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

AUTOR(ES)

MacMillan, Andrew M.

FONTE

The National Academy of Sciences of the USA

RESUMO

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.

Documentos Relacionados

• Characterization of U2AF6, a Splicing Factor Related to U2AF35
• Dual Function for U2AF35 in AG-Dependent Pre-mRNA Splicing
• Evidence for Substrate-Specific Requirement of the Splicing Factor U2AF35 and for Its Function after Polypyrimidine Tract Recognition by U2AF65
• The 35-kDa mammalian splicing factor SC35 mediates specific interactions between U1 and U2 small nuclear ribonucleoprotein particles at the 3' splice site.
• Interaction between Subunits of Heterodimeric Splicing Factor U2AF Is Essential In Vivo