Characterization of Pentatricopeptide Repeat (PPR) Proteins in Trypanosoma Brucei
Author | : |
Publisher | : |
Total Pages | : 88 |
Release | : 2007 |
ISBN-10 | : OCLC:213063405 |
ISBN-13 | : |
Rating | : 4/5 (05 Downloads) |
Book excerpt: Pentatricopeptide repeat proteins (PPRs) are one of the largest newly identified protein families. PPR is characterized by the presence of a tandemly repeated 35 amino acids motif. It is extremely abundant in plants, and found in many organisms including mammals. PPRs are said to play essential roles in mitochondria, probably via binding to organelle transcripts. A number of recent reports characterizing PPR proteins indicate that PPR are important regulatory proteins that play essential roles in polycistronic RNA processing, RNA stability, and translation of special transcripts [2, 64]. To date, there are 23 PPRs identified in T.brucei by Mingler et al. [2] and 28 distinct PPRs detected by Pusnik et al. [1] by using different bioinformatics approaches. T.brucei encodes far more PPRs than the other non-plant organisms such as mammals. Trypanosomes undergoes a complex life cycle and thus it need to adapt to the changes of environment where it resides. T.brucei undergoes different morphological forms, metabolic changes, and alterations in gene expression during the changes of environment between insect and mammalian hosts. Thus, T.brucei serves as a good model to study the function of PPRs. We utilized tetracycline (tet)-regulated RNA interference (RNAi) to study the function of two T.brucei PPR proteins, TbPPR6 and TbPPR7. Northern blot analysis shows that TbPPR6 (Tb11.01.7930) was down-regulated by 49% while TbPPR7 (Tb927.3.4550) showed a decrease level of 23% in tetracycline induction RNAi cells. Knock-down of TbPPR6 caused a slow growth phenotype, while TbPPR7 knock-down caused only a minor slow growth phenotype, suggesting that TbPPR6 and TbPPR7 are essential for optimal growth of T.brucei . Poisoned primer extension assays demonstrated that depletion of TbPPR6 and TbPPR7 to the levels described above did not affect the mRNAs analyzed. However, there may be a modest effect on COI stability, particularly after depletion of TbPPR7. To characterize the RNA binding properties of TbPPR4 (Tb10.70.5780), recombinant TbPPR4 full length (PTbPR4-FL), and two of its truncated versions (TbPPR4-1, TbPPR4-0) were expressed and purified. Homopolymer binding assays show that TbPPR4-FL preferentially binds poly-(G) agarose beads over the other homopolymers. We further characterized the TbPPR4-RNA interaction by changing the salt concentration (NaCl) of the binding buffer. Increasing salt concentration in the binding buffer decreases the binding ability of TbPPR4-FL to poly-(G) agarose beads, indicating that electrostatic interactions play a prominent role in the binding of TbPPR4-FL. Interactions such as hydrogen bonding and hydrophobic interactions may also involve in the interactions of TbPPR4-FL with poly-(G). Titration experiments of TbPPR4-FL, TbPPR4-1, and TbPPR4-0 show that TbPPR4-FL binds significantly better than TbPPR4-1 and TbPPR4-0 while TbPPR4-1 binds better than TbPR4-0 to poly-(G). Thus, PPRs with increasing numbers of PPR motifs have higher affinity to RNA. Our studies add to the growing literature that defines PPR motifs as RNA binding motifs, and suggest that TbPPR6 and TbPPR7 maybe play a role in mitochondrial RNA processing and/or stability.