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. 2002 Dec;22(23):8241-53.
doi: 10.1128/MCB.22.23.8241-8253.2002.

Stable mRNP formation and export require cotranscriptional recruitment of the mRNA export factors Yra1p and Sub2p by Hpr1p

Daniel Zenklusen  1 , Patrizia Vinciguerra, Jean-Christophe Wyss, Françoise Stutz
Affiliations

Stable mRNP formation and export require cotranscriptional recruitment of the mRNA export factors Yra1p and Sub2p by Hpr1p

Daniel Zenklusen et al. Mol Cell Biol. 2002 Dec.

Abstract

Yra1p/REF participates in mRNA export by recruiting the export receptor Mex67p to messenger ribonucleoprotein (mRNP) complexes. Yra1p also binds Sub2p, a DEAD box ATPase/RNA helicase implicated in splicing and required for mRNA export. We identified genetic and physical interactions between Yra1p, Sub2p, and Hpr1p, a protein involved in transcription elongation whose deletion leads to poly(A)(+) RNA accumulation in the nucleus. By chromatin immunoprecipitation (ChIP) experiments, we show that Hpr1p, Sub2p, and Yra1p become associated with active genes during transcription elongation and that Hpr1p is required for the efficient recruitment of Sub2p and Yra1p. The data indicate that transcription and export are functionally linked and that mRNA export defects may be due in part to inefficient loading of essential mRNA export factors on the growing mRNP. We also identified functional interactions between Yra1p and the exosome components Rrp45p and Rrp6p. We show that yra1, sub2, and Deltahpr1 mutants all present defects in mRNA accumulation and that deletion of RRP6 in yra1 mutants restores normal mRNA levels. The data support the hypothesis that an exosome-dependent surveillance mechanism targets improperly assembled mRNPs for degradation.

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Figures

FIG. 1.
FIG. 1.
(A) yra1ΔRBD is synthetically lethal with a mutation in HPR1. Strain sl57 (yra1ΔRBD hpr1-57 〈pCH1122 YRA1 URA ADE3〉) was transformed with empty vector or YRA1 and HPR1 gene constructs (pFS2525 and pFS2576). Transformants were streaked on 5-FOA plates to select against wild-type pCH1122-YRA1 and examine rescue of the synthetic phenotype. (B) Various yra1 mutants are synthetically lethal with Δhpr1. The YRA1 shuffle (FSY1026) or YRA1 shuffle Δhpr1 (FSY1655) strains were transformed with vector or plasmids encoding wild-type or mutant Yra1p (LEU2 CEN). Transformants were streaked on 5-FOA plates and incubated at 25 or 37°C for 4 days. (C) sub2 mutants are synthetically lethal with Δhpr1. The SUB2 shuffle (DLY23) or the SUB2 shuffle Δhpr1 (FSY1664) strains were transformed with empty vector or plasmids encoding wild-type or mutant Sub2p (LEU2 CEN). Transformants were analyzed as described for panel B.
FIG. 2.
FIG. 2.
(A) Hpr1p, Sub2p, and Yra1p physically interact in vivo. Total extracts were prepared from the indicated ProtA-tagged strains and purified on IgG beads. Copurifying proteins were eluted with high salt levels. Total extracts (input; lanes 1 to 6) or eluted proteins (eluates; lanes 7 to 12) were examined by Western blotting with antibodies against Yra1p, Yra2p, or Hpr1p. A nontagged (no tag) strain was used as a control for nonspecific binding to IgG beads. The antibodies against Hpr1p also recognize the ProtA-Sub2 and Hpr1-ProtA fusions present in input samples. (B) Sub2p interacts with GST-Hpr1p. GST alone or GST-Hpr1p (pFS2642) was coexpressed in E. coli with plasmid pFS2633 encoding nontagged Sub2p (lanes 4 and 6) or an empty vector (lanes 5 and 7). Total extracts were purified on glutathione beads, and the selected material was fractionated by SDS-PAGE (lanes 4 to 7) and Coomassie stained. A size marker (lane 1) and aliquots from total lysates expressing GST-Hpr1p plus Sub2p (lane 2) or GST-Hpr1p alone (lane 3) were run in parallel. (C) Sub2p interacts with Yra1p and Yra2p in E. coli. GST-Yra1p (pFS1853), GST-Yra2p (pFS2113), or GST alone was immobilized on beads and incubated with extracts from E. coli transformed with vector (lanes 4 to 6) or plasmid pFS2676 expressing a His6-Sub2p-tagged protein (lanes 7 to 9). Material associated with the beads (lanes 4 to 9) and aliquots of His6-Sub2p extract (lane 2) or His6-Sub2p purified on Ni-nitrilotriacetic acid beads (lane 3) were fractionated by SDS-PAGE followed by Coomassie staining.
FIG. 3.
FIG. 3.
Hpr1p, Sub2p, and Yra1p associate with the PMA1 gene during transcription elongation. (A) Diagram of the constitutively expressed PMA1 gene. Primer sets were designed to amplify 100- to 130-bp-long fragments corresponding to the PMA1 promoter (pro), to the 5′, middle (m), and 3′ coding regions, or to the 3′ UTR. An additional primer pair specific for a nontranscribed intergenic region (int) devoid of an ORF served as the internal background control. As shown in panels B, C, D, and G, a cross-linked and sonicated extract from wild-type cells grown at 25°C was immunoprecipitated with antibodies specific for TBP, the CTD of RNA polymerase II, the 80-kDa cap binding protein (Cbp80), and Yra1p, respectively. Coprecipitating DNA was amplified by quantitative real-time PCR with the primers indicated in panel A, as described in Materials and Methods. The relative abundance of the different PMA1 gene segments in each immunoprecipitate was expressed as a severalfold increase with respect to the nontranscribed intergenic region value, arbitrarily set to 1. The indicated values correspond to the means of at least two independent experiments. (E and F) Cross-linked and sonicated extracts were prepared from strains expressing Hpr1-ProtA (FSY1525) or ProtA-Sub2p (FSY1473) and affinity purified on IgG beads. Copurifying DNA fragments were quantified as described above.
FIG. 4.
FIG. 4.
The efficient recruitment of Sub2p and Yra1p to the transcribing PMA1 gene requires Hpr1p. (A) Strains expressing ProtA-Sub2p in the presence (FSY1473) or absence (FSY1656) of Hpr1p were grown at 25°C and analyzed as described for Fig. 3F. (B) W303 wild-type and Δhpr1 (FSY1624) strains were shifted to 37°C for 20 min prior to cross-linking. Sonicated extracts were immunoprecipitated with antibodies against Yra1p, and coprecipitating DNA fragments were analyzed as described for Fig. 3G.
FIG. 5.
FIG. 5.
GFP-yra1-8 is not recruited to the PMA1 gene, exhibits a poly(A)+ RNA export defect, and sequesters HSP104 transcripts within nuclear foci. (A) W303 wild-type and GFP-yra1-8 strains were shifted to 37°C for 20 min prior to cross-linking. Sonicated extracts were immunoprecipitated with antibodies against Yra1p, and coprecipitating DNA fragments were analyzed as described for Fig. 3G. (B) The GFP-yra1-8 mutant strain was grown at 25°C and shifted to 37°C for 1 h or to 42°C for 30 min. After fixation, the distributions of poly(A)+ RNA or HSP104 mRNAs were examined by in situ hybridization with Cy3-labeled oligo(dT)55 or HSP104-specific oligonucleotide probes, as indicated. DAPI (4′,6′-diamidino-2-phenylindole) staining was used to verify the nuclear localization of the HSP104 and poly(A)+ RNA signals.
FIG. 6.
FIG. 6.
Yrap1 genetically and physically interacts with components of the exosome. (A) yra1ΔRBD is synthetically lethal with a mutation in RRP45. Strain sl5 (yra1ΔRBD rrp45-1 〈pCH1122-YRA1 URA ADE3〉) was transformed with empty vector or YRA1 and RRP45 gene constructs (pFS2525 and pFS2575). Transformants were streaked on 5-FOA as described for Fig. 1A. (B) Mutations in YRA1 are synthetically lethal with Δrrp6. The YRA1 shuffle (FSY1026) or YRA1 shuffle Δrrp6 (FSY1621) strains were transformed with vector or plasmids encoding wild-type or mutant Yra1p (LEU2 CEN). Transformants were streaked on 5-FOA plates and analyzed as described for panel A. (C) Yra1p physically interacts with components of the exosome. Total extracts were prepared from a nontagged strain (no tag) or strains expressing Rrp45-ProtA (FSY1476) or Rrp6-ProtA (FSY1657) and purified on IgG beads (lanes 1 to 6). Copurifying proteins were eluted with high levels of salt. Total extracts (input; lanes 1 to 3) or eluted proteins (eluates; lanes 4 to 6) were examined by Western blotting with antibodies against Yra1p and Hpr1p.
FIG. 7.
FIG. 7.
yra1, sub2, and Δhpr1 mutants induce defects in mRNA accumulation. (A) Northern blot analysis of PMA1 mRNAs in wild-type or GFP-yra1-8, sub2-201, and Δhpr1 mutant strains grown at 25°C (lanes 1 to 4) or shifted to 37°C for 45 min (lanes 5 to 8). The PMA1 signals were normalized to 18S rRNA and expressed as a percentage of that of the wild type, as indicated (% wt). (B) Wild-type or GFP-yra1-8 (FSY1568), sub2-201 (FSY1613), and Δhpr1 (FSY1624) mutant strains, transformed with plasmids (2μm URA3) containing the β-galactosidase or YAT1 genes driven by a galactose-inducible promoter, were grown to mid-log phase in selective medium containing 2% lactate-2% glycerol-0.05% glucose and induced with 3% galactose-1% raffinose for 150 min at 25 or 37°C, as shown above the lanes. Total RNA was analyzed by primer extension with 32P-labeled oligonucleotides specific for β-galactosidase, YAT1, or the endogenous GAL1 gene transcripts, as indicated. A primer specific for U1 snRNA was added to the reactions as an internal control for loading. The primer-extended bands were quantified with an Instant Imager apparatus and normalized to U1 snRNA. The transcript levels in mutant strains were expressed as a percentage of that of the wild type, as indicated at the bottom of each gel (% wt). (C) The YRA1 (FSY1026) or YRA1 Δrrp6 (FSY1621) strain was shuffled, as described for Fig. 6B, with plasmid Lac111-YRA1 Gen (pFS2525; lanes 1 and 2) or Lac111-yra1-8 (pFS2328; lanes 3 and 4) and transformed with the galactose-inducible β-galactosidase reporter construct (pLGSD5). Transformants were grown, and RNAs were analyzed as for panel B, except that the galactose induction was performed at 30°C.
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