Structure and Function of RNA Processing Machines

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. Research accomplishments from the past year are summarized below. During the production of ribosomes the pre-rRNA is processed by a series of endo and exo-ribonucleases to facilitate the removal of the external and internal transcribed spacers. Removal of the ITS2 (internal transcribed spacer 2) is initiated by the endoribonuclease Las1. We recently established that Las1 is reliant on its binding partner the polynucleotide kinase, Grc3 (Nol9) for nuclease activity but how this complex gets recruited to the ITS2 remains unknown. Previous work in mammalian cells has shown that Las1 and Grc3 form part of a larger complex with several other ribosome assembly factors (PELP1, WDR18, TEX10, and SENP3) called the RNA rixosome. We reconstituted the RNA rixosome and discovered that two complex members, PELP1 and WDR18 form a stable subcomplex with one another. PELP1 (Proline-, Glutamic acid-, Leucine-rich protein 1) is a large scaffolding protein that has been implicated in many cellular activities beyond ribosome assembly including steroid receptor (SR) coactivation and heterochromatin maintenance activities. The N-terminal domain of PELP1, which is required for association with WDR18 contains several signaling motifs including eleven LxxLL and three PxxP (x is any amino acid) motifs both known for mediating steroid receptor (SR) signaling. To shed light on the structure and function of PELP1s signaling motifs we determined the cryo-EM structure of PELP1 bound to WDR18 revealing that WDR18 and PELP1 assemble into an interconnected tetramer composed of two copies of PELP1 and two copies of WDR18. Surprisingly the structure also revealed that the signaling motifs within PELP1 are not in a conformation that would support binding other factors, suggesting that WDR18 may direct PELP1 towards its role in ribosome production. Another major accomplishment over the past year was focused on pre-tRNA processing. Similar to the pre-rRNA, pre-tRNAs undergo a series of processing steps before they can be charged with their cognate amino acid. A subset of human tRNAs contain introns that must be removed to properly form the anticodon stem loop. Removal of these introns is facilitated by the tRNA splicing endonuclease (TSEN) complex. This tetrameric complex is composed of four subunits including the nucleases TSEN2 and TSEN34, and the structural subunits TSEN15 and TSEN54. All four subunits of this complex are essential for life and mutations within the complex are association with a group of rare neurodevelopmental disorders known as pontocerebellar hypoplasia (PCH). A lack of structures of any eukaryotic TSEN complex has hindered our understanding of how the complex recognizes and processes pre-tRNA. We determined a cryo-EM structure of the human TSEN complex bound to an intron containing pre-tRNA. This structure revealed the overall architecture of the complex including the arrangement of the four subunits and the extensive pre-tRNA interface formed by the TSEN54 subunit. Finally, the structure also enabled us to map the PCH causing variants onto the structure providing new insight into how these mutations interfere with TSEN function.