CUS2, a yeast homolog of human Tat-SF1, rescues function of misfolded U2 through an unusual RNA recognition motif

Abstract

A screen for suppressors of a U2 snRNA mutation identified CUS2, an atypical member of the RNA recognition motif (RRM) family of RNA binding proteins. CUS2 protein is associated with U2 RNA in splicing extracts and interacts with PRP11, a subunit of the conserved splicing factor SF3a. Absence of CUS2 renders certain U2 RNA folding mutants lethal, arguing that a normal activity of CUS2 is to help refold U2 into a structure favorable for its binding to SF3b and SF3a prior to spliceosome assembly. Both CUS2 function in vivo and the in vitro RNA binding activity of CUS2 are disrupted by mutation of the first RRM, suggesting that rescue of misfolded U2 involves the direct binding of CUS2. Human Tat-SF1, reported to stimulate Tat-specific, transactivating region-dependent human immunodeficiency virus transcription in vitro, is structurally similar to CUS2. Anti-Tat-SF1 antibodies coimmunoprecipitate SF3a66 (SAP62), the human homolog of PRP11, suggesting that Tat-SF1 has a parallel function in splicing in human cells.

FIG. 1

(A) Secondary structure model of the 5′ end of yeast U2 snRNA. Mutations G53A, C62U, A61Δ, tmC (68ACACG72), tmC′ (80CGCUGU85), smC (tmC plus tmC′), ΔC (deletion of nucleotides 69 to 85), and SMB (58AAC60 plus 99UUG101) discussed in the text are indicated. (B) Suppression of G53A and C62U by different CUS2 alleles. Strain BJ81 was transformed with either wild-type (wt) U2 (top half of plate) or the cold-sensitive G53A mutation (bottom half) and the indicated CUS2 allele on a plasmid. Strains were streaked onto glucose-containing medium and incubated at 18°C for 5 days. (C) Enhancement of U2 mutations by the cus2::HIS3 disruption. Strain D2 (top half of plate) or D2CUS2KO (bottom half) was transformed with a plasmid carrying the indicated U2 allele and streaked on 5-fluoroorotic acid at 26°C to shuffle out the wild-type U2 gene on pCH1122. Growth was for 4 days.

FIG. 2

Splicing defects caused by U2-G53A are suppressed or enhanced by different CUS2 alleles. Yeast strains carrying the indicated alleles of U2 and CUS2 were grown at 30°C and shifted to 18°C for 4 h before extraction of RNA and primer extension with a labeled oligonucleotide complementary to U3 snRNA. Samples were normalized to contain the same amount of spliced U3 so that the level of unspliced U3 indicates splicing inhibition. Lane 1, U2-G53A with cus2::HIS3; lane 2, U2-G53A with CUS2⁺; lane 3, U2-G53A with CUS2-9; lane 4, U2-G53A with CUS2-25; lane 5, wild-type U2 with cus2::HIS3; lane 6, wild-type U2 with CUS2⁺.

FIG. 3

Coimmunoprecipitation of snRNA with tagged-CUS2. Extracts prepared from strains carrying untagged (lanes 1 to 4) or tagged (lanes 5 to 8) CUS2 protein were incubated with anti-HA antibody 12CA5 bound to PAS and washed with NET buffer containing 50 mM (lanes 1 and 5), 100 mM (lanes 2 and 6), 150 mM (lanes 3 and 7), and 200 mM (lanes 4 and 8) NaCl. RNA was extracted from the immunoprecipitate and used as the template for a reverse transcription reaction using a mixture of 5′-end-labeled oligonucleotides complementary to U1, U2, U5, and U6 snRNAs. The right-hand lane is a sample from a reverse transcription reaction using total yeast RNA as the template.

FIG. 4

CUS2 interacts with PRP11 in vivo. Plasmids designed to express fusion proteins of URA3 with the GAL4 DNA binding domain (left) or CUS2 with the GAL4 DNA binding domain (right) were cotransformed into yeast Y190 with plasmids designed to express fusion proteins of the GAL4 activation domain with the proteins indicated at the left. After selection for both plasmids, independent transformants were arrayed on the plate and assayed for β-galactosidase by an X-Gal overlay method. Strains that express β-galactosidase turn blue (shown as black in this photograph).

FIG. 5

CUS2 is structurally homologous to Tat-SF1. (A) Schematic comparison of CUS2 and Tat-SF1. Tat-SF1 is more than twice as large as CUS2, but most of this difference resides in the C-terminal acidic domain that is greatly shortened in CUS2. The unfilled regions share a charged nature but little sequence identity. (B) “Stitched” BLAST results generated by comparison of CUS2 with UAP2 and the first 420 residues of Tat-SF1. Each RRM is designated between angle brackets with RNP-1 and RNP-2 within each RRM indicated. Identical and similar amino acids are boxed in black and gray, respectively. The positions of the Y48D mutation, the C-terminal truncation (I∗), and CUS2-9 (L284F) and CUS2-25 (D282N) suppressor substitutions are shown.

FIG. 6

Coimmunoprecipitation of SF3a66 with anti-Tat-SF1 antibody; Western blot of protein preparations from HeLa nuclear extract probed with anti-SF3a66 MAb. NaCl washes (150 mM) of nuclear extract were incubated with PAS bound with preimmune serum (lane 3), anti-Tat-SF1 antibody (lane 4), or anti-SF3a60 antibody (lane 5). Samples (with Sepharose) were boiled and separated on an SDS–10% polyacrylamide gel. Lane 2 is total nuclear extract (NE) not purified via PAS. Positions of molecular mass standards (m) are indicated in kilodaltons.

FIG. 7

CUS2 rescue of U2 G53A requires the C-terminal tail where the suppressor mutations are located and an RNA binding activity supported by RRM1. (A) Enhancement of U2 G53A mutation by mutant CUS2 alleles. Strain RP01 was transformed with plasmids expressing U2 G53A and the indicated CUS2 mutant alleles. Strains were streaked onto glucose-containing medium and incubated at 18°C for 5 days. wt, wild type; term, terminus. (B) His₆-CUS2 binds RNA in vitro. U2 RNA (20 fmol) and CUS2 (wild type) or Y48D protein were mixed, incubated in 10 μl, and then run on a native polyacrylamide gel. Lanes 1 to 5, U2 plus CUS2; lanes 6 to 8, U2 plus Y48D. C and F, RNA-protein complex and free U2 RNA, respectively.

FIG. 8

Model for CUS2 action in preparing U2 snRNP for recruitment to the spliceosome. The U2 allele specificity of CUS2 suppression and enhancement suggests a close interaction with stem IIa, as shown. Newly synthesized U2, U2 snRNA emerging from the splicing pathway, or otherwise misfolded U2 interacts with CUS2 and is refolded. ySF3b and ySF3a are the S. cerevisiae protein complexes homologous to the human splicing factors SF3b and SF3a (39). Protein-protein interactions (35, 39, 43) (Fig. 4) are indicated by small gray boxes. The interaction between CUS2 and PRP11 may assist the recruitment of ySF3a to the U2 snRNP as indicated. The ySF3b subunit labeled “155?” is a yeast open reading frame homologous to a vertebrate SF3b subunit suggested to be SF3b155/SAP155 (58a). Once completely assembled, the U2 snRNP can bind to the commitment complex. Given the interaction between the S. pombe homologs of CUS2 and U2AF (47), CUS2 could have a role in U2 snRNP binding to the commitment complex through MUD2 (1).