Limits...
The chromatin scaffold protein SAFB1 localizes SUMO-1 to the promoters of ribosomal protein genes to facilitate transcription initiation and splicing.

Liu HW, Banerjee T, Guan X, Freitas MA, Parvin JD - Nucleic Acids Res. (2015)

Bottom Line: In this study, we found that SUMO-1 marks the promoters of ribosomal protein genes via modification of the Scaffold Associated Factor B (SAFB) protein, and the SUMOylated SAFB stimulated both the binding of RNA polymerase to promoters and pre-mRNA splicing.Depletion of SAFB decreased RNA polymerase II binding to promoters and nuclear processing of the mRNA, though mRNA stability was not affected.This study reveals an unexpected role of SUMO-1 and SAFB in the stimulatory coupling of promoter binding, transcription initiation and RNA processing.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Informatics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA.

Show MeSH
SAFB depletion does not affect the stability of the precursor or mature mRNA of RPL26 gene. (A–C) HeLa cells were transfected with SAFB or control siRNA for 48 h and then treated with either 2 μg/ml of actinomycin D (ActD) or DMSO as a control. The amount of RPL26 mRNA was monitored by qRT-PCR (panel A). Results show the concentrations of RNA species as the average of three independent replicates normalized to the concentration of the RNA in the DMSO vehicle sample. The abundance of intron-containing RPL26 pre-mRNA was monitored by qRT-PCR (panel B), and immunoblots from SAFB depletions were assessed (panel C). (D–E) The growth of HeLa cells following depletion of SAFB or SUMO-1 was assessed. Forty-eight hours post transfection with the siRNA, 10 000 cells were plated per well on day 0. Cell number was counted on days 1–4, as indicated. Results from three independent replicates are shown (panel D). T-test analysis was used to evaluate whether the growth rates were significantly different, and the p-value for comparing SUMO-1 depletion to the control depletion was 0.392 and for SAFB 0.153. Immunoblots taken at day 0 are shown (panel E).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4402547&req=5

Figure 6: SAFB depletion does not affect the stability of the precursor or mature mRNA of RPL26 gene. (A–C) HeLa cells were transfected with SAFB or control siRNA for 48 h and then treated with either 2 μg/ml of actinomycin D (ActD) or DMSO as a control. The amount of RPL26 mRNA was monitored by qRT-PCR (panel A). Results show the concentrations of RNA species as the average of three independent replicates normalized to the concentration of the RNA in the DMSO vehicle sample. The abundance of intron-containing RPL26 pre-mRNA was monitored by qRT-PCR (panel B), and immunoblots from SAFB depletions were assessed (panel C). (D–E) The growth of HeLa cells following depletion of SAFB or SUMO-1 was assessed. Forty-eight hours post transfection with the siRNA, 10 000 cells were plated per well on day 0. Cell number was counted on days 1–4, as indicated. Results from three independent replicates are shown (panel D). T-test analysis was used to evaluate whether the growth rates were significantly different, and the p-value for comparing SUMO-1 depletion to the control depletion was 0.392 and for SAFB 0.153. Immunoblots taken at day 0 are shown (panel E).

Mentions: We have shown previously that SUMO-1 marks the chromatin just upstream of the transcription start site of constitutive housekeeping genes and that the SUMO-1 mark stimulated transcription. In addition, the SUMO-1 mark was also found enriched on exons in the human genome, suggesting a potential role for facilitating splicing (8). Given that SAFB interacts with the CTD of RNAPII (13), that SAFB1 is involved in recruitment of SUMO-1 and RNAPII on the promoters (Figures 3 and 4), and that our mass spectrometry results indicated multiple SUMOylated splicing factors (Supplementary Table S1), we tested whether SAFB depletion may affect mRNA expression of the RP genes at the level of pre-mRNA splicing. We investigated the RNA processing of two RP genes, RPL26 and RPL7a, by quantifying RNA containing the exon-exon junction for spliced mRNA in the nucleus, and we quantified the abundance of the intron-exon junctions for measuring pre-mRNA concentration. We are confident that the PCR product from the unspliced pre-mRNA did not result from contamination of genomic DNA since the samples were thoroughly treated with DNase and since the pre-mRNA decreased over time following actinomycin D treatment (Figure 6), and such a decrease over time is inconsistent with genomic DNA contamination. The RT-qPCR analysis showed that depletion of either SUMO-1 or SAFB did not affect the abundance of the primary transcripts relative to the control in the nucleus (Figure 5A), but the spliced mRNA purified from the nucleus was less abundant in SUMO-1 or SAFB depleted cells. This result suggested that SUMO-1 and SAFB are involved in mRNA processing (Figure 5B). It was surprising that the decrease in splicing did not result in an excess accumulation of nuclear pre-mRNA. We suggest that the decrease in initiation (Figure 1) was balanced by the decrease in RNA processing (Figure 5B) to yield little change to the unspliced pre-mRNA (Figure 5A). We also tested the mature mRNA in the cytosol, and the RP genes were reduced due to either SUMO-1 or SAFB depletion (Figure 5C).


The chromatin scaffold protein SAFB1 localizes SUMO-1 to the promoters of ribosomal protein genes to facilitate transcription initiation and splicing.

Liu HW, Banerjee T, Guan X, Freitas MA, Parvin JD - Nucleic Acids Res. (2015)

SAFB depletion does not affect the stability of the precursor or mature mRNA of RPL26 gene. (A–C) HeLa cells were transfected with SAFB or control siRNA for 48 h and then treated with either 2 μg/ml of actinomycin D (ActD) or DMSO as a control. The amount of RPL26 mRNA was monitored by qRT-PCR (panel A). Results show the concentrations of RNA species as the average of three independent replicates normalized to the concentration of the RNA in the DMSO vehicle sample. The abundance of intron-containing RPL26 pre-mRNA was monitored by qRT-PCR (panel B), and immunoblots from SAFB depletions were assessed (panel C). (D–E) The growth of HeLa cells following depletion of SAFB or SUMO-1 was assessed. Forty-eight hours post transfection with the siRNA, 10 000 cells were plated per well on day 0. Cell number was counted on days 1–4, as indicated. Results from three independent replicates are shown (panel D). T-test analysis was used to evaluate whether the growth rates were significantly different, and the p-value for comparing SUMO-1 depletion to the control depletion was 0.392 and for SAFB 0.153. Immunoblots taken at day 0 are shown (panel E).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4402547&req=5

Figure 6: SAFB depletion does not affect the stability of the precursor or mature mRNA of RPL26 gene. (A–C) HeLa cells were transfected with SAFB or control siRNA for 48 h and then treated with either 2 μg/ml of actinomycin D (ActD) or DMSO as a control. The amount of RPL26 mRNA was monitored by qRT-PCR (panel A). Results show the concentrations of RNA species as the average of three independent replicates normalized to the concentration of the RNA in the DMSO vehicle sample. The abundance of intron-containing RPL26 pre-mRNA was monitored by qRT-PCR (panel B), and immunoblots from SAFB depletions were assessed (panel C). (D–E) The growth of HeLa cells following depletion of SAFB or SUMO-1 was assessed. Forty-eight hours post transfection with the siRNA, 10 000 cells were plated per well on day 0. Cell number was counted on days 1–4, as indicated. Results from three independent replicates are shown (panel D). T-test analysis was used to evaluate whether the growth rates were significantly different, and the p-value for comparing SUMO-1 depletion to the control depletion was 0.392 and for SAFB 0.153. Immunoblots taken at day 0 are shown (panel E).
Mentions: We have shown previously that SUMO-1 marks the chromatin just upstream of the transcription start site of constitutive housekeeping genes and that the SUMO-1 mark stimulated transcription. In addition, the SUMO-1 mark was also found enriched on exons in the human genome, suggesting a potential role for facilitating splicing (8). Given that SAFB interacts with the CTD of RNAPII (13), that SAFB1 is involved in recruitment of SUMO-1 and RNAPII on the promoters (Figures 3 and 4), and that our mass spectrometry results indicated multiple SUMOylated splicing factors (Supplementary Table S1), we tested whether SAFB depletion may affect mRNA expression of the RP genes at the level of pre-mRNA splicing. We investigated the RNA processing of two RP genes, RPL26 and RPL7a, by quantifying RNA containing the exon-exon junction for spliced mRNA in the nucleus, and we quantified the abundance of the intron-exon junctions for measuring pre-mRNA concentration. We are confident that the PCR product from the unspliced pre-mRNA did not result from contamination of genomic DNA since the samples were thoroughly treated with DNase and since the pre-mRNA decreased over time following actinomycin D treatment (Figure 6), and such a decrease over time is inconsistent with genomic DNA contamination. The RT-qPCR analysis showed that depletion of either SUMO-1 or SAFB did not affect the abundance of the primary transcripts relative to the control in the nucleus (Figure 5A), but the spliced mRNA purified from the nucleus was less abundant in SUMO-1 or SAFB depleted cells. This result suggested that SUMO-1 and SAFB are involved in mRNA processing (Figure 5B). It was surprising that the decrease in splicing did not result in an excess accumulation of nuclear pre-mRNA. We suggest that the decrease in initiation (Figure 1) was balanced by the decrease in RNA processing (Figure 5B) to yield little change to the unspliced pre-mRNA (Figure 5A). We also tested the mature mRNA in the cytosol, and the RP genes were reduced due to either SUMO-1 or SAFB depletion (Figure 5C).

Bottom Line: In this study, we found that SUMO-1 marks the promoters of ribosomal protein genes via modification of the Scaffold Associated Factor B (SAFB) protein, and the SUMOylated SAFB stimulated both the binding of RNA polymerase to promoters and pre-mRNA splicing.Depletion of SAFB decreased RNA polymerase II binding to promoters and nuclear processing of the mRNA, though mRNA stability was not affected.This study reveals an unexpected role of SUMO-1 and SAFB in the stimulatory coupling of promoter binding, transcription initiation and RNA processing.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Informatics, Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA.

Show MeSH