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Colloquium on Molecular Kinesis in Cellular Function and Plasticity
Fig. 3. Assembly and transport of the BR RNP particle and its relation to a number of BR RNA-associated proteins. The BR particle is assembled on the gene (left), passes through the nucleoplasm, unfolds, and translocates through the nuclear pore (middle). On the cytoplasmic side, the BR RNP fibril becomes engaged in protein synthesis and the polysomes anchor at the endoplasmic reticulum (right). The tripartite nuclear pore complex with its central channel is seen in black and its nuclear and cytoplasmic fibers are presented in pink. The BR gene with its five exons is displayed above the BR particle scheme, and the flow patterns of the BR RNA-associated proteins are outlined below. snRNP, small nuclear RNP. Modified from ref. 8; printed with permission from Elsevier Science.
As an example of a protein flow analysis, I have chosen the immunoelectron microscopic analysis of a cap-binding protein, CBP20 (27). CBP20 is known to bind to the 5' end of the transcript in a cap-binding complex (CBC) together with another protein, CBP80 (28). An antibody raised against the human CBP20 was applied in the study of the BR particle. Cryosections through salivary gland cells were prepared and challenged with the anti-CBP20 antibody and subsequently with a secondary antibody coupled to gold. As shown in Fig. 2, the gold particles are present in the proximal portions of the active BR gene (Fig. 2A) as well as in the distal portions (Fig. 2B and C). In the almost finished BR particles it can be seen that the gold is at the 5' end of the particle (Fig. 2B; cf. schematic drawing in Fig. 3)—i.e., the position of the cap structure. Furthermore, it was noted that there is no increase in binding during the course of transcription, suggesting that the protein is added to the cap structure almost immediately upon initiation of transcription. BR particles released into the nucleoplasm are also labeled with gold (Fig. 2E and F). Finally, during translocation through the nuclear pore, the leading 5' end of the BR particle is labeled and the gold can also be seen on the cytoplasmic side of the nuclear pore complex (Fig. 2G and H). Further out in the cytoplasm, there are no gold particles. We conclude that CBP20 is added cotranscriptionally and remains associated with the particle to and through the nuclear pore. On the cytoplasmic side, it is released from the particle and probably returns to the nucleus. These data are in good agreement with the observation that CBPs are shuttling proteins (28).
During the last couple of years a number of various RNA-binding proteins have been studied, and our results are summed up in Fig. 3. The flow patterns of the proteins are presented below the morphological description of the assembly and transport of the BR particle; the exon-intron organization of the BR gene is shown above. It is evident that the various proteins show quite different behavior during gene expression. Thus, not only the particle’s morphology but also its protein composition during the transport from the gene to the cytoplasm is drastically changed.
Spliceosome Assembly and Disassembly
As a marker for Spliceosome components we chose the snRNP proteins and used a monoclonal anti-snRNP antibody (Y12) to perform immunoelectron microscopy experiments (29). When the growing BR RNP products were studied in situ, it was noted that the snRNP proteins were present mainly in the proximal portion and only to a minor extent in the middle and distal portions of the active gene. Furthermore, nucleoplasmic BR particles, isolated, unfolded, and spread on a grid surface, showed labeling only at one end of the transcript, presumably the 3' end. Thus, the snRNPs do not associate along the whole pre-mRNP fibril but rather bind to the 5' and 3' ends—i.e., the regions containing introns. These results nicely agree with an earlier analysis carried out at the RNA level, showing that the three 5' end introns are spliced concomitantly with transcription in the promoter-proximal third of the gene, whereas the 3' intron is spliced mainly posttranscriptionally (30). We conclude that the observed discontinuous distribution of snRNP proteins along the pre-mRNP fibril implies that spliceosomes both assemble and disassemble rapidly on the RNP fibril.
Proteins Confined to the Nucleus
Two of the studied proteins, hrp45 (31) and hrp23 (32), proved to be confined to the cell nucleus. The hrp45 protein contains two amino-terminal RNP-consensus RNA-binding domains (RBDs) and a carboxyl-terminal region rich in arginine-serine dipeptide repeats (RS domain), an organization characteristic of