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. 2015 Nov 9;211(3):569–586. doi: 10.1083/jcb.201506048

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© 2015 Schreiber et al.

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Figure 3. — TRIM3 is present in mRNP particles but is not essential for mRNP particle trafficking. (A) TRIM3 interacts with PURA in an RNA-dependent manner. TRIM3 was immunoprecipitated from hippocampal synapse-enriched fractions and samples were immunoblotted (IB) and stained for TRIM3 and PURA. PURA was detected in RNase inhibitor–treated samples, but not in RNase-treated samples. Samples prepared from Trim3^−/− mice served as negative control for the immunoprecipitation. (B) TRIM3 and PURA do not interact when expressed in HEK293 cells. HEK293 cells were cotransfected with full-length TRIM3 and GFP-PURA. TRIM3 and PURA were immunoprecipitated from lysates, immunoblotted and stained for TRIM3 and GFP (PURA). PURA did not coimmunoprecipitate with TRIM3, and TRIM3 did not coimmunoprecipiate with PURA. (C and D) PURA levels are not altered in Trim3^−/− mice. Hippocampal synapse-enriched fractions were prepared from wild-type and Trim3^−/− mice under control conditions (home cage) and 2 h after contextual fear conditioning (shock). Samples were immunoblotted and stained for PURA. Normalized PURA levels did not differ between conditions (means ± SEM, n = 4 per genotype). (E) TRIM3 does not alter PURA levels in HEK293 cells. HEK293 cells were cotransfected with PURA and TRIM3, PURA and ΔRBCC-TRIM3, or PURA alone. No differences in PURA levels were observed at any time point after transfection. (F) TRIM3 is not essential for PURA trafficking. Example time-lapse images of a mobile GFP-PURA cluster (red arrow) over a timespan of 90 s are shown. Bars, 2 µm. (G and H) Trim3^−/− neurons and wild-type control neurons expressed equal amounts of PURA clusters (G) and had equal fractions of mobile clusters (H). (I) Trim3^−/− neurons and wild-type control neurons expressed equal amounts of short-distance and long-distance clusters. (J and K) Long-distance PURA clusters show slightly increased kinetics in Trim3^−/− neurons. The total distance moved (J) did not change, but the maximum velocity (K) and the maximum distance reached from origin (L) were significantly increased in Trim3^−/− neurons (means ± SEM, two-tailed t test, *, P < 0.05, n = 27/40 clusters per genotype). (M–O) Short-distance PURA clusters show unaltered kinetics in Trim3^−/− neurons. The total distance moved (M), the maximum velocity (N), and the maximum distance reached from origin (O) did not differ significantly in Trim3^−/− neurons compared with wild-type neurons (means ± SEM, n = 76/80 clusters per genotype).