The essential protein Mtr4 is a conserved Ski2-like RNA helicase that maintains the integrity of nuclear RNA by promoting the 3' end decay of a wide variety of RNA substrates. Mtr4 activates the multi-protein exosome in RNA processing, surveillance, and turnover pathways by unwinding secondary structure and/or displacing associated proteins from RNA substrates. While Mtr4 may be able to promote decay independently, it is often associated with large multi-protein assemblies. Specifically, Mtr4 is the largest member of the TRAMP (Trf4/Air2/Mtr4 polyadenylation) complex which targets a plethora of RNA substrates for degradation by appending them with small (~5nt) poly(A) tails via the polymerase activity of Trf4. Mtr4 preferentially binds and unwinds RNAs with short poly(A) tails. Notably, the mechanism by which Mtr4 recognizes the length and identity of the RNA 3' end is coupled to the modulation of poly(A) polymerase activity of Trf4. The lack of structural data for Mtr4 and associated complexes severely limits the understanding of Mtr4 function. Particularly, it is unclear how Mtr4 senses RNA features, acts on RNA substrates, delivers RNA substrates to the exosome, and assembles into larger protein complexes. Presented here is the x-ray crystal structure of Mtr4 combined with detailed structural and biochemical analysis of the enzyme. The structure reveals that Mtr4 contains a four domain helicase core that is conserved in other RNA helicases and a unique arch-like RNA binding domain that is required for the in vivo processing of 5.8S rRNA. Furthermore, kinetic and in vivo analysis of conserved residues implicated in the poly(A) sensing mechanism demonstrates that ratchet helix residues regulate RNA unwinding and impact RNA sequence specificity. A comparison of the apo Mtr4 structure with the RNA/ADP bound structure (determined elsewhere) provides a view of the range of motion that individual domains of Mtr4 adopt upon substrate binding as well as the possible conformations that occur during RNA translocation. These studies provide an important framework for understanding the fundamental role of Mtr4 in exosome-mediated RNA decay, and more broadly describe common themes in architecture and function of the Ski2-like helicase family.

There are numerous RNAs transcribed in the cell that are not directly involved in protein translation. Maintaining proper levels of RNA is crucial for cell viability, making RNA surveillance an essential process (equivalent to regulating protein levels). Mtr4 is an essential RNA helicase that activates exosome-mediated 3'-5' turnover in RNA processing mechanisms. Mtr4 has several binding partners, with the most prominent one being the complex Trf4/5-Air1/2-Mtr4 polyadenylating (TRAMP) complex. The polyadenylation and unwinding activity of TRAMP is modulated by a sensing mechanism in Mtr4 that detects both length and identity of 3'-end poly(A) tails. While it has been known that Mtr4 has an unwinding preference for substrates with a 3' poly(A) tail and a length of approximately 5 nucleotides, the mechanistic detail is unclear. It is also unclear what structural features of Mtr4 contribute to this sensing function. By using x-ray crystal structures of Mtr4, a ratchet helix was identified to interact with RNA substrates. Significant conservation of this ratchet helix along the RNA binding path was observed, similar to conservation patterns throughout Ski2-like and DEAH/RHA-box helicases. Structural characterization revealed a novel arch domain shown to bind structured RNAs, which may aid in cooperative RNA recognition in conjunction with the ratchet helix. In this thesis we demonstrate that the conserved residues at the third (R1030) and fourth (E1033) turns of the Mtr4 ratchet helix uniquely influence RNA unwinding rates. Furthermore, when mutated, ratchet helix positions confer slow growth phenotypes to Saccharomyces cerevisiae and are synthetically lethal in an Mtr4-archless background. The unwinding activity of these mutants when in the TRAMP complex alters the unwinding rates of Mtr4, and in some instances recovers substrate specificity. Our findings demonstrate the importance of R1030 and E1033 for helicase activity, and additionally link the arch domain of Mtr4 in essential unwinding events.

RNA is a molecular messenger of the cell, essential to many cellular pathways and processes. In order to maintain functionality, RNA is processed and modified by protein complexes such as the exosome and associated proteins. The exosome-mediated RNA processing or degradation both require a Ski-2 like helicase to function. One such helicase is the Frequency-interacting RNA Helicase (FRH), an essential RNA helicase from Neurospora Crassa. FRH is homologous to the Saccharomyces cerevisiae Mtr4 from the Ski2-like family of RNA helicases. Sequence alignments between FRH and Ski2-like family helicases predicted FRH to share the helicase core domains and the inserted arch domain a characteristic of the Mtr4-like proteins in this protein family. FRH is also a main component of the circadian oscillation pathway in N. crassa. The participation of FRH in circadian oscillation is not a shared role across RNA helicases. FRH forms a link between two major cellular pathways providing a unique system to study RNA surveillance. Here we present the 3.51Å and 3.25Å crystal structures of FRH which supports structural prediction by maintaining the core architecture found in Ski2-like helicases. These similarities are accompanied by significant flexibility of the arch domain and revealed a unique homodimer. Other known Ski2-like helicases have not been observed to form dimers and function biologically as monomers. Furthermore, the initial characterization of helicase activity of FRH on a poly-adenylated RNA substrate is presented. Also explored is the evidence of a dimer through crosslinking and size exclusion chromatography assays.

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