Limits...
The chromatin remodeling and mRNA splicing functions of the Brahma (SWI/SNF) complex are mediated by the SNR1/SNF5 regulatory subunit.

Zraly CB, Dingwall AK - Nucleic Acids Res. (2012)

Bottom Line: The Drosophila Brahma (Brm) SWI/SNF complex assists in reprogramming and coordinating gene expression in response to ecdysone hormone signaling at critical points during development.Unexpectedly, we found that incorporation of a loss of function SNR1 subunit led to alterations in RNA polymerase elongation, pre-mRNA splicing regulation and chromatin accessibility of ecdysone hormone regulated genes, revealing that SNR1 functions to restrict BRM-dependent nucleosome remodeling activities downstream of the promoter region.Our results reveal critically important roles of the SNR1/SNF5 subunit and the Brm chromatin remodeling complex in transcription regulation during elongation by RNA Polymerase II and completion of pre-mRNA transcripts that are dependent on hormone signaling in late development.

View Article: PubMed Central - PubMed

Affiliation: Cardinal Bernardin Cancer Center, Oncology Institute, Stritch School of Medicine, Loyola University of Chicago, 2160 S. First Avenue, Maywood, IL 60153, USA.

ABSTRACT
Nucleosome remodeling catalyzed by the ATP-dependent SWI/SNF complex is essential for regulated gene expression. Transcriptome profiling studies in flies and mammals identified cell cycle and hormone responsive genes as important targets of remodeling complex activities. Loss of chromatin remodeling function has been linked to developmental abnormalities and aggressive cancers. The Drosophila Brahma (Brm) SWI/SNF complex assists in reprogramming and coordinating gene expression in response to ecdysone hormone signaling at critical points during development. We used RNAi knockdown in cultured cells and transgenic flies, and conditional mutant alleles to identify unique and important functions of two conserved Brm complex core subunits, SNR1/SNF5 and BRM/SNF2-SWI2, on target gene regulation. Unexpectedly, we found that incorporation of a loss of function SNR1 subunit led to alterations in RNA polymerase elongation, pre-mRNA splicing regulation and chromatin accessibility of ecdysone hormone regulated genes, revealing that SNR1 functions to restrict BRM-dependent nucleosome remodeling activities downstream of the promoter region. Our results reveal critically important roles of the SNR1/SNF5 subunit and the Brm chromatin remodeling complex in transcription regulation during elongation by RNA Polymerase II and completion of pre-mRNA transcripts that are dependent on hormone signaling in late development.

Show MeSH

Related in: MedlinePlus

SNR1 negatively regulates expression and splicing of Eig71Eh transcripts. (A) Developmental splicing regulation of Eig71Eh transcripts. Agarose gel analysis of RT-PCR products from WT and snr1E1 using Eig71Eh primers designed to detect spliced and unspliced Eig71Eh transcripts. Samples were normalized to a control transcript (ribosomal protein gene rp49; data not shown). Note Eig71Eh transcript is not expressed during the BG stage. (B) Comparison of Eig71Eh RNA splice forms. qRT-PCR analysis of snr1E1 and WT described in (A). Graph indicates fold abundance of spliced form in snr1E1 mutant compared to WT (+/+). (C and D) PolII Ser2P-CTD predominates on Eig71Eh 3′ exon in snr1E1 mutants. ChIP/qPCR of 5′ (C) and 3′ regions (D) using antibodies to Ser2P-CTD and Ser5P-CTD PolII on EP stage chromatin from snr1E1 and WT.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3401471&req=5

gks288-F4: SNR1 negatively regulates expression and splicing of Eig71Eh transcripts. (A) Developmental splicing regulation of Eig71Eh transcripts. Agarose gel analysis of RT-PCR products from WT and snr1E1 using Eig71Eh primers designed to detect spliced and unspliced Eig71Eh transcripts. Samples were normalized to a control transcript (ribosomal protein gene rp49; data not shown). Note Eig71Eh transcript is not expressed during the BG stage. (B) Comparison of Eig71Eh RNA splice forms. qRT-PCR analysis of snr1E1 and WT described in (A). Graph indicates fold abundance of spliced form in snr1E1 mutant compared to WT (+/+). (C and D) PolII Ser2P-CTD predominates on Eig71Eh 3′ exon in snr1E1 mutants. ChIP/qPCR of 5′ (C) and 3′ regions (D) using antibodies to Ser2P-CTD and Ser5P-CTD PolII on EP stage chromatin from snr1E1 and WT.

Mentions: In human cell lines, the SWI/SNF complex regulates RNA splicing by decreasing the PolII transcription elongation rate resulting in increased accumulation of PolII on variant exons and by interacting directly with splicing factors (8,9). Since a decreased elongation rate represents a form of RNA polymerase pausing, we looked in snr1E1 mutants for altered splicing regulation. Our expectation was that if PolII was not appropriately paused in the snr1E1 mutant, an increased transcription elongation rate might result in a pool of RNA species with retained introns. Examination of Eig RNA accumulation at developmental stages subject to ecdysone regulation revealed that predominantly non-spliced RNA first accumulates at the WPP stage in WT animals (Figure 4A). Conversion to a smaller spliced form is most evident during the later EP stage even though a non-spliced RNA subpopulation persists. No RNA is detected later in pupariation (see LP, Figure 3C). Thus, the ratio of spliced to non-spliced Eig RNA steadily increases over developmental time, indicating that Eig splicing is developmentally regulated. Unexpectedly and in contrast to WT, analysis of RNA accumulation in the snr1E1 mutant revealed that the spliced form predominated at all developmental stages where the transcript was detected (Figure 4A and B). The largest differential between snr1E1 and WT was measured at the WPP stage, in which we observed 79-fold increased spliced product and 23-fold greater total expression. By early pupal development, the difference in spliced product in snr1E1 is 11-fold over WT with 28-fold greater total expression. Thus, the enhanced intron removal of Eig71Eh and Eig71Ei RNAs in the snr1E1 mutant reveals an unanticipated role for SNR1 in regulating splicing/RNA maturation.Figure 4.


The chromatin remodeling and mRNA splicing functions of the Brahma (SWI/SNF) complex are mediated by the SNR1/SNF5 regulatory subunit.

Zraly CB, Dingwall AK - Nucleic Acids Res. (2012)

SNR1 negatively regulates expression and splicing of Eig71Eh transcripts. (A) Developmental splicing regulation of Eig71Eh transcripts. Agarose gel analysis of RT-PCR products from WT and snr1E1 using Eig71Eh primers designed to detect spliced and unspliced Eig71Eh transcripts. Samples were normalized to a control transcript (ribosomal protein gene rp49; data not shown). Note Eig71Eh transcript is not expressed during the BG stage. (B) Comparison of Eig71Eh RNA splice forms. qRT-PCR analysis of snr1E1 and WT described in (A). Graph indicates fold abundance of spliced form in snr1E1 mutant compared to WT (+/+). (C and D) PolII Ser2P-CTD predominates on Eig71Eh 3′ exon in snr1E1 mutants. ChIP/qPCR of 5′ (C) and 3′ regions (D) using antibodies to Ser2P-CTD and Ser5P-CTD PolII on EP stage chromatin from snr1E1 and WT.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3401471&req=5

gks288-F4: SNR1 negatively regulates expression and splicing of Eig71Eh transcripts. (A) Developmental splicing regulation of Eig71Eh transcripts. Agarose gel analysis of RT-PCR products from WT and snr1E1 using Eig71Eh primers designed to detect spliced and unspliced Eig71Eh transcripts. Samples were normalized to a control transcript (ribosomal protein gene rp49; data not shown). Note Eig71Eh transcript is not expressed during the BG stage. (B) Comparison of Eig71Eh RNA splice forms. qRT-PCR analysis of snr1E1 and WT described in (A). Graph indicates fold abundance of spliced form in snr1E1 mutant compared to WT (+/+). (C and D) PolII Ser2P-CTD predominates on Eig71Eh 3′ exon in snr1E1 mutants. ChIP/qPCR of 5′ (C) and 3′ regions (D) using antibodies to Ser2P-CTD and Ser5P-CTD PolII on EP stage chromatin from snr1E1 and WT.
Mentions: In human cell lines, the SWI/SNF complex regulates RNA splicing by decreasing the PolII transcription elongation rate resulting in increased accumulation of PolII on variant exons and by interacting directly with splicing factors (8,9). Since a decreased elongation rate represents a form of RNA polymerase pausing, we looked in snr1E1 mutants for altered splicing regulation. Our expectation was that if PolII was not appropriately paused in the snr1E1 mutant, an increased transcription elongation rate might result in a pool of RNA species with retained introns. Examination of Eig RNA accumulation at developmental stages subject to ecdysone regulation revealed that predominantly non-spliced RNA first accumulates at the WPP stage in WT animals (Figure 4A). Conversion to a smaller spliced form is most evident during the later EP stage even though a non-spliced RNA subpopulation persists. No RNA is detected later in pupariation (see LP, Figure 3C). Thus, the ratio of spliced to non-spliced Eig RNA steadily increases over developmental time, indicating that Eig splicing is developmentally regulated. Unexpectedly and in contrast to WT, analysis of RNA accumulation in the snr1E1 mutant revealed that the spliced form predominated at all developmental stages where the transcript was detected (Figure 4A and B). The largest differential between snr1E1 and WT was measured at the WPP stage, in which we observed 79-fold increased spliced product and 23-fold greater total expression. By early pupal development, the difference in spliced product in snr1E1 is 11-fold over WT with 28-fold greater total expression. Thus, the enhanced intron removal of Eig71Eh and Eig71Ei RNAs in the snr1E1 mutant reveals an unanticipated role for SNR1 in regulating splicing/RNA maturation.Figure 4.

Bottom Line: The Drosophila Brahma (Brm) SWI/SNF complex assists in reprogramming and coordinating gene expression in response to ecdysone hormone signaling at critical points during development.Unexpectedly, we found that incorporation of a loss of function SNR1 subunit led to alterations in RNA polymerase elongation, pre-mRNA splicing regulation and chromatin accessibility of ecdysone hormone regulated genes, revealing that SNR1 functions to restrict BRM-dependent nucleosome remodeling activities downstream of the promoter region.Our results reveal critically important roles of the SNR1/SNF5 subunit and the Brm chromatin remodeling complex in transcription regulation during elongation by RNA Polymerase II and completion of pre-mRNA transcripts that are dependent on hormone signaling in late development.

View Article: PubMed Central - PubMed

Affiliation: Cardinal Bernardin Cancer Center, Oncology Institute, Stritch School of Medicine, Loyola University of Chicago, 2160 S. First Avenue, Maywood, IL 60153, USA.

ABSTRACT
Nucleosome remodeling catalyzed by the ATP-dependent SWI/SNF complex is essential for regulated gene expression. Transcriptome profiling studies in flies and mammals identified cell cycle and hormone responsive genes as important targets of remodeling complex activities. Loss of chromatin remodeling function has been linked to developmental abnormalities and aggressive cancers. The Drosophila Brahma (Brm) SWI/SNF complex assists in reprogramming and coordinating gene expression in response to ecdysone hormone signaling at critical points during development. We used RNAi knockdown in cultured cells and transgenic flies, and conditional mutant alleles to identify unique and important functions of two conserved Brm complex core subunits, SNR1/SNF5 and BRM/SNF2-SWI2, on target gene regulation. Unexpectedly, we found that incorporation of a loss of function SNR1 subunit led to alterations in RNA polymerase elongation, pre-mRNA splicing regulation and chromatin accessibility of ecdysone hormone regulated genes, revealing that SNR1 functions to restrict BRM-dependent nucleosome remodeling activities downstream of the promoter region. Our results reveal critically important roles of the SNR1/SNF5 subunit and the Brm chromatin remodeling complex in transcription regulation during elongation by RNA Polymerase II and completion of pre-mRNA transcripts that are dependent on hormone signaling in late development.

Show MeSH
Related in: MedlinePlus