Cus2 enforces the first ATP-dependent step of splicing by binding to yeast SF3b1 through a UHM–ULM interaction

Abstract

Stable recognition of the intron branchpoint (BP) by the U2 snRNP to form the pre-spliceosome is the first ATP-dependent step of splicing. Genetic and biochemical data from yeast indicate that Cus2 aids U2 snRNA folding into the stem IIa conformation prior to pre-spliceosome formation. Cus2 must then be removed by an ATP-dependent function of Prp5 before assembly can progress. However, the location from which Cus2 is displaced and the nature of its binding to the U2 snRNP are unknown. Here, we show that Cus2 contains a conserved UHM (U2AF homology motif) that binds Hsh155, the yeast homolog of human SF3b1, through a conserved ULM (U2AF ligand motif). Mutations in either motif block binding and allow pre-spliceosome formation without ATP. A 2.0 Å resolution structure of the Hsh155 ULM in complex with the UHM of Tat-SF1, the human homolog of Cus2, and complementary binding assays show that the interaction is highly similar between yeast and humans. Furthermore, we show that Tat-SF1 can replace Cus2 function by enforcing ATP dependence of pre-spliceosome formation in yeast extracts. Cus2 is removed before pre-spliceosome formation, and both Cus2 and its Hsh155 ULM binding site are absent from available cryo-EM structure models. However, our data are consistent with the apparent location of the disordered Hsh155 ULM between the U2 stem-loop IIa and the HEAT repeats of Hsh155 that interact with Prp5. We propose a model in which Prp5 uses ATP to remove Cus2 from Hsh155 such that extended base-pairing between U2 snRNA and the intron BP can occur.

Structural basis of intron selection by U2 snRNP in the presence of covalent inhibitors

“…The transition from the BSL to the precursor U2/intron duplex is likely facilitated by the stepwise action of PRP5/DDX46, TAT-SF1, and SF3A2 (Supplementary Fig. 4 ) 23 , 24 , 62 . Studies in yeast indicate that Prp5p’s (human PRP5/DDX46) ATPase activity is required to displace Cus2p (human TAT-SF1) from SF3B1 62 .…”

“…4 ) 23 , 24 , 62 . Studies in yeast indicate that Prp5p’s (human PRP5/DDX46) ATPase activity is required to displace Cus2p (human TAT-SF1) from SF3B1 62 . Consequently, it has been proposed that Prp5-dependent destabilization of Cus2p results in BSL’s disruption, thereby enabling the extended pairing between U2 and the intron, during prespliceosome’s formation 62 .…”

Identification of herboxidiene features that mediate conformation-dependent SF3B1 interactions to inhibit splicing

“…Additionally, the protein HTATSF1 contacts both sides of the SF3B1 clamp, suggesting that it may serve as a brace for the open conformation. In addition to the structures, genetic and biochemical evidence show that both proteins are displaced prior to completion of A-complex assembly 16,33,34 . It is possible that either the positioning or removal of DDX46 and HTATSF1, and thus SF3B1 conformation, are dependent on ATP hydrolysis.…”

Impact of cancer-associated mutations in Hsh155/SF3b1 HEAT repeats 9-12 on pre-mRNA splicing in Saccharomyces cerevisiae

“…SF3b1, the largest subunit of SF3b subcomplex of the U2 snRNP, is known to play a primary role in intron recognition during spliceosome assembly [6][7][8][9][10][11]. The N-terminal domain (NTD) of human SF3b1 contains regions which interact with the U2AF, Tat-SF1, and SF3b14 splicing factors (Fig 1A) [6,[11][12][13]. The C-terminal domain of SF3b1 is composed of 20 HEAT domain repeats (HR) [6,11].…”