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Novel cis-trans interactions are involved in post-transcriptional regulation of cyclin-dependent kinase inhibitor p21WAF1/CIP1 mRNA.

Zhang L, Wali A, Fontana JA, Dawson MI, Rishi AK - J Mol Signal (2010)

Bottom Line: CD437 treatment of cells resulted in elevated binding of ~85 kD and ~55 kD cytoplasmic proteins with putative CD437-responsive sequences.Treatment of cells with ActD or CHX prior to CD437 exposure did not abrogate RNA-protein interactions.However, treatment of cytoplasmic protein extracts with proteinase K or alkaline phosphatase resulted in loss of RNA-protein interactions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Karmanos Cancer Institute, Department of Internal Medicine, Wayne State University and John D, Dingell VA Medical Center, Room B4325, 4646 John R, Detroit, MI 48201, USA. Rishia@Karmanos.org.

ABSTRACT

Background: A variety of pathways target CDKI p21WAF1/CIP1 expression at transcriptional, post-transcriptional as well as translational levels. We previously found that cell growth suppressing retinoid CD437 enhanced expression of p21WAF1/CIP1 and DNA damage inducible GADD45 proteins in part by elevating their mRNA stability.

Results: Here, we investigated molecular mechanisms of CD437-dependent post-transcriptional regulation of p21WAF1/CIP1 expression. By utilizing MDA-MB-468 HBC cells expressing chimeric rabbit beta-globin-p21WAF1/CIP1 transcripts we mapped multiple CD437-responsive sequences located within positions 1195 to 1795 of the 3'-untranslated region of p21WAF1/CIP1 mRNA. Several cytoplasmic proteins present in MDA-MB-468, MCF-7 HBC as well as HL-60R leukemia cells bound specifically, in vitro, with these CD437-responsive sequences. CD437 treatment of cells resulted in elevated binding of ~85 kD and ~55 kD cytoplasmic proteins with putative CD437-responsive sequences. A 12 nt RNA sequence (5'-UGUGGUGGCACA-3') present within CD437-responsive region of p21WAF1/CIP1 mRNA displayed specific and elevated binding with the above noted proteins. Treatment of cells with ActD or CHX prior to CD437 exposure did not abrogate RNA-protein interactions. However, treatment of cytoplasmic protein extracts with proteinase K or alkaline phosphatase resulted in loss of RNA-protein interactions.

Conclusions: CD437 regulates cell growth in part by regulating stability of p21WAF1/CIP1 mRNA that involves specific RNA-protein interactions that are phosphorylation-dependent, while not requiring nascent transcription or protein synthesis.

No MeSH data available.


Related in: MedlinePlus

CD437 regulation of chimeric rabbit β-globin-p21WAF1/CIP1 mRNA. Two independent sublines derived from transfection of RGB-WAF (581-2004) plasmid [31.4(A4) and 31.4(C5)] or RBG plasmid [29.6(1) and 29.6(2)] were grown either in the absence (-) or presence (+) of 1 μM CD437 for 48 hours. Total RNAs were prepared and expression of RBG transcripts analyzed by northern blot hybridization as described [12]. Levels of RNA loading in each lane were assessed by signals from hybridization with ribosomal phosphoprotein 36B4 cDNA [18]
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Figure 1: CD437 regulation of chimeric rabbit β-globin-p21WAF1/CIP1 mRNA. Two independent sublines derived from transfection of RGB-WAF (581-2004) plasmid [31.4(A4) and 31.4(C5)] or RBG plasmid [29.6(1) and 29.6(2)] were grown either in the absence (-) or presence (+) of 1 μM CD437 for 48 hours. Total RNAs were prepared and expression of RBG transcripts analyzed by northern blot hybridization as described [12]. Levels of RNA loading in each lane were assessed by signals from hybridization with ribosomal phosphoprotein 36B4 cDNA [18]

Mentions: To determine whether sequences present in the p21WAF1/CIP1 3'-UTR cause instability of heterologous rabbit βglobin (RBG) mRNA, as well as harbor CD437-responsive elements, we utilized MDA-MB-468 HBC sublines that stably express RBG or RBG-WAF (581-2004) transcripts in the following experiments. First, the cells were either untreated or treated with 1 μM CD437 followed by analysis of expression of RBG or RBG-WAF transcripts by northern blot hybridization as described in the methods. CD437 treatment resulted in elevated levels of RBG-WAF (581-2004) transcripts, while no such increase in expression of RBG mRNA was noted (figure 1). Next, multiple, independent sublines expressing RBG or RBG-WAF (581-2004) transcripts were separately incubated in the presence or absence of CD437 for 40 hours, followed by their treatments with 4 μg/ml ActD for additional 2, 4, 6, 8, or 10 hours. Total cellular RNAs were isolated and expression of RBG and RBG-WAF (581-2004) transcripts analyzed by northern blot as in Methods. As shown in figure 2A, CD437 treatments enhanced expression of chimeric RBG-WAF (581-2004) transcripts. The expression of RBG mRNA, on the other hand, was unaltered following similar treatments with ActD in the absence or presence of CD437 (data not shown). Next, the northern blot data from two and four independent sublines expressing RBG and RBG-WAF (581-2004) mRNAs, respectively, that were treated with ActD in the absence or presence of CD437 as in figure 2A, was utilized in calculating rate of decay of the RBG and RBG-WAF (581-2004) transcripts. The half-life (t½) of RBG and RBG-WAF (581-2004) transcripts was calculated as described before [12]. The RBG-WAF (581-2004) transcripts displayed a half-life of ~4 h in comparison to RBG transcripts that were found to be very stable (figures 2B and 2C). Thus, the data in figure 2 suggest that the 3'-UTR of p21WAF1/CIP1 contains sequences that affect stability of the RBG transcripts in the HBC cells. Further, CD437 treatment of HBC cells expressing RBG-WAF (581-2004) transcripts resulted in significant enhancement (~4-fold) of the stability of the chimeric transcripts (figure 2B). Together, data in figures 1 and 2 demonstrate that the ~1.4 kb long 3'-UTR of p21WAF1/CIP1 mRNA contains elements that regulate stability of RBG mRNAs in HBC cells dependent as well as independent of CD437.


Novel cis-trans interactions are involved in post-transcriptional regulation of cyclin-dependent kinase inhibitor p21WAF1/CIP1 mRNA.

Zhang L, Wali A, Fontana JA, Dawson MI, Rishi AK - J Mol Signal (2010)

CD437 regulation of chimeric rabbit β-globin-p21WAF1/CIP1 mRNA. Two independent sublines derived from transfection of RGB-WAF (581-2004) plasmid [31.4(A4) and 31.4(C5)] or RBG plasmid [29.6(1) and 29.6(2)] were grown either in the absence (-) or presence (+) of 1 μM CD437 for 48 hours. Total RNAs were prepared and expression of RBG transcripts analyzed by northern blot hybridization as described [12]. Levels of RNA loading in each lane were assessed by signals from hybridization with ribosomal phosphoprotein 36B4 cDNA [18]
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: CD437 regulation of chimeric rabbit β-globin-p21WAF1/CIP1 mRNA. Two independent sublines derived from transfection of RGB-WAF (581-2004) plasmid [31.4(A4) and 31.4(C5)] or RBG plasmid [29.6(1) and 29.6(2)] were grown either in the absence (-) or presence (+) of 1 μM CD437 for 48 hours. Total RNAs were prepared and expression of RBG transcripts analyzed by northern blot hybridization as described [12]. Levels of RNA loading in each lane were assessed by signals from hybridization with ribosomal phosphoprotein 36B4 cDNA [18]
Mentions: To determine whether sequences present in the p21WAF1/CIP1 3'-UTR cause instability of heterologous rabbit βglobin (RBG) mRNA, as well as harbor CD437-responsive elements, we utilized MDA-MB-468 HBC sublines that stably express RBG or RBG-WAF (581-2004) transcripts in the following experiments. First, the cells were either untreated or treated with 1 μM CD437 followed by analysis of expression of RBG or RBG-WAF transcripts by northern blot hybridization as described in the methods. CD437 treatment resulted in elevated levels of RBG-WAF (581-2004) transcripts, while no such increase in expression of RBG mRNA was noted (figure 1). Next, multiple, independent sublines expressing RBG or RBG-WAF (581-2004) transcripts were separately incubated in the presence or absence of CD437 for 40 hours, followed by their treatments with 4 μg/ml ActD for additional 2, 4, 6, 8, or 10 hours. Total cellular RNAs were isolated and expression of RBG and RBG-WAF (581-2004) transcripts analyzed by northern blot as in Methods. As shown in figure 2A, CD437 treatments enhanced expression of chimeric RBG-WAF (581-2004) transcripts. The expression of RBG mRNA, on the other hand, was unaltered following similar treatments with ActD in the absence or presence of CD437 (data not shown). Next, the northern blot data from two and four independent sublines expressing RBG and RBG-WAF (581-2004) mRNAs, respectively, that were treated with ActD in the absence or presence of CD437 as in figure 2A, was utilized in calculating rate of decay of the RBG and RBG-WAF (581-2004) transcripts. The half-life (t½) of RBG and RBG-WAF (581-2004) transcripts was calculated as described before [12]. The RBG-WAF (581-2004) transcripts displayed a half-life of ~4 h in comparison to RBG transcripts that were found to be very stable (figures 2B and 2C). Thus, the data in figure 2 suggest that the 3'-UTR of p21WAF1/CIP1 contains sequences that affect stability of the RBG transcripts in the HBC cells. Further, CD437 treatment of HBC cells expressing RBG-WAF (581-2004) transcripts resulted in significant enhancement (~4-fold) of the stability of the chimeric transcripts (figure 2B). Together, data in figures 1 and 2 demonstrate that the ~1.4 kb long 3'-UTR of p21WAF1/CIP1 mRNA contains elements that regulate stability of RBG mRNAs in HBC cells dependent as well as independent of CD437.

Bottom Line: CD437 treatment of cells resulted in elevated binding of ~85 kD and ~55 kD cytoplasmic proteins with putative CD437-responsive sequences.Treatment of cells with ActD or CHX prior to CD437 exposure did not abrogate RNA-protein interactions.However, treatment of cytoplasmic protein extracts with proteinase K or alkaline phosphatase resulted in loss of RNA-protein interactions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Karmanos Cancer Institute, Department of Internal Medicine, Wayne State University and John D, Dingell VA Medical Center, Room B4325, 4646 John R, Detroit, MI 48201, USA. Rishia@Karmanos.org.

ABSTRACT

Background: A variety of pathways target CDKI p21WAF1/CIP1 expression at transcriptional, post-transcriptional as well as translational levels. We previously found that cell growth suppressing retinoid CD437 enhanced expression of p21WAF1/CIP1 and DNA damage inducible GADD45 proteins in part by elevating their mRNA stability.

Results: Here, we investigated molecular mechanisms of CD437-dependent post-transcriptional regulation of p21WAF1/CIP1 expression. By utilizing MDA-MB-468 HBC cells expressing chimeric rabbit beta-globin-p21WAF1/CIP1 transcripts we mapped multiple CD437-responsive sequences located within positions 1195 to 1795 of the 3'-untranslated region of p21WAF1/CIP1 mRNA. Several cytoplasmic proteins present in MDA-MB-468, MCF-7 HBC as well as HL-60R leukemia cells bound specifically, in vitro, with these CD437-responsive sequences. CD437 treatment of cells resulted in elevated binding of ~85 kD and ~55 kD cytoplasmic proteins with putative CD437-responsive sequences. A 12 nt RNA sequence (5'-UGUGGUGGCACA-3') present within CD437-responsive region of p21WAF1/CIP1 mRNA displayed specific and elevated binding with the above noted proteins. Treatment of cells with ActD or CHX prior to CD437 exposure did not abrogate RNA-protein interactions. However, treatment of cytoplasmic protein extracts with proteinase K or alkaline phosphatase resulted in loss of RNA-protein interactions.

Conclusions: CD437 regulates cell growth in part by regulating stability of p21WAF1/CIP1 mRNA that involves specific RNA-protein interactions that are phosphorylation-dependent, while not requiring nascent transcription or protein synthesis.

No MeSH data available.


Related in: MedlinePlus