Reconstitution of the complete pathway of ITS2 processing at the pre-ribosome

Lisa Fromm,Elena Conti,Sebastian Falk,Jan Michael Schuller,Ed Hurt,Dirk Flemming,Matthias Thoms

doi:10.1038/s41467-017-01786-9

Lisa Fromm, Elena Conti + Show 5 more

Open Access

https://doi.org/10.1038/s41467-017-01786-9

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Abstract

Removal of internal transcribed spacer 2 (ITS2) from pre-ribosomal RNA is essential to make functional ribosomes. This complicated processing reaction begins with a single endonucleolytic cleavage followed by exonucleolytic trimming at both new cleavage sites to generate mature 5.8S and 25S rRNA. We reconstituted the 7S→5.8S processing branch within ITS2 using purified exosome and its nuclear cofactors. We find that both Rrp44’s ribonuclease activities are required for initial RNA shortening followed by hand over to the exonuclease Rrp6. During the in vitro reaction, ITS2-associated factors dissociate and the underlying ‘foot’ structure of the pre-60S particle is dismantled. 7S pre-rRNA processing is independent of 5S RNP rotation, but 26S→25S trimming is a precondition for subsequent 7S→5.8S processing. To complete the in vitro assay, we reconstituted the entire cycle of ITS2 removal with a total of 18 purified factors, catalysed by the integrated activities of the two participating RNA-processing machines, the Las1 complex and nuclear exosome.

Highlights

Removal of internal transcribed spacer 2 (ITS2) from pre-ribosomal RNA is essential to make functional ribosomes
This complicated process starts in the nucleolus with transcription of a large ribosomal RNA precursor, called 35S in yeast and 47S pre-rRNA in humans, which consists of mature 18S, 5.8S and 25S/28S rRNA, and intervening RNA sequences, 5′ and 3′ external transcribed spacers (5′-ETS, 3′-ETS), and internal transcribed spacers 1 and 2 (ITS1, ITS2) that are removed at distinct points of the pathway[1, 2]
In our in vitro assays, we observed that the majority of 7S prerRNA is processed to 5.8S rRNA, but a minor signal pool is detected above the 5.8S signal

Summary

Introduction

Removal of internal transcribed spacer 2 (ITS2) from pre-ribosomal RNA is essential to make functional ribosomes. Eukaryotic ribosomes are formed through a series of consecutive assembly and maturation reactions, which include folding, modification and processing of the pre-rRNA, and incorporation of ~80 ribosomal proteins, which leads to mature and functional ribosomes This complicated process starts in the nucleolus with transcription of a large ribosomal RNA precursor, called 35S in yeast and 47S pre-rRNA in humans, which consists of mature 18S, 5.8S and 25S/28S rRNA, and intervening RNA sequences, 5′ and 3′ external transcribed spacers (5′-ETS, 3′-ETS), and internal transcribed spacers 1 and 2 (ITS1, ITS2) that are removed at distinct points of the pathway[1, 2]. This provides a rationale for why ribosomes carrying 6S pre-rRNA are functional[2] (see Supplementary Fig. 1)

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