Unique Interactions of the Nuclear Export Receptors TbMex67 and TbMtr2 with Components of the 5S Ribonuclear Particle in Trypanosoma brucei.

Eukaryotic ribosome biogenesis is a complicated and highly conserved biological process. A critical step in ribosome biogenesis is the translocation of the immature ribosomal subunits from the nucleoplasm, across the nucleopore complex, to the cytoplasm where they undergo final maturation. Many nonribosomal proteins are needed to facilitate export of the ribosomal subunits, and one complex participating in export of the pre-60S in Saccharomyces cerevisiae is the heterodimer Mex67-Mtr2. In Trypanomsoma brucei, the process of ribosome biogenesis differs from the yeast process in key steps and is not yet fully characterized. However, our laboratory has previously identified the trypanosome-specific proteins P34/P37 and has shown that P34/P37 are necessary for the formation of the 5S ribonuclear particle (RNP) and for the nuclear export of the pre-60S subunit. We have also shown that loss of TbMex67 or TbMtr2 leads to aberrant ribosome formation, rRNA processing, and polysome formation in T. brucei In this study, we characterize the interaction of TbMex67 and TbMtr2 with the components of the 5S RNP (P34/P37, L5 and 5S rRNA) of the 60S subunit. We demonstrate that TbMex67 directly interacts with P34 and L5 proteins as well as 5S rRNA, while TbMtr2 does not. Using protein sequence alignments and structure prediction modeling, we show that TbMex67 lacks the amino acids previously shown to be essential for binding to 5S rRNA in yeast and in general aligns more closely with the human orthologue (NXF1 or TAP). This work suggests that the T. brucei Mex67-Mtr2 binds ribosomal cargo differently from the yeast system.IMPORTANCETrypanosoma brucei is the causative agent for both African sleeping sickness in humans and nagana in cattle. Ribosome biogenesis in these pathogens requires both conserved and trypanosome-specific proteins to coordinate in a complex pathway. We have previously shown that the trypanosome-specific proteins P34/P37 are essential to the interaction of the TbNmd3-TbXpoI export complex with the 60S ribosomal subunits, allowing their translocation across the nuclear envelope. Our recent studies show that the trypanosome orthologues of the auxiliary export proteins TbMex67-TbMtr2 are required for ribosome assembly, proper rRNA processing, and polysome formation. Here we show that TbMex67-TbMtr2 interact with members of the 60S ribosomal subunit 5S RNP. Although TbMex67 has a unique structure among the Mex67 orthologues and forms unique interactions with the 5S RNP, particularly with trypanosome-specific P34/P37, it performs a conserved function in ribosome assembly. These unique structures and parasite-specific interactions may provide new therapeutic targets against this important parasite.

The Nuclear Export Receptors TbMex67 and TbMtr2 Are Required for Ribosome Biogenesis in Trypanosoma brucei.

Ribosomal maturation is a complex and highly conserved biological process involving migration of a continuously changing RNP across multiple cellular compartments. A critical point in this process is the translocation of individual ribosomal subunits (60S and 40S) from the nucleus to the cytoplasm, and a number of export factors participate in this process. In this study, we characterize the functional role of the auxiliary export receptors TbMex67 and TbMtr2 in ribosome biogenesis in the parasite Trypanosoma brucei We demonstrate that depletion of each of these proteins dramatically impacts the steady-state levels of other proteins involved in ribosome biogenesis, including the trypanosome-specific factors P34 and P37. In addition, we observe that the loss of TbMex67 or TbMtr2 leads to aberrant ribosome formation, rRNA processing, and polysome formation. Although the TbMex67-TbMtr2 heterodimer is structurally distinct from Mex67-Mtr2 complexes previously studied, our data show that they retain a conserved function in ribosome biogenesis.IMPORTANCE The nuclear export of ribosomal subunits (60S and 40S) depends in part on the activity of the essential auxiliary export receptors TbMtr2 and TbMex67. When these proteins are individually depleted from the medically and agriculturally significant parasite Trypanosoma brucei, distinct alterations in the processing of the rRNAs of the large subunit (60S) are observed as well as aberrations in the assembly of functional ribosomes (polysomes). We also established that TbMex67 and TbMtr2 interact directly or indirectly with the protein components of the 5S RNP, including the trypanosome-specific P34/P37. The critical role that TbMex67 and TbMtr2 play in this essential biological process together with their parasite-specific interactions may provide new therapeutic targets against this important parasite.