Fig. 7
Ar-Larp retrogradely transports tRNAs from the cytoplasm to the nucleus, resulting in cell cycle arrest. a The tRNA distribution in HeLa overexpressing GFP-Ar-Larp (white arrows indicate typical cells) or GFP alone. tRNAs were detected by FISH using oligonucleotide probes complementary to tRNA sequences. Green, GFP-fused Ar-Larp and GFP alone. Red, tRNAs detected with a rhodamine-conjugated anti-digoxigenin (anti-DIG) antibody. Blue, nuclei counterstained with DAPI. Scale bar = 50 μm. b Detection of Ar-Larp-tRNA complexes by co-immunoprecipitation using an anti-Ar-Larp antibody and probes specific to the tRNAs. Northern blot analysis of tRNAs from the pellets (co-IP) or total extract fractions (Input) of HeLa cells overexpressing GFP-Ar-Larp. Lane 1, HeLa cells overexpressing GFP-Ar-Larp; lane 2, control cells overexpressing GFP alone. c Analysis of the retrograde transport of tRNA from the cytoplasm to the nuclei in Ar-Larp-overexpressing HeLa cells. HeLa cells were treated with ActD (5 μM) to block tRNA synthesis for 2 h after Ar-Larp was expressed for 8 h. The nuclear and cytoplasmic distributions of tRNA in HeLa cells were then detected by FISH using oligonucleotide probes (tRNALeu) complementary to the tRNA sequences at 18, 24, and 36 h after Ar-Larp expression (white arrow indicates typical cells). Cells expressing GFP alone and two Ar-Larp mutants were used as controls. Mutant 1 lacked the domain between RRM1 and RRM2, and therefore was not retained in the nucleus. Mutant 2 could accumulate and be retained in the nucleus, but lacked the RNA-binding domain. Green, GFP-fused Ar-Larp and GFP alone. Red, tRNAs detected with a rhodamine-conjugated anti-DIG antibody. Blue, nuclei counterstained with DAPI. Scale bar = 50 μm. d The distribution of tRNAs in cells at each stage during diapause formation (the stages are indicated in Fig. 1a and in pseudodiapause cysts induced by Ar-Larp RNA interference. Dashed line circles indicate cells in each stage and pseudodiapause cysts. Scale bar = 50 μm