Deletion of MUD2, the yeast homolog of U2AF65, can bypass the requirement for sub2, an essential spliceosomal ATPase - PubMed (original) (raw)

Deletion of MUD2, the yeast homolog of U2AF65, can bypass the requirement for sub2, an essential spliceosomal ATPase

A L Kistler et al. Genes Dev. 2001.

Abstract

Mammalian U2AF65 and UAP56 are required for prespliceosome (PS) formation. We tested the predictions that the yeast UAP56 homolog, SUB2, is required for the same step and functions collaboratively with MUD2, the yeast homolog of U2AF65. Unexpectedly, sub2-1 extracts accumulate PS-like complexes. Moreover, deletion of MUD2 exacerbates the cs phenotype of sub2 alleles yet suppresses both the ts sub2-1 and the lethal Deltasub2 phenotypes. We propose that Sub2 functionally interacts with Mud2 both before and after PS formation. In the absence of Mud2, Sub2 function becomes dispensable.

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Figures

Figure 1

Figure 1

Sub2 is highly homologous to UAP56. Alignment of UAP56 and Sub2. Sequences were aligned with CLUSTALW and MacBoxShade. Dark gray boxes highlight identical amino acids, light gray boxes highlight similar residues. Thick black bars above the aligned sequences highlight the seven conserved motifs (I, Ia, II–VI) of helicase superfamily 2. Mutations present in sub2-1 (plain letters) and sub2-5 (circled letters) are indicated below the altered residue.

Figure 2

Figure 2

Biochemical phenotype of heat-inactivated sub2-1 mutant. (A) Time course of in vitro splicing activity of sub2-1 extracts. sub2-1 (lanes 1–8) or SUB2 (lanes 9–16) extracts were preincubated at 37°C (lanes 1–4 and 9–12) or on ice (lanes 5–8 and 13–16) for 15 min and then assayed for splicing activity with radiolabeled pre-actin RNA at 23°C for 0, 8, 16, and 30 min. (B) Spliceosome assembly phenotype of sub2-1 extract. Spliceosome assembly of 37°C preincubated samples from the 16-min timepoint shown in A (sub2-1, lane 3 and SUB2, lane 11) was assessed by native gel analysis (Cheng and Abelson 1986). Spliceosome complexes corresponding to H (heterogenous), PS (prespliceosome), and S (spliceosome) are indicated at left; lane 1, sub2-1; lane 2, SUB2. (C) Chase of splicing defect in sub2-1 extract. Heat-preincubated sub2-1 (lanes 1, 3–5) or U2-inactivated extracts (lanes 2,6) were incubated with radiolabeled preactin RNA in a two-step incubation schematized in the box above the gel. Extracts used in each stage of the incubation experiment are indicated above each lane. sub2-1Δ, 37°C preincubated extract; U2-, U2 oligo-inactivated extract; sub2-1+, mock- (ice) preincubated extract; U2+, mock oligo-inactivated extract.

Figure 3

Figure 3

Impact of Δ_mud2_ on the lethality of Δ_sub2_. Growth on 5-FOA-LEU media of Δ_sub2MUD2/pSUB-URA3 CEN_ strain (left side of plate) compared with growth of Δ_sub2_Δ_mud2/pSUB2-URA3 CEN_ strain (right side of plate) transformed with either pSUB2-LEU2 CEN (upper segment), pMUD2-LEU2 CEN (middle segment), or pLEU2 CEN (lower segment) plasmids.

Figure 4

Figure 4

Models for a Sub2-Mud2-dependent splicing pathway and a Sub2-Mud2-independent splicing pathway. Spliceosomal complexes formed during the early ATP-independent and ATP-dependent stages of splicing are diagrammed. CC1, commitment complex 1; CC2, commitment complex 2; PS, prespliceosome. Circled B, BBP; Circled M, Mud2. The U1 snRNP, U2 snRNP, and Sub2, Prp5, and Cus2 are denoted by labeled shapes. Hypothesized defects in Sub2 functions by different sub2 alleles are indicated at right in the Sub2–Mud2 dependent pathway model. Small arrows from Prp5 and Sub2 indicate activity on potential substrates in the presence of ATP. A possible scenario for the functions of both Prp5 and Sub2 during the ATP-dependent conversion of CC2 to PS complex is shown surrounded by brackets in the Sub2–Mud2 dependent pathway model. Potentially less stable binding of BBP with BPS in the absence of Mud2 is denoted by a dotted outline for the BBP symbol in the Sub2–Mud2 independent pathway.

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