Sebastian Falk

The exosome-binding factors Rrp6 and Rrp47 form a composite surface for recruiting the Mtr4 helicase.

The exosome is a conserved multi‐subunit ribonuclease complex that functions in 3′ end processing, turnover and surveillance of nuclear and cytoplasmic RNAs. In the yeast nucleus, the 10‐subunit core complex of the exosome (Exo‐10) physically and functionally interacts with the Rrp6 exoribonuclease and its associated cofactor Rrp47, the helicase Mtr4 and Mpp6. Here, we show that binding of Mtr4 to Exo‐10 in vitro is dependent upon both Rrp6 and Rrp47, whereas Mpp6 binds directly and independently of other cofactors. Crystallographic analyses reveal that the N‐terminal domains of Rrp6 and Rrp47 form a highly intertwined structural unit. Rrp6 and Rrp47 synergize to create a composite and conserved surface groove that binds the N‐terminus of Mtr4. Mutation of conserved residues within Rrp6 and Mtr4 at the structural interface disrupts their interaction and inhibits growth of strains expressing a C‐terminal GFP fusion of Mtr4. These studies provide detailed structural insight into the interaction between the Rrp6–Rrp47 complex and Mtr4, revealing an important link between Mtr4 and the core exosome.

The conformational plasticity of eukaryotic RNA‐dependent ATPases

RNA helicases are present in all domains of life and participate in almost all aspects of RNA metabolism, from transcription and processing to translation and decay. The diversity of pathways and substrates that they act on is reflected in the diversity of their individual functions, structures, and mechanisms. However, RNA helicases also share hallmark properties. At the functional level, they promote rearrangements of RNAs and RNP particles by coupling nucleic acid binding and release with ATP hydrolysis. At the molecular level, they contain two domains homologous to the bacterial RecA recombination protein. This conserved catalytic core is flanked by additional domains, which typically regulate the ATPase activity in cis. Binding to effector proteins targets or regulates the ATPase activity in trans. Structural and biochemical studies have converged on the plasticity of RNA helicases as a fundamental property that is used to control their timely activation in the cell. In this review, we focus on the conformational regulation of conserved eukaryotic RNA helicases.

The RNA helicase FRH is an ATP-dependent regulator of CK1a in the circadian clock of Neurospora crassa

The Neurospora clock protein FRQ forms a complex with casein kinase 1a (CK1a) and FRH, a DEAD box-containing RNA helicase with a clock-independent essential function in RNA metabolism. In the course of a circadian period, FRQ is progressively hyperphosphorylated and eventually degraded. Timed hyperphosphorylation of FRQ is crucial for timekeeping of the clock. Here we show that the ATPase activity of FRH attenuates the kinetics of CK1a-mediated hyperphosphorylation of FRQ. Hyperphosphorylation of FRQ is strictly dependent on site-specific recruitment of a CK1a molecule that is activated upon binding. The FRH ATPase cycle regulates the access of CK1a to phosphorylation sites in FRQ in cis, suggesting that FRH is an ATP-dependent remodelling factor acting on the protein complex. We show that the affinity of CK1a for FRQ decreases with increasing FRQ phosphorylation, suggesting functional inactivation of FRQ in the negative feedback loop of the circadian clock before and independent of its degradation.

Mpp6 Incorporation in the Nuclear Exosome Contributes to RNA Channeling through the Mtr4 Helicase

Summary The RNA-degrading exosome mediates the processing and decay of many cellular transcripts. In the yeast nucleus, the ubiquitous 10-subunit exosome core complex (Exo-9–Rrp44) functions with four conserved cofactors (Rrp6, Rrp47, Mtr4, and Mpp6). Biochemical and structural studies to date have shed insights into the mechanisms of the exosome core and its nuclear cofactors, with the exception of Mpp6. We report the 3.2-Å resolution crystal structure of a S. cerevisiae Exo-9–Mpp6 complex, revealing how linear motifs in the Mpp6 middle domain bind Rrp40 via evolutionary conserved residues. In particular, Mpp6 binds near a tryptophan residue of Rrp40 that is mutated in human patients suffering from pontocerebellar hypoplasia. Using biochemical assays, we show that Mpp6 is required for the ability of Mtr4 to extend the trajectory of an RNA entering the exosome core, suggesting that it promotes the channeling of substrates from the nuclear helicase to the processive RNase.

Structure of the RBM7–ZCCHC8 core of the NEXT complex reveals connections to splicing factors

The eukaryotic RNA exosome participates extensively in RNA processing and degradation. In human cells, three accessory factors (RBM7, ZCCHC8 and hMTR4) interact to form the nuclear exosome targeting (NEXT) complex, which directs a subset of non-coding RNAs for exosomal degradation. Here we elucidate how RBM7 is incorporated in the NEXT complex. We identify a proline-rich segment of ZCCHC8 as the interaction site for the RNA-recognition motif (RRM) of RBM7 and present the crystal structure of the corresponding complex at 2.0 Å resolution. On the basis of the structure, we identify a proline-rich segment within the splicing factor SAP145 with strong similarity to ZCCHC8. We show that this segment of SAP145 not only binds the RRM region of another splicing factor SAP49 but also the RRM of RBM7. These dual interactions of RBM7 with the exosome and the spliceosome suggest a model whereby NEXT might recruit the exosome to degrade intronic RNAs.

Structural insights into the interaction of the nuclear exosome helicase Mtr4 with the pre-ribosomal protein Nop53

The nuclear exosome and the associated RNA helicase Mtr4 participate in the processing of several ribonucleoprotein particles (RNP), including the maturation of the large ribosomal subunit (60S). S. cerevisiae Mtr4 interacts directly with Nop53, a ribosomal biogenesis factor present in late pre-60S particles containing precursors of the 5.8S rRNA. The Mtr4-Nop53 interaction plays a pivotal role in the maturation of the 5.8S rRNA, providing a physical link between the nuclear exosome and the pre-60S RNP. An analogous interaction between Mtr4 and another ribosome biogenesis factor, Utp18, directs the exosome to an earlier preribosomal particle. Nop53 and Utp18 contain a similar Mtr4-binding motif known as the arch-interacting motif (AIM). Here, we report the 3.2 Å resolution crystal structure of S. cerevisiae Mtr4 bound to the interacting region of Nop53, revealing how the KOW domain of the helicase recognizes the AIM sequence of Nop53 with a network of hydrophobic and electrostatic interactions. The AIM-interacting residues are conserved in Mtr4 and are not present in the related cytoplasmic helicase Ski2, rationalizing the specificity and versatility of Mtr4 in the recognition of different AIM-containing proteins. Using nuclear magnetic resonance (NMR), we show that the KOW domain of Mtr4 can simultaneously bind an AIM-containing protein and a structured RNA at adjacent surfaces, suggesting how it can dock onto RNPs. The KOW domains of exosome-associated helicases thus appear to have evolved from the KOW domains of ribosomal proteins and to function as RNP-binding modules in the context of the nuclear exosome.

Das RNA-Exosom – eine molekulare Maschine für den RNA-Abbau

The exosome is an evolutionary conserved macromolecular complex that degrades RNAs from the 3′ end. It is present both in the cytoplasm and the nucleus, where it degrades different types of RNAs in yeast, human cells and other eukaryotes studied to date. The exosome is involved in the turnover of mRNAs, ncRNA and tRNAs, but also in the surveillance of faulty pre-mRNAs. In addition to its degradative function it is also involved in the controlled processing of e. g. rRNAs.