Structure of the nuclear exosome component Rrp6p

reveals an interplay between the active site and the HRDC domain

Søren F. Midtgaard*†, Jannie Assenholt†, Anette Thyssen Jonstrup*†, Lan B. Van*†, Torben Heick Jensen†, and Ditlev E. Brodersen*†‡*Centre for Structural Biology, Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10c, DK-8000 Aarhus C, Denmark; and †Centre formRNP Biogenesis and Metabolism, Department of Molecular Biology, University of Aarhus, C. F. Møllers Alle´ , bygn. 130, DK-8000 Aarhus C, Denmark

PNAS., Vol. 103, pp.11898-11903, 2006

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INTRODUCTION3 additional proteins to yeast nuclear core exosome:Rrp6p (human PM/Scl-100):RNase D-type enzymeMtr4p : DEAD-box RNA helicaseRrp47 : less well characterized

Trends Biochem Sci., 2002,27,11-18.

Rrp47

The nuclear exosome is essential for maturation of eukaryotic ribosomal RNAs.

Trends Cell Biol., 2002,12,90-96.

Many small nucleolar RNAs (snoRNAs) depend on the nuclear exosome during their maturation.

Deletion of Rrp6p in yeast also leads to accumulation of extended forms of both polycistronic snoRNAs and the independently transcribed snoRNAs,

The EMBO Journal ,1999,18,5399–5410.

Mol. Cell. Biol.,, 2000, 20, 441-452.

Domain structure of yeast Rrp6p

RNase D domain

RNase D domain : similar to bacterial RNase D, DEDD nucleases, DNA/RNA

DEDD nucleases : four conserved acidic residues required for nucleic acid degradation in the 3’-5’ direction (ex. PARN, Klenow fragment ), two divalent metal ion, DEDDy subgroup

HRDC domain : helicase and RNase D C-terminal domain, a putative nucleic acids binding domain, specificity

N-terminal domain : absent in bacterial RNase D

METHODS AND RESULTSProtein construct used in crystallization : 129-536 (Y361A)

Overall structure:

Rrp6p Requires both Mn2+ and Zn2+ to Bind Nucleotides.

Binding of a nucleotide in the active site is associated with a closure of the active-site cleft.

Rrp6p Rrp6p-AMP

Recognition of the 3’ Ribonucleotide.

RNA Processing by Rrp6p Is Affected by N-Terminal Anchoring of the HRDC Domain.

D457A mutation rendered Rrp6p incapable of proper 3'-end trimming of stable RNAs while retaining the exonucleolytic activity.

The N-terminal-HRDC interaction

The importance of the D457–Q133/N142 interaction on Rrp6p RNA-processing efficiency.

Correct anchoring of the HRDC domain is required to maintain efficient processing of some substrates by Rrp6p but that the defects in RNA processing are not rate-limiting for growth.

SUMMARY AND DISCUSSION1. The only characterized structure of a eukaryotic exosome comp

onent.2. Structural homology to the prokaryotic RNase D enzyme.

N-terminal extension : may participate in anchoring of Rrp6p to core exosome.

3. Substrate specificity : H241 ( N-terminal domain ) K342 ( RNase D domain )

4. The exonuclease-HRDC contact affects processing of some substrates by Rrp6p, such as snR40.