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Efficient transcription through an intron requires the binding of an Sm-type U1 snRNP with intact stem loop II to the splice donor.

Alexander MR, Wheatley AK, Center RJ, Purcell DF - Nucleic Acids Res. (2010)

Bottom Line: Position and sequence context for U1-binding is crucial because a promoter proximal intron placed upstream of the mutated SD failed to rescue transcription.Furthermore, U1-rescue was independent of promoter and exon sequence and is partially replaced by the transcription elongation activator Tat, pointing to an intron-localized block in transcriptional elongation.Thus, transcriptional coupling of U1 snRNA binding to the SD may licence the polymerase for transcription through the intron.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Melbourne, Melbourne 3010, Australia.

ABSTRACT
The mechanism behind the positive action of introns upon transcription and the biological significance of this positive feedback remains unclear. Functional ablation of splice sites within an HIV-derived env cDNA significantly reduced transcription that was rescued by a U1 snRNA modified to bind to the mutated splice donor (SD). Using this model we further characterized both the U1 and pre-mRNA structural requirements for transcriptional enhancement. U1 snRNA rescued as a mature Sm-type snRNP with an intact stem loop II. Position and sequence context for U1-binding is crucial because a promoter proximal intron placed upstream of the mutated SD failed to rescue transcription. Furthermore, U1-rescue was independent of promoter and exon sequence and is partially replaced by the transcription elongation activator Tat, pointing to an intron-localized block in transcriptional elongation. Thus, transcriptional coupling of U1 snRNA binding to the SD may licence the polymerase for transcription through the intron.

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U1 snRNA enhancement of transcription requires the Sm domain and stem loop II (A) U1 snRNA secondary structure and location of U170k, U1A, U1C and Sm proteins in the small ribonucleoprotein (snRNP). Mutations to disrupt binding of U170k, U1A, Sm are shown in red as well as the deletion made to stem loop II in blue. (B) Sm domain and stem loop II are important for U1 snRNA rescue. Wild-type U1 snRNA and the four U1 snRNA body (nt 12–164) mutants with either a wild-type or 5′arm (nt 1–11) mutant were co-transfected with a gp140uncGFP reporter (shown) containing the SD4SA7 mutation in addition to 20 ng of pCMV-Tat and 100 ng of pCMV-Rev. Values represent the mean of the fold difference between the wild-type and mut5′arm from four transfections performed on different days. Error bars represent the standard deviation of the mean.
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Figure 3: U1 snRNA enhancement of transcription requires the Sm domain and stem loop II (A) U1 snRNA secondary structure and location of U170k, U1A, U1C and Sm proteins in the small ribonucleoprotein (snRNP). Mutations to disrupt binding of U170k, U1A, Sm are shown in red as well as the deletion made to stem loop II in blue. (B) Sm domain and stem loop II are important for U1 snRNA rescue. Wild-type U1 snRNA and the four U1 snRNA body (nt 12–164) mutants with either a wild-type or 5′arm (nt 1–11) mutant were co-transfected with a gp140uncGFP reporter (shown) containing the SD4SA7 mutation in addition to 20 ng of pCMV-Tat and 100 ng of pCMV-Rev. Values represent the mean of the fold difference between the wild-type and mut5′arm from four transfections performed on different days. Error bars represent the standard deviation of the mean.

Mentions: U1 snRNA exists as a highly structured RNA bound to proteins forming a small nuclear ribonucleoprotein (snRNP) (Figure 3A). We mutated the binding sites for U170k, U1A and Sm to assess the relative importance of these proteins in enhancement of transcription by U1 snRNA. We used a fluorescent Env-GFP fusion protein reporter, gp140uncGFP, where the coding sequence of GFP was placed in-frame with an un-cleaved and truncated form of Env. The Env protein was truncated immediately prior to the transmembrane domain to create a soluble Env analogue and facilitate the correct folding and fluorescence of GFP. This truncation of Env does not compromise the rev responsive element (RRE), which lies between the end of gp120 and gp140, or the splicing signals at SA7. The gp160 cleavage motif was mutated to prevent disassociation into gp120 and gp41-GFP (28). Fluorescence-activated cell sorting (FACS) of cells transfected with gp140uncGFP containing the SD4SA7 mutation was used to report the fold-increase in expression induced by U1 snRNA which could bind to mutSD4SA7 env mRNA through mut5′arm. This enabled a quantitative assessment of mutations made to the U1 snRNA body. The wild-type U1 body with mutant 5′ arm increased expression from the SD4SA7 mutant reporter by 2.4-fold (2.4 ± 0.2, n = 4) (Figure 3B), confirming the western blot data (Figure 2B, lowest panel, compare lanes 4 and 5).Figure 3.


Efficient transcription through an intron requires the binding of an Sm-type U1 snRNP with intact stem loop II to the splice donor.

Alexander MR, Wheatley AK, Center RJ, Purcell DF - Nucleic Acids Res. (2010)

U1 snRNA enhancement of transcription requires the Sm domain and stem loop II (A) U1 snRNA secondary structure and location of U170k, U1A, U1C and Sm proteins in the small ribonucleoprotein (snRNP). Mutations to disrupt binding of U170k, U1A, Sm are shown in red as well as the deletion made to stem loop II in blue. (B) Sm domain and stem loop II are important for U1 snRNA rescue. Wild-type U1 snRNA and the four U1 snRNA body (nt 12–164) mutants with either a wild-type or 5′arm (nt 1–11) mutant were co-transfected with a gp140uncGFP reporter (shown) containing the SD4SA7 mutation in addition to 20 ng of pCMV-Tat and 100 ng of pCMV-Rev. Values represent the mean of the fold difference between the wild-type and mut5′arm from four transfections performed on different days. Error bars represent the standard deviation of the mean.
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Related In: Results  -  Collection

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Figure 3: U1 snRNA enhancement of transcription requires the Sm domain and stem loop II (A) U1 snRNA secondary structure and location of U170k, U1A, U1C and Sm proteins in the small ribonucleoprotein (snRNP). Mutations to disrupt binding of U170k, U1A, Sm are shown in red as well as the deletion made to stem loop II in blue. (B) Sm domain and stem loop II are important for U1 snRNA rescue. Wild-type U1 snRNA and the four U1 snRNA body (nt 12–164) mutants with either a wild-type or 5′arm (nt 1–11) mutant were co-transfected with a gp140uncGFP reporter (shown) containing the SD4SA7 mutation in addition to 20 ng of pCMV-Tat and 100 ng of pCMV-Rev. Values represent the mean of the fold difference between the wild-type and mut5′arm from four transfections performed on different days. Error bars represent the standard deviation of the mean.
Mentions: U1 snRNA exists as a highly structured RNA bound to proteins forming a small nuclear ribonucleoprotein (snRNP) (Figure 3A). We mutated the binding sites for U170k, U1A and Sm to assess the relative importance of these proteins in enhancement of transcription by U1 snRNA. We used a fluorescent Env-GFP fusion protein reporter, gp140uncGFP, where the coding sequence of GFP was placed in-frame with an un-cleaved and truncated form of Env. The Env protein was truncated immediately prior to the transmembrane domain to create a soluble Env analogue and facilitate the correct folding and fluorescence of GFP. This truncation of Env does not compromise the rev responsive element (RRE), which lies between the end of gp120 and gp140, or the splicing signals at SA7. The gp160 cleavage motif was mutated to prevent disassociation into gp120 and gp41-GFP (28). Fluorescence-activated cell sorting (FACS) of cells transfected with gp140uncGFP containing the SD4SA7 mutation was used to report the fold-increase in expression induced by U1 snRNA which could bind to mutSD4SA7 env mRNA through mut5′arm. This enabled a quantitative assessment of mutations made to the U1 snRNA body. The wild-type U1 body with mutant 5′ arm increased expression from the SD4SA7 mutant reporter by 2.4-fold (2.4 ± 0.2, n = 4) (Figure 3B), confirming the western blot data (Figure 2B, lowest panel, compare lanes 4 and 5).Figure 3.

Bottom Line: Position and sequence context for U1-binding is crucial because a promoter proximal intron placed upstream of the mutated SD failed to rescue transcription.Furthermore, U1-rescue was independent of promoter and exon sequence and is partially replaced by the transcription elongation activator Tat, pointing to an intron-localized block in transcriptional elongation.Thus, transcriptional coupling of U1 snRNA binding to the SD may licence the polymerase for transcription through the intron.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Melbourne, Melbourne 3010, Australia.

ABSTRACT
The mechanism behind the positive action of introns upon transcription and the biological significance of this positive feedback remains unclear. Functional ablation of splice sites within an HIV-derived env cDNA significantly reduced transcription that was rescued by a U1 snRNA modified to bind to the mutated splice donor (SD). Using this model we further characterized both the U1 and pre-mRNA structural requirements for transcriptional enhancement. U1 snRNA rescued as a mature Sm-type snRNP with an intact stem loop II. Position and sequence context for U1-binding is crucial because a promoter proximal intron placed upstream of the mutated SD failed to rescue transcription. Furthermore, U1-rescue was independent of promoter and exon sequence and is partially replaced by the transcription elongation activator Tat, pointing to an intron-localized block in transcriptional elongation. Thus, transcriptional coupling of U1 snRNA binding to the SD may licence the polymerase for transcription through the intron.

Show MeSH