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An ordered pathway of snRNP binding during mammalian pre‐mRNA splicing complex assembly.
Author(s) -
Bindereif A.,
Green M. R.
Publication year - 1987
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1987.tb02520.x
Subject(s) - snrnp , rna splicing , biology , splice , genetics , rna , gene
We have studied the assembly, composition and structure of splicing complexes using biotin‐avidin affinity chromatography and RNase protection assays. We find that U1, U2, U4, U5 and U6 snRNPs associate with the pre‐mRNA and are in the mature, functional complex. Association of U1 snRNP with the pre‐mRNA is rapid and ATP independent; binding of all other snRNPs occurs subsequently and is ATP dependent. Efficient binding of U1 and U2 snRNPs requires a 5′ splice site or a 3′ splice site/branch point region, respectively. Both sequence elements are required for efficient U4, U5 and U6 snRNP binding. Mutant RNA substrates containing only a 5′ splice site or a 3′ splice site/branch point region are assembled into ‘partial’ splicing complexes, which contain a subset of these five snRNPs. RNase protection experiments indicate that in contrast to U1 and U2 snRNPs, U4, U5 and U6 snRNPs do not contact the pre‐mRNA. Based upon the time course of snRNP binding and the composition of sucrose gradient fractionated splicing complexes we suggest an assembly pathway proceeding from a 20S (U1 snRNP only) through a 40S (U1 and U2 snRNPs) to the functional 60S splicing complex (U1, U2, U4, U5 and U6 snRNPs).