Isolation and characterization of Dcp1p, the yeast mRNA decapping enzyme

AbstractNonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway involved in many cellular pathways and crucial for telomere maintenance and embryo development. Core NMD factors Upf1, Upf2 and Upf3 are conserved from yeast to mammals, but a universal NMD model is lacking. We used affinity purification coupled with mass spectrometry and an improved data analysis protocol to obtain the first large-scale quantitative characterization of yeast NMD complexes in yeast (112 experiments). Unexpectedly, we identified two distinct complexes associated with Upf1: Detector (Upf1/2/3) and Effector. Effector contained the mRNA decapping enzyme, together with Nmd4 and Ebs1, two proteins that globally affected NMD and were critical for RNA degradation mediated by the Upf1 C-terminal helicase region. The fact that Nmd4 association to RNA was dependent on Detector components and the similarity between Nmd4/Ebs1 and mammalian Smg5-7 proteins suggest that in all eukaryotes NMD operates through successive Upf1-bound Detector and Effector complexes.

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Functional characterization of the mammalian mRNA decapping enzyme hDcp2

RNA ◽

10.1261/rna.5690503 ◽

2003 ◽

Vol 9 (9) ◽

pp. 1138-1147 ◽

Cited By ~ 80

Author(s):

C. PICCIRILLO

Keyword(s):

Functional Characterization ◽

Mrna Decapping ◽

Decapping Enzyme

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Characterization of a Second Vaccinia Virus mRNA-Decapping Enzyme Conserved in Poxviruses

Journal of Virology ◽

10.1128/jvi.01668-07 ◽

2007 ◽

Vol 81 (23) ◽

pp. 12973-12978 ◽

Cited By ~ 63

Author(s):

Susan Parrish ◽

Bernard Moss

Keyword(s):

Vaccinia Virus ◽

Point Mutations ◽

Mrna Turnover ◽

Lower Sensitivity ◽

Nudix Hydrolase ◽

Viral Mrnas ◽

Mrna Decapping ◽

Optimal Activity ◽

Decapping Enzyme

ABSTRACT Vaccinia virus (VACV) encodes enzymes that cap the 5′ end of viral mRNAs, which enhances their stability and translation. Nevertheless, recent studies demonstrated that the VACV D10 protein (VACV-WR_115) decaps mRNA, an enzymatic activity not previously shown to be encoded by a virus. The decapping activity of D10 is dependent on a Nudix hydrolase motif that is also present in the VACV D9 protein (VACV-WR_114), which shares 25% sequence identity with D10. Here, we showed that a purified recombinant VACV D9 fusion protein also decaps mRNA and that this activity was abolished by point mutations in the Nudix hydrolase motif. Decapping was specific for a methylated cap attached to RNA and resulted in the liberation of m7GDP. D9 differed from D10 in requiring a longer capped RNA substrate for optimal activity, having greater sensitivity to inhibition by uncapped RNA, and having lower sensitivity to inhibition by nucleotide cap analogs unattached to RNA. Since D9 is expressed early in infection and D10 late, we suggest that the two proteins enhance mRNA turnover and manipulate gene expression in a complementary and overlapping manner.

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In vitro characterization of the yeast DcpS, a scavenger mRNA decapping enzyme

10.1101/644492 ◽

2019 ◽

Author(s):

Madalee G. Wulf ◽

John Buswell ◽

Siu-Hong Chan ◽

Nan Dai ◽

Katherine Marks ◽

...

Keyword(s):

Reaction Conditions ◽

Mrna Decapping ◽

Rna Transcripts ◽

In Vitro Characterization ◽

Eukaryotic Mrnas ◽

Phosphate Linkage ◽

Decapping Enzyme

AbstractEukaryotic mRNAs are modified at their 5’ end early during transcription by the addition of N7-methylguanosine (m7G), which forms the “cap” on the first 5’ nucleotide. Identification of the 5’ nucleotide on mRNA is necessary for determination of the Transcription Start Site (TSS). We explored the effect of various reaction conditions on the activity of the yeast scavenger mRNA decapping enzyme DcpS (yDcpS) and examined decapping of 30 chemically distinct cap structures varying the state of methylation, sugar, phosphate linkage, and base composition on 25mer RNA oligonucleotides. Contrary to the generally accepted belief that DcpS enzymes only decap short oligonucleotides, we found that yDcpS efficiently decaps RNA transcripts as long as 1400 nucleotides. Further, we validated the application of yDcpS for enriching capped RNA using a strategy of specifically tagging the 5’ end of capped RNA by first decapping and then recapping it with an affinity-tagged guanosine nucleotide.

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