A Sub-Element in PRE enhances nuclear export of intronless mRNAs by recruiting the TREX complex via ZC3H18.
We found that PRE drastically enhances the cytoplasmic accumulation of cDNA transcripts independent of any viral protein.Together, our data indicate that SEP1 enhances mRNA export by recruiting TREX via ZC3H18.The new mRNA export factors that we identified might be involved in cap- and splicing-dependent TREX recruitment to cellular mRNAs.
Affiliation: Shanghai Key Laboratory of Molecular Andrology, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
- Cell Nucleus/metabolism*
- Nucleocytoplasmic Transport Proteins/metabolism*
- RNA, Messenger/metabolism*
- RNA, Viral/metabolism*
- RNA-Binding Proteins/metabolism*/physiology
- Active Transport, Cell Nucleus
- DNA, Complementary/metabolism
- HeLa Cells
- Nuclear Proteins/metabolism
- RNA Caps/metabolism
- RNA Transport
- Ribonucleoproteins/isolation & purification
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Figure 4: ZC3H18 plays key roles in SEP1-dependent mRNA export. (A) HeLa cells were infected with lenti-viruses expressing the indicated shRNAs, and 96 h later, total RNAs were extracted followed by RT-PCR analyses to determine knockdown efficiencies. RT products of the control knockdown (10%, 30% and 100%) were used for PCR. (B) Cells described in (A) were transfected with the cG-SEP1 construct at 72 h post-infection, followed by FISH analysis 24 h later to detect the cG-SEP1 mRNA. The FISH probe was same as that used in Figure 1. (C) 12 pmol of 32P-labeled SEP1 and two control RNAs were incubated with 0, 12, 40 and 120 nmol of purified GST-ZC3H18-ZD (Z-ZD). The protein–RNA complexes were fractionated on a 1% agarose gel and visualized using autoradiography. The position of free RNAs and protein-bound RNAs are indicated. The left panel shows coomassie staining of the purified Z-ZD protein. (D) 32P-labeled SEP1 (12 pmol) were incubated with 200 nmol of purified GST or GST-Z-ZD protein followed by electrophoresis mobility shift assay similar to (C). The left panel shows coomassie staining of the purified GST and GST-Z-ZD protein. (E) Plasmids encoding the indicated Flag-tagged proteins were transfected into HeLa cells, and 24 h later, the cells were harvested and RNase A-treated lysates were used for Flag-IPs followed by western analyses using Thoc2 and Flag antibodies. (F) The pcDNA3-ZC3H18 plasmid was transfected into HeLa cells, and 24 h later, the cells were harvested and RNase A-treated lysates were used for IPs using Thoc2 and eIF4A3 antibodies followed by western analyses with indicated antibodies. (G) HeLa cells were treated with control or ZC3H18 siRNA. Forty-eight hours later, cG-SEP1, pSUPER-tRNA and VSV M plasmids were co-transfected into knockdown cells. Twenty-four hours later, cells were harvested and in vivo RNA immunoprecipitation was carried out using indicated antibodies. Immunoprecipitated RNAs were analyzed by RT-qPCR. The bars indicate the ratio of the IP efficiency of cG-SEP1 mRNA relative to that of tRNA. The relative IP efficiencies from control knockdown cells were considered as 100%. The bars and error bars represent average values and standard deviations from three independent experiments. Immunoprecipitated proteins were analyzed by westerns (left panel).
We next examined the roles of other SEP1-associating proteins in SEP1-mediated mRNA export. To this end, we carried out RNAi of these proteins, including Brr2, KIAA1429, Acinus, ARS2, hnRNPL, RBM15, RBMX, SAFB2 and ZC3H18. RT-PCR analyses showed that mRNA levels of the target genes were reduced at least 70% (Figure 4A). When RBMX, SAFB2 as well as previously reported TREX-interacting proteins were knocked down, the nucleocytoplasmic distribution of the cG-SEP1 mRNA was not significantly affected (Figure 4B). However, unexpectedly, knockdown of ZC3H18, which was not known to interact with TREX, significantly inhibited nuclear export of the cG-SEP1 mRNA. To confirm this result, we used three different siRNAs that efficiently silenced the expression of ZC3H18 (Supplementary Figure S3). Similar to shRNA-treated cells, the cG-SEP1 mRNA was largely nuclear in ZC3H18 siRNA-treated cells. Therefore, we concluded that ZC3H18 is required for SEP1-mediated mRNA export.