PTH mRNA colocalization with microtubules is dependent upon LC8. (a) Purified calf brain microtubule preparations, which contained MAPs, were assembled in the presence of paclitaxel and GTP and incubated with total RNA from rat thyroparathyroid tissue. Polymerized microtubules were sedimented, and the RNA, which was extracted from both the supernatants (S) and the pellets (P), was assayed for PTH mRNA by Northern blot analysis. PTH mRNA was present in the polymerized microtubule pellet and not in the supernatant. When increasing concentrations of LC8 were added to the RNA-microtubule preparation, there was a shift of PTH mRNA from the pellet to the supernatant, indicating that LC8 mediates the association of PTH mRNA to microtubules. (b) In vitro transcribed RNA for PTH or the CaSR 3′-UTR were incubated either without (–) or with (+) polymerized calf brain microtubule preparations that contained MAPs, and the samples were centrifuged to separate the microtubule pellet (P) from the supernatant (S). The RNA from both fractions was analyzed by Northern blot analysis (top 2 panels). When no microtubules were added, PTH mRNA was in the supernatant. With polymerized microtubules, PTH mRNA was present in the microtubule pellet, and the CaSR 3′-UTR RNA was present in the supernatant. Western blot analysis of samples of each fraction with antibody 74-1 to dynein IC 74 is shown in the bottom panel. IC was present in the microtubule pellet. (c) Cytosolic protein extracts derived from rat thyroparathyroid tissue were incubated without or with paclitaxel and GTP to polymerize endogenous microtubules, with or without ATP (5 mM) and NaCl (1 M), and the samples were centrifuged to separate the microtubule pellet (P) from the supernatant (S). The samples were then examined for PTH and ribosomal RNA using Northern blot analysis. In the absence of paclitaxel and GTP, both PTH and ribosomal RNA (28S and 18S) were present in the supernatant. When the microtubules were polymerized by the addition of paclitaxel and GTP, the PTH RNA was present in the microtubule pellet and the ribosomal RNA was still present in the supernatant. Addition of ATP and NaCl, or ATP alone, which specifically elute the dynein from the microtubules, displaced PTH mRNA to the supernatant. Addition of NaCl alone had no effect. This indicates that PTH mRNA associates with dynein in an ATP-dependent manner. Western blot analysis as in b showed that IC was present in the microtubule pellet and was eluted by the addition of ATP and NaCl.