The ribosome assembly factor Nop53 controls association of the RNA exosome with pre-60S particles in yeast

Leidy Paola P Cepeda,Felipe F.M Bagatelli,Renata M Santos,Marlon D.M Santos,Fabio C.S Nogueira,Carla C Oliveira

doi:10.1074/jbc.ra119.010193

Abstract

Eukaryotic ribosomal biogenesis is a high-energy-demanding and complex process that requires hundreds of trans-acting factors to dynamically build the highly-organized 40S and 60S subunits. Each ribonucleoprotein complex comprises specific rRNAs and ribosomal proteins that are organized into functional domains. The RNA exosome complex plays a crucial role as one of the pre-60S-processing factors, because it is the RNase responsible for processing the 7S pre-rRNA to the mature 5.8S rRNA. The yeast pre-60S assembly factor Nop53 has previously been shown to associate with the nucleoplasmic pre-60S in a region containing the "foot" structure assembled around the 3' end of the 7S pre-rRNA. Nop53 interacts with 25S rRNA and with several 60S assembly factors, including the RNA exosome, specifically, with its catalytic subunit Rrp6 and with the exosome-associated RNA helicase Mtr4. Nop53 is therefore considered the adaptor responsible for recruiting the exosome complex for 7S processing. Here, using proteomics-based approaches in budding yeast to analyze the effects of Nop53 on the exosome interactome, we found that the exosome binds pre-ribosomal complexes early during the ribosome maturation pathway. We also identified interactions through which Nop53 modulates exosome activity in the context of 60S maturation and provide evidence that in addition to recruiting the exosome, Nop53 may also be important for positioning the exosome during 7S processing. On the basis of these findings, we propose that the exosome is recruited much earlier during ribosome assembly than previously thought, suggesting the existence of additional interactions that remain to be described.

Highlights

Eukaryotic ribosomal biogenesis is a high-energy– demanding and complex process that requires hundreds of trans-acting factors to dynamically build the highly-organized 40S and 60S subunits
To investigate in more detail the role played by Nop53 in exosome recruitment to pre-60S for processing of the prerRNA 7S, we tested its interaction with core exosome subunits and with the exosome cofactor Mpp6
Based on the available structure of the nuclear exosome [12] showing that Rrp45 and Mpp6 are exposed on the same side of the complex, it is conceivable to hypothesize that Nop53 could recruit the 14-subunit exosome and be important for the positioning of the exosome on the pre-60S particle during 7S pre-rRNA processing

Summary

The abbreviations used are

5Ј-ETS, 5Ј-external transcribed spacer; FDR, false-discovery rate; PDB, Protein Data Bank; PMSF, phenylmethylsulfonyl fluoride; snoRNP, small nucleolar ribonucleoprotein; HRP, horseradish peroxidase; PSM, peptide spectrum match; LTQ, linear trap quadropole orbitrap velos mass spectrometer; TAP, tandem affinity purification. The determination of Mtr structure revealed that it interacts with the other two subunits of the TRAMP complex through its DEXH domain [20], whereas its KOW domain is involved in the interaction with the pre60S trans-acting factor Nop53 [17]. We show evidence that Nop may be involved in the recruitment and in positioning of the nuclear exosome on the pre-60S particle for processing of 7S pre-rRNA, based on the observation that, in addition to its direct interaction with Rrp and Mtr, Nop interacts with the core subunit Rrp and the exosome cofactor Mpp. We show that Nop depletion affects the exosome interactome, enriching components of early pre-ribosomal subunits

Results

Discussion

Experimental procedures