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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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Proposed model for U1 snRNA enhancement of transcription. (A) Transcription of pre-mRNA containing the SD4-SA7 intron is inhibited in the absence of U1 snRNA recruitment by SD4. (B) When U1 snRNA binds at the SD, transcription through the SD4-SA7 intron is efficient, possibly due to an increase in the processivity of elongation via P-TEFb phosphorylation of the Pol II CTD at Serine 2.
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Figure 7: Proposed model for U1 snRNA enhancement of transcription. (A) Transcription of pre-mRNA containing the SD4-SA7 intron is inhibited in the absence of U1 snRNA recruitment by SD4. (B) When U1 snRNA binds at the SD, transcription through the SD4-SA7 intron is efficient, possibly due to an increase in the processivity of elongation via P-TEFb phosphorylation of the Pol II CTD at Serine 2.

Mentions: The molecular determinants allowing U1 snRNA enhancement of transcription are unclear, but the data presented could result from a transcriptional pause somewhere within the intron, which is overcome by the recruitment of an Sm-type U1 snRNA with intact stem loop II to the intron-bordering SD as modelled in Figure 7. A non-mutually exclusive possibility is that formation of the RNA:RNA helix between U1mut5′arm and mutSD4 neutralizes a transcriptional defect induced by mutSD4. Given the central role of phosphorylation of the CTD of Pol II in transcriptional control and the observed partial rescue of the splice mutant by HIV Tat, our data also raises the possibility that U1 snRNA–binding induces an increase in the phosphorylation at Ser 2, suggesting that U1 snRNA may provide positive feedback to the elongating Pol II during transcription through this intron-containing mRNA.Figure 7.


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)

Proposed model for U1 snRNA enhancement of transcription. (A) Transcription of pre-mRNA containing the SD4-SA7 intron is inhibited in the absence of U1 snRNA recruitment by SD4. (B) When U1 snRNA binds at the SD, transcription through the SD4-SA7 intron is efficient, possibly due to an increase in the processivity of elongation via P-TEFb phosphorylation of the Pol II CTD at Serine 2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875018&req=5

Figure 7: Proposed model for U1 snRNA enhancement of transcription. (A) Transcription of pre-mRNA containing the SD4-SA7 intron is inhibited in the absence of U1 snRNA recruitment by SD4. (B) When U1 snRNA binds at the SD, transcription through the SD4-SA7 intron is efficient, possibly due to an increase in the processivity of elongation via P-TEFb phosphorylation of the Pol II CTD at Serine 2.
Mentions: The molecular determinants allowing U1 snRNA enhancement of transcription are unclear, but the data presented could result from a transcriptional pause somewhere within the intron, which is overcome by the recruitment of an Sm-type U1 snRNA with intact stem loop II to the intron-bordering SD as modelled in Figure 7. A non-mutually exclusive possibility is that formation of the RNA:RNA helix between U1mut5′arm and mutSD4 neutralizes a transcriptional defect induced by mutSD4. Given the central role of phosphorylation of the CTD of Pol II in transcriptional control and the observed partial rescue of the splice mutant by HIV Tat, our data also raises the possibility that U1 snRNA–binding induces an increase in the phosphorylation at Ser 2, suggesting that U1 snRNA may provide positive feedback to the elongating Pol II during transcription through this intron-containing mRNA.Figure 7.

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
Related in: MedlinePlus