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Isoforms of U1-70k control subunit dynamics in the human spliceosomal U1 snRNP.

Hernández H, Makarova OV, Makarov EM, Morgner N, Muto Y, Krummel DP, Robinson CV - PLoS ONE (2009)

Bottom Line: Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle.Results also show that unstructured post-translationally modified C-terminal tails are responsible for the dynamics of Sm-B/B' and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo.These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
Most human protein-encoding genes contain multiple exons that are spliced together, frequently in alternative arrangements, by the spliceosome. It is established that U1 snRNP is an essential component of the spliceosome, in human consisting of RNA and ten proteins, several of which are post-translationally modified and exist as multiple isoforms. Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle. Using mass spectrometry we investigate the composition and dynamics of the native human U1 snRNP and compare native and recombinant complexes to isolate the effects of various subunits and isoforms on the overall stability. Our data reveal differential incorporation of four protein isoforms and dynamic interactions of subunits U1-A, U1-C and Sm-B/B'. Results also show that unstructured post-translationally modified C-terminal tails are responsible for the dynamics of Sm-B/B' and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo. These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.

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Related in: MedlinePlus

Monitoring changes in the ratio of the U1-70k isoforms in charge states of the intact spectrum and dissociation products.All peaks are scaled according to the ratio of the U1-70k isoforms(red arrows) to enable direct comparison irrespective of the presence of Sm-B/B'. The ratio of isoform 2: isoform 1 in the intact complex is (70∶30) (a). For loss of U1-C the isoform 2 : isoform 1 ratio decreases to 39∶61 (b) while loss of Sm-B/B' leads to an increase in the ratio 23∶77 (c). Subsequent loss of U1-C from the complex in which Sm-B/B' is absent reveals the same isoform ratio (23∶77) as observed for the loss of Sm-B/B'.
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pone-0007202-g005: Monitoring changes in the ratio of the U1-70k isoforms in charge states of the intact spectrum and dissociation products.All peaks are scaled according to the ratio of the U1-70k isoforms(red arrows) to enable direct comparison irrespective of the presence of Sm-B/B'. The ratio of isoform 2: isoform 1 in the intact complex is (70∶30) (a). For loss of U1-C the isoform 2 : isoform 1 ratio decreases to 39∶61 (b) while loss of Sm-B/B' leads to an increase in the ratio 23∶77 (c). Subsequent loss of U1-C from the complex in which Sm-B/B' is absent reveals the same isoform ratio (23∶77) as observed for the loss of Sm-B/B'.

Mentions: To determine whether or not the dissociation of subunits Sm-B/B' and U1-C is affected by the presence of different U1-70k isoforms we monitored the population of isoforms in the stripped complexes formed in the gas phase. The ratio of the two isoforms (U1-70k isoform 1 and isoform 2) in the intact complex was determined above as 70∶30 (figure S3 and figure 5a). At high activation energy dissociation of Sm-B/B' occurs with equal propensity from both U1-70k isoforms, the initial ratio is therefore maintained (70∶30) (figure S4). At lower activation energy, when Sm-B/B' is dissociated from the complex however an increase in the disparity between the two populations is observed (isoform 1: isoform 2, 77∶23, figure 5b and figure S5). This is an intriguing result which we interpret in terms of a reduced tendency for dissociation of Sm-B/B' from the U1-70k isoform 2. Turning our attention to loss of U1-C directly from the intact complex, rather than an increase in the ratio, we observe a clear decrease (61∶39 isoforms 1∶2, figure 5c and figure S6). This is attributed to a greater tendency for U1-70k isoform 1 to retain U1-C than isoform 2 (figure 5c). Considering sequential loss of Sm-B/B' followed by U1-C we find that the increased ratio (77∶23) is maintained in this product, consistent with our observation for loss of Sm-B/B' alone. Overall therefore we can conclude that the subunit interfaces between U1-C and Sm-B/B' are enhanced by interaction with U1-70k isoforms 1 and 2 respectively.


Isoforms of U1-70k control subunit dynamics in the human spliceosomal U1 snRNP.

Hernández H, Makarova OV, Makarov EM, Morgner N, Muto Y, Krummel DP, Robinson CV - PLoS ONE (2009)

Monitoring changes in the ratio of the U1-70k isoforms in charge states of the intact spectrum and dissociation products.All peaks are scaled according to the ratio of the U1-70k isoforms(red arrows) to enable direct comparison irrespective of the presence of Sm-B/B'. The ratio of isoform 2: isoform 1 in the intact complex is (70∶30) (a). For loss of U1-C the isoform 2 : isoform 1 ratio decreases to 39∶61 (b) while loss of Sm-B/B' leads to an increase in the ratio 23∶77 (c). Subsequent loss of U1-C from the complex in which Sm-B/B' is absent reveals the same isoform ratio (23∶77) as observed for the loss of Sm-B/B'.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2747018&req=5

pone-0007202-g005: Monitoring changes in the ratio of the U1-70k isoforms in charge states of the intact spectrum and dissociation products.All peaks are scaled according to the ratio of the U1-70k isoforms(red arrows) to enable direct comparison irrespective of the presence of Sm-B/B'. The ratio of isoform 2: isoform 1 in the intact complex is (70∶30) (a). For loss of U1-C the isoform 2 : isoform 1 ratio decreases to 39∶61 (b) while loss of Sm-B/B' leads to an increase in the ratio 23∶77 (c). Subsequent loss of U1-C from the complex in which Sm-B/B' is absent reveals the same isoform ratio (23∶77) as observed for the loss of Sm-B/B'.
Mentions: To determine whether or not the dissociation of subunits Sm-B/B' and U1-C is affected by the presence of different U1-70k isoforms we monitored the population of isoforms in the stripped complexes formed in the gas phase. The ratio of the two isoforms (U1-70k isoform 1 and isoform 2) in the intact complex was determined above as 70∶30 (figure S3 and figure 5a). At high activation energy dissociation of Sm-B/B' occurs with equal propensity from both U1-70k isoforms, the initial ratio is therefore maintained (70∶30) (figure S4). At lower activation energy, when Sm-B/B' is dissociated from the complex however an increase in the disparity between the two populations is observed (isoform 1: isoform 2, 77∶23, figure 5b and figure S5). This is an intriguing result which we interpret in terms of a reduced tendency for dissociation of Sm-B/B' from the U1-70k isoform 2. Turning our attention to loss of U1-C directly from the intact complex, rather than an increase in the ratio, we observe a clear decrease (61∶39 isoforms 1∶2, figure 5c and figure S6). This is attributed to a greater tendency for U1-70k isoform 1 to retain U1-C than isoform 2 (figure 5c). Considering sequential loss of Sm-B/B' followed by U1-C we find that the increased ratio (77∶23) is maintained in this product, consistent with our observation for loss of Sm-B/B' alone. Overall therefore we can conclude that the subunit interfaces between U1-C and Sm-B/B' are enhanced by interaction with U1-70k isoforms 1 and 2 respectively.

Bottom Line: Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle.Results also show that unstructured post-translationally modified C-terminal tails are responsible for the dynamics of Sm-B/B' and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo.These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
Most human protein-encoding genes contain multiple exons that are spliced together, frequently in alternative arrangements, by the spliceosome. It is established that U1 snRNP is an essential component of the spliceosome, in human consisting of RNA and ten proteins, several of which are post-translationally modified and exist as multiple isoforms. Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle. Using mass spectrometry we investigate the composition and dynamics of the native human U1 snRNP and compare native and recombinant complexes to isolate the effects of various subunits and isoforms on the overall stability. Our data reveal differential incorporation of four protein isoforms and dynamic interactions of subunits U1-A, U1-C and Sm-B/B'. Results also show that unstructured post-translationally modified C-terminal tails are responsible for the dynamics of Sm-B/B' and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo. These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.

Show MeSH
Related in: MedlinePlus