Cajal bodies (CBs) are evolutionarily conserved nuclear organelles that contain many factors involved in the transcription and processing of RNA. in CBs fluorescence recovery was markedly slower than in the nucleoplasm and there were at least three kinetic components. The recovery rate within CBs was independent of bleach spot diameter and could not be attributed to high CB viscosity or density. We propose that binding to other molecules and possibly assembly into larger complexes are the rate-limiting steps for FRAP of U7 coilin and TBP inside CBs. GV endogenous U7 snRNA is localized primarily in the CBs (Wu and Gall 1993 Wu et al. 1996 Here we show that fluorescein-U7 that has been injected into the oocyte Rabbit Polyclonal to Doublecortin. cytoplasm like endogenous U7 can be immunoprecipitated from GV extracts by mAb Y12 (Fig. 1 A). Y12 recognizes a symmetrical dimethylarginine epitope found on several Sm proteins and on coilin (Brahms et al. 2001 Hebert et al. 2002 Fluorescein-U7 that has been recovered from I-BET-762 injected oocytes migrates at the expected molecular weight on a Northern blot and shows little sign of degradation after prolonged incubation inside cells (Fig. 1 B). Finally when fluorescein-U7 snRNA is injected into the oocyte cytoplasm and GVs are subsequently isolated for cytological analysis one sees brightly fluorescent CBs against a low level of nucleoplasmic staining (Fig. 1 C). In contrast when fluorescein-U7 is injected directly into oil-isolated GVs fluorescence is barely detectable in CBs even after overnight incubation (Fig. 1 C). These results suggest that under standard conditions of cytoplasmic injection fluorescein-U7 transcripts associate properly with Sm proteins in the cytoplasm before they enter the nucleus where they are stable. Fluorescein-U7 snRNPs are then concentrated in CBs in a pattern that is indistinguishable from that of the endogenous snRNP. Figure 1. When injected into the cytoplasm fluorescein-U7 snRNA is assembled into a stable snRNP complex that is targeted to the nucleoplasm and to CBs. (A) Autoradiograph of 32P-labeled snRNAs I-BET-762 that were injected into the cytoplasm recovered the next day from … Coilin was originally identified on I-BET-762 the basis of autoimmune sera that specifically stained CBs (then called coiled bodies; Andrade et al. 1991 Ra?ka et al. 1991 Earlier studies suggested that coilin might be involved in the transport of snRNPs from the cytoplasm to CBs I-BET-762 (Bauer and Gall 1997 Bellini and Gall 1998 This view is now strongly supported by evidence from coilin knockout mice (Tucker et al. 2001 and biochemical data showing an association between coilin and SMN the spinal muscular atrophy gene (Hebert et al. 2001 2002 In our experiments in vitro-transcribed GFP-coilin mRNA was efficiently translated when injected into the oocyte cytoplasm. The fusion protein isolated from injected cells after overnight incubation migrated at the anticipated molecular weight on Western blots and reacted with antibodies against both coilin and GFP (Fig. 2 A). Consistent with previous observations (Wu et al. 1994 Handwerger et al. 2002 the fusion protein is readily targeted to CBs as shown by the appearance of fluorescent CBs within several hours after injection of the mRNA (Fig. 2 B). These data indicate that the fusion protein is stable in the GV over the time course of our experiments and that GFP does not disrupt the targeting of coilin to CBs. Figure 2. GFP-coilin is efficiently translated by oocytes and is targeted to CBs. (A) Western blot of GV proteins from control oocytes I-BET-762 (lane 1) or oocytes injected with GFP-coilin mRNA (lanes 2 and 3) stained with an antibody against … Human GFP-TBP mRNA was also robustly translated by oocytes. The fusion protein migrated at the anticipated molecular weight on Western blots and was recognized by antibodies against GFP and human TBP (Fig. 3 A). Although the anti-TBP antibody did not react convincingly with endogenous TBP on Western blots it stained CBs more brightly than other structures in control GV spread preparations (Fig. 3 B). In spreads of GVs from injected oocytes GFP fluorescence was also detected primarily in CBs (Fig. 3 B). Finally the axes and lateral loops of the lampbrush chromosomes appeared fully.