Structural Mechanisms of Ribosome Assembly

National Institute Of Environmental Health Sciences

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Abstract

Protein translation by the ribosome is essential for cellular life but the molecular mechanisms governing how ribosomes are made are poorly defined. Ribosome biogenesis is a major consumer of cellular energy and an incredibly complex pathway involving hundreds of trans-acting factors. We take a multidisciplinary approach to study macromolecular machines required for ribosome assembly and pre-tRNA processing. Our efforts of the last year were focused on three main projects. (1) RNase PNK: RNase PNK is a multi-enzyme complex composed of the endoribonuclease Las1 (Las1L in mammals) and the polynucleotide kinase Grc3 (Nol9 in mammals). RNase PNK initiates processing of the internal transcribed spacer 2 (ITS2) during ribosome assembly. The Las1 nuclease cuts the ITS2 at the C2 site and then the 5 cleavage product is phosphorylated by the Grc3 kinase. Cleavage at the C2 site is an essential step during ribosome assembly because it separates the precursors of the 5.8S and 25S rRNA, triggers the further processing of the 5 end of the 26S pre-rRNA, and primes pre-60S particles for transit from the nucleolus to the nucleoplasm. While it is now established that Las1 is the C2 endoribonuclease the molecular mechanisms governing C2 cleavage are unclear. Mutations in the mammalian Las1 gene have also been linked to neurological dysfunction, underscoring the need to further understand the activity of this enzyme. We have established that there is functional cross-talk between the nuclease and kinase domains of Las1 and Grc3 allowing for an exquisite level of control of enzymatic activity. We are continuing to use cryo-EM, biochemistry, and cell biology to define the mechanisms of RNA recruitment and processing by RNase PNK. (2) Rix7 AAA-ATPase: Rix7 is a AAA-ATPase that is required for maturation of the large ribosomal subunit. Our working hypothesis is that Rix7 utilizes the power of ATP hydrolysis to facilitate the removal of assembly factors from early pre-ribosome particles. We recently determined a high-resolution cryo-EM structure of Rix7 that revealed it assembles into an asymmetric homo-hexamer with a central channel through which substrates are unfolded. The structure also revealed how inter and intra-molecular communication networks support crosstalk between the different AAA domains to facilitate processing substrate translocation. (3) TSEN Complex: The tRNA splicing endonuclease complex (TSEN) is a heterotetrametric complex required to facilitate the removal of introns from intro-containing tRNAs. Mutations within all four subunits of the TSEN complex are associated with neurodegenerative disorders, however the mechanisms underlying disease pathogenesis are unknown. We developed a recombinant system to purify the human TSEN complex. In collaboration with Greg Materas Lab from UNC Chapel Hill, we were able to show that the polynucleotide kinase Clp1, a known interaction partner of the TSEN complex, plays a regulatory role in tRNA splicing. We are currently taking a structure-function approach to define how the TSEN complex binds and cleaves pre-tRNAs.