Limits...
A Sub-Element in PRE enhances nuclear export of intronless mRNAs by recruiting the TREX complex via ZC3H18.

Chi B, Wang K, Du Y, Gui B, Chang X, Wang L, Fan J, Chen S, Wu X, Li G, Cheng H - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

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

Purification and identification of SEP1-interacting proteins. (A) Schematic of the RNA substrates that were used for RNP purifications. Each stem loop indicates an MS2 binding site. (B and C) In vitro transcribed, 32P-labeled SEP1 and its reverse complement sequence (rSEP1) containing three MS2 binding sites were incubated with MS2-MBP followed by incubation with HeLa nuclear extract for 1 h. The mixtures were separated by gel filtration and purified using amylose resin. The purified RNPs were then separated by SDS-PAGE followed by silver-staining (B) and western analyses (C) using the indicated antibodies. RNAs that were present in the purified RNPs were visualized using autoradiography and are shown in the left panel in (B). The proteins present in the bands that are indicated in the right panel in (B) were identified using mass spectrometry. The UAP/URH antibody detects both UAP56 and URH49. (D) In vitro transcribed, 32P-labeled SEP1 and rSEP1 were incubated in HeLa nuclear extract under splicing conditions for 1 h followed by RNA IPs using the indicated antibodies. The control was an antibody to fSAP130. One-fourth of the input was loaded. The graph shows the quantification of IP efficiencies. U/U indicates UAP56/URH49. The bars indicate the average ratios of IP efficiencies for rSEP1 relative to those for SEP1 for three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4066777&req=5

Figure 2: Purification and identification of SEP1-interacting proteins. (A) Schematic of the RNA substrates that were used for RNP purifications. Each stem loop indicates an MS2 binding site. (B and C) In vitro transcribed, 32P-labeled SEP1 and its reverse complement sequence (rSEP1) containing three MS2 binding sites were incubated with MS2-MBP followed by incubation with HeLa nuclear extract for 1 h. The mixtures were separated by gel filtration and purified using amylose resin. The purified RNPs were then separated by SDS-PAGE followed by silver-staining (B) and western analyses (C) using the indicated antibodies. RNAs that were present in the purified RNPs were visualized using autoradiography and are shown in the left panel in (B). The proteins present in the bands that are indicated in the right panel in (B) were identified using mass spectrometry. The UAP/URH antibody detects both UAP56 and URH49. (D) In vitro transcribed, 32P-labeled SEP1 and rSEP1 were incubated in HeLa nuclear extract under splicing conditions for 1 h followed by RNA IPs using the indicated antibodies. The control was an antibody to fSAP130. One-fourth of the input was loaded. The graph shows the quantification of IP efficiencies. U/U indicates UAP56/URH49. The bars indicate the average ratios of IP efficiencies for rSEP1 relative to those for SEP1 for three independent experiments.

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.


A Sub-Element in PRE enhances nuclear export of intronless mRNAs by recruiting the TREX complex via ZC3H18.

Chi B, Wang K, Du Y, Gui B, Chang X, Wang L, Fan J, Chen S, Wu X, Li G, Cheng H - Nucleic Acids Res. (2014)

Purification and identification of SEP1-interacting proteins. (A) Schematic of the RNA substrates that were used for RNP purifications. Each stem loop indicates an MS2 binding site. (B and C) In vitro transcribed, 32P-labeled SEP1 and its reverse complement sequence (rSEP1) containing three MS2 binding sites were incubated with MS2-MBP followed by incubation with HeLa nuclear extract for 1 h. The mixtures were separated by gel filtration and purified using amylose resin. The purified RNPs were then separated by SDS-PAGE followed by silver-staining (B) and western analyses (C) using the indicated antibodies. RNAs that were present in the purified RNPs were visualized using autoradiography and are shown in the left panel in (B). The proteins present in the bands that are indicated in the right panel in (B) were identified using mass spectrometry. The UAP/URH antibody detects both UAP56 and URH49. (D) In vitro transcribed, 32P-labeled SEP1 and rSEP1 were incubated in HeLa nuclear extract under splicing conditions for 1 h followed by RNA IPs using the indicated antibodies. The control was an antibody to fSAP130. One-fourth of the input was loaded. The graph shows the quantification of IP efficiencies. U/U indicates UAP56/URH49. The bars indicate the average ratios of IP efficiencies for rSEP1 relative to those for SEP1 for three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4066777&req=5

Figure 2: Purification and identification of SEP1-interacting proteins. (A) Schematic of the RNA substrates that were used for RNP purifications. Each stem loop indicates an MS2 binding site. (B and C) In vitro transcribed, 32P-labeled SEP1 and its reverse complement sequence (rSEP1) containing three MS2 binding sites were incubated with MS2-MBP followed by incubation with HeLa nuclear extract for 1 h. The mixtures were separated by gel filtration and purified using amylose resin. The purified RNPs were then separated by SDS-PAGE followed by silver-staining (B) and western analyses (C) using the indicated antibodies. RNAs that were present in the purified RNPs were visualized using autoradiography and are shown in the left panel in (B). The proteins present in the bands that are indicated in the right panel in (B) were identified using mass spectrometry. The UAP/URH antibody detects both UAP56 and URH49. (D) In vitro transcribed, 32P-labeled SEP1 and rSEP1 were incubated in HeLa nuclear extract under splicing conditions for 1 h followed by RNA IPs using the indicated antibodies. The control was an antibody to fSAP130. One-fourth of the input was loaded. The graph shows the quantification of IP efficiencies. U/U indicates UAP56/URH49. The bars indicate the average ratios of IP efficiencies for rSEP1 relative to those for SEP1 for three independent experiments.
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.

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.

View Article: PubMed Central - PubMed

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