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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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Comparison of spectra recorded for the U1 snRNP isolated from HeLa cells and a recombinant human U1 snRNP complex reconstituted in vitro.(a) The high m/z region of recombinant U1 snRNP reconstituted with all subunits except U1-C and with truncated Sm-B/B', shows remaining stripped complexes after loss of U1-A, Sm-E and Sm-F (or Sm-G* - masses of Sm-F and tagged recombinant Sm-G* cannot be distinguished (table S7) (b) U1 snRNP from HeLa cells showing predominantly loss of Sm-B/B'. Inset shows the low m/z region containing an extended distribution of charge states from dissociated full-length Sm-B/B'. Solution and MS conditions (a) 850 mM ammonium acetate. LCT: capillary: 1.5 kV, cone: 200 V, extractor: 75 V, source readback: 8.4 mbar, ToF readback: 2.1×10−6 mbar. (b) 200 mM ammonium acetate. QToF2: capillary: 1.5 kV, cone: 200 V, extractor: 0 V, collision cell voltage: 160 V, source transfer region readback: 7.1×10−3 mbar, ToF readback: 1.3×10−6 mbar.
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pone-0007202-g004: Comparison of spectra recorded for the U1 snRNP isolated from HeLa cells and a recombinant human U1 snRNP complex reconstituted in vitro.(a) The high m/z region of recombinant U1 snRNP reconstituted with all subunits except U1-C and with truncated Sm-B/B', shows remaining stripped complexes after loss of U1-A, Sm-E and Sm-F (or Sm-G* - masses of Sm-F and tagged recombinant Sm-G* cannot be distinguished (table S7) (b) U1 snRNP from HeLa cells showing predominantly loss of Sm-B/B'. Inset shows the low m/z region containing an extended distribution of charge states from dissociated full-length Sm-B/B'. Solution and MS conditions (a) 850 mM ammonium acetate. LCT: capillary: 1.5 kV, cone: 200 V, extractor: 75 V, source readback: 8.4 mbar, ToF readback: 2.1×10−6 mbar. (b) 200 mM ammonium acetate. QToF2: capillary: 1.5 kV, cone: 200 V, extractor: 0 V, collision cell voltage: 160 V, source transfer region readback: 7.1×10−3 mbar, ToF readback: 1.3×10−6 mbar.

Mentions: The most intriguing result observed here however is the dissociation of Sm-B/B' (23.7/24.7 kDa), U1-C (17.4 kDa) and U1-A (31.2 kDa) in preference to all other smaller subunits in the Sm ring (with the exception of Sm-E). Sm-B/B' and U1-A are approximately three and four times larger than Sm-G respectively. Sub-stoichiometric binding and ready dissociation of U1-A are common observations in many of our spectra, implying labile association of this subunit with the intact particle. Moreover, unfolding does not appear to be a prerequisite of its gas or solution phase dissociation (figure S2). By contrast Sm-B/B' and U1-C undergo significant unfolding and appear to adopt multiple conformations, prompting their ready dissociation from the U1 snRNP complex both in solution and gas phases. Increasing further the activation energy applied to the intact cellular complex generates greater dissociation of protein subunits and higher intensity series for stripped complexes (figure 4 right) compared with experiments at lower activation energies. Three charge state series are identified, assigned to loss of U1-C and Sm-B/B' and to loss of both Sm-B/B' and Sm-E. The series assigned to loss of Sm-B/B' extend across at least eight charge states, in two different distributions, implying a remarkable ability of Sm-B/B' to accommodate charge prior to dissociation. The Sm-B/B' released under these conditions has an average charge state of 17/18+, in accord with dissociation in the gas phase (figure 1) but distinct from the complete unfolding observed in solution (figure S2). Interestingly these results also highlight the formation of a surprisingly stable complex, that of a partly formed 6-membered ring.


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)

Comparison of spectra recorded for the U1 snRNP isolated from HeLa cells and a recombinant human U1 snRNP complex reconstituted in vitro.(a) The high m/z region of recombinant U1 snRNP reconstituted with all subunits except U1-C and with truncated Sm-B/B', shows remaining stripped complexes after loss of U1-A, Sm-E and Sm-F (or Sm-G* - masses of Sm-F and tagged recombinant Sm-G* cannot be distinguished (table S7) (b) U1 snRNP from HeLa cells showing predominantly loss of Sm-B/B'. Inset shows the low m/z region containing an extended distribution of charge states from dissociated full-length Sm-B/B'. Solution and MS conditions (a) 850 mM ammonium acetate. LCT: capillary: 1.5 kV, cone: 200 V, extractor: 75 V, source readback: 8.4 mbar, ToF readback: 2.1×10−6 mbar. (b) 200 mM ammonium acetate. QToF2: capillary: 1.5 kV, cone: 200 V, extractor: 0 V, collision cell voltage: 160 V, source transfer region readback: 7.1×10−3 mbar, ToF readback: 1.3×10−6 mbar.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2747018&req=5

pone-0007202-g004: Comparison of spectra recorded for the U1 snRNP isolated from HeLa cells and a recombinant human U1 snRNP complex reconstituted in vitro.(a) The high m/z region of recombinant U1 snRNP reconstituted with all subunits except U1-C and with truncated Sm-B/B', shows remaining stripped complexes after loss of U1-A, Sm-E and Sm-F (or Sm-G* - masses of Sm-F and tagged recombinant Sm-G* cannot be distinguished (table S7) (b) U1 snRNP from HeLa cells showing predominantly loss of Sm-B/B'. Inset shows the low m/z region containing an extended distribution of charge states from dissociated full-length Sm-B/B'. Solution and MS conditions (a) 850 mM ammonium acetate. LCT: capillary: 1.5 kV, cone: 200 V, extractor: 75 V, source readback: 8.4 mbar, ToF readback: 2.1×10−6 mbar. (b) 200 mM ammonium acetate. QToF2: capillary: 1.5 kV, cone: 200 V, extractor: 0 V, collision cell voltage: 160 V, source transfer region readback: 7.1×10−3 mbar, ToF readback: 1.3×10−6 mbar.
Mentions: The most intriguing result observed here however is the dissociation of Sm-B/B' (23.7/24.7 kDa), U1-C (17.4 kDa) and U1-A (31.2 kDa) in preference to all other smaller subunits in the Sm ring (with the exception of Sm-E). Sm-B/B' and U1-A are approximately three and four times larger than Sm-G respectively. Sub-stoichiometric binding and ready dissociation of U1-A are common observations in many of our spectra, implying labile association of this subunit with the intact particle. Moreover, unfolding does not appear to be a prerequisite of its gas or solution phase dissociation (figure S2). By contrast Sm-B/B' and U1-C undergo significant unfolding and appear to adopt multiple conformations, prompting their ready dissociation from the U1 snRNP complex both in solution and gas phases. Increasing further the activation energy applied to the intact cellular complex generates greater dissociation of protein subunits and higher intensity series for stripped complexes (figure 4 right) compared with experiments at lower activation energies. Three charge state series are identified, assigned to loss of U1-C and Sm-B/B' and to loss of both Sm-B/B' and Sm-E. The series assigned to loss of Sm-B/B' extend across at least eight charge states, in two different distributions, implying a remarkable ability of Sm-B/B' to accommodate charge prior to dissociation. The Sm-B/B' released under these conditions has an average charge state of 17/18+, in accord with dissociation in the gas phase (figure 1) but distinct from the complete unfolding observed in solution (figure S2). Interestingly these results also highlight the formation of a surprisingly stable complex, that of a partly formed 6-membered ring.

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