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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

Nano ESI mass spectra of the cellular U1 snRNP showing intact U1 snRNP as well a series of dissociated individual proteins (blue) and the U1snRNA after release from the complex (beige).The charge states assigned to the intact complex correspond in mass to: 247.2, 248.2 and 249.1 kDa. An expansion of the charge states assigned to the intact complex reveals at least three different forms (a–c) (green). Dissociation of individual proteins from the intact complex gives rise to doublets (g–h) and triplets (d–f) in the peaks assigned to the sub-complex products (green). A schematic model of the U1 snRNP architecture is shown [17] where iso1/2 indicates U1-70k isoform 1 or 2 and B/B' refers to Sm-B or Sm-B'. MS (QToF2) conditions: capillary: 1.5 kV, cone: 200 V, extractor: 0 V, collision cell voltage: 100 V, source transfer region readback: 7.1×10−3 mbar, ToF readback: 1.3×10−6 mbar. Inset conditions as main spectrum except collision cell voltage 130 V.
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pone-0007202-g001: Nano ESI mass spectra of the cellular U1 snRNP showing intact U1 snRNP as well a series of dissociated individual proteins (blue) and the U1snRNA after release from the complex (beige).The charge states assigned to the intact complex correspond in mass to: 247.2, 248.2 and 249.1 kDa. An expansion of the charge states assigned to the intact complex reveals at least three different forms (a–c) (green). Dissociation of individual proteins from the intact complex gives rise to doublets (g–h) and triplets (d–f) in the peaks assigned to the sub-complex products (green). A schematic model of the U1 snRNP architecture is shown [17] where iso1/2 indicates U1-70k isoform 1 or 2 and B/B' refers to Sm-B or Sm-B'. MS (QToF2) conditions: capillary: 1.5 kV, cone: 200 V, extractor: 0 V, collision cell voltage: 100 V, source transfer region readback: 7.1×10−3 mbar, ToF readback: 1.3×10−6 mbar. Inset conditions as main spectrum except collision cell voltage 130 V.

Mentions: To define the composition of native human U1 snRNP initially we carried out a proteomic analysis. All ten of the anticipated proteins were detected and additionally two from the U2 snRNP (Sm-A' and Sm-B″, see supplementary text S1). Subsequently, we determined the masses of the intact U1 proteins after chromatographic separation of the denatured complex and electrospray ionisation (ESI) MS. We repeated the separation process to determine the amino acid sequence of tryptic peptides enabling us to identify the Sm proteins and to correlate identity with intact masses [26], [31] (table S1). Three of the subunits (U1-70k, Sm-D1 and Sm-D2) were not observed using this approach. Phosphorylation of U1-70k and its removal with U1 snRNA has been suggested previously for the absence of U1-70k in preparations of cellular extracts [32]. However since U1-70k, Sm-D1 and Sm-D2 were readily identified in our proteomics analysis we used database values, considering the two U1-70k isoforms with RNA binding domains: U1-70k isoforms 1 and 2 (table S1) [21], [22]. The mass of the snRNA component of the U1 snRNP complex was determined after a phenol/chloroform extraction of the nucleic acid followed by an ethanol precipitation (supplementary text S1). Re-suspension in aqueous buffer and MS confirms the presence of one species with a mass determined experimentally (53250±22 Da) close to that predicted for the established sequence (53271 Da) [33] (figure 1 inset). Summation of the masses of the 10 protein subunit, seven determined empirically and three from databases, together with the lowest mass protein isoforms and the measured mass of the snRNA, leads to the lowest calculated mass for the intact complex as 245806 Da (table S2).


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)

Nano ESI mass spectra of the cellular U1 snRNP showing intact U1 snRNP as well a series of dissociated individual proteins (blue) and the U1snRNA after release from the complex (beige).The charge states assigned to the intact complex correspond in mass to: 247.2, 248.2 and 249.1 kDa. An expansion of the charge states assigned to the intact complex reveals at least three different forms (a–c) (green). Dissociation of individual proteins from the intact complex gives rise to doublets (g–h) and triplets (d–f) in the peaks assigned to the sub-complex products (green). A schematic model of the U1 snRNP architecture is shown [17] where iso1/2 indicates U1-70k isoform 1 or 2 and B/B' refers to Sm-B or Sm-B'. MS (QToF2) conditions: capillary: 1.5 kV, cone: 200 V, extractor: 0 V, collision cell voltage: 100 V, source transfer region readback: 7.1×10−3 mbar, ToF readback: 1.3×10−6 mbar. Inset conditions as main spectrum except collision cell voltage 130 V.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0007202-g001: Nano ESI mass spectra of the cellular U1 snRNP showing intact U1 snRNP as well a series of dissociated individual proteins (blue) and the U1snRNA after release from the complex (beige).The charge states assigned to the intact complex correspond in mass to: 247.2, 248.2 and 249.1 kDa. An expansion of the charge states assigned to the intact complex reveals at least three different forms (a–c) (green). Dissociation of individual proteins from the intact complex gives rise to doublets (g–h) and triplets (d–f) in the peaks assigned to the sub-complex products (green). A schematic model of the U1 snRNP architecture is shown [17] where iso1/2 indicates U1-70k isoform 1 or 2 and B/B' refers to Sm-B or Sm-B'. MS (QToF2) conditions: capillary: 1.5 kV, cone: 200 V, extractor: 0 V, collision cell voltage: 100 V, source transfer region readback: 7.1×10−3 mbar, ToF readback: 1.3×10−6 mbar. Inset conditions as main spectrum except collision cell voltage 130 V.
Mentions: To define the composition of native human U1 snRNP initially we carried out a proteomic analysis. All ten of the anticipated proteins were detected and additionally two from the U2 snRNP (Sm-A' and Sm-B″, see supplementary text S1). Subsequently, we determined the masses of the intact U1 proteins after chromatographic separation of the denatured complex and electrospray ionisation (ESI) MS. We repeated the separation process to determine the amino acid sequence of tryptic peptides enabling us to identify the Sm proteins and to correlate identity with intact masses [26], [31] (table S1). Three of the subunits (U1-70k, Sm-D1 and Sm-D2) were not observed using this approach. Phosphorylation of U1-70k and its removal with U1 snRNA has been suggested previously for the absence of U1-70k in preparations of cellular extracts [32]. However since U1-70k, Sm-D1 and Sm-D2 were readily identified in our proteomics analysis we used database values, considering the two U1-70k isoforms with RNA binding domains: U1-70k isoforms 1 and 2 (table S1) [21], [22]. The mass of the snRNA component of the U1 snRNP complex was determined after a phenol/chloroform extraction of the nucleic acid followed by an ethanol precipitation (supplementary text S1). Re-suspension in aqueous buffer and MS confirms the presence of one species with a mass determined experimentally (53250±22 Da) close to that predicted for the established sequence (53271 Da) [33] (figure 1 inset). Summation of the masses of the 10 protein subunit, seven determined empirically and three from databases, together with the lowest mass protein isoforms and the measured mass of the snRNA, leads to the lowest calculated mass for the intact complex as 245806 Da (table S2).

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