Trypanosoma brucei PUF9 regulates mRNAs for proteins involved in replicative processes over the cell cycle.
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.
Archer
Stuart K
SK
Zentrum für Molekulare Biologie Heidelberg, DKFZ-ZMBH Allianz, Heidelberg, Germany. s.archer@zmbh.uni-heidelberg.de
Luu
Van-Duc
VD
de Queiroz
Rafael A
RA
Brems
Stefanie
S
Clayton
Christine
C
D000818:Animals; D002453:Cell Cycle / Q000502:physiology; D016195:G2 Phase; D007150:Immunohistochemistry; D020411:Oligonucleotide Array Sequence Analysis; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D016196:S Phase; D014346:Trypanosoma brucei brucei / Q000235:genetics*
D015800:Protozoan Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins