RBP16 is a multifunctional gene regulatory protein involved in editing and stabilization of specific mitochondrial mRNAs in Trypanosoma brucei

Abstract

RBP16 is a Trypanosoma brucei mitochondrial RNA-binding protein that associates with guide RNAs (gRNAs), mRNAs, and ribosomal RNAs. Based on its inclusion in the multifunctional Y-box protein family and its ability to bind multiple RNA classes, we hypothesized that RBP16 plays a role in diverse aspects of mitochondrial gene regulation. To gain insight into RBP16 function, we generated cells expressing less than 10% of wild-type RBP16 levels by tetracycline-regulated RNA interference (RNAi). Poisoned primer extension analyses revealed that edited, but not unedited, CYb mRNA is reduced by ∼98% in tetracycline-induced RBP16 RNAi cells, suggesting that RBP16 is critical for CYb RNA editing. The down-regulation of CYb editing in RBP16 RNAi transfectants apparently entails a defect in gRNA utilization, as gCYb[560] abundance is similar in uninduced and induced cells. We observed a surprising degree of specificity regarding the ability of RBP16 to modulate editing, as editing of mRNAs other than CYb is not significantly affected upon RBP16 disruption. However, the abundance of the never edited mitochondrial RNAs COI and ND4 is reduced by 70%-80% in RBP16 RNAi transfectants, indicating an additional role for RBP16 in the stabilization of these mRNAs. Analysis of RNAs bound to RBP16 immunoprecipitated from wild-type cells reveals that RBP16 is associated with multiple gRNA sequence classes in vivo, including those whose abundance and usage appear unaffected by RBP16 disruption. Overall, our results indicate that RBP16 is an accessory factor that regulates the editing and stability of specific populations of mitochondrial mRNAs.

Evidence for U-tail Stabilization of gRNA/mRNA Interactions in Kinetoplastid RNA Editing

“…It may be that RNA editing of developmentally regulated transcripts requires the participation of specific protein factors that allow efficient gRNA/mRNA annealing. A number of gRNA binding proteins have been characterized that accelerate the hybridization of cognate guide RNA/pre-edited mRNA pairs [43][44][45][46]. Recently, Pelletier and Read [46] showed that RNAi knockdowns of the Y-box protein, RBP16, led to a 98% reduction in the levels of edited CYb mRNA.…”

Targeted depletion of a mitochondrial nucleotidyltransferase suggests the presence of multiple enzymes that polymerize mRNA 3′ tails in Trypanosoma brucei mitochondria

“…For northern blot analysis of edited RPS12 mRNA, a radiolabeled DNA probe corresponding to the fulllength fully edited RPS12 sequence was generated by PCR reaction using oligonucleotides CR6-5′T7 and CR6-3′E with [α-32 P]dATP, and hybridization was performed as described previously (17). Poisoned primer extensions using oligonucleotides Tub-RT, COI-RT, ND1-RT, ND4-RT, CO2-RT, CYb-RT, A6-3′NE, ND7-RT and COIII-3′NE were performed with 20 μg of total RNA as described previously (43). Gels were analyzed by phosphoimager analysis on a Bio-Rad Personal FX Phosphoimager using Quantity One software.…”

Trypanosoma brucei RNA editing protein TbMP42 (band VI) is crucial for the endonucleolytic cleavages but not the subsequent steps of U-deletion and U-insertion

“…The final column summarizes the demonstrated (and proposed) roles in editing, as cited in the Introduction and the above references. mHel61p helicase of the z20S complex may aid removal of gRNAs after editing (Missel et al 1997); also proteins separate from the z20S complex can affect editing, including TbMP108/KRET1 (that adds U-tails onto gRNAs) (Aphasizhev et al 2002(Aphasizhev et al , 2003c, TbgBP21 and TbgBP25 (that stimulate RNA annealing) (Müller et al 2001;Müller and Göringer 2002;Aphasizhev et al 2003b;Schumacher et al 2006), REAP1 (a putative mRNA-binding protein) , and RBP16 (a CYb RNA factor) (Pelletier and Read 2003). …”