Regulated alternative polyadenylation is an important feature of gene expression, but how gene transcription rate affects this process remains to be investigated. polo is a cell-cycle gene that uses two poly(A) signals in the 3 0 untranslated region (UTR) to produce alternative messenger RNAs that differ in their 3 0 UTR length. Using a mutant Drosophila strain that has a lower transcriptional elongation rate, we show that transcription kinetics can determine alternative poly(A) site selection. The physiological consequences of incorrect polo poly(A) site choice are of vital importance; transgenic flies lacking the distal poly(A) signal cannot produce the longer transcript and die at the pupa stage due to a failure in the proliferation of the precursor cells of the abdomen, the histoblasts. This is due to the low translation efficiency of the shorter transcript produced by proximal poly(A) site usage. Our results show that correct polo poly(A) site selection functions to provide the correct levels of protein expression necessary for histoblast proliferation, and that the kinetics of RNA polymerase II have an important role in the mechanism of alternative polyadenylation.

Background: PEX5 binds newly synthesized peroxisomal proteins in the cytosol and releases them in the organelle matrix. Results: PEX5 binds monomeric catalase and releases it in the presence of PEX14. Conclusion: PEX14 participates in the cargo release step. Significance: Knowing how PEX5 interacts with cargo proteins and which factors disrupt this interaction are crucial for understanding this protein sorting pathway.

Background:The mammalian deubiquitinase that hydrolyzes the ubiquitin-PEX5 thioester conjugate was unknown. Results: USP9X was found to be the most active deubiquitinase acting on ubiquitin-PEX5. Conclusion:We propose that USP9X participates in the PEX5-mediated peroxisomal protein import pathway. Significance: The unbiased biochemical strategy described here will be useful to identify deubiquitinases acting on other substrates.

Background: How the soluble receptor PEX5 delivers its cargoes to the peroxisome remains largely unknown. Results: Cargo translocation occurs after docking of the receptor at the peroxisome and before any ATP-dependent step. Conclusion: Translocation is concomitant with PEX5 insertion into the docking/translocation machinery. Significance: These results support a model in which cargoes are pushed across the peroxisomal membrane by PEX5.