Dual function for U2AF(35) in AG-dependent pre-mRNA splicing

Abstract

The splicing factor U2AF is required for the recruitment of U2 small nuclear RNP to pre-mRNAs in higher eukaryotes. The 65-kDa subunit of U2AF (U2AF(65)) binds to the polypyrimidine (Py) tract preceding the 3' splice site, while the 35-kDa subunit (U2AF(35)) contacts the conserved AG dinucleotide at the 3' end of the intron. It has been shown that the interaction between U2AF(35) and the 3' splice site AG can stabilize U2AF(65) binding to weak Py tracts characteristic of so-called AG-dependent pre-mRNAs. U2AF(35) has also been implicated in arginine-serine (RS) domain-mediated bridging interactions with splicing factors of the SR protein family bound to exonic splicing enhancers (ESE), and these interactions can also stabilize U2AF(65) binding. Complementation of the splicing activity of nuclear extracts depleted of U2AF by chromatography in oligo(dT)-cellulose requires, for some pre-mRNAs, only the presence of U2AF(65). In contrast, splicing of a mouse immunoglobulin M (IgM) M1-M2 pre-mRNA requires both U2AF subunits. In this report we have investigated the sequence elements (e.g., Py tract strength, 3' splice site AG, ESE) responsible for the U2AF(35) dependence of IgM. The results indicate that (i) the IgM substrate is an AG-dependent pre-mRNA, (ii) U2AF(35) dependence correlates with AG dependence, and (iii) the identity of the first nucleotide of exon 2 is important for U2AF(35) function. In contrast, RS domain-mediated interactions with SR proteins bound to the ESE appear to be dispensable, because the purine-rich ESE present in exon M2 is not essential for U2AF(35) activity and because a truncation mutant of U2AF(35) consisting only of the pseudo-RNA recognition motif domain and lacking the RS domain is active in our complementation assays. While some of the effects of U2AF(35) can be explained in terms of enhanced U2AF(65) binding, other activities of U2AF(35) do not correlate with increased cross-linking of U2AF(65) to the Py tract. Collectively, the results argue that interaction of U2AF(35) with a consensus 3' splice site triggers events in spliceosome assembly in addition to stabilizing U2AF(65) binding, thus revealing a dual function for U2AF(35) in pre-mRNA splicing.

FIG. 1

Pre-mRNA splicing substrates. (A) Schematic representation of AdML, IgM, and mutant splicing substrates. Black boxes and thick lines represent the AdML exons and intron, respectively; white boxes and thin lines depict the IgM M1-M2 exons and intron. yBP indicates the yeast consensus BP sequence, TACTAAC. (B) Sequence of splicing substrates at the 3′ splice site, including BP, Py tract, and part of the 3′ exon (in capitals). The BP in the wild-type substrates is underlined; sequences in bold indicate mutated nucleotides.

FIG. 2

In vitro splicing reconstitution assay. Radioactively labeled pre-mRNAs were incubated under splicing conditions, and RNAs were isolated and fractionated by electrophoresis on denaturing 13% polyacrylamide gels. Pre-mRNAs were incubated in HeLa nuclear extract (NE) or odtΔNE in the absence or presence of recombinant GST-U2AF⁶⁵ and His-U2AF³⁵ (for protein concentrations, see below). Splicing substrates and products are indicated schematically on the left of each panel, represented as in Fig. 1A. (A) AdML pre-mRNA, with 90 nM GST-U2AF⁶⁵ in lanes 3 and 4 and 210 nM His-U2AF³⁵ in lane 4; (B) mouse IgM M1-M2 minigene, with 90nM GST-U2AF⁶⁵ in lanes 3 and 4 and 210 nM His-U2AF³⁵ in lane 4 and 90, 180, and 270 nM GST-U2AF⁶⁵ in lanes 7, 8, and 9, respectively; (C) 5′ AdML-IgM, a chimeric RNA comprising the 5′ half of the AdML and the 3′ half of the IgM substrate, with 90nM GST-U2AF⁶⁵ in lanes 3 and 4 and 210 nM His-U2AF³⁵ in lane 4.

FIG. 3

A U-rich Py tract, but not a consensus BP, renders IgM U2AF³⁵ independent. Radioactively labeled yBP-IgM (A) or Py AdML-IgM RNA (B) was incubated in HeLa nuclear extract (NE) or odtΔNE in the absence or presence of 90 nM GST-U2AF⁶⁵ and 210 nM U2AF³⁵ and were analyzed as described for Fig. 2. Splicing products and intermediates are indicated on the left of each panel and are represented as in Fig. 1A.

FIG. 4

IgM is an AG-dependent substrate. (A) Splicing assays. The splicing substrates indicated at the top of each panel were incubated in HeLa nuclear extract (NE) in the presence or absence of ATP or in odtΔNE in the presence of recombinant U2AF subunits (90 nM U2AF⁶⁵, 210 nM U2AF³⁵) as indicated. Products were analyzed as described for Fig. 2. IgM splicing products are indicated on the left, and AdML splicing products are indicated on the right. (B) Spliceosome assembly assay. IgM and 3′ss GA/C-IgM RNAs were incubated in HeLa nuclear extract (NE) in the absence or presence of ATP. IgM was also incubated in odtΔNE (right panel) in the presence of recombinant U2AF subunits or the U2AF-containing column eluate (GUA). After incubation for 20 min the mixtures were loaded onto native polyacrylamide composite gels to separate ATP-independent hnRNP complexes (complex H) from ATP-dependent prespliceosomes (complex A) and two conformations of the spliceosome (complex B/C). wt, wild type.

FIG. 5

Cross-linking of U2AF⁶⁵ to IgM and 3′ss GA/C-IgM. The radioactively labeled 3′-half RNAs of IgM and 3′ splice site mutants (3′ ss AG/C-IgM and 3′ss GA/C-IgM) were incubated under splicing condition in HeLa nuclear extracts (NE) or odtΔNE, with or without GST-U2AF⁶⁵ (concentrations [conc.] are indicated above each lane) or 210 nM His-U2AF³⁵. The mixtures were irradiated with UV light and U2AF⁶⁵ immunoprecipitated with specific anti-U2AF⁶⁵ antibodies. The precipitates were fractionated on sodium dodecyl sulfate–10% polyacrylamide gels, and the dried gels were exposed to a phosphorimager screen. The positions of GST-U2AF⁶⁵, endogenous U2AF⁶⁵, and U2AF³⁵ are indicated on the left. Quantification of the phosphorimager signals corresponding to GST-U2AF⁶⁵ cross-linking is shown in the lower panel. The signal obtained in odtΔNE without added protein was used as background, and the value was deducted from values obtained for GST-U2AF⁶⁵ cross-linking. The value for 90 nM GST-U2AF⁶⁵ was set to 300 arbitrary scan units, and the remaining values were scaled accordingly to be able to directly compare them. wt, wild type.

FIG. 6

A consensus 3′ splice site, but not the ESE, is required for optimal U2AF³⁵ function on IgM. (A) Splicing complementation assay using 3′ exon AdML 3′ss AG/C-IgM and 3′ exon AdML 3′ss AG/G-IgM pre-mRNA substrates in HeLa nuclear extract (NE) or odtΔNE supplemented with 90 nM GST-U2AF⁶⁵ and 210 nM His-U2AF³⁵ as indicated above each lane. After incubation the RNA was isolated and splicing products were separated on denaturing 13% polyacrylamide gels. (B) Cross-linking of U2AF⁶⁵ in the reactions shown in panel A. The 3′-half RNAs corresponding to 3′ exon AdML 3′ss AG/C-IgM and 3′ exon AdML 3′ss AG/G-IgM were incubated under the same conditions as those described for panel A. The mixtures were then irradiated with UV light, and U2AF⁶⁵ was immunoprecipitated with specific anti-U2AF⁶⁵ antibodies. Precipitated proteins were separated on sodium dodecyl sulfate–10% polyacrylamide gels and exposed to a phosphorimager screen. The positions of GST-U2AF⁶⁵, U2AF⁶⁵, and U2AF³⁵ are indicated on the left. The lower panel shows a quantification of the signals corresponding to U2AF⁶⁵. (C) Splicing complementation assay using the 3′ss AG/C-IgM pre-mRNA performed as described for panel A.

FIG. 7

The U2AF³⁵ ψRRM is sufficient to provide U2AF³⁵ activity for splicing of IgM in oligo(dT)-depleted nuclear extracts. Radioactively labeled IgM RNA was incubated in NE or odtΔNE supplemented with either U2AF⁶⁵ alone, with additional U2AF³⁵ ΨRRM, or with full-length U2AF³⁵ and was analyzed as described for Fig. 2. A schematic representation of the U2AF³⁵ domain structure and the truncation mutant are shown on the right.