Stoichiometries of U2AF35, U2AF65 and U2 snRNP reveal new early spliceosome assembly pathways
| Autor: | Lucy P. Eperon, Igor Vorechovsky, Robert Weinmeister, Jana Kralovicova, Li Chen, Andrew J. Hudson, Ian C. Eperon |
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| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
RNA Splicing Plasma protein binding Tropomyosin Biology Models Biological Cell Line Ribonucleoprotein U1 Small Nuclear 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Adenosine Triphosphate Genetics RNA Precursors Humans splice snRNP Ribonucleoprotein Intron Splicing Factor U2AF Introns Phosphoric Monoester Hydrolases Cell biology Survival of Motor Neuron 2 Protein 030104 developmental biology chemistry Multiprotein Complexes RNA splicing Spliceosomes RNA RNA Splice Sites Adenosine triphosphate 030217 neurology & neurosurgery Small nuclear RNA Protein Binding |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| Popis: | The selection of 3΄ splice sites (3΄ss) is an essential early step in mammalian RNA splicing reactions, but the processes involved are unknown. We have used single molecule methods to test whether the major components implicated in selection, the proteins U2AF35 and U2AF65 and the U2 snRNP, are able to recognize alternative candidate sites or are restricted to one pre-specified site. In the presence of adenosine triphosphate (ATP), all three components bind in a 1:1 stoichiometry with a 3΄ss. Pre-mRNA molecules with two alternative 3΄ss can be bound concurrently by two molecules of U2AF or two U2 snRNPs, so none of the components are restricted. However, concurrent occupancy inhibits splicing. Stoichiometric binding requires conditions consistent with coalescence of the 5΄ and 3΄ sites in a complex (I, initial), but if this cannot form the components show unrestricted and stochastic association. In the absence of ATP, when complex E forms, U2 snRNP association is unrestricted. However, if protein dephosphorylation is prevented, an I-like complex forms with stoichiometric association of U2 snRNPs and the U2 snRNA is base-paired to the pre-mRNA. Complex I differs from complex A in that the formation of complex A is associated with the loss of U2AF65 and 35. |
| Databáze: | OpenAIRE |
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