The splicing factor UAP56/HEL/Sub2p is essential for mRNA export [1-4]. It has been proposed [1,2] that UAP56/HEL/Sub2p interacts with the pre-mRNA during splicing and recruits the export factor Aly/REF/Yra1 (reviewed in [5]) to the spliced mRNA. However, UAP56/HEL/Sub2p also participates in the transport of intronless mRNAs, and thus its role in export is not necessarily coupled to splicing [2-4]. Here, we characterize the HEL protein of Chironomus tentans and we analyze in situ the interaction of HEL with a natural export substrate, the Balbiani ring pre-messenger ribonucleoprotein (BR pre-mRNP, reviewed in [6]). Using immunoelectron microscopy, we show that HEL binds to the BR pre-mRNP cotranscriptionally and that incorporation of HEL into the pre-mRNP is independent of the location of introns along the BR pre-mRNA. We also show that HEL accompanies the BR mRNP to the nuclear pore and is released from the BR mRNP during translocation to the cytoplasm. Aly/REF is also released from the BR mRNP during translocation but after dissociation of HEL. In summary, we have shown that binding of HEL to the BR pre-mRNA occurs independently of splicing, and we have established the point in the export pathway at which HEL and Aly/REF interact with the mRNP.