The TREX complex (TREX) plays key roles in nuclear export of mRNAs. However, little is known about its transcriptome-wide binding targets. We used individual cross-linking and immunoprecipitation (iCLIP) to identify the binding sites of ALYREF, an mRNA export adaptor in TREX, in human cells. Consistent with previous in vitro studies, ALYREF binds to a region near the 5′ end of the mRNA in a CBP80-dependent manner. Unexpectedly, we identified PABPN1-dependent ALYREF binding near the 3′ end of the mRNA. Furthermore, the 3′ processing factor CstF64 directly interacts with ALYREF and is required for the overall binding of ALYREF on the mRNA. In addition, we found that numerous middle exons harbor ALYREF binding sites and identified ALYREF-binding motifs that promote nuclear export of intronless mRNAs. Together, our study defines enrichment of ALYREF binding sites at the 5′ and the 3′ regions of the mRNA in vivo, identifies export-promoting ALYREF-binding motifs, and reveals CstF64- and PABPN1-mediated coupling of mRNA nuclear export to 3′ processing.