Structural insights into the interaction of the nuclear exosome helicase Mtr4 with the preribosomal protein Nop53

Abstract

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 exosomeassociated helicases thus appear to have evolved from the KOW domains of ribosomal proteins and to function as RNPbinding modules in the context of the nuclear exosome.

Cryo-EM structure of an early precursor of large ribosomal subunit reveals a half assembled intermediate

“…The foot structure is similar with that in the nucleoplasmic pre-60S structures (16)(17)(18), except that Nop53 is not present ( Figure 3B). Nop53 recruits the exosome for processing 7S pre-rRNA (29,30). Nop53 is of low abundance in Rpf1-TAP particle ( Figure S1) and should associate at a later stage.…”

Substrate Specificity of the TRAMP Nuclear Surveillance Complexes

“…2C). This might not have been anticipated, since the arch domain was implicated in targeting Mtr4 to degrade specific pre-rRNA spacer regions that are independent of TRAMP 15, 16 . Higher correlations between different TRAMP components were observed for oligo(A) RNAs than considering all reads (Figs.…”

“…However, a more likely interaction is between Air2 and the Arch. Indeed, in the crystal structure, the N-terminus from the adjacent Air2 molecule in the crystal lattice was shown to contact the Mtr4 Arch at the same sites as the AIM of Nop53 15, 22 , through residues that are conserved in Air1. Association of Air2 and Air1 with the Arch domain, might require conformational changes in which the Arch moves toward the helicase core, switching Mtr4 (and the TRAMP) to a closed conformation.…”

“…These results strongly indicate that substrate specificity can be provided by Trf4 and Trf5, while Air1 and Air2 appear to be largely interchangeable. This was unexpected, since it had previously been anticipated that RNA binding specificity would largely be determined by Air1 and Air2, which are Zn-knuckle RNA BPs 26, 40 , or by specific interactions of AIM-containing proteins with the Arch domain of Mtr4 15, 16 .…”

“…It was previously suggested that TRAMP recruitment to substrates would be driven by Air1/2 through their RNA binding activities 4, 11 (reviewed in 12 ). In addition, Mtr4 includes a distinctive “Arch” or “KOW” domain, consisting of a beta-barrel stalk inserted into the typical DExH core 13–15 . The Arch domain is specifically bound by ribosomal biogenesis factors carrying an A rch Interacting M otif (AIM), enabling direct Mtr4 recruitment to pre-rRNAs 16 .…”