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

Abstract

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.

Figure 1

SC35 functionally substitutes for U2AF⁶⁵ in the reconstitution of pre-mRNA splicing in U2AF-depleted extracts. (A) Splicing of PIPβG pre-mRNA in: mock depleted extract (lane 1); U2AF-depleted extract (lane 2); and U2AF⁶⁵-reconstituted (lane 3) and SC35-reconstituted (lane 4) U2AF-depleted extract. (B) Western analysis with anti-U2AF⁶⁵ antibody of mock depleted (lane 1) and U2AF-depleted (lane 2) extract. (C) Western analysis of (Left) crude lysate of SC35 overexpression with α-p50 (anti-Drosophila U2AF⁵⁰ antibody) and anti-SC35 antibody (mAb104) and (Right) purified SC35 with α-p50 and anti-SC35 antibody (mAb104).

Figure 2

SC35 reconstitution of splicing in U2AF-depleted reactions is substrate-specific. Splicing of PIPβG pre-mRNA in mock (lane 1), U2AF-depleted (lane 2), U2AF⁶⁵-reconstituted (lane 3), and SC35-reconstituted (lane 4) extracts. Splicing of PIP85.A in mock (lane 5), U2AF-depleted (lane 6), U2AF⁶⁵ reconstituted (lane 7) and SC35 reconstituted (lane 8) extracts.

Figure 3

SC35 reconstitutes pre-mRNA splicing in U2AF-depleted extracts dependent on the presence of functional U1 snRNP. Splicing of PIPβG pre-mRNA in: nuclear extract (lane 1); nuclear extract preblocked with α-U1 oligonucleotide (lane 2); nuclear extract preblocked with α-U1 oligonucleotide and supplemented with SC35 (lane 3); U2AF-depleted extract supplemented with U2AF⁶⁵ (lane 4); U2AF-depleted extract supplemented with U2AF⁶⁵ and preblocked with α-U1 oligonucleotide (lane 5); U2AF-depleted extract supplemented with U2AF⁶⁵, preblocked with α-U1 oligonucleotide, and supplemented with SC35 (lane 6); U2AF-depleted extract supplemented with SC35 (lane 7);and U2AF-depleted extract blocked with α-U1 oligonucleotide and supplemented with SC35 (lane 8).

Figure 4

Three distinct pathways resulting in spliceosome assembly. Under typical splicing conditions, both U2AF and U1 snRNP are required for spliceosome assembly with a network of stabilizing interactions between U2AF, U1 snRNP, and SR proteins (B; ref. 20). In a substrate-specific manner, excess SC35 reconstitutes splicing in U2AF-depleted reactions in a U1 snRNP-dependent pathway (A) and in U1 snRNP-depleted reactions in a U2AF-dependent pathway (C).