Molecular Basis for poly(A) RNP Architecture and Recognition by the Pan2-Pan3 Deadenylase.

The stability of eukaryotic mRNAs is dependent on a ribonucleoprotein (RNP) complex of poly(A)-binding proteins (PABPC1/Pab1) organized on the poly(A) tail. This poly(A) RNP not only protects mRNAs from premature degradation but also stimulates the Pan2-Pan3 deadenylase complex to catalyze the first step of poly(A) tail shortening. We reconstituted this process in vitro using recombinant proteins and show that Pan2-Pan3 associates with and degrades poly(A) RNPs containing two or more Pab1 molecules. The cryo-EM structure of Pan2-Pan3 in complex with a poly(A) RNP composed of 90 adenosines and three Pab1 protomers shows how the oligomerization interfaces of Pab1 are recognized by conserved features of the deadenylase and thread the poly(A) RNA substrate into the nuclease active site. The structure reveals the basis for the periodic repeating architecture at the 3' end of cytoplasmic mRNAs. This illustrates mechanistically how RNA-bound Pab1 oligomers act as rulers for poly(A) tail length over the mRNAs' lifetime.

Concerted structural rearrangements enable RNA channeling into the cytoplasmic Ski238-Ski7-exosome assembly.

The Ski2-Ski3-Ski8 (Ski238) helicase complex directs cytoplasmic mRNAs toward the nucleolytic exosome complex for degradation. In yeast, the interaction between Ski238 and exosome requires the adaptor protein Ski7. We determined different cryo-EM structures of the Ski238 complex depicting the transition from a rigid autoinhibited closed conformation to a flexible active open conformation in which the Ski2 helicase module has detached from the rest of Ski238. The open conformation favors the interaction of the Ski3 subunit with exosome-bound Ski7, leading to the recruitment of the exosome. In the Ski238-Ski7-exosome holocomplex, the Ski2 helicase module binds the exosome cap, enabling the RNA to traverse from the helicase through the internal exosome channel to the Rrp44 exoribonuclease. Our study pinpoints how conformational changes within the Ski238 complex regulate exosome recruitment for RNA degradation. We also reveal the remarkable conservation of helicase-exosome RNA channeling mechanisms throughout eukaryotic nuclear and cytoplasmic exosome complexes.

The yeast ski complex: crystal structure and RNA channeling to the exosome complex.

The Ski complex is a conserved multiprotein assembly required for the cytoplasmic functions of the exosome, including RNA turnover, surveillance, and interference. Ski2, Ski3, and Ski8 assemble in a tetramer with 1:1:2 stoichiometry. The crystal structure of an S. cerevisiae 370 kDa core complex shows that Ski3 forms an array of 33 TPR motifs organized in N-terminal and C-terminal arms. The C-terminal arm of Ski3 and the two Ski8 subunits position the helicase core of Ski2 centrally within the complex, enhancing RNA binding. The Ski3 N-terminal arm and the Ski2 insertion domain allosterically modulate the ATPase and helicase activities of the complex. Biochemical data suggest that the Ski complex can thread RNAs directly to the exosome, coupling the helicase and the exoribonuclease through a continuous RNA channel. Finally, we identify a Ski8-binding motif common to Ski3 and Spo11, rationalizing the moonlighting properties of Ski8 in mRNA decay and meiosis.

Structure of a Cytoplasmic 11-Subunit RNA Exosome Complex.

The RNA exosome complex associates with nuclear and cytoplasmic cofactors to mediate the decay, surveillance, or processing of a wide variety of transcripts. In the cytoplasm, the conserved core of the exosome (Exo10) functions together with the conserved Ski complex. The interaction of S. cerevisiae Exo10 and Ski is not direct but requires a bridging cofactor, Ski7. Here, we report the 2.65 Å resolution structure of S. cerevisiae Exo10 bound to the interacting domain of Ski7. Extensive hydrophobic interactions rationalize the high affinity and stability of this complex, pointing to Ski7 as a constitutive component of the cytosolic exosome. Despite the absence of sequence homology, cytoplasmic Ski7 and nuclear Rrp6 bind Exo10 using similar surfaces and recognition motifs. Knowledge of the interacting residues in the yeast complexes allowed us to identify a splice variant of human HBS1-Like as a Ski7-like exosome-binding protein, revealing the evolutionary conservation of this cytoplasmic cofactor.

The molecular architecture of the TRAMP complex reveals the organization and interplay of its two catalytic activities.

The TRAMP complex is involved in the nuclear surveillance and turnover of noncoding RNAs and intergenic transcripts. TRAMP is associated with the nuclear exosome and consists of a poly(A)polymerase subcomplex (Trf4-Air2) and a helicase (Mtr4). We found that N-terminal low-complexity regions of Trf4 and Air2 bind Mtr4 in a cooperative manner. The 2.4 Å resolution crystal structure of the corresponding ternary complex reveals how Trf4 and Air2 wrap around the DExH core of the helicase. Structure-based mutations on the DExH core impair binding to Trf4 and Air2, and also to Trf5 and Air1. The combination of structural, biochemical, and biophysical data suggests that the poly(A)polymerase core of Trf4-Air2 is positioned below the base of the helicase, where the unwound 3' end of an RNA substrate is expected to emerge. The results reveal conceptual similarities between the two major regulators of the exosome, the nuclear TRAMP and cytoplasmic Ski complexes.

Distinct and evolutionary conserved structural features of the human nuclear exosome complex.

The nuclear RNA exosome complex mediates the processing of structured RNAs and the decay of aberrant non-coding RNAs, an important function particularly in human cells. Most mechanistic studies to date have focused on the yeast system. Here, we reconstituted and studied the properties of a recombinant 14-subunit human nuclear exosome complex. In biochemical assays, the human exosome embeds a longer RNA channel than its yeast counterpart. The 3.8 Å resolution cryo-EM structure of the core complex bound to a single-stranded RNA reveals that the RNA channel path is formed by two distinct features of the hDIS3 exoribonuclease: an open conformation and a domain organization more similar to bacterial RNase II than to yeast Rrp44. The cryo-EM structure of the holo-complex shows how obligate nuclear cofactors position the hMTR4 helicase at the entrance of the core complex, suggesting a striking structural conservation from lower to higher eukaryotes.

Ser/Thr/Tyr protein phosphorylation in the archaeon Halobacterium salinarum--a representative of the third domain of life.

In the quest for the origin and evolution of protein phosphorylation, the major regulatory post-translational modification in eukaryotes, the members of archaea, the "third domain of life", play a protagonistic role. A plethora of studies have demonstrated that archaeal proteins are subject to post-translational modification by covalent phosphorylation, but little is known concerning the identities of the proteins affected, the impact on their functionality, the physiological roles of archaeal protein phosphorylation/dephosphorylation, and the protein kinases/phosphatases involved. These limited studies led to the initial hypothesis that archaea, similarly to other prokaryotes, use mainly histidine/aspartate phosphorylation, in their two-component systems representing a paradigm of prokaryotic signal transduction, while eukaryotes mostly use Ser/Thr/Tyr phosphorylation for creating highly sophisticated regulatory networks. In antithesis to the above hypothesis, several studies showed that Ser/Thr/Tyr phosphorylation is also common in the bacterial cell, and here we present the first genome-wide phosphoproteomic analysis of the model organism of archaea, Halobacterium salinarum, proving the existence/conservation of Ser/Thr/Tyr phosphorylation in the "third domain" of life, allowing a better understanding of the origin and evolution of the so-called "Nature's premier" mechanism for regulating the functional properties of proteins.

Single step protocol to purify recombinant proteins with low endotoxin contents.

Endotoxin is an unwanted by product of recombinant proteins purified from Escherichia coli. The inherent toxicity of endotoxins makes their removal an important step for the proteins' application in several biological assays and for safe parenteral administration. The method described in this paper is a one-step protocol which is effective at removing tightly bound endotoxin from over-expressed tagged proteins in E. coli. We combined affinity chromatography with a non-ionic detergent washing step, to remove most of the endotoxin contaminants from the end product. An endotoxin reduction of less than 4 to 0.2 EU mg(-1) was achieved with protein recovery close to a yield 100%. As this new protocol requires only one step to simultaneously purify tagged proteins and eliminate endotoxins, it represents a substantial advantage in time, effort, and expense.