tnc. Results are reported as the fraction of luciferase emission detected in cells transfected with pShag driven by different pol III type 3 promoters (U6; NDUFS4; NF1; Park2; Runx2; Tnc) with respect to the no promoter control (NP). Data are reported as mean values ± standard deviation resulting of three determinations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3472747&req=5

f1-ijms-13-11323: Promoter activity transfection assay of five novel pol III type 3 promoters in cells of different origin: (A) NCTC (Murine Liver); (B) SH-SY5Y (Human Neuroblastoma); (C) HeLa (Human Cervical Cancer) and; (D) U2OS (Osteosarcoma) cell lines. NP, pShag-No promoter negative control; U6, pShag-U6 positive control; NDUFS4, pShag-NDUFS4; NF1, pShag-NF1; Park2, pShag-Park2; Runx2, pShag-Runx2; Tnc, pShag-Tnc. Results are reported as the fraction of luciferase emission detected in cells transfected with pShag driven by different pol III type 3 promoters (U6; NDUFS4; NF1; Park2; Runx2; Tnc) with respect to the no promoter control (NP). Data are reported as mean values ± standard deviation resulting of three determinations.
Mentions: In order to assess whether the predicted novel putative transcription units are indeed transcribed, we selected five elements, mapped in five protein-coding genes of interest (NF1, Neurofibromin 1 NM_001042492; Park2, parkin NM_004562; TNC, Tenascin C NM_002160; Runx2, Runt-related transcription factor 2 NM_004348; NDUFS4, NADH dehydrogenase Fe-S protein 4 NM_002495) as experimental models to test the transcriptional activity of their pol III type 3 promoters in vitro, in different cell lines. To detect quantitatively the transcription rate of the novel elements we fused their pol III type 3 promoters to a luciferase silencer hairpin (pSHAG-NF1/-Park2/-TNC/-Runx2/- NDUFS4, hereafter referred to as pS-NF1/-Park2/-TNC/-Runx2/-NDUFS4). In this condition, if the promoter is active in a specific cell line, the transcription of the hairpin drives the post-transcriptional silencing of a co-transfected luciferase cDNA and, ultimately, leads to the decrease of luciferase signal; on the contrary, an unaltered luminescent signal would indicate that the luciferase is not silenced and thus the promoter of the specific ncRNA is not actively transcribed in the cell line tested. Although a different experimental approach such as in vitro transcription and/or primer extension is needed to unambiguously validate the transcriptional activity of these putative promoters, as shown in Figure 1, they affect luciferase activity in a cell type-specific manner, suggesting specific biological roles associated with their corresponding protein-coding genes. Accordingly, the Runx2-associated ncRNA is actively transcribed in Murine Liver NCTC and in osteosarcoma Osteosarcoma U2OS cell lines (respectively 80% and 72% decrease of luciferase emission in cells transfected with pShag-Runx2), in which the Runx2 protein-coding gene (that encodes a nuclear protein essential for osteoblastic differentiation and skeletal morphogenesis) is expressed at high levels. Similarly, in neuroblastoma SH-SY5Y cells we detected the highest expression level of the park2-associated ncRNA (77% decrease of luciferase emission in cells SH-SY5Y transfected with pShag-Park2) that maps in the Park2 protein (parkin, NM_004562) [20].

Bottom Line: The investigation of a subset of these elements showed that they play relevant roles in physiology and/or pathology.The bioinformatic analysis of a subset of these elements that map in introns of protein-coding genes in antisense configuration suggest their association with alternative splicing, similarly to other recently characterized small RNAs.Interestingly, the analysis of the transcriptional activity of these novel promoters shows that they are active in a cell-type specific manner, in accordance with the emerging body of evidence of a tissue/cell-specific activity of pol III.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Medicine (DiMES), University of Genoa, 16132 Genoa, Italy; E-Mails: sgarritano@biologia.unipi.it (S.G.); arianna.gigoni@hotmail.it (A.G.); delfinacosta@gmail.com (D.C.); paolo.malatesta@unige.it (P.M.); ranieri.cancedda@unige.it (R.C.).

ABSTRACT
We recently identified a novel dataset of snRNA-like trascriptional units in the human genome. The investigation of a subset of these elements showed that they play relevant roles in physiology and/or pathology. In this work we expand our collection of small RNAs taking advantage of a newly developed algorithm able to identify genome sequence stretches with RNA polymerase (pol) III type 3 promoter features thus constituting putative pol III binding sites. The bioinformatic analysis of a subset of these elements that map in introns of protein-coding genes in antisense configuration suggest their association with alternative splicing, similarly to other recently characterized small RNAs. Interestingly, the analysis of the transcriptional activity of these novel promoters shows that they are active in a cell-type specific manner, in accordance with the emerging body of evidence of a tissue/cell-specific activity of pol III.

Show MeSH
Related in: MedlinePlus