SC35-mediated reconstitution of splicing in U2AF-depleted nuclear extract

Andrew M. Macmillan, Patrick S. Mccaw, John D. Crispino, Phillip A. Sharp*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

9 Scopus citations


Assembly of the mammalian spliceosome is known to proceed in an ordered fashion through several discrete complexes, but the mechanism of this assembly process may not be universal. In an early step, pre-mRNAs are committed to the splicing pathway through association with U1 small nuclear ribonucleoprotein (snRNP) and non-snRNP splicing factors, including U2AF and members of the SR protein family. As a means of studying the steps of spliceosome assembly, we have prepared HeLa nuclear extracts specifically depleted of the splicing factor U2AF. Surprisingly, the SR protein SC35 can functionally substitute for U2AF65 in the reconstitution of pre-mRNA splicing in U2AF-depleted extracts. This reconstitution is substrate- specific and is reminiscent of the SC35-mediated reconstitution of splicing in extracts depleted of U1 snRNP. However, SC35 reconstitution of splicing in U2AF-depleted extracts is dependent on the presence of functional U1 snRNP. These observations suggest that there are at least three distinguishable mechanisms for the binding of U2 snRNP to the pre-mRNA, including U2AF- dependent and -independent pathways.

Original languageEnglish (US)
Pages (from-to)133-136
Number of pages4
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number1
StatePublished - Jan 7 1997

ASJC Scopus subject areas

  • General


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