Intra- and Intermolecular Regulatory Interactions in Upf1, the RNA Helicase Central to Nonsense-Mediated mRNA Decay in Yeast

Abstract

RNA helicases are involved in almost every aspect of RNA metabolism, yet very little is known about the regulation of this class of enzymes. In Saccharomyces cerevisiae, the stability and translational fidelity of nonsense-containing mRNAs are controlled by the group I RNA helicase Upf1 and the proteins it interacts with, Upf2 and Upf3. Combining the yeast two-hybrid system with genetic analysis, we show here that the cysteine-and histidine-rich (CH) domain and the RNA helicase domain of yeast Upf1 can engage in two new types of molecular interactions: an intramolecular interaction between these two domains and self-association of each of these domains. Multiple observations indicate that these molecular interactions are crucial for Upf1 regulation. First, coexpression of the CH domain and the RNA helicase domain in trans can reconstitute Upf1 function in both promoting nonsense-mediated mRNA decay (NMD) and preventing nonsense suppression. Second, mutations that disrupt Upf1 intramolecular interaction cause loss of Upf1 function. These mutations weaken Upf2 interaction and, surprisingly, promote Upf1 selfassociation. Third, the genetic defects resulting from deficiency in Upf1 intramolecular interaction or RNA binding are suppressed by expression of Upf2. Collectively, these data reveal a set of sequential molecular interactions and their roles in regulating Upf1 function during activation of NMD and suggest that cis intramolecular interaction and trans self-association may be general mechanisms for regulation of RNA helicase functions. E ukaryotic cells have evolved multiple quality control mechanisms to ensure the fidelity of gene expression (1-3). One of these mechanisms, nonsense-mediated mRNA decay (NMD), which operates during mRNA translation, targets transcripts containing a premature termination codon (PTC) (4). This mRNA decay pathway ensures rapid degradation of PTC-containing transcripts and thus prevents the cell from accumulating truncated and potentially deleterious polypeptides (5, 6). NMD also targets a subset of functionally relevant wild-type mRNAs (7-9), suggesting that this decay pathway has a substantial role in posttranscriptional gene regulation and likely controls important cellular functions.From yeast to humans, NMD requires a set of conserved regulatory factors, the Upf proteins: Upf1, Upf2, and Upf3 (4, 7). These factors interact with each other and appear to constitute the core NMD machinery in eukaryotic cells (10-13). Deletion or silencing of each of the genes encoding these factors selectively stabilizes PTC-containing transcripts and other NMD substrates (9, 11, 13-15). In multicellular organisms, NMD also requires additional regulatory factors, including Smg1 and Smg5 to Smg7 (4, 7). These factors control Upf1 phosphorylation and dephosphorylation, a cycle that, in turn, controls several important Upf1 functions during NMD, including translation repression (16), remodeling of terminating messenger ribonucleoprotein particles (mRNPs) (17), and recruitment of the decay enzymes (1...

Control of mRNA decapping by positive and negative regulatory elements in the Dcp2 C-terminal domain

“…S4C), indicating that the observed Dcp2:Upf1 interaction is not bridged by any of these factors. This region of Upf1 is also involved in Upf2 binding (He et al 1997) and self-association (He et al 2013), suggesting that it may play a role in sequential molecular interactions during activation of NMD.…”

“…Since both Edc3 and Upf1 can self-associate (Decker et al 2007;He et al 2013), we utilized two-hybrid tester strains with and without complete deletions of EDC3 or UPF1. Full-length Edc3 interacted strongly with Dcp2 in edc3Δ cells and this interaction was eliminated by deletion of Edc3's Lsm domain and substantially diminished by deletion of the YjeF-N domain (Supplemental Fig.…”

“…The occurrence of functions independent of Dcp2 interaction, and the ability of these factors to bind directly to their substrate mRNAs (Chowdhury et al 2007;Johansson et al 2007;He et al 2014), suggests that the assembly of the active decapping complexes most likely occurs on mRNAs and that Edc3 and Upf1 function in recruiting the decapping enzyme to their targeted mRNAs. Since both Edc3 and Upf1 self-associate and the self-association of these factors appears to be critical for their respective decay activities (He et al 2013(He et al , 2014, it is possible that the presumptive upstream functions for Edc3 and Upf1 are carried out by distinct Edc3 or Upf1 molecules other than those in complexes with the decapping enzyme. In this scenario, the decapping enzyme would be recruited to the respective decay substrates by either Edc3 or Upf1 dimerization.…”

“…Plasmids harboring the upf1::HIS3, dcp2::HIS3, dcp2-N245::Kan MX6, and edc3::URA3 alleles, or containing UPF1 fragments fused to either the GAL4 DNA-binding domain (DB) or activation domain (AD), or containing EDC3 fragments fused to GAL4-AD were described previously (He et al 1997(He et al , 2013(He et al , 2014. Details of plasmid construction are described in Supplemental Information.…”

Regulation of Natural mRNAs by the Nonsense-Mediated mRNA Decay Pathway

“…Upf2p and Upf3p are responsible for regulating Upf1p function. Upf1p is a group 1 RNA helicase with ATPase activity (24)(25)(26). S. cerevisiae Upf1p has been shown to interact with Upf2p, which in turn interacts with Upf3p (18,27).…”