APOBEC1 is a cytidine deaminase that edits messenger RNAs and was the first enzyme in the APOBEC family to be functionally characterized. Under appropriate conditions APOBEC1 also deaminates deoxycytidine in single-stranded DNA (ssDNA). The other ten members of the APOBEC family have not been fully characterized however several have deoxycytidine deaminase activity on ssDNAs. Despite the nucleic acid substrate preferences of different APOBEC proteins, a common feature appears to be their intrinsic ability to bind to RNA as well as to ssDNA. RNA binding to APOBEC proteins together with protein-protein interactions, post-translation modifications and subcellular localization serve as biological modulators controlling the DNA mutagenic activity of these potentially genotoxic proteins.

P34 and P37 are two previously identified RNA binding proteins in the flagellate protozoan Trypanosoma brucei. RNA interference studies have determined that the proteins are involved in and essential for ribosome biogenesis. The proteins interact with the 5S rRNA with nearly identical binding characteristics. We have shown that this interaction is achieved mainly through the LoopA region of the RNA, but P34 and P37 also protect the L5 binding site located on LoopC. We now provide evidence to show that these factors form a novel pre-ribosomal particle through interactions with both 5S rRNA and the L5 ribosomal protein. Further in silico and in vitro analysis of T. brucei L5 indicates a lower affinity for 5S rRNA than expected, based on other eukaryotic L5 proteins. We hypothesize that P34 and P37 complement L5 and bridge the interaction with 5S rRNA, stabilizing it and aiding in the early steps of ribosome biogenesis.

Cytidine deaminases have important roles in the regulation of nucleoside/deoxynucleoside pools for DNA and RNA synthesis. The APOBEC family of cytidine deaminases (named after the first member of the family that was described, Apolipoprotein B mRNA Editing Catalytic Subunit 1, a.k.a. APOBEC1 or A1) is a fascinating group of mutagenic proteins that use RNA and single stranded DNA (ssDNA) as substrates for their cytidine or deoxycytidine deaminase activities. APOBEC proteins and base-modification nucleic acid editing have been the subject of numerous publications, reviews and speculation. These proteins play diverse roles in host cell defense, protecting cells from invading genetic material, enabling the acquired immune response to antigens and changing protein expression at the level of the genetic code in mRNA or DNA. The amazing power these proteins have for interphase cell functions relies on structural and biochemical properties that are beginning to be understood. At the same time, the substrate selectivity of each member in the family and their regulation remains to be elucidated. This review of the APOBEC family will focus on an open question in regulation, namely what role the interactions of these proteins with RNA have in editing substrate recognition or allosteric regulation of DNA mutagenic and host defense activities.

We previously identified two Trypanosoma brucei RNA binding proteins, P34 and P37, and determined that they are essential for proper ribosomal assembly in this organism. Loss of these proteins via RNA interference is lethal and causes a decrease in both 5S rRNA levels and formation of 80S ribosomes, concomitant with a decrease in total cellular protein synthesis. These data suggest that these proteins are involved at some point in the ribosomal biogenesis pathway. In the current study, we have performed subcellular fractionation in conjunction with immune capture experiments specific for 60S ribosomal proteins and accessory factors in order to determine when and where P34 and P37 are involved in the ribosomal biogenesis pathway. These studies demonstrate that P34 and P37 associate with the 60S ribosomal subunit at the stage of the nucleolar 90S particle and remain associated subsequent to nuclear export. In addition, P34 and P37 associate with conserved 60S ribosomal subunit nuclear export factors exportin 1 and Nmd3, suggesting that they are components of the 60S ribosomal subunit nuclear export complex in T. brucei. Most significantly, the pre-60S complex does not associate with exportin 1 or Nmd3 in the absence of P34 and P37. These results demonstrate that, although T. brucei 60S ribosomal subunits utilize a nuclear export complex similar to that described for other organisms, trypanosome-specific factors are essential to the process.

We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, which have been shown to interact with a family of nucleolar phosphoproteins, NOPP44/46, in Trypanosoma brucei. These proteins are nearly identical, the major difference being an 18-amino-acid insert in the N terminus of p37. In order to characterize the interaction between p34 and p37 and NOPP44/46, we have utilized an RNA interference (RNAi) cell line that specifically targets p34 and p37. Within these RNAi cells, we detected a disruption of a higher-molecular-weight complex containing NOPP44/46, as well as a dramatic increase in nuclear NOPP44/46 protein levels. We demonstrated that no change occurred in NOPP44/46 mRNA steadystate levels or stability, nor was there a change in cellular protein levels. These results led us to investigate whether p34 and p37 regulate NOPP44/46 cellular localization. Examination of the p34 and p37 amino acid sequences revealed a leucine-rich nuclear export signal, which interacts with the nuclear export factor exportin 1. Immune capture experiments demonstrated that p34, p37, and NOPP44/46 associate with exportin 1. When these experiments were performed with p34/p37 RNAi cells, NOPP44/46 no longer associated with exportin 1. Sequential immune capture experiments demonstrated that p34, p37, NOPP44/46, and exportin 1 exist in a common complex. Inhibiting exportin 1-mediated nuclear export led to an increase in nuclear NOPP44/46 proteins, indicating that they are exported from the nucleus via this pathway. Together, our results demonstrate that p34 and p37 regulate NOPP44/46 cellular localization by facilitating their association with exportin 1.African trypanosomes are the causative agents of African sleeping sickness in humans and nagana in cattle (22). This parasite continues to pose a serious threat to human health and to cause devastating economic losses (25). African trypanosomiasis is a reemerging infectious disease with estimates ranging between 300,000 and 500,000 new cases each year (34). This rise in infection rates is due, in part, to an increase in parasite drug resistance and vector pesticide resistance.In previous work from our laboratory, two Trypanosoma brucei RNA binding proteins, p34 and p37, were identified and characterized (39, 40). The only major difference between them is an 18-amino-acid insert located within the amino terminus of p37 that is absent in p34. These proteins contain two RNA binding domains (RBD) within the central coding region, and they have been shown to interact with 5S rRNA (31). In addition to the interaction with 5S rRNA, p34 and p37 have been found to interact with a family of nucleolar phosphoproteins, NOPP44/46 (30). These abundant proteins have been identified and characterized by M. Parsons and colleagues (9,10,27,29). An interaction(s) between p34 and p37 and NOPP44/46 was mapped through yeast two-hybrid analysis and protein affinity chromatography and was shown to be mediated via the RNA binding domain of p34 and p37 (30). Thus, th...