Limits...
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.

Show MeSH
A three-step model improves the regulation of alternative splicing. (A) A three-step model improves the regulation of cotranscriptional splicing events. (A, top) Scheme of a cotranscriptional alternative splicing event regulated by the elongation rate of RNA polymerase II. Splicing occurs at splice acceptor SA1 only when the splicing reaction is completed before splice acceptor SA2 is synthesized. When both SA2 and SA1 are present on the pre-mRNA, splicing always occurs at SA2. When the polymerase has a fast elongation rate, 25% of the splicing events take place at site SA1, whereas when the polymerase elongates slowly, 75% of the pre-mRNAs are spliced at SA1. (A, bottom) Calculated curves of the appearance of the spliced product as a function of time after transcription (seconds), for models having a single (left) or three limiting steps (right), and optimized to fit the MINX kinetics. The vertical lines point to the elongation time required to obtain 25% and 75% of splicing at SA1. (B) A three-step model improves the regulation of posttranscriptional splicing events. (B, top) Schematics of a posttranscriptional alternative splicing event. Product A is produced constitutively with n rate-limiting steps, whereas product B is produced with a single rate-limiting step that is regulated. (B, bottom) Calculated curves of the appearance of the spliced product A as a function of the rate of the competing splicing event B, for models having a single (left) or three limiting steps (right), and optimized to fit the MINX kinetics. The vertical lines point to the rates δ required to obtain 25% and 75% of product A.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: A three-step model improves the regulation of alternative splicing. (A) A three-step model improves the regulation of cotranscriptional splicing events. (A, top) Scheme of a cotranscriptional alternative splicing event regulated by the elongation rate of RNA polymerase II. Splicing occurs at splice acceptor SA1 only when the splicing reaction is completed before splice acceptor SA2 is synthesized. When both SA2 and SA1 are present on the pre-mRNA, splicing always occurs at SA2. When the polymerase has a fast elongation rate, 25% of the splicing events take place at site SA1, whereas when the polymerase elongates slowly, 75% of the pre-mRNAs are spliced at SA1. (A, bottom) Calculated curves of the appearance of the spliced product as a function of time after transcription (seconds), for models having a single (left) or three limiting steps (right), and optimized to fit the MINX kinetics. The vertical lines point to the elongation time required to obtain 25% and 75% of splicing at SA1. (B) A three-step model improves the regulation of posttranscriptional splicing events. (B, top) Schematics of a posttranscriptional alternative splicing event. Product A is produced constitutively with n rate-limiting steps, whereas product B is produced with a single rate-limiting step that is regulated. (B, bottom) Calculated curves of the appearance of the spliced product A as a function of the rate of the competing splicing event B, for models having a single (left) or three limiting steps (right), and optimized to fit the MINX kinetics. The vertical lines point to the rates δ required to obtain 25% and 75% of product A.

Mentions: Next, we used values of the best fits to simulate the production of the spliced product (Figs. 4 and S2 G). With a single limiting step, the appearance of the spliced mRNA follows an exponential curve. However, with three limiting steps, the appearance of the spliced message has a more sigmoidal shape (Fig. 4 A). These kinetics can be understood from the viewpoint of a stochastic model. In the case of a single limiting step, some molecules will be spliced quickly, and others slowly, because of the stochasticity of the single step. In contrast, if there are several successive rate-limiting steps occurring at a similar rate, the stochastic nature of each event will be averaged by the larger number of steps involved. Each molecule will take a similar time to splice, thereby mimicking a deterministic process. Thus, the number of steps influences the distribution of splicing times around the mean. This is confirmed by calculating the probability of splicing as a function of time, which shows that the probability function peaks more closely around the mean when the number of limiting steps increases (Fig. S2 F). To provide a quantitative measure of the variation among individual molecules, we defined the kinetic noise as the standard deviation of the splicing time, for a population of molecules, divided by the mean splicing time. The splicing noise is inversely proportional to the root square of the number of steps, and thus decreases with additional steps (see Materials and methods).


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)

A three-step model improves the regulation of alternative splicing. (A) A three-step model improves the regulation of cotranscriptional splicing events. (A, top) Scheme of a cotranscriptional alternative splicing event regulated by the elongation rate of RNA polymerase II. Splicing occurs at splice acceptor SA1 only when the splicing reaction is completed before splice acceptor SA2 is synthesized. When both SA2 and SA1 are present on the pre-mRNA, splicing always occurs at SA2. When the polymerase has a fast elongation rate, 25% of the splicing events take place at site SA1, whereas when the polymerase elongates slowly, 75% of the pre-mRNAs are spliced at SA1. (A, bottom) Calculated curves of the appearance of the spliced product as a function of time after transcription (seconds), for models having a single (left) or three limiting steps (right), and optimized to fit the MINX kinetics. The vertical lines point to the elongation time required to obtain 25% and 75% of splicing at SA1. (B) A three-step model improves the regulation of posttranscriptional splicing events. (B, top) Schematics of a posttranscriptional alternative splicing event. Product A is produced constitutively with n rate-limiting steps, whereas product B is produced with a single rate-limiting step that is regulated. (B, bottom) Calculated curves of the appearance of the spliced product A as a function of the rate of the competing splicing event B, for models having a single (left) or three limiting steps (right), and optimized to fit the MINX kinetics. The vertical lines point to the rates δ required to obtain 25% and 75% of product A.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: A three-step model improves the regulation of alternative splicing. (A) A three-step model improves the regulation of cotranscriptional splicing events. (A, top) Scheme of a cotranscriptional alternative splicing event regulated by the elongation rate of RNA polymerase II. Splicing occurs at splice acceptor SA1 only when the splicing reaction is completed before splice acceptor SA2 is synthesized. When both SA2 and SA1 are present on the pre-mRNA, splicing always occurs at SA2. When the polymerase has a fast elongation rate, 25% of the splicing events take place at site SA1, whereas when the polymerase elongates slowly, 75% of the pre-mRNAs are spliced at SA1. (A, bottom) Calculated curves of the appearance of the spliced product as a function of time after transcription (seconds), for models having a single (left) or three limiting steps (right), and optimized to fit the MINX kinetics. The vertical lines point to the elongation time required to obtain 25% and 75% of splicing at SA1. (B) A three-step model improves the regulation of posttranscriptional splicing events. (B, top) Schematics of a posttranscriptional alternative splicing event. Product A is produced constitutively with n rate-limiting steps, whereas product B is produced with a single rate-limiting step that is regulated. (B, bottom) Calculated curves of the appearance of the spliced product A as a function of the rate of the competing splicing event B, for models having a single (left) or three limiting steps (right), and optimized to fit the MINX kinetics. The vertical lines point to the rates δ required to obtain 25% and 75% of product A.
Mentions: Next, we used values of the best fits to simulate the production of the spliced product (Figs. 4 and S2 G). With a single limiting step, the appearance of the spliced mRNA follows an exponential curve. However, with three limiting steps, the appearance of the spliced message has a more sigmoidal shape (Fig. 4 A). These kinetics can be understood from the viewpoint of a stochastic model. In the case of a single limiting step, some molecules will be spliced quickly, and others slowly, because of the stochasticity of the single step. In contrast, if there are several successive rate-limiting steps occurring at a similar rate, the stochastic nature of each event will be averaged by the larger number of steps involved. Each molecule will take a similar time to splice, thereby mimicking a deterministic process. Thus, the number of steps influences the distribution of splicing times around the mean. This is confirmed by calculating the probability of splicing as a function of time, which shows that the probability function peaks more closely around the mean when the number of limiting steps increases (Fig. S2 F). To provide a quantitative measure of the variation among individual molecules, we defined the kinetic noise as the standard deviation of the splicing time, for a population of molecules, divided by the mean splicing time. The splicing noise is inversely proportional to the root square of the number of steps, and thus decreases with additional steps (see Materials and methods).

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