Trypanosoma brucei PUF9 Regulates mRNAs for Proteins Involved in Replicative Processes over the Cell Cycle

PLoS Pathogens

Rafael A. de Queiroz¹  Stefanie Brems¹  Stuart K. Archer²  Van-Duc Luu²  Christine Clayton² 

[1] Deutsches Krebsforschungszentrum, DKFZ-ZMBH Allianz, Heidelberg, Germany;Zentrum für Molekulare Biologie Heidelberg, DKFZ-ZMBH Allianz, Heidelberg, Germany

关键词: Kinetoplasts;    Cell cycle;    cell division;    RNA interference;    Messenger RNA;    Untranslated regions;    Northern blot;    Trypanosoma;    Trypanosoma brucei gambiense;   

DOI  :  10.1371/journal.ppat.1000565

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

Many genes that are required at specific points in the cell cycle exhibit cell cycle–dependent expression. In the early-diverging model eukaryote and important human pathogen Trypanosoma brucei, regulation of gene expression in the cell cycle and other processes is almost entirely post-transcriptional. Here, we show that the T. brucei RNA-binding protein PUF9 stabilizes certain transcripts during S-phase. Target transcripts of PUF9—LIGKA, PNT1 and PNT2—were identified by affinity purification with TAP-tagged PUF9. RNAi against PUF9 caused an accumulation of cells in G2/M phase and unexpectedly destabilized the PUF9 target mRNAs, despite the fact that most known Puf-domain proteins promote degradation of their target mRNAs. The levels of the PUF9-regulated transcripts were cell cycle dependent, peaking in mid- to late- S-phase, and this effect was abolished when PUF9 was targeted by RNAi. The sequence UUGUACC was over-represented in the 3′ UTRs of PUF9 targets; a point mutation in this motif abolished PUF9-dependent stabilization of a reporter transcript carrying the PNT1 3′ UTR. LIGKA is involved in replication of the kinetoplast, and here we show that PNT1 is also kinetoplast-associated and its over-expression causes kinetoplast-related defects, while PNT2 is localized to the nucleus in G1 phase and redistributes to the mitotic spindle during mitosis. PUF9 targets may constitute a post-transcriptional regulon, encoding proteins involved in temporally coordinated replicative processes in early G2 phase.

【 授权许可】

CC BY