Assembly of an export-competent mRNP is needed for efficient release of the 3'-end processing complex after polyadenylation

Abstract

Before polyadenylated mRNA is exported from the nucleus, the 3'-end processing complex is removed by a poorly described mechanism. In this study, we asked whether factors involved in mRNP maturation and export are also required for disassembly of the cleavage and polyadenylation complex. An RNA immunoprecipitation assay monitoring the amount of the cleavage factor (CF) IA component Rna15p associated with poly(A)(+) RNA reveals defective removal of Rna15p in mutants of the nuclear export receptor Mex67p as well as other factors important for assembly of an export-competent mRNP. In contrast, Rna15p is not retained in mutants of export factors that function primarily on the cytoplasmic side of the nuclear pore. Consistent with a functional interaction between Mex67p and the 3'-end processing complex, a mex67 mutant accumulates unprocessed SSA4 transcripts and exhibits a severe growth defect when this mutation is combined with mutation of Rna15p or another CF IA subunit, Rna14p. RNAs that become processed in a mex67 mutant have longer poly(A) tails both in vivo and in vitro. This influence of Mex67p on 3'-end processing is conserved, as depletion of its human homolog, TAP/NXF1, triggers mRNA hyperadenylation. Our results indicate a function for nuclear mRNP assembly factors in releasing the 3'-end processing complex once polyadenylation is complete.

FIG. 1.

Increased retention of Rna15p on polyadenylated transcripts in certain export mutants. (A) Rna15p is retained on polyadenylated GAL7 transcript in hpr1Δ, tho2Δ, sub2-201, mex67-5, mex67-6, mtr2-26, GFP-yra1-8, and sac3Δ but not in npl3-3 or rat7-1 mutant cells. Cells were grown in liquid YPGal medium at 25°C and then shifted to 37°C for 1 h before formaldehyde cross-linking. Whole-cell lysates from isogenic WT or mutant strains were incubated with Rna15p antiserum to immunoprecipitate the cross-linked protein-RNA complex. The immunoprecipitated RNA was reverse transcribed with an oligo(dT) primer in the presence of reverse transcriptase (+). To control for DNA contamination, the reverse transcriptase was also omitted from some samples (−). Each lane shows the radioactive PCR signal with a primer set within the GAL7 coding region after reverse transcription. The lanes labeled “Input” show RT-PCR amplification of input RNA from each experiment. The results for each mutant and WT control are representative of at least three independent experiments, except for hpr1Δ, which was analyzed twice. (B) Rna15p is retained on polyadenylated SSA4 transcript in the mex67-5 mutant. The RNA immunoprecipitation assay was performed as in Fig. 1A, but a primer set upstream of the SSA4 poly(A) site was used in the PCR. Immunoprecipitation with preimmune serum (pre) is shown as a control. (C) Western blotting of cell lysates with antibody against the CF IA subunit Rna15p, the CPF subunit Pta1p, or the Rpb3p subunit of RNAPII. Extract was prepared from WT, mex67-5, and mex67-6 strains after shifting to 37°C for 1 h. (D) Pta1p is retained on polyadenylated RNA in the mex67-6 mutant. The RNA immunoprecipitation assay was performed as for panel B but with Pta1p antiserum instead of Rna15p antiserum. Pta1p levels on polyadenylated RNAs were increased for both GAL7 (top) and SSA4 (bottom) transcripts. Immunoprecipitation with preimmune serum (lanes marked “pre”) is shown as a control.

FIG. 2.

Genetic interactions and shared in vivo phenotypes of mex67 and CF IA mutants. (A) Pairing of mex67-6 with rna14-3 (left) or rna15-2 (right) causes synthetic lethality at 30°C. Cells from WT, mex67-6, rna15-2, rna14-3, mex67-6/rna15-2, and mex67-6/rna14-3 strains were grown in liquid YPD medium at 24°C, after which 10-fold serial dilutions were spotted on YPD plates and incubated for 2 to 3 days at the indicated temperatures. (B) Schematic of the RNase H/Northern blotting strategy to examine SSA4 transcripts. Treatment with oligonucleotide SC3 and RNase H in the 3′ end of the SSA4 open reading frame shortens transcripts and increases resolution. Additional treatment with oligo(dT) shortens the poly(A) tails, resulting in a 218-nucleotide product if all poly(A) is removed. Treatment with oligonucleotide SC128 at the beginning of the downstream RTT105 gene trims the longest read-through RNAs to 387 nucleotides. The position of the probe used for Northern blotting is also indicated. (C) Northern blot analysis of SSA4 transcripts from WT, rna15-2, mex67-5, and mex67-6 cells grown at 25°C and then shifted to 37°C for 15 or 60 min. RNAs were treated with the indicated oligonucleotides and RNase H. The blot was hybridized with a probe complementary to sequences upstream of the SSA4 poly(A) site. The positions of hyperadenylated and normal-length poly(A)⁺ RNAs, poly(A)-depleted RNA (SC3/dT), and transcripts that extend beyond the SSA4 poly(A) site (read-through RNAs) and their derivatives after treatment with oligonucleotide SC128 (SC3/SC128) are indicated.

FIG. 3.

Detection of SSA4 transcripts in fixed cells by RNA FISH analysis. WT, mex67-5, mex67-6, rna15-2, and rna14-3 cells were grown at 25°C and then shifted to 37°C for 15 or 60 min. The positions of the FISH probes for detection of SSA4 RNAs containing sequence upstream (KD199/KD200) or downstream (THJ366/THJ367) of the poly(A) site are indicated in the diagram at the top. Cells were costained with DAPI (4′,6-diamidino-2-phenylindole) and overlaid as indicated.

FIG. 4.

Analysis of in vitro coupled cleavage and polyadenylation reactions in mex67-5 extract. (A) Extract from mex67-5 cells processes RNA efficiently but gives longer poly(A) tails than that of WT extract. Cells were grown in liquid YPD at 25°C and then shifted to 37°C for 1 h before extracts were prepared. Processing reactions were performed by incubation with a radioactively labeled precursor RNA containing the GAL7 poly(A) site (Pre) at 30°C for the indicated times. (B) Tails are not shortened in mex67-5 extract if processing is performed at 37°C instead of 30°C. The slight decrease in processing efficiency in the mex67-5 extract at 37°C in this experiment is not reproducible. (C) The PAN nuclease trims tails synthesized in vitro. Extracts were prepared from WT and mex67-5 cells expressing tandem affinity purification-tagged Pan2p, and PAN was removed by incubation with IgG beads. (Left) Western blotting analysis of Pan2 in mock-depleted (+) or PAN-depleted (−) extract. (Right) Processing in extracts with or without PAN. Reactions were performed for 120 min at 30°C. (D) The mex67-5 mutation does not cause an increased association of Rna15p with RNAs that are polyadenylated in vitro. Processing reaction mixtures were incubated at 30°C for 30 min, and RNAs were immunoprecipitated using Rna15p antibody (+) or preimmune serum (−).

FIG. 5.

The mRNA hyperadenylation phenotype is conserved in human cells depleted for NXF1/TAP. (A) Northern blotting analysis was done with total RNA isolated from HEK293T cells either mock-transfected (lane 1) or transfected with a plasmid expressing enhanced green fluorescent protein (EGFP) mRNA (lanes 2 and 3). In addition, cells were treated with either control (lanes 1 and 2) or NXF1/TAP (lane 3) siRNA. The blots were hybridized with probes directed against EGFP RNA or β-actin (control). Western blotting analysis used an antibody specific for NXF1/TAP or an antibody against hnRNP C as a loading control. Intervening lanes not relevant to this study were removed. (B) EGFP RNAs are hyperadenylated upon NXF1/TAP downregulation. RNase H/Northern blotting analysis of the 3′ ends of the EGFP transcripts from panel A. Positions of oligonucleotides used for the RNase H digestions (dT₁₈ and r-t) are indicated on top of the panel. Lanes 1 and 4, no oligonucleotide; lanes 2 and 5, oligo(dT)₁₈; lanes 3 and 6, oligonucleotide complementary to sequence downstream of the poly(A) site. (C) MS2 RNA FISH of U2OS cells stably expressing HIV-1 transcripts containing 24 MS2 sites. Cells were treated with either control siRNAs or siRNAs targeting TAP/NXF1. Cells were costained with DAPI as indicated. Arrows indicate nuclear dots.

FIG. 6.

Model depicting the assembly of an export-competent mRNP. As is evident from recent reviews (13, 43, 55, 67), the process of mRNA export is more complex than portrayed here, and for simplicity, only factors examined in this study are indicated. The THO complex is thought to help recruit both Sub2p and Mex67p to the transcribed gene. The 3′-end processing factors, CPF and CF IA, interact with the phosphorylated CTD of the elongating RNAP II. CF IA recruits Yra1p to the elongation complex, and perhaps concurrently with polyadenylation, a reorganization occurs in which Yra1p is transferred to Sub2p and then to the Mex67p/Mtr2p complex. Npl3p is also recruited cotranscriptionally and exits the nucleus as part of an mRNP containing Mex67p/Mtr2p and other export factors. Sac3p, as part of the TREX2 complex, interacts with Mex67p and the nuclear pore. After passage through the pore, Dbp5p, Gle1p, and Rat7p are important for remodeling the mRNP on the cytoplasmic side. In our study, mutation of the factors shown in light blue and green caused retention of the Rna15p subunit of CF IA on polyadenylated mRNA, while those indicated in brown did not.