cytoplasmic side, and protein synthesis is initiated (Fig. 3) (19). The translocation process has been studied in detail, and several discrete steps have been elucidated: binding of the BR RNP particle to the nucleoplasmic fibers of the nuclear pore complex, docking of the particle in front of the central channel of the pore complex, unrolling of the ribbon and translocation of the RNP complex with its 5' end in the lead through the channel, exit of the unfolded RNP fibril into the cytoplasm, and formation of a polysome just outside the pore (8). Thus, the translocation of the BR RNP particle appears to be an ordered process with several well-defined stages. Furthermore, the spectacular conformational changes of the BR particle indicate that the process is quite dynamic, which is further supported by the observation that during translocation the BR particle loses proteins while others are presumably added (see below).
Evidently proteins become associated with the RNA concomitant with transcription. In fact, the proteins seem to bind to the growing RNA molecule in the immediate vicinity of the RNA polymerase. Several questions are close at hand: What proteins are associated with the RNP particle? Are the proteins simply packaging proteins, or do they also play other functional roles? It has been estimated that there are 400–500 average-sized protein molecules in a BR particle (20).
It is well established that pre-mRNA is associated with many different proteins, usually designated hnRNP proteins (heterogeneous nuclear RNP proteins) (21). For example, in humans there are 30 major hnRNP proteins and a large number of minor ones (22). As a rule, the proteins can bind to a broad range of different sequences, some with higher affinity, others with lower affinity (21). Thus, as the hnRNP proteins show sequence preference in their interaction with RNA, they are likely to be nonrandomly bound to pre-mRNA. It has been directly shown in reconstitution experiments that each different RNA species is associated with a unique combination of hnRNP proteins (23). These studies were performed under conditions for binding sites and, therefore, resemble the in vivo situation in the cell nucleus. Furthermore, the hnRNP protein compositions at various puffs
on polytene chromosomes in Drosophila (24) and Chironomus (25) differ quantitatively but also qualitatively, suggesting that each type of transcript binds a specific subset of hnRNP proteins. It is, therefore, an interesting possibility that the hnRNP proteins are not only unspecific RNA packaging proteins but also capable of exerting specific, transcript-related functions. To test such a hypothesis, it is attractive to study the protein set-up of individual specific transcripts and relate the individual proteins to the fate of the transcript.
It would have been most satisfactory if the proteins in the BR particles could have been studied by a direct approach. It is true that the BR particles can be isolated as a 300S fraction (20), but the quantities are not sufficient to allow a direct biochemical characterization. Instead, we adopted an indirect approach devised by Dreyfuss and coworkers (26). Nuclear RNA-binding proteins were isolated from C. tentans cultured cells by single-stranded DNA-Sepharose affinity chromatography and were used to raise monoclonal antibodies in mice. A collection of such antibodies was obtained (25). Antibodies that showed high specificity in Western blot experiments and bound to the BRs in immunocytochemical experiments were selected for further experiments. The antibodies were used to characterize the corresponding proteins by cDNA cloning and to study the fate of the proteins during the assembly and transport of the BR particle by using immunocytochemical and immunoelectron microscopy experiments.