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

Jason Talkish,Clara L Kielkopf,Justin R Leach,Manuel Ares,Haller Igel,Oarteze Hunter,Steven W Horner,Nazish N Jeffery,Jermaine L Jenkins

doi:10.1261/rna.070649.119

Jason Talkish, Clara L Kielkopf + Show 7 more

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https://doi.org/10.1261/rna.070649.119

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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.

Highlights

Pre-messenger RNA splicing is a dynamic process catalyzed by the ribonucleoprotein complex known as the spliceosome
Primary sequence comparisons identified yeast Cus2 and mammalian Tat-SF1 as U2AF homology motif (UHM)-containing proteins (Kielkopf et al 2001, 2004). Both proteins contain sequences indicating an RNA recognition motif (RRM)-like fold, and characteristic residues known to mediate contact with U2AF ligand motif (ULM) are found in appropriate positions (Fig. 1A,C)
We show that Cus2 and its human homolog Tat-SF1 bind the core U2 small nuclear ribonucleoproteins (snRNP) protein Hsh155 through conserved UHM and ULM domain surfaces (Figs. 1–4) in vivo (Fig. 1E) and in vitro with high affinity (Figs. 1F, 2)

Summary

Introduction

Pre-messenger RNA (pre-mRNA) splicing is a dynamic process catalyzed by the ribonucleoprotein complex known as the spliceosome. Subunits of the spliceosome known as small nuclear ribonucleoproteins (snRNP)s, each named for the snRNA they carry, assemble in an ordered pathway of events. Many of these events are regulated in an ATP-dependent manner by DExD/H proteins dedicated to specific steps in splicing (Chang et al 2013; Liu and Cheng 2015). Upon ATP binding or hydrolysis, DExD/H proteins alter the binding of other components of the splicing machinery, promoting conformational changes within the RNA and protein components These changes lead to release of some proteins and recruitment of others to the spliceosome, ensuring the fidelity and directionality of spliceosome assembly and catalysis (Koodathingal and Staley 2013). This challenge is more daunting given that several key spliceosomal components are part of early complexes that await structure determination or are not RNA (2019) 25:1020–1037; Published by Cold Spring Harbor Laboratory Press for the RNA Society

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