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Real-time imaging of cotranscriptional splicing reveals a kinetic model that reduces noise: implications for alternative splicing regulation.

Schmidt U, Basyuk E, Robert MC, Yoshida M, Villemin JP, Auboeuf D, Aitken S, Bertrand E - J. Cell Biol. (2011)

Bottom Line: All small nuclear ribonucleoproteins (snRNPs) are loaded on nascent pre-mRNAs, and spliceostatin A inhibits splicing but not snRNP recruitment.Each pre-mRNA molecule is predicted to require a similar time to splice, reducing kinetic noise and improving the regulation of alternative splicing.This model is relevant to other kinetically controlled processes acting on few molecules.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut de Génétique Moléculaire de Montpellier, Centre National de la Recherche Scientifique UMR 5535, 34293 Montpellier Cedex 5, France.

ABSTRACT
Splicing is a key process that expands the coding capacity of genomes. Its kinetics remain poorly characterized, and the distribution of splicing time caused by the stochasticity of single splicing events is expected to affect regulation efficiency. We conducted a small-scale survey on 40 introns in human cells and observed that most were spliced cotranscriptionally. Consequently, we constructed a reporter system that splices cotranscriptionally and can be monitored in live cells and in real time through the use of MS2-GFP. All small nuclear ribonucleoproteins (snRNPs) are loaded on nascent pre-mRNAs, and spliceostatin A inhibits splicing but not snRNP recruitment. Intron removal occurs in minutes and is best described by a model where several successive steps are rate limiting. Each pre-mRNA molecule is predicted to require a similar time to splice, reducing kinetic noise and improving the regulation of alternative splicing. This model is relevant to other kinetically controlled processes acting on few molecules.

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Co-transcriptional splicing and characterization of the MINX reporter cell lines. (A) RT-PCR assay to determine whether introns splice cotranscriptionally or not. A hypothetical gene is schematized with the position of the primers used for RT and the competitive PCR. (B) Scheme of the MINX constructs. SAmut_MS2in contains a single AG to GG mutation at the splice acceptor site. The green, red, and blue bars represent the positions of the FISH probes, and the distance is indicated in nucleotides. (C) RNA polymerase II accumulates at the reporter transcription site. WT_MS2in stable cells were transfected with Tat and processed for in situ hybridization with an MS2 probe (red), and for immunofluorescence against the large subunit of RNA polymerase II (RPB1, green). Insets show enlarged images. Bar, 10 µm. (D) WT_MS2in is spliced cotranscriptionally. WT_MS2in or SAmut_MS2in cells were treated with SSA when indicated, and processed for in situ hybridization with probes recognizing the intron (MS2, red), exon 2 (LacZ, blue), or the spliced junction (spliced, green). Bar, 10 µm. Right panels show intensity line scans of the images, using the lines defined by the arrowheads. (E) Quantification of cotranscriptional splicing by RT-PCR. The indicated cell lines were treated as in D, and total RNAs were extracted and RT-PCR amplified with the indicated primers (see scheme, the scissors depict the 3′ end cleavage site). The position of the primers used for reverse transcription are also indicated. pdT, oligo dT primer; 3′ uncleaved primer, primer located downstream the polyadenylation site. Spliced RNAs: 317 bp; unspliced RNAs: 671 bp.
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fig1: Co-transcriptional splicing and characterization of the MINX reporter cell lines. (A) RT-PCR assay to determine whether introns splice cotranscriptionally or not. A hypothetical gene is schematized with the position of the primers used for RT and the competitive PCR. (B) Scheme of the MINX constructs. SAmut_MS2in contains a single AG to GG mutation at the splice acceptor site. The green, red, and blue bars represent the positions of the FISH probes, and the distance is indicated in nucleotides. (C) RNA polymerase II accumulates at the reporter transcription site. WT_MS2in stable cells were transfected with Tat and processed for in situ hybridization with an MS2 probe (red), and for immunofluorescence against the large subunit of RNA polymerase II (RPB1, green). Insets show enlarged images. Bar, 10 µm. (D) WT_MS2in is spliced cotranscriptionally. WT_MS2in or SAmut_MS2in cells were treated with SSA when indicated, and processed for in situ hybridization with probes recognizing the intron (MS2, red), exon 2 (LacZ, blue), or the spliced junction (spliced, green). Bar, 10 µm. Right panels show intensity line scans of the images, using the lines defined by the arrowheads. (E) Quantification of cotranscriptional splicing by RT-PCR. The indicated cell lines were treated as in D, and total RNAs were extracted and RT-PCR amplified with the indicated primers (see scheme, the scissors depict the 3′ end cleavage site). The position of the primers used for reverse transcription are also indicated. pdT, oligo dT primer; 3′ uncleaved primer, primer located downstream the polyadenylation site. Spliced RNAs: 317 bp; unspliced RNAs: 671 bp.

Mentions: Although cotranscriptional splicing has been demonstrated in human cells, it is not clear whether this is a predominant mode of splicing or not. To address this question, we chose a set of 40 constitutive and alternatively spliced human introns and determined with a quantitative assay whether they splice cotranscriptionally or not. To detect nascent RNAs still attached to chromatin, we used an RT primer situated beyond the 3′ cleavage and polyadenylation site. Then, we performed competitive RT-PCR assays with primers that amplified simultaneously the spliced and the unspliced RNAs for the last two introns of the selected genes (Fig. 1 A). Because the distance the reverse transcription can extend is limited, we selected introns that were <3 kb from the cleavage and polyadenylation site. Remarkably, most introns were spliced completely or nearly completely before 3′ end maturation (Table I). This was also the case for four large introns (>1 kb) and for introns that showed alternative splicing. Only one intron was found to splice predominantly posttranscriptionally. Although this set of introns is small and biased for short introns, these data nevertheless suggest that cotranscriptional splicing is frequent in human cells. We thus developed a model system that splices cotranscriptionally and that can be monitored in living cells.


Real-time imaging of cotranscriptional splicing reveals a kinetic model that reduces noise: implications for alternative splicing regulation.

Schmidt U, Basyuk E, Robert MC, Yoshida M, Villemin JP, Auboeuf D, Aitken S, Bertrand E - J. Cell Biol. (2011)

Co-transcriptional splicing and characterization of the MINX reporter cell lines. (A) RT-PCR assay to determine whether introns splice cotranscriptionally or not. A hypothetical gene is schematized with the position of the primers used for RT and the competitive PCR. (B) Scheme of the MINX constructs. SAmut_MS2in contains a single AG to GG mutation at the splice acceptor site. The green, red, and blue bars represent the positions of the FISH probes, and the distance is indicated in nucleotides. (C) RNA polymerase II accumulates at the reporter transcription site. WT_MS2in stable cells were transfected with Tat and processed for in situ hybridization with an MS2 probe (red), and for immunofluorescence against the large subunit of RNA polymerase II (RPB1, green). Insets show enlarged images. Bar, 10 µm. (D) WT_MS2in is spliced cotranscriptionally. WT_MS2in or SAmut_MS2in cells were treated with SSA when indicated, and processed for in situ hybridization with probes recognizing the intron (MS2, red), exon 2 (LacZ, blue), or the spliced junction (spliced, green). Bar, 10 µm. Right panels show intensity line scans of the images, using the lines defined by the arrowheads. (E) Quantification of cotranscriptional splicing by RT-PCR. The indicated cell lines were treated as in D, and total RNAs were extracted and RT-PCR amplified with the indicated primers (see scheme, the scissors depict the 3′ end cleavage site). The position of the primers used for reverse transcription are also indicated. pdT, oligo dT primer; 3′ uncleaved primer, primer located downstream the polyadenylation site. Spliced RNAs: 317 bp; unspliced RNAs: 671 bp.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3105549&req=5

fig1: Co-transcriptional splicing and characterization of the MINX reporter cell lines. (A) RT-PCR assay to determine whether introns splice cotranscriptionally or not. A hypothetical gene is schematized with the position of the primers used for RT and the competitive PCR. (B) Scheme of the MINX constructs. SAmut_MS2in contains a single AG to GG mutation at the splice acceptor site. The green, red, and blue bars represent the positions of the FISH probes, and the distance is indicated in nucleotides. (C) RNA polymerase II accumulates at the reporter transcription site. WT_MS2in stable cells were transfected with Tat and processed for in situ hybridization with an MS2 probe (red), and for immunofluorescence against the large subunit of RNA polymerase II (RPB1, green). Insets show enlarged images. Bar, 10 µm. (D) WT_MS2in is spliced cotranscriptionally. WT_MS2in or SAmut_MS2in cells were treated with SSA when indicated, and processed for in situ hybridization with probes recognizing the intron (MS2, red), exon 2 (LacZ, blue), or the spliced junction (spliced, green). Bar, 10 µm. Right panels show intensity line scans of the images, using the lines defined by the arrowheads. (E) Quantification of cotranscriptional splicing by RT-PCR. The indicated cell lines were treated as in D, and total RNAs were extracted and RT-PCR amplified with the indicated primers (see scheme, the scissors depict the 3′ end cleavage site). The position of the primers used for reverse transcription are also indicated. pdT, oligo dT primer; 3′ uncleaved primer, primer located downstream the polyadenylation site. Spliced RNAs: 317 bp; unspliced RNAs: 671 bp.
Mentions: Although cotranscriptional splicing has been demonstrated in human cells, it is not clear whether this is a predominant mode of splicing or not. To address this question, we chose a set of 40 constitutive and alternatively spliced human introns and determined with a quantitative assay whether they splice cotranscriptionally or not. To detect nascent RNAs still attached to chromatin, we used an RT primer situated beyond the 3′ cleavage and polyadenylation site. Then, we performed competitive RT-PCR assays with primers that amplified simultaneously the spliced and the unspliced RNAs for the last two introns of the selected genes (Fig. 1 A). Because the distance the reverse transcription can extend is limited, we selected introns that were <3 kb from the cleavage and polyadenylation site. Remarkably, most introns were spliced completely or nearly completely before 3′ end maturation (Table I). This was also the case for four large introns (>1 kb) and for introns that showed alternative splicing. Only one intron was found to splice predominantly posttranscriptionally. Although this set of introns is small and biased for short introns, these data nevertheless suggest that cotranscriptional splicing is frequent in human cells. We thus developed a model system that splices cotranscriptionally and that can be monitored in living cells.

Bottom Line: All small nuclear ribonucleoproteins (snRNPs) are loaded on nascent pre-mRNAs, and spliceostatin A inhibits splicing but not snRNP recruitment.Each pre-mRNA molecule is predicted to require a similar time to splice, reducing kinetic noise and improving the regulation of alternative splicing.This model is relevant to other kinetically controlled processes acting on few molecules.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut de Génétique Moléculaire de Montpellier, Centre National de la Recherche Scientifique UMR 5535, 34293 Montpellier Cedex 5, France.

ABSTRACT
Splicing is a key process that expands the coding capacity of genomes. Its kinetics remain poorly characterized, and the distribution of splicing time caused by the stochasticity of single splicing events is expected to affect regulation efficiency. We conducted a small-scale survey on 40 introns in human cells and observed that most were spliced cotranscriptionally. Consequently, we constructed a reporter system that splices cotranscriptionally and can be monitored in live cells and in real time through the use of MS2-GFP. All small nuclear ribonucleoproteins (snRNPs) are loaded on nascent pre-mRNAs, and spliceostatin A inhibits splicing but not snRNP recruitment. Intron removal occurs in minutes and is best described by a model where several successive steps are rate limiting. Each pre-mRNA molecule is predicted to require a similar time to splice, reducing kinetic noise and improving the regulation of alternative splicing. This model is relevant to other kinetically controlled processes acting on few molecules.

Show MeSH