Isoforms of U1-70k control subunit dynamics in the human spliceosomal U1 snRNP.
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.
Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.
Helena
H
Hernández
Olga V
OV
Makarova
Evgeny M
EM
Makarov
Nina
N
Morgner
Yutaka
Y
Muto
Daniel Pomeranz
DP
Krummel
Carol V
CV
Robinson
D002384:Catalysis; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008968:Molecular Conformation; D020033:Protein Isoforms; D011499:Protein Processing, Post-Translational; D020836:Protein Structure, Quaternary; D040901:Proteomics / Q000379:methods; D012313:RNA / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D017381:Spliceosomes / Q000378:metabolism*
D020033:Protein Isoforms; D012342:RNA, Small Nuclear; D011994:Recombinant Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C490688:SNRNP70 protein, human; D012313:RNA