The Mtr4 ratchet helix and arch domain both function to promote RNA unwinding

Abstract

Mtr4 is a conserved Ski2-like RNA helicase and a subunit of the TRAMP complex that activates exosome-mediated 3′-5′ turnover in nuclear RNA surveillance and processing pathways. Prominent features of the Mtr4 structure include a four-domain ring-like helicase core and a large arch domain that spans the core. The ‘ratchet helix’ is positioned to interact with RNA substrates as they move through the helicase. However, the contribution of the ratchet helix in Mtr4 activity is poorly understood. Here we show that strict conservation along the ratchet helix is particularly extensive for Ski2-like RNA helicases compared to related helicases. Mutation of residues along the ratchet helix alters in vitro activity in Mtr4 and TRAMP and causes slow growth phenotypes in vivo. We also identify a residue on the ratchet helix that influences Mtr4 affinity for polyadenylated substrates. Previous work indicated that deletion of the arch domain has minimal effect on Mtr4 unwinding activity. We now show that combining the arch deletion with ratchet helix mutations abolishes helicase activity and produces a lethal in vivo phenotype. These studies demonstrate that the ratchet helix modulates helicase activity and suggest that the arch domain plays a previously unrecognized role in unwinding substrates.

Retinitis Pigmentosa Mutations in Bad Response to Refrigeration 2 (Brr2) Impair ATPase and Helicase Activity

“…The arginine is not within hydrogen bonding distance of the RNA and may be acting as an electrostatic shield for the highly negative RNA backbone. Structures of the close homolog Hel308 bound to DNA (16) mutations in Brr2 (17). Interestingly, the severity of phenotypes is inverted compared with Brr2 with respect to the position of the mutations along the ratchet helix.…”

“…Although analogous positions along the ratchet helix in Mtr4 and Hel308 have been structurally shown to interact directly with the nucleic acid, both proteins have different conserved amino acids in each position that result in different types of nucleic acids interactions. The analogous region in the ratchet helix of Hel308 uses tryptophan to form base-stacking interactions with its bound DNA (16), whereas the Mtr4 ratchet helix residues form hydrogen bonds with its bound RNA (17). The interactions between the Brr2 ratchet helix and U4 RNA could affect either the binding or movement of RNA through the Brr2 RNA binding channel as U4/U6 snRNAs are unwound during spliceosome assembly.…”

“…In homologous helicases of Brr2 the ratchet helix has been shown to contact the single strand of nucleic acid that helicases track along as they unwind duplex regions (16,17). Several mutations associated with the human disease RP that results in degenerative blindness are found within the ratchet helix of Brr2's catalytically active first helicase cassette.…”

“…1B) (4,14,15). The Sec63 domain has a ratchet ␣-helix that has been shown to be important for RNA binding and helicase activity in other SF2 family members (16,17). However, the importance of the ratchet helix has not yet been characterized for Brr2 RNA helicase and ATPase activity.…”

Functions and regulation of the Brr2 RNA helicase during splicing

“…However, additional domains are restricted to individual proteins or sub-groups of helicases. E.g., while the Ski2-like DNA helicase, Hel308, contains a HLH domain (which improves coupling of ATPase and unwinding activities 67 ) following the ratchet domain as also seen in Brr2, the Ski2-like RNA helicase, Mtr4, contains an arch-like domain inserted into its core (which aids in the recognition of specific substrates 68,69 and modulates helicase activity 70 ). The DEAH/RHA helicases Prp2, Prp16, Prp22 and Prp43, in contrast, contain a C-terminal OBfold, 58,59,71,72 which in Prp43 stimulates RNA binding and ATPase activity.…”