Structure and Function of RNA Processing Machines

National Institute Of Environmental Health Sciences

Investigators

Linked publications, trials & patents

Abstract

Ribosomes are large ribonucleoprotein complexes that carry out the fundamental role of translating mRNA into protein yet the molecular mechanisms governing ribosome assembly remain poorly understood. Eukaryotic ribosome assembly is a complex and energetically demanding process that relies on hundreds of trans-acting assembly factors, including many RNA processing machines. The majority of the pre-rRNA is transcribed as a polycistronic precursor that includes the 18S, 5.8S, and 28S rRNA as well as 4 spacer sequences. There are two external spacers, known as the 5’ and 3’ ETS, and two internal spacers known as the ITS1 and ITS2. Removal of these spacer sequences is a critical part of the assembly pathway and is coordinated by a series of endo and exo-ribonucleases. ITS2 removal is initiated by the endoribonuclease Las1. We previously established that Las1 forms a tetrameric assembly with the polynucleotide kinase Grc3 (Nol9 in humans) that we termed RNase PNK. Together these two enzymes coordinate the cleavage and phosphorylation of the ITS2, triggering its subsequent decay by exonucleases. RNase PNK forms a larger complex with a group of other ribosome assembly factors (PELP1, WDR18, TEX10, SENP3, and MDN1) known as the RNA rixosome. We used reconstitution studies combined with structural and functional approaches to develop an integrative structural model of the human rixosome complex. We discovered that PELP1 is the central scaffold of the rixosome complex. The N-terminal domain of PELP1 along with the structural subunits WDR18 and TEX10 form the structural core of the rixosome to which the enzymatic subunits are flexibly tethered. The C-terminal IDR (intrinsically disordered region) of PELP1 specifically mediates association with the AAA-ATPase MDN1 and the SUMO protease SENP3. We determined the crystal structure of SENP3 bound to a short linear motif (SLIM) of the PELP1 IDR and established that this SLIM is important for SUMO protease activity. Collectively this work establishes how PELP1 mediates both the modular assembly and enzymatic activity of the rixosome. Analogous to pre-rRNA processing, pre-tRNAs must undergo a series of processing steps before they can be charged with their cognate amino acid and deliver amino acids to the ribosome during translation. Across eukaryotes a subset of pre-tRNAs contain introns within the anticodon stem loop. While the exact purpose of these introns remains unclear they must be removed to form the mature anticodon stem loop. In humans, removal of tRNA introns is catalyzed by the tRNA splicing endonuclease (TSEN) complex. This essential tetrameric complex is composed to two nuclease subunits (TSEN2 and TSEN34) and two structural subunits (TSEN54 and TSEN15). Mutations within the complex are associated with a group of rare neurodevelopmental disorders known as pontocerebellar hypoplasia (PCH). We recently determined a cryo-EM structure of the human TSEN complex bound to an intron containing pre-tRNA in the pre-cleavage state, revealing the overall architecture of the complex and the mode of tRNA recognition. We are continuing to explore the molecular mechanisms of human tRNA splicing through structural and biochemical approaches.