Abstract

RNA binding proteins are involved in many aspects of RNA metabolism. In Trypanosoma brucei, our laboratory has identified two trypanosome-specific RNA binding proteins P34 and P37 that are involved in the maturation of the 60S subunit during ribosome biogenesis. These proteins are part of the T. brucei 5S ribonucleoprotein particle (5S RNP) and P34 binds to 5S ribosomal RNA (rRNA) and ribosomal protein L5 through its N-terminus and its RNA recognition motif (RRM) domains. We generated truncated P34 proteins to determine these domains’ interactions with 5S rRNA and L5. Our analyses demonstrate that RRM1 of P34 mediates the majority of binding with 5S rRNA and the N-terminus together with RRM1 contribute the most to binding with L5. We determined that the consensus ribonucleoprotein (RNP) 1 and 2 sequences, characteristic of canonical RRM domains, are not fully conserved in the RRM domains of P34. However, the aromatic amino acids previously described to mediate base stacking interactions with their RNA target are conserved in both of the RRM domains of P34. Surprisingly, mutation of these aromatic residues did not disrupt but instead enhanced 5S rRNA binding. However, we identified four arginine residues located in RRM1 of P34 that strongly impact L5 binding. These mutational analyses of P34 suggest that the binding site for 5S rRNA and L5 are near each other and specific residues within P34 regulate the formation of the 5S RNP. These studies show the unique way that the domains of P34 mediate binding with the T. brucei 5S RNP.

Highlights

  • Ribosome biogenesis is a conserved cellular process that is essential in all organisms

  • Previous studies from our laboratory showed that the RNA recognition motif (RRM) domains and the N-terminal domain of P34 are important in mediating interactions with 5S ribosomal RNA (rRNA) and that the C-terminal domain alone did not bind to 5S rRNA [19]

  • We generated truncated P34 proteins that consisted of the N-terminus with the RRM domains, the RRM domains together, and the RRM domains individually in order to further characterize how these domains of P34 mediate binding with 5S rRNA (Fig 1)

Read more

Summary

Introduction

Ribosome biogenesis is a conserved cellular process that is essential in all organisms. This process requires the coordination of ribosomal RNAs (rRNA), ribosomal proteins and over 200 accessory factors to form functional ribosomes [1, 2]. The 35S precursor rRNA is subsequently cleaved and processed into rRNA components that together with ribosomal and non-ribosomal proteins form the 43S and 66S pre-ribosomal subunits [10]. These two subunits are further modified and are exported to the cytoplasm as 60S and pre-40S pre-ribosomal subunits. The pre-40S undergoes its final maturation steps and joins with the mature 60S to form the 80S ribosome that performs protein synthesis [10]

Methods
Results
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call