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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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Related in: MedlinePlus

Engineering a U1 snRNA dependent expression system using the HIV unspliced env mRNA. (A) Diagram of the pHIV-Env model construct. An HIV U3 region promoter (grey box) drives transcription through R and U5 regions, all three of which make up the long terminal repeat (LTR). The cDNA has the SD1/SA4b intron removed and contains two exons, shown in black rectangles and one intron bounded by SD4 and SA7. The Env (gp160) open reading frame (ORF) expressed from the unspliced mRNA is shown, the Rev ORF expressed from spliced mRNA is also shown, Vpu and Nef ORFs are present but do not express significant protein in this spliced isoform. (B) Mutations made to SD4 shown in red, a second cryptic SD SD4x overlaps the minimal upstream ORF (uORF) and Vpu start. (C) Use of SD4x was prevented by mutations shown in red at the 3′ splice site, SA7. The branch point (BP), polypyrimidine tract (PPT) and cryptic acceptors were mutated in addition to SA7 itself. All mutations introduced were silent with respect to the Env-coding sequence. (D) Splice site mutations ablated splicing. RT–PCR using Odp.2 and Odp.40 was performed on RNA extracted from cells transfected with the indicated mutant. PCR products were extracted from the gel, cloned and sequenced. Sequence of SA7x is shown to the right. (E) The spliced product of the pHIV-Env transcript makes the Rev protein which controls the export of unspliced 4-kb mRNA and hence Env protein expression. The amino acid sequence is shown at the SD4/SA7 exon/exon junction. The Rev ORF was inactivated using a stop codon at Rev amino acid 38. This mutant was assessed by northern blotting of 4-kb (unspliced) and 2-kb (spliced) mRNA present in nuclear (N), nuclear-associated rough endoplasmic reticulum (R) and cytoplasmic (C) fractions. The 4- and 2-kb probe was made using Odp.1409 and Odp.1410. Probes were designed to bind to gapdh (Acc: NM_002046) for loading, U6 snRNA (Acc: X07425), 7SL (Acc:NR_002715) and mitochondrial tRNAlys (Acc:X93334, tRNA 14) to control for the efficiency of N, R and C fractions respectively.
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Figure 1: Engineering a U1 snRNA dependent expression system using the HIV unspliced env mRNA. (A) Diagram of the pHIV-Env model construct. An HIV U3 region promoter (grey box) drives transcription through R and U5 regions, all three of which make up the long terminal repeat (LTR). The cDNA has the SD1/SA4b intron removed and contains two exons, shown in black rectangles and one intron bounded by SD4 and SA7. The Env (gp160) open reading frame (ORF) expressed from the unspliced mRNA is shown, the Rev ORF expressed from spliced mRNA is also shown, Vpu and Nef ORFs are present but do not express significant protein in this spliced isoform. (B) Mutations made to SD4 shown in red, a second cryptic SD SD4x overlaps the minimal upstream ORF (uORF) and Vpu start. (C) Use of SD4x was prevented by mutations shown in red at the 3′ splice site, SA7. The branch point (BP), polypyrimidine tract (PPT) and cryptic acceptors were mutated in addition to SA7 itself. All mutations introduced were silent with respect to the Env-coding sequence. (D) Splice site mutations ablated splicing. RT–PCR using Odp.2 and Odp.40 was performed on RNA extracted from cells transfected with the indicated mutant. PCR products were extracted from the gel, cloned and sequenced. Sequence of SA7x is shown to the right. (E) The spliced product of the pHIV-Env transcript makes the Rev protein which controls the export of unspliced 4-kb mRNA and hence Env protein expression. The amino acid sequence is shown at the SD4/SA7 exon/exon junction. The Rev ORF was inactivated using a stop codon at Rev amino acid 38. This mutant was assessed by northern blotting of 4-kb (unspliced) and 2-kb (spliced) mRNA present in nuclear (N), nuclear-associated rough endoplasmic reticulum (R) and cytoplasmic (C) fractions. The 4- and 2-kb probe was made using Odp.1409 and Odp.1410. Probes were designed to bind to gapdh (Acc: NM_002046) for loading, U6 snRNA (Acc: X07425), 7SL (Acc:NR_002715) and mitochondrial tRNAlys (Acc:X93334, tRNA 14) to control for the efficiency of N, R and C fractions respectively.

Mentions: Mutations were introduced into pHIV-Env (Figure 1A) to prevent U1 snRNA binding at the SD4 site (Figure 1B). This construct expresses HIV Env, Rev and minimal amounts of Vpu protein (25). The extent of splicing of the env mRNA was used as a surrogate measure of U1 snRNA binding. Assessment of splicing by RT–PCR showed cryptic splicing induced by a single mutation at SD4 (Figure 1D, lane mutSD4). The cryptic donor site, SD4x, which was previously identified upon mutation of SD4 (33) was confirmed by sequencing of the spliced cDNA product. Inactivating SD4x was complicated by the overlapping minimal uORF and Vpu start codon that may influence Env translation (34). However, we could prevent the use of SD4x through a series of mutations at SA7 and adjacent cryptic acceptor sites, SA7c, a and b (35). The AG dinucleotide at SA7, branch point (BP), and polypyrimidine tract (PPT) which bind U2AF35, SF1 (36) and U2AF65 (37) respectively, were mutated to prevent formation of the early spliceosome complex (38). This effectively ablated splicing as shown by the absence of 2-kb spliced mRNA (Figure 1D). The cryptic site used by the SA7 mutant was also identified by sequencing and was found to be 20 nt downstream from SA7 (Figure 1D).Figure 1.


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)

Engineering a U1 snRNA dependent expression system using the HIV unspliced env mRNA. (A) Diagram of the pHIV-Env model construct. An HIV U3 region promoter (grey box) drives transcription through R and U5 regions, all three of which make up the long terminal repeat (LTR). The cDNA has the SD1/SA4b intron removed and contains two exons, shown in black rectangles and one intron bounded by SD4 and SA7. The Env (gp160) open reading frame (ORF) expressed from the unspliced mRNA is shown, the Rev ORF expressed from spliced mRNA is also shown, Vpu and Nef ORFs are present but do not express significant protein in this spliced isoform. (B) Mutations made to SD4 shown in red, a second cryptic SD SD4x overlaps the minimal upstream ORF (uORF) and Vpu start. (C) Use of SD4x was prevented by mutations shown in red at the 3′ splice site, SA7. The branch point (BP), polypyrimidine tract (PPT) and cryptic acceptors were mutated in addition to SA7 itself. All mutations introduced were silent with respect to the Env-coding sequence. (D) Splice site mutations ablated splicing. RT–PCR using Odp.2 and Odp.40 was performed on RNA extracted from cells transfected with the indicated mutant. PCR products were extracted from the gel, cloned and sequenced. Sequence of SA7x is shown to the right. (E) The spliced product of the pHIV-Env transcript makes the Rev protein which controls the export of unspliced 4-kb mRNA and hence Env protein expression. The amino acid sequence is shown at the SD4/SA7 exon/exon junction. The Rev ORF was inactivated using a stop codon at Rev amino acid 38. This mutant was assessed by northern blotting of 4-kb (unspliced) and 2-kb (spliced) mRNA present in nuclear (N), nuclear-associated rough endoplasmic reticulum (R) and cytoplasmic (C) fractions. The 4- and 2-kb probe was made using Odp.1409 and Odp.1410. Probes were designed to bind to gapdh (Acc: NM_002046) for loading, U6 snRNA (Acc: X07425), 7SL (Acc:NR_002715) and mitochondrial tRNAlys (Acc:X93334, tRNA 14) to control for the efficiency of N, R and C fractions respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 1: Engineering a U1 snRNA dependent expression system using the HIV unspliced env mRNA. (A) Diagram of the pHIV-Env model construct. An HIV U3 region promoter (grey box) drives transcription through R and U5 regions, all three of which make up the long terminal repeat (LTR). The cDNA has the SD1/SA4b intron removed and contains two exons, shown in black rectangles and one intron bounded by SD4 and SA7. The Env (gp160) open reading frame (ORF) expressed from the unspliced mRNA is shown, the Rev ORF expressed from spliced mRNA is also shown, Vpu and Nef ORFs are present but do not express significant protein in this spliced isoform. (B) Mutations made to SD4 shown in red, a second cryptic SD SD4x overlaps the minimal upstream ORF (uORF) and Vpu start. (C) Use of SD4x was prevented by mutations shown in red at the 3′ splice site, SA7. The branch point (BP), polypyrimidine tract (PPT) and cryptic acceptors were mutated in addition to SA7 itself. All mutations introduced were silent with respect to the Env-coding sequence. (D) Splice site mutations ablated splicing. RT–PCR using Odp.2 and Odp.40 was performed on RNA extracted from cells transfected with the indicated mutant. PCR products were extracted from the gel, cloned and sequenced. Sequence of SA7x is shown to the right. (E) The spliced product of the pHIV-Env transcript makes the Rev protein which controls the export of unspliced 4-kb mRNA and hence Env protein expression. The amino acid sequence is shown at the SD4/SA7 exon/exon junction. The Rev ORF was inactivated using a stop codon at Rev amino acid 38. This mutant was assessed by northern blotting of 4-kb (unspliced) and 2-kb (spliced) mRNA present in nuclear (N), nuclear-associated rough endoplasmic reticulum (R) and cytoplasmic (C) fractions. The 4- and 2-kb probe was made using Odp.1409 and Odp.1410. Probes were designed to bind to gapdh (Acc: NM_002046) for loading, U6 snRNA (Acc: X07425), 7SL (Acc:NR_002715) and mitochondrial tRNAlys (Acc:X93334, tRNA 14) to control for the efficiency of N, R and C fractions respectively.
Mentions: Mutations were introduced into pHIV-Env (Figure 1A) to prevent U1 snRNA binding at the SD4 site (Figure 1B). This construct expresses HIV Env, Rev and minimal amounts of Vpu protein (25). The extent of splicing of the env mRNA was used as a surrogate measure of U1 snRNA binding. Assessment of splicing by RT–PCR showed cryptic splicing induced by a single mutation at SD4 (Figure 1D, lane mutSD4). The cryptic donor site, SD4x, which was previously identified upon mutation of SD4 (33) was confirmed by sequencing of the spliced cDNA product. Inactivating SD4x was complicated by the overlapping minimal uORF and Vpu start codon that may influence Env translation (34). However, we could prevent the use of SD4x through a series of mutations at SA7 and adjacent cryptic acceptor sites, SA7c, a and b (35). The AG dinucleotide at SA7, branch point (BP), and polypyrimidine tract (PPT) which bind U2AF35, SF1 (36) and U2AF65 (37) respectively, were mutated to prevent formation of the early spliceosome complex (38). This effectively ablated splicing as shown by the absence of 2-kb spliced mRNA (Figure 1D). The cryptic site used by the SA7 mutant was also identified by sequencing and was found to be 20 nt downstream from SA7 (Figure 1D).Figure 1.

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