Testing the putative effect of Dicer-substrate siRNAs in regulating gene expression at transcriptional level

Müge KOVANCILAR; Mehmet Nejat DALAY; Uğur DELİGEZER*

Makale detayları

Testing the putative effect of Dicer-substrate siRNAs in regulating gene expression at transcriptional level

Yazarlar:

Müge KOVANCILAR | Mehmet Nejat DALAY | Uğur DELİGEZER*

Yayıncı:

TUBITAK

Dergi:

Turkish Journal of Biology 2011 Vol.35 Issue 4, pp.399-404

Belge Tipi:

Makale

Yayın Tarihi:

3/2011

Belge Dili:

Türkçe

Document ID (Org No.):

2000812817

Sıklık:

İki Aylık

eISSN:

1303-6092

pISSN:

1300-0152

Anahtar kelimeler:

Özet:

 

RNA interference (RNAi) pathway is a gene silencing process during which small double-stranded RNA (dsRNA) molecules trigger the degradation of homologous RNA targets. Small interfering RNAs (siRNAs) are the mediators of the RNAi that can be induced in vitro and in vivo by direct application of chemically synthesized siRNAs.  Recently, promoter-targeted small non-coding RNAs have been described to be capable of regulating gene expression at the transcriptional level. In the present study we tested the hypothesis that Dicer-substrate siRNAs (D-siRNAs), which trigger gene silencing through intrinsic RNAi pathway and are therefore more potent in gene knockdown than traditionally used 21-23mer siRNAs, may also display regulatory effects at the transcriptional level. Synthetic 27mer D-siRNAs targeting the promoter/early coding region of the CDKN2A gene were designed and transfected into HeLa cells, and 48 h post-transfection the CDKN2A expression was analyzed in quantitative real-time PCR using the house-keeping G6PDH gene as reference. In comparison to the mutant version, the CDKN2A gene was effectively knocked-down by the D-siRNA, but no evidence of transcriptional regulation was found. We conclude that the D-siRNA-induced suppression is likely to occur after transcription.

 

RNA interference (RNAi) pathway is a gene silencing process during which small double-stranded RNA (dsRNA) molecules trigger the degradation of homologous RNA targets. Small interfering RNAs (siRNAs) are the mediators of the RNAi that can be induced in vitro and in vivo by direct application of chemically synthesized siRNAs.  Recently, promoter-targeted small non-coding RNAs have been described to be capable of regulating gene expression at the transcriptional level. In the present study we tested the hypothesis that Dicer-substrate siRNAs (D-siRNAs), which trigger gene silencing through intrinsic RNAi pathway and are therefore more potent in gene knockdown than traditionally used 21-23mer siRNAs, may also display regulatory effects at the transcriptional level. Synthetic 27mer D-siRNAs targeting the promoter/early coding region of the CDKN2A gene were designed and transfected into HeLa cells, and 48 h post-transfection the CDKN2A expression was analyzed in quantitative real-time PCR using the house-keeping G6PDH gene as reference. In comparison to the mutant version, the CDKN2A gene was effectively knocked-down by the D-siRNA, but no evidence of transcriptional regulation was found. We conclude that the D-siRNA-induced suppression is likely to occur after transcription.

Makale detayları

Testing the putative effect of Dicer-substrate siRNAs in regulating gene expression at transcriptional level

Yazarlar:

Müge KOVANCILAR | Mehmet Nejat DALAY | Uğur DELİGEZER*

Yayıncı:

TUBITAK

Dergi:

Turkish Journal of Biology 2011 Vol.35 Issue 4, pp.399-404

Belge Tipi:

Makale

Yayın Tarihi:

3/2011

Belge Dili:

Türkçe

Document ID (Org No.):

2000812817

Sıklık:

İki Aylık

eISSN:

1303-6092

pISSN:

1300-0152

Key words: RNA interference

Dicer-substrate siRNAs

transcriptional silencing

CDKN2A

Sayı

Makale detayları

Testing the putative effect of Dicer-substrate siRNAs in regulating gene expression at transcriptional level

Yazarlar: Müge KOVANCILAR | Mehmet Nejat DALAY | Uğur DELİGEZER*

Yayıncı: TUBITAK

Dergi: Turkish Journal of Biology 2011 Vol.35 Issue 4, pp.399-404

Belge Tipi: Makale

Yayın Tarihi: 3/2011

Belge Dili: Türkçe

Document ID (Org No.): 2000812817

Sıklık: İki Aylık

eISSN: 1303-6092

pISSN: 1300-0152

Key words: RNA interference

Dicer-substrate siRNAs

transcriptional silencing

CDKN2A

Sayı

Yazarlar:

Müge KOVANCILAR | Mehmet Nejat DALAY | Uğur DELİGEZER*

Yayıncı:

TUBITAK

Dergi:

Turkish Journal of Biology 2011 Vol.35 Issue 4, pp.399-404

Belge Tipi:

Makale

Yayın Tarihi:

3/2011

Belge Dili:

Türkçe

Document ID (Org No.):

2000812817

Sıklık:

İki Aylık

eISSN:

1303-6092

pISSN:

1300-0152