Genetic and Biochemical Characterization of Mutations in the ATPase and Helicase Regions of the Upf1 Protein

Abstract

mRNA degradation is an important control point in the regulation of gene expression and has been linked to the process of translation. One clear example of this linkage is the nonsense-mediated mRNA decay pathway, in which nonsense mutations in a gene can reduce the abundance of the mRNA transcribed from that gene. For the yeast Saccharomyces cerevisiae, the Upf1 protein (Upf1p), which contains a cysteine-and histidine-rich region and nucleoside triphosphate hydrolysis and helicase motifs, was shown to be a trans-acting factor in this decay pathway. Biochemical analysis of the wild-type Upf1p demonstrates that it has RNA-dependent ATPase, RNA helicase, and RNA binding activities. A UPF1 gene disruption results in stabilization of nonsensecontaining mRNAs, leading to the production of enough functional product to overcome an auxotrophy resulting from a nonsense mutation. A genetic and biochemical study of the UPF1 gene was undertaken in order to understand the mechanism of Upf1p function in the nonsense-mediated mRNA decay pathway. Our analysis suggests that Upf1p is a multifunctional protein with separable activities that can affect mRNA turnover and nonsense suppression. Mutations in the conserved helicase motifs of Upf1p that inactivate its mRNA decay function while not allowing suppression of leu2-2 and tyr7-1 nonsense alleles have been identified. In particular, one mutation located in the ATP binding and hydrolysis motif of Upf1p that changed the aspartic and glutamic acid residues to alanine residues (DE572AA) lacked ATPase and helicase activities, and the mutant formed a Upf1p:RNA complex in the absence of ATP; surprisingly, however, the Upf1p:RNA complex dissociated as a consequence of ATP binding. This result suggests that ATP binding, independent of its hydrolysis, can modulate Upf1p:RNA complex formation for this mutant protein. The role of the RNA binding activity of Upf1p in modulating nonsense suppression is discussed.Many studies have indicated that the processes of mRNA turnover and translation are directly linked, and understanding this relationship is critical in elucidating the mechanism of mRNA decay (13,38,43,44,50,53). cis-acting sequences that promote instability of mRNAs have been identified in 5Ј untranslated regions, protein-coding regions, and 3Ј untranslated regions of transcripts. Recent results demonstrate that ongoing translation is required for certain elements to be able to promote accelerated mRNA degradation (13,38,43,44,50). The role of translation in determining mRNA decay rates is direct. Certain instability elements identified in protein-coding regions and 3Ј untranslated regions require translation of their respective protein-coding regions in order for the decay determinants to be active (1,5,7,10,19,20,32,40,42,55,64,65).One clear example of the relationship between translation and mRNA turnover is the observation that nonsense mutations in a gene can reduce the abundance of its mRNA in a process termed nonsense-mediated mRNA decay (reviewed in references 38, 43, and 63)...

Mtt1 is a Upf1-like helicase that interacts with the translation termination factors and whose overexpression can modulate termination efficiency

“…Previous results demonstrated that the Hcs2/Hel1p/ Mtt1p exhibited 59 r 39 DNA helicase activity (Biswas et al+, 1995;Bean & Matson, 1997)+ We have observed RNA-dependent ATPase activity in vitro, supporting a role for Mtt1p in RNA-dependent processes (data not shown), demonstrating that, like the Upf1 protein from this family, Mtt1 has both DNA-and RNA-dependent ATPase activity in vitro (Czaplinski et al+, 1995, Weng et al+, 1996a, 1996b)+ A comparison of the MTT1 and UPF1 genes identified several regions of similarity+ Both proteins contain a cysteine-histidine-rich region near the amino terminal end of the protein and harbor all of the motifs characteristic of a superfamily group I helicase (Fig+ 1)+ The primary sequences of the cysteinehistidine-rich regions of UPF1 and MTT1 are not very homologous; however, it is conceivable that these cysteine-histidine-rich regions form an unidentified type of cysteine-histidine-rich protein structure+ Interestingly, mutations in the cysteine-histidine-rich region of FIGURE 6. Mtt1p is associated with polyribosomes+ Polysome extracts of cells transformed with pG-1FLAGMTT1 were prepared and centrifuged through a 7-47% linear sucrose gradient+ An identical sample was treated with RNAse A and centrifuged in an identical manner+ A 254 was monitored as gradient fractions were collected+ The absorbance profiles are located at the top of each panel+ Total protein in each fraction was TCA precipitated and analyzed by SDS-PAGE followed by western blotting using anti-FLAG M2 monoclonal antibody as described previously (Czaplinski et al+, 1995)+ UPF1 have been shown previously to increase programmed Ϫ1 frameshifting efficiencies and promote nonsense suppression (Cui et al+, 1996;Weng et al+, 1996b)+…”

“…The results presented here describe a potential cellular role for the Mtt1p, a previously characterized helicase with significant homology to the Upf1p, a factor involved in regulating both translation termination and NMD (Czaplinski et al+, 1995(Czaplinski et al+, , 1998Weng et al+, 1996aWeng et al+, , 1996bWeng et al+, , 1998+ Evidence is presented indicating that Mtt1p is a second member of the Upf1-like family of helicases and has a role in modulating translation termination+ Interestingly, comparison of the MTT1 gene with other superfamily group I helicases identified unique features that tag this as a specific subfamily of superfamily group I helicases, possibly being involved in either RNA-dependent or RNA/DNA-dependent processes (Fig+ 1)+ As will be discussed below, these results suggest that a subset of the superfamily group I (Upf1p family) of RNA helicases are involved in modulating the efficiency of the translation termination process+…”

“…Yeast media, transformations, RNA isolation, blotting, and hybridization were as described (Scheistl & Geitz 1989;Rose et al+, 1990;Hagan et al+, 1995;Weng et al+, 1996a)+ Strains constructed for this study MTT1 was disrupted from PLY22 (a his4-38 SUF1-1 ura3-52 met14 ) and PLY36 (a his4-38 SUF1-1 ura3-52 met14 upf1-2) to assay for frameshift suppression+ To assay nonsense suppression of leu2-2 and tyr7-1, strain KC2 was created by crossing PLY146 (a ura3-52 leu2-2(UGA) tyr7-1(UAG) trp1 upf1::URA3; Leeds et al+, 1991Leeds et al+, , 1992 with PLY22, selecting diploids on ϪuraϪtrp media, sporulating, and assaying random spores for auxotrophy on Ϫura, Ϫtrp, Ϫleu, and Ϫtyr media independently+ Subsequent analysis of strain KC2 (a ura3-52 leu2-2(UGA) tyr7-1(UAG) trp1 his4-38 met14 ) demonstrated that it also retained the met14 and his4-38 markers, but not SUF1-1. Deletion of UPF1 from KC2 was as described (pKOM; Cui et al+, 1995)+ To disrupt MTT1 (mtt1::hisGURA3hisG), pKOMTT1 was digested with PvuII and transformed into yeast+ To confirm the knockout of MTT1, genomic DNA was digested with XhoI and a 2+2-kb XhoIXbaI probe fragment was excised from pUC19MTT1+ Wildtype MTT1 gives a 20-kb genomic fragment, whereas mtt1::hisGURA3hisG gives an ;6+2-kb fragment+ After selection on 5-FOA to remove hisGURA3 (mtt1::hisG), the probe yields an ;3+5-kb fragment+…”

“…Previous results demonstrated that the Upf1p has a role in regulating both mRNA turnover and translation termination (Weng et al+, 1996a(Weng et al+, , 1996b(Weng et al+, , 1998Czaplinski et al+, 1998)+ The effect of mtt1⌬ on NMD was examined by monitoring the abundance of the CYH2 precursor and mature mRNA, which encodes a ribosomal protein+ The inefficiently spliced CYH2 precursor, which contains an intron near the 59 end, is a naturally occurring substrate for the nonsense-mediated mRNA decay pathway (He et al+, 1993)+ The results demonstrated that the steady-state levels of CYH2 precursor and CYH2 mRNA were equivalent to that found in a wild-type strain (Fig+ 3, compare lanes 1 and 3)+ The CYH2 precursor abundance increases in a upf1⌬ strain, which is expected in this strain, as the Upf1p is required to promote NMD (Fig+ 3, compare lanes 1 and 2)+ The abundance of the wild-type CYH2 transcript, which is not a substrate of the NMD pathway, was equivalent in all strains tested (Fig+ 3)+ Furthermore, the abundance of the transcripts that are substrates for the NMD pathway are not affected to any greater extent in a upf1⌬ mtt1⌬ strain versus only a upf1⌬ strain (Fig+ 3, compare lanes 2 and 4)+ pG-1MTT1 did not alter the levels of nonsense containing transcripts, or complement deletion of UPF1 (data not shown), demonstrating that MTT1 plays an independent role in translation termination+…”

Erratum to: Structure and Mechanisms of SF1 DNA Helicases

“…The D and E residues coordinate the ATP-associated Mg 2+ and activate the attacking water molecule, respectively [109,110]. Mutation of these residues reduces ATPase and helicase reactivity [111][112][113].…”