Structural and functional characterization of the yeast Ski2-Ski3-Ski8 complex

The Ski2-Ski3-Ski8 (SKI) complex is a conserved multi-protein
assembly required for the cytoplasmic functions of the exosome,
including messenger RNA (mRNA) turnover, surveillance and
interference. The helicase Ski2, the tetratricopeptide repeat (TPR)
protein Ski3 and the �-propeller Ski8 assemble in a heterotetramer
with 1:1:2 stoichiometry. While the function of the Ski2-Ski3-Ski8
complex as a general cofactor of the cytoplasmic exosome has been
well established, it remains largely unclear how it contributes to
the regulation of the exosome. The PhD thesis at hand addresses
this question by investigating the structural and biochemical
properties of the Ski2-Ski3-Ski8 complex. Solving the crystal
structure of the 113 kDa helicase region of S. cerevisiae Ski2 by
experimental phasing revealed the presence of a canonical DExH core
and an atypical accessory domain that is inserted in the helicase
core. This insertion domain binds ribonucleic acid (RNA)
unspeci�cally and is located at the RNA entry site of the helicase
core. The overall architecture of Ski2 including the presence of an
accessory domain is similar to the structure of the related
helicase Mtr4, but the structural and biochemical properties of the
accessory domains from both proteins are di�erent. The Ski2
insertion domain is not required for formation of the
Ski2-Ski3-Ski8 complex. Its removal allowed to crystallize a
Ski2�insert-Ski3-Ski8 complex from S. cerevisiae, and the crystal
structure of this 370 kDa core complex was determined
experimentally. It shows that Ski3 forms an array of 33 TPR motifs,
creating a sca�old for the other subunits. Ski3 and the two Ski8
subunits bind the helicase core of Ski2 and position it centrally
within the complex. This creates an extended internal RNA channel
and modulates the enzymatic properties of the Ski2 helicase. Both
Ski8 subunits are bound through a structurally conserved motif. A
similar motif is present and functional in yeast Spo11, a protein
that initiates deoxyribonucleic acid (DNA) double strand breaks
during meiotic recombination. Association of Ski8 to either complex
is mutually exclusive, rationalizing how Ski8 can perform its two
distinct roles in mRNA metabolism and meiotic recombination.
Biochemical studies suggest that the SKI complex can thread RNAs
directly to the exosome, coupling the helicase and the
exoribonuclease through a continuous channel of 43-44 nucleotides
length. Finally, an internal regulatory mechanism in the
Ski2-Ski3-Ski8 complex was identi�ed. Both the Ski2-insertion
domain and the Ski3 N-terminus cooperate to inhibit ATPase and
helicase activity of Ski2 when bound in the SKI complex. Thus, the
SKI complex regulates exosome activity in two ways. First by a
direct substrate channeling mechanism to the exosome that connects
helicase and nuclease activities of both complexes which may
activate the exosome towards certain substrates. Second, by an
inhibitory mechanism that regulates substrate access to the
helicase complex, which is a prerequisite for controlling the
exosome's substrate speci�city. This doctoral thesis provides the
�rst structural description of the entire yeast SKI complex and
identi�es two mechanisms that may contribute to regulation of the
activity of the cytoplasmic exosome.