The total disappearance of radioactivity within the clusters of interchromatin granules after a chase of 90 min strongly suggests that Us11 RNA and poly(A)+ RNA detected by in situ hybridization in these structures (3) are transiently stored at these sites before being transported to the cytoplasm. two monoclonal antibodies: mAb H5, which recognizes the hyperphosphorylated form of the carboxy-terminal domain (CTD) of the molecule, and mAb 7C2, which recognizes both its hyperphosphorylated and unphosphorylated forms. The two mAbs bind to the newly formed Us11 transcription factories and the clusters of interchromatin granules of transfected cells. In control cells, however, clusters of interchromatin granules were labeled with mAb H5 but not with mAB 7C2. Taken together, our data demonstrate the involvement of the clusters of interchromatin granules in the intranuclear migration of Us11 RNA in transient expression. They also suggest the occurrence of changes in the accessibility of the RNA polymerase II CTD upon expression of the Us11 gene after transfection by exposing some epitopes, otherwise masked in nontransfected cells. polytene chromosomes have been reported to be transcribed by such a mixture (55). Also Zeng et al. (56) demonstrated that RNA polymerase IIo and IIa can be photo-crosslinked EMD638683 S-Form to nascent RNA, which suggests that multiple phosphorylated forms of RNA polymerase II molecules are engaged in transcription. Further EMD638683 S-Form studies are needed to substantiate this possibility. Pulse-chase experiments undertaken to visualize the EMD638683 S-Form migration route followed by newly synthesized RNA revealed that the RNA molecules synthesized during the pulse with tritiated uridine leave the foci of intermingled Us11 RNP fibrils (i.e., the viral transcription factories) to accumulate for a limited period of time within the clusters of interchromatin granules that are not involved in transcription. The total disappearance of radioactivity within the clusters of interchromatin granules after a chase of 90 min strongly suggests that Us11 RNA and poly(A)+ RNA detected by in situ hybridization in these structures (3) are transiently stored at these sites before being transported to the cytoplasm. However, we cannot exclude that these polyadenylated viral molecules, or only a fraction of them, might be degraded within the clusters of interchromatin granules. The present data are in line with our previous results showing that RNA polymerase II transcripts enter the clusters of interchromatin granules before their transport to the cytoplasm and/or their decay (52). This is true for control cells but also for cells with a high level of RNA polymerase II transcription, such as cells lytically infected with either adenovirus (39) or HSV-1 (4), and for cells transiently expressing Us11 gene [(3), this report]. Although they are not transcription sites, clusters of interchromatin granules in control and transfected cells contain large amounts of hyperphosphorylated RNA polymerase II as revealed by their intense labeling following the use of mAb H5. This is in line with recent studies that demonstrate a functional interaction between the hyperphosphorylated CTD of the RNA polymerase II and splicing factors [(13,31) for review see (50)] even though this association can occur without transcription (28). This is also in good agreement with studies carried EMD638683 S-Form out at optical level, which reveal that the colocalization of the hyperphosphorylated form of the large subunit of RNA polymerase II with splicing components occurs in interconnected speckle domains (6,7,32) corresponding to the perichromatin fibrils and clusters of interchromatin granules and excluding the coiled bodies (13). Without totally excluding possible masking of the epitopes, we could not detect in the interchromatin granule-associated zones any labeling corresponding to the Ho form of the RNA polymerase II. The latter and splicing components, therefore, apparently only colocalize in one of the three accumulation sites of spliceosome components (51). However, some authors using different monoclonal antibodies only detect a diffuse fluorescence corresponding to perichromatin fibrils and no fluorescence over speckle domains corresponding to clusters of interchromatin granules (9,26). An unexpected and exciting result is the observation of an intense binding of mAb 7C2 to some clusters of interchromatin granules of transfected cells. An explanation for these conflicting results obtained by us and GYPA others would be that the different epitopes of the CTD of the RNA polymerase II are not equally accessible to the specific antibodies when the molecule is in the clusters of interchromatin granules. Therefore, the unusual labeling by the mAb 7C2 of clusters of interchromatin granules might reflect the unmasking of the corresponding epitope when cells are expressing the Us11 gene, although the reasons for such a better accessibility in cells transiently expressing Us11 gene, but not in cells lytically infected with HSV-1 (4), remain unknown. Because the mAb 7C2 recognizes both phosphorylated and unphosphorylated CTD of RNA polymerase.