A Sub-Element in PRE enhances nuclear export of intronless mRNAs by recruiting the TREX complex via ZC3H18.
Bottom Line: 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.Show MeSH
Related in: MedlinePlus
Mentions: To identify the cellular proteins that specifically associate with the SEP1 RNA, SEP1 and its reverse complement sequence (rSEP1) were fused to three MS2 binding sites and in vitro transcribed (Figure 2A). These RNAs were incubated with MS2-MBP, followed by incubation with HeLa nuclear extract under splicing conditions for 60 min. RNPs formed on the SEP1 and rSEP1 RNAs were isolated by gel filtration followed by affinity purification using amylose resin. Proteins from equivalent amounts of RNPs were separated by SDS-PAGE followed by silver staining. As shown in Figure 2B, multiple proteins were specifically present in the SEP1 RNP. These proteins were identified using mass spectrometry. Strikingly, most of these proteins were TREX components or putative TREX-interacting proteins that include Brr2, KIAA1429, Acinus, ARS2, RBM15 and hnRNPL (Figure 2B, labeled underline) (3,9,31). In addition, three putative RNA-binding proteins SAFB2, ZC3H18 and RBMX were also specifically detected in the SEP1 RNP. To examine whether the entire TREX complex associates with the SEP1 RNA, western analyses were carried out on SEP1 and rSEP1 RNPs using antibodies to TREX proteins. CBP80 was used as a loading control, and eIF4AIII, which is a component of the EJC, was used to determine the specificity of the purifications. As shown in Figure 2C, CBP80 was present equally in the SEP1 and rSEP1 RNP, whereas eIF4AIII was absent from both RNPs. Significantly, UAP56/URH49 was readily detected in the SEP1 RNP, but not in the rSEP1 RNP (Figure 2C). The amounts of Thoc2, Thoc5 and Aly present in the SEP1 RNP were also significantly more than those in the rSEP1 RNP. These results indicate that the entire TREX complex efficiently associated with the SEP1 RNA.
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.