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The Polycomb protein and E3 ubiquitin ligase Ring1B harbors an IRES in its highly conserved 5' UTR.

Boutsma E, Noback S, van Lohuizen M - PLoS ONE (2008)

Bottom Line: Here we report that Ring1B belongs to the exclusive group of proteins that for their translation depend on a stable 5' UTR sequence in their mRNA known as an Internal Ribosome Entry Site (IRES).Although our findings indicate Ring1B can be translated under conditions where cap-dependent translation is impaired, we found the Ring1B IRES to be cap-dependent.This raises the possibility that translational control of Ring1B is a multi-layered process and that translation of Ring1B needs to be maintained under varying conditions, which is in line with its essential role as an E3 ligase for monoubiquitination of histone H2A in the PRC1 Polycomb protein complex.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Genetics, Netherlands Cancer Institute, Amsterdam, The Netherlands.

ABSTRACT
Ring1B is an essential member of the highly conserved Polycomb group proteins, which orchestrate developmental processes, cell growth and stem cell fate by modifying local chromatin structure. Ring1B was found to be the E3 ligase that monoubiquitinates histone H2A, which adds a new level of chromatin modification to Polycomb group proteins. Here we report that Ring1B belongs to the exclusive group of proteins that for their translation depend on a stable 5' UTR sequence in their mRNA known as an Internal Ribosome Entry Site (IRES). In cell transfection assays the Ring1B IRES confers significantly higher expression levels of Ring1B than a Ring1B cDNA without the IRES. Also, dual luciferase assays show strong activity of the Ring1B IRES. Although our findings indicate Ring1B can be translated under conditions where cap-dependent translation is impaired, we found the Ring1B IRES to be cap-dependent. This raises the possibility that translational control of Ring1B is a multi-layered process and that translation of Ring1B needs to be maintained under varying conditions, which is in line with its essential role as an E3 ligase for monoubiquitination of histone H2A in the PRC1 Polycomb protein complex.

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The Ring1B 5′ UTR does not have cryptic promoter activity.To test cryptic promoter activity of the Ring1B 5′ UTR, the Ring1B and EMCV IRES dual luciferase constructs were cloned into a pcDNA3.1 vector without CMV promoter (A). The constructs correspond to the constructs depicted in figure 3A, with Renilla luciferase 5′ of the IRES and Firefly luciferase 3′ of the IRES. The y-axis shows the absolute counts of Firefly luciferase (cap-dependent) and Renilla luciferase (IRES-dependent) after transient transfection in COS7 cells. In the constructs without promoter, no mRNA is produced, therefore no translation occurs and no luciferase activity can be measured, indicating the Ring1B 5′ UTR does not contain a cryptic promoter. pEGFP vector was cotransfected in COS7 cells and showed equal fluorescence in every transfection. In a bicistronic construct with the EGFP ORF downstream of the Ring1B IRES we could clearly detect EGFP fluorescence with fluorescent microscopy (B, Ring1B IRES only) as well as EGFP protein on Western blot (C). The Ring1B IRES (IRESR) seems to give a slightly higher expression than the EMCV IRES (IRESE), but we did observe some variation in this in our different experiments. On average we would say the EGFP levels were equal. pEGFP served as a positive control, empty pcDNA3.1 as a negative control, and tubulin as a loading control.
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pone-0002322-g004: The Ring1B 5′ UTR does not have cryptic promoter activity.To test cryptic promoter activity of the Ring1B 5′ UTR, the Ring1B and EMCV IRES dual luciferase constructs were cloned into a pcDNA3.1 vector without CMV promoter (A). The constructs correspond to the constructs depicted in figure 3A, with Renilla luciferase 5′ of the IRES and Firefly luciferase 3′ of the IRES. The y-axis shows the absolute counts of Firefly luciferase (cap-dependent) and Renilla luciferase (IRES-dependent) after transient transfection in COS7 cells. In the constructs without promoter, no mRNA is produced, therefore no translation occurs and no luciferase activity can be measured, indicating the Ring1B 5′ UTR does not contain a cryptic promoter. pEGFP vector was cotransfected in COS7 cells and showed equal fluorescence in every transfection. In a bicistronic construct with the EGFP ORF downstream of the Ring1B IRES we could clearly detect EGFP fluorescence with fluorescent microscopy (B, Ring1B IRES only) as well as EGFP protein on Western blot (C). The Ring1B IRES (IRESR) seems to give a slightly higher expression than the EMCV IRES (IRESE), but we did observe some variation in this in our different experiments. On average we would say the EGFP levels were equal. pEGFP served as a positive control, empty pcDNA3.1 as a negative control, and tubulin as a loading control.

Mentions: Several recent publications state it is hard to discern IRES activity from cryptic promoter activity in GC-rich leader sequences [56], [57]. To exclude this possibility, we cut out the CMV promoter from our Ring1B expression constructs (figure 2A, construct nr. 5) and tested these for Ring1B expression. As expected, no Ring1B protein could be detected on Western blot (figure 2B, lane 5). In addition, we cloned the dual luciferase construct including the Ring1B and, as a control, EMCV IRES in a promoterless pcDNA3.1 construct to test cryptic promoter activity (figure 3A, construct 3). As in our experiment with the promoterless Ring1B construct, which gave no Ring1B expression, the promoterless dual luciferase constructs gave neither Renilla luciferase activity nor Firefly luciferase activity (figure 4A).


The Polycomb protein and E3 ubiquitin ligase Ring1B harbors an IRES in its highly conserved 5' UTR.

Boutsma E, Noback S, van Lohuizen M - PLoS ONE (2008)

The Ring1B 5′ UTR does not have cryptic promoter activity.To test cryptic promoter activity of the Ring1B 5′ UTR, the Ring1B and EMCV IRES dual luciferase constructs were cloned into a pcDNA3.1 vector without CMV promoter (A). The constructs correspond to the constructs depicted in figure 3A, with Renilla luciferase 5′ of the IRES and Firefly luciferase 3′ of the IRES. The y-axis shows the absolute counts of Firefly luciferase (cap-dependent) and Renilla luciferase (IRES-dependent) after transient transfection in COS7 cells. In the constructs without promoter, no mRNA is produced, therefore no translation occurs and no luciferase activity can be measured, indicating the Ring1B 5′ UTR does not contain a cryptic promoter. pEGFP vector was cotransfected in COS7 cells and showed equal fluorescence in every transfection. In a bicistronic construct with the EGFP ORF downstream of the Ring1B IRES we could clearly detect EGFP fluorescence with fluorescent microscopy (B, Ring1B IRES only) as well as EGFP protein on Western blot (C). The Ring1B IRES (IRESR) seems to give a slightly higher expression than the EMCV IRES (IRESE), but we did observe some variation in this in our different experiments. On average we would say the EGFP levels were equal. pEGFP served as a positive control, empty pcDNA3.1 as a negative control, and tubulin as a loading control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2386971&req=5

pone-0002322-g004: The Ring1B 5′ UTR does not have cryptic promoter activity.To test cryptic promoter activity of the Ring1B 5′ UTR, the Ring1B and EMCV IRES dual luciferase constructs were cloned into a pcDNA3.1 vector without CMV promoter (A). The constructs correspond to the constructs depicted in figure 3A, with Renilla luciferase 5′ of the IRES and Firefly luciferase 3′ of the IRES. The y-axis shows the absolute counts of Firefly luciferase (cap-dependent) and Renilla luciferase (IRES-dependent) after transient transfection in COS7 cells. In the constructs without promoter, no mRNA is produced, therefore no translation occurs and no luciferase activity can be measured, indicating the Ring1B 5′ UTR does not contain a cryptic promoter. pEGFP vector was cotransfected in COS7 cells and showed equal fluorescence in every transfection. In a bicistronic construct with the EGFP ORF downstream of the Ring1B IRES we could clearly detect EGFP fluorescence with fluorescent microscopy (B, Ring1B IRES only) as well as EGFP protein on Western blot (C). The Ring1B IRES (IRESR) seems to give a slightly higher expression than the EMCV IRES (IRESE), but we did observe some variation in this in our different experiments. On average we would say the EGFP levels were equal. pEGFP served as a positive control, empty pcDNA3.1 as a negative control, and tubulin as a loading control.
Mentions: Several recent publications state it is hard to discern IRES activity from cryptic promoter activity in GC-rich leader sequences [56], [57]. To exclude this possibility, we cut out the CMV promoter from our Ring1B expression constructs (figure 2A, construct nr. 5) and tested these for Ring1B expression. As expected, no Ring1B protein could be detected on Western blot (figure 2B, lane 5). In addition, we cloned the dual luciferase construct including the Ring1B and, as a control, EMCV IRES in a promoterless pcDNA3.1 construct to test cryptic promoter activity (figure 3A, construct 3). As in our experiment with the promoterless Ring1B construct, which gave no Ring1B expression, the promoterless dual luciferase constructs gave neither Renilla luciferase activity nor Firefly luciferase activity (figure 4A).

Bottom Line: Here we report that Ring1B belongs to the exclusive group of proteins that for their translation depend on a stable 5' UTR sequence in their mRNA known as an Internal Ribosome Entry Site (IRES).Although our findings indicate Ring1B can be translated under conditions where cap-dependent translation is impaired, we found the Ring1B IRES to be cap-dependent.This raises the possibility that translational control of Ring1B is a multi-layered process and that translation of Ring1B needs to be maintained under varying conditions, which is in line with its essential role as an E3 ligase for monoubiquitination of histone H2A in the PRC1 Polycomb protein complex.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Genetics, Netherlands Cancer Institute, Amsterdam, The Netherlands.

ABSTRACT
Ring1B is an essential member of the highly conserved Polycomb group proteins, which orchestrate developmental processes, cell growth and stem cell fate by modifying local chromatin structure. Ring1B was found to be the E3 ligase that monoubiquitinates histone H2A, which adds a new level of chromatin modification to Polycomb group proteins. Here we report that Ring1B belongs to the exclusive group of proteins that for their translation depend on a stable 5' UTR sequence in their mRNA known as an Internal Ribosome Entry Site (IRES). In cell transfection assays the Ring1B IRES confers significantly higher expression levels of Ring1B than a Ring1B cDNA without the IRES. Also, dual luciferase assays show strong activity of the Ring1B IRES. Although our findings indicate Ring1B can be translated under conditions where cap-dependent translation is impaired, we found the Ring1B IRES to be cap-dependent. This raises the possibility that translational control of Ring1B is a multi-layered process and that translation of Ring1B needs to be maintained under varying conditions, which is in line with its essential role as an E3 ligase for monoubiquitination of histone H2A in the PRC1 Polycomb protein complex.

Show MeSH
Related in: MedlinePlus