Abstract
The 5' messenger RNA (mRNA) cap structure enhances translation and protects the transcript against exonucleolytic degradation. During mRNA turnover, this cap is removed from the mRNA. This decapping step is catalyzed by the Scavenger Decapping Enzyme (DcpS), in case the mRNA has been exonucleolyticly shortened from the 3' end by the exosome complex. Here, we show that DcpS only processes mRNA fragments that are shorter than three nucleotides in length. Based on a combination of methyl transverse relaxation optimized (TROSY) NMR spectroscopy and X-ray crystallography, we established that the DcpS substrate length-sensing mechanism is based on steric clashes between the enzyme and the third nucleotide of a capped mRNA. For longer mRNA substrates, these clashes prevent conformational changes in DcpS that are required for the formation of a catalytically competent active site. Point mutations that enlarge the space for the third nucleotide in the mRNA body enhance the activity of DcpS on longer mRNA species. We find that this mechanism to ensure that the enzyme is not active on translating long mRNAs is conserved from yeast to humans. Finally, we show that the products that the exosome releases after 3' to 5' degradation of the mRNA body are indeed short enough to be decapped by DcpS. Our data thus directly confirms the notion that mRNA products of the exosome are direct substrates for DcpS. In summary, we demonstrate a direct relationship between conformational changes and enzyme activity that is exploited to achieve substrate selectivity.
Highlights
The 5′ messenger RNA cap structure enhances translation and protects the transcript against exonucleolytic degradation
A final and irreversible way to terminate gene expression is the degradation of an messenger RNA (mRNA) transcript
An mRNA molecule is divided into the mRNA body that includes the coding region as well as the 3′ and 5′ UTRs, the 3′ poly(A)-tail, and the protecting 5′ cap structure
Summary
The 5′ messenger RNA (mRNA) cap structure enhances translation and protects the transcript against exonucleolytic degradation. In the 3′ to 5′ pathway (Fig. 1A), the mRNA body is processively hydrolyzed by the 10-component cytoplasmic exosome complex (exo-10; exo-9 plus Rrp44/Dis3), followed by the removal of the cap structure of the remaining short RNA fragment by the Scavenger Decapping enzyme DcpS (Dcs1p in yeast) [9, 10]. Our data show that DcpS is only active on mRNA that have undergone prior processing by the exosome This DcpS selection mechanism is conserved from yeast to humans and is caused by the inability of the enzyme to undergo structural changes that are required for the formation of a catalytically active state around long mRNA transcripts. The enzyme undergoes a see-saw motion to simultaneously release the products from one binding site and capture a substrate in the other active site (Fig. 1A)
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