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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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Recruitment of spliceosomal components to the MINX transcription site. (A–C) Recruitment of snRNPs. WT_MS2in and SAmut_MS2in cells were transfected with Tat vector and processed for multicolor FISH experiments with probes against LacZ in exon 2 (red), and each of the snRNAs (green). In B, cells were treated with SSA. Insets show enlarged views. (D) SF3b155 recruitment in SSA-treated cells. WT_MS2in cells were transfected with vectors expressing Tat and MS2–GFP (green), treated with SSA for 3 h, and processed for immunofluorescence against SF3B155 (red). Bars, 10 µm.
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fig2: Recruitment of spliceosomal components to the MINX transcription site. (A–C) Recruitment of snRNPs. WT_MS2in and SAmut_MS2in cells were transfected with Tat vector and processed for multicolor FISH experiments with probes against LacZ in exon 2 (red), and each of the snRNAs (green). In B, cells were treated with SSA. Insets show enlarged views. (D) SF3b155 recruitment in SSA-treated cells. WT_MS2in cells were transfected with vectors expressing Tat and MS2–GFP (green), treated with SSA for 3 h, and processed for immunofluorescence against SF3B155 (red). Bars, 10 µm.

Mentions: Next, we determined whether splicing factors were recruited to the MINX transcription site using two-color FISH. In untreated cells, all snRNAs were detected at the transcription site, but accumulation was weak (Fig. 2 A). In contrast, in cells treated with SSA for 3 h, all the snRNA accumulated at the MINX transcription site at high levels (Fig. 2 B). This suggested that the weak accumulation observed in untreated cells was caused by the high efficiency of the splicing reaction, as the snRNPs resided only shortly on the pre-mRNA to complete splicing. This is consistent with previous studies of spliceosome assembly in fixed and live cells, which estimated that spliceosomal components resided on pre-mRNAs for 15–30 s on average (Wetterberg et al., 2001; Huranová et al., 2010). Interestingly, SF3b155, the known spliceostatin target, was also recruited in cells treated with the drug (Fig. 2 D). Our data thus suggest that SSA does not block spliceosome assembly, but inhibits spliceosome activation, which is in agreement with in vitro data (Roybal and Jurica, 2010). We next analyzed the splice acceptor mutant. Similar to SSA-treated cells, we found that all snRNAs were enriched at the transcription site of the mutant reporter, including U1 and U4 (Fig. 2 C). Altogether, this demonstrated that the spliceosome was assembled cotranscriptionally on the MINX reporter, and that SSA and mutating the splice acceptor site impaired splicing catalysis but not recruitment of spliceosomal components.


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)

Recruitment of spliceosomal components to the MINX transcription site. (A–C) Recruitment of snRNPs. WT_MS2in and SAmut_MS2in cells were transfected with Tat vector and processed for multicolor FISH experiments with probes against LacZ in exon 2 (red), and each of the snRNAs (green). In B, cells were treated with SSA. Insets show enlarged views. (D) SF3b155 recruitment in SSA-treated cells. WT_MS2in cells were transfected with vectors expressing Tat and MS2–GFP (green), treated with SSA for 3 h, and processed for immunofluorescence against SF3B155 (red). Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig2: Recruitment of spliceosomal components to the MINX transcription site. (A–C) Recruitment of snRNPs. WT_MS2in and SAmut_MS2in cells were transfected with Tat vector and processed for multicolor FISH experiments with probes against LacZ in exon 2 (red), and each of the snRNAs (green). In B, cells were treated with SSA. Insets show enlarged views. (D) SF3b155 recruitment in SSA-treated cells. WT_MS2in cells were transfected with vectors expressing Tat and MS2–GFP (green), treated with SSA for 3 h, and processed for immunofluorescence against SF3B155 (red). Bars, 10 µm.
Mentions: Next, we determined whether splicing factors were recruited to the MINX transcription site using two-color FISH. In untreated cells, all snRNAs were detected at the transcription site, but accumulation was weak (Fig. 2 A). In contrast, in cells treated with SSA for 3 h, all the snRNA accumulated at the MINX transcription site at high levels (Fig. 2 B). This suggested that the weak accumulation observed in untreated cells was caused by the high efficiency of the splicing reaction, as the snRNPs resided only shortly on the pre-mRNA to complete splicing. This is consistent with previous studies of spliceosome assembly in fixed and live cells, which estimated that spliceosomal components resided on pre-mRNAs for 15–30 s on average (Wetterberg et al., 2001; Huranová et al., 2010). Interestingly, SF3b155, the known spliceostatin target, was also recruited in cells treated with the drug (Fig. 2 D). Our data thus suggest that SSA does not block spliceosome assembly, but inhibits spliceosome activation, which is in agreement with in vitro data (Roybal and Jurica, 2010). We next analyzed the splice acceptor mutant. Similar to SSA-treated cells, we found that all snRNAs were enriched at the transcription site of the mutant reporter, including U1 and U4 (Fig. 2 C). Altogether, this demonstrated that the spliceosome was assembled cotranscriptionally on the MINX reporter, and that SSA and mutating the splice acceptor site impaired splicing catalysis but not recruitment of spliceosomal components.

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