Abstract
Ribosome biogenesis is a highly complex and conserved cellular process that is responsible for making ribosomes. During this process, there are several assembly steps that function as regulators to ensure proper ribosome formation. One of these steps is the assembly of the 5S ribonucleoprotein particle (5S RNP) in the central protuberance of the 60S ribosomal subunit. In eukaryotes, the 5S RNP is composed of 5S rRNA, ribosomal proteins L5 and L11, and assembly factors Rpf2 and Rrs1. Our laboratory previously showed that in Trypanosoma brucei, the 5S RNP is composed of 5S rRNA, L5, and trypanosome-specific RNA binding proteins P34 and P37. In this study, we characterize an additional component of the 5S RNP, the T.brucei homolog of Rpf2. This is the first study to functionally characterize interactions mediated by Rpf2 in an organism other than fungi. T. brucei Rpf2 (TbRpf2) was identified from tandem affinity purification using extracts prepared from protein A-tobacco etch virus (TEV)-protein C (PTP)-tagged L5, P34, and P37 cell lines, followed by mass spectrometry analysis. We characterized the binding interactions between TbRpf2 and the previously characterized members of the T.brucei 5S RNP. Our studies show that TbRpf2 mediates conserved binding interactions with 5S rRNA and L5 and that TbRpf2 also interacts with trypanosome-specific proteins P34 and P37. We performed RNA interference (RNAi) knockdown of TbRpf2 and showed that this protein is essential for the survival of the parasites and is critical for proper ribosome formation. These studies provide new insights into a critical checkpoint in the ribosome biogenesis pathway in T.brucei. IMPORTANCETrypanosoma brucei is the parasitic protozoan that causes African sleeping sickness. Ribosome assembly is essential for the survival of this parasite through the different host environments it encounters during its life cycle. The assembly of the 5S ribonucleoprotein particle (5S RNP) functions as one of the regulatory checkpoints during ribosome biogenesis. We have previously characterized the 5S RNP in T.brucei and showed that trypanosome-specific proteins P34 and P37 are part of this complex. In this study, we characterize for the first time the interactions of the homolog of the assembly factor Rpf2 with members of the 5S RNP in another organism besides fungi. Our studies show that Rpf2 is essential in T.brucei and that it forms unique interactions within the 5S RNP, particularly with P34 and P37. These studies have identified parasite-specific interactions that can potentially function as new therapeutic targets against sleeping sickness.
Highlights
Ribosome biogenesis is a highly complex and conserved cellular process that is responsible for making ribosomes
Our laboratory previously showed that in T. brucei the 5S ribonucleoprotein particle (5S RNP) is composed of 5S rRNA, L5, and trypanosome-specific RNA binding proteins P34 and P37 [15, 16]
These results show that recombinant T. brucei ribosome production factor 2 (Rpf2) (TbRpf2) protein is functional in the absence of regulator of ribosome synthesis 1 (Rrs1) and is able to bind to 5S rRNA
Summary
Ribosome biogenesis is a highly complex and conserved cellular process that is responsible for making ribosomes. We performed RNA interference (RNAi) knockdown of TbRpf and showed that this protein is essential for the survival of the parasites and is critical for proper ribosome formation These studies provide new insights into a critical checkpoint in the ribosome biogenesis pathway in T. brucei. Ribosome biogenesis begins in the nucleolus with the transcription of a 35S rRNA precursor by RNA polymerase I [8] This precursor is processed into three of the four rRNA species (18S, 5.8S, and 25S) and joins together with ribosomal proteins and accessory factors to form the 60S and 40S preribosomal subunits [6, 9]. To determine the complexity of the 5S RNP in T. brucei, we performed tandem affinity purification using protein A-tobacco etch virus (TEV)-protein C (PTP)tagged L5, P34, and P37 proteins From these studies, we identified the T. brucei homolog of the Rpf protein as well as the homologs of ribosomal protein L11 and Rrs. We characterize T. brucei Rpf (TbRpf2) to determine whether it is an essential protein and to identify the conserved and unique features of this protein in T. brucei
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