Discovery of an mRNA 5'-end capping quality control mechanism

Updated Jan. 2023

mRNAs are capped at the 5'-end (m7Gppp) during the early stage of Pol II transcription. The cap protects the mRNA and is essential for translation.

The capping process involves 3 steps: The primary transcript from Pol II, with a 5' triphosphate group (pppRNA), is converted to ppRNA. GMP is then linked to the RNA, through a 5'-5' connection, to make the capped RNA (GpppRNA). Finally, a methyltransferase methylates the 7 position of guanine to produce the mature cap (m7GpppRNA).

It is generally believed that capping is a default process, in that it always proceeds to completion. A quality control mechanism for 5' capping was not known.

In our studies on the Rat1-Rai1 complex, we unexpectedly discovered that Rai1 has an active site of its own. Biochemical experiments demonstrated that Rai1 possesses RNA 5' pyrophosphohydrolase (PPH) activity, providing the first hint that there may be involved in 5' capping quality control in eukaryotes.

Rai1 has sequence homologs from yeast to humans, and the mammalian homolog is known as Dom3Z. However, the sequence conservation is very weak, only a few residues are strictly conserved among these proteins.

Major findings from this project

There is a large pocket in the structures of Rai1 and Dom3Z, and the few residues that are conserved between Rai1 and Dom3Z are located in this pocket.

Most importantly, there is a cluster of four acidic residues (Glu150, Glu199, Asp201, and Glu239 in Rai1) at the bottom of the pocket, and three of them (Glu150, Asp201, and Glu239), together with the main chain carbonyl of Leu240 and two water molecules, form the octahedral coordination sphere of a divalent cation (Mg2+ or Mn2+).

The structural information therefore suggests that Rai1 and Dom3Z may have catalytic functions.

Biochemical studies demonstrate that Rai1 does possess catalytic activity, acting as a pyrophosphohydrolase (PPH) on RNA substrates with a 5' triphosphate.

Rai1 also has decapping activity towards RNA substrates with a 5' (unmethylated) cap. The activity towards properly capped RNA (with a 5' methylated cap) is much weaker.

In contrast to the classical decapping enzyme (Dcp2), Rai1 releases the entire cap (GpppG) from the RNA, while Dcp2 releases GDP (Gpp) from the RNA.

Aberrantly capped mRNAs, due to a temperature-sensitive defect in the methyltransferase (abd1-5), are stabilized in yeast cells lacking Rai1.

Yeast cells under nutritional stress (glucose deprivation or amino acid starvation) accumulate defectively capped mRNAs in the absence of Rai1.

Rai1 (and Dom3Z) has important roles in mRNA quality surveillance, promoting the degradation of mRNAs with defective 5' caps. This is the first demonstration of an mRNA 5' capping quality control mechanism.

The Rai1 homolog in yeast (YDR370C) has decapping as well as 5'-3' exoribonuclease activities, Dxo1. It however lacks PPH activity.

Yeast cells lacking both Rai1 and Dxo1 show capping defects even under normal growth conditions.

Mouse Dom3Z has PPH, decapping and distributive 5'-3' exoribonuclease activities. It has been renamed as DXO (decapping exonuclease).

The decapping activity of DXO does not discriminate between methylated and unmethylated caps in vitro. However, cap binding proteins protect the mature, methylated caps from being decapped by DXO.

The crystal structures of DXO in complex with a 5-mer RNA oligo, pU5, as well as a 6-mer oligo with phosphorothioate linkages, pU(S)6, have been determined. The structure of DXO in complex with the methylated cap analog m7GpppG has also been determined.

In the presence of the pU5 oligo, a second metal ion is bound in the active site. A terminal oxygen atom of the 5' phosphate group of the RNA is a bridging ligand between the two metal ions.

The pU5 oligo is bound as a product of the reaction, the RNA body with a 5' phosphate. The active site pocket is only large enough to accommodate single-stranded RNA.

The pU(S)6 oligo mimics the substrate, with the 5' phosphate group recognized by conserved residues in DXO.

The m7GpppG cap analog is bound on the other side of the active site from the pU5 oligo.

The same active site carries out all three apparently distinct catalytic activities, PPH, decapping, and 5'-3' exoribonuclease. The different binding modes of the substrates dictate the outcome of the reaction.

Knockdown of DXO levels in 293 cells leads retention of all introns in three randomly chosen pre-mRNAs, providing a link between capping quality and splicing at all exon-intron junctions.

The catalytic activities of a collection of Rai1 homologs have been characterized. They show highly diverse activity profiles.

Crystal structures of two of these homologs bound to RNA oligos show differences in RNA binding modes, which may be related to their different activity profiles.

A new catalytic activity for these enzymes, 5'-end triphosphonucleotide hydrolase (TPH), has been identified.

DXO/Rai1 enzymes have deNADding activity, and can remove NAD from NAD-capped RNAs. has been identified.

The crystal structures of mouse DXO/Rai1 in complex with 3'-phosphate NAD clearly define the molecular mechanism for their deNADding activity.

NAD-capped mRNAs and snoRNAs have been identified in mammalian cells.

DXO also has activity toward RNA with 5' hydroxyl group. The binding mode of the 5' hydroxyl RNA suggests that DXO releases a 5' hydrolxyl dinucleotide as the first product, a 5' hydroxyl dinucleotide hydrolase (HDH) activity.

Biochemical studies have confirmed that the first product released is a dinucleotide.

DXO may help with the degradation of RNA with 5' hydroxyl group.

DXO can also remove 5' FAD and dephospho-CoA caps on RNAs, giving rise to deFADding and deCoAping activities.

Structures of DXO and Rai1 in complex with FAD and CoA have been determined, revealing the catalytic mechanisms of these activities.

DXO/Rai1 family enzymes have activities against RNAs with diverse 5' modifications, and help mediate their degradation.

Mammalian Nudix enzymes can have deNADding, deFADding, and deCoAping activities.

Publications from this project

Xiang S, Cooper-Morgan A, Jiao X, Kiledjian M, Manley JL, Tong L. (2009). Structure and function of the 5'->3' exoribonuclease Rat1 and its activating partner Rai1. Nature, 458, 784-788. Reprint(PDF)
X. Jiao, S. Xiang, C.-S. Oh, C.E. Martin, L. Tong & M. Kiledjian. (2010). Identification of a quality-control mechanism for eukaryotic mRNA 5'-end capping. Nature, 467, 608-611. Reprint(PDF)
J.H. Chang,* X. Jiao,* K. Chiba, C.-S. Oh, C.E. Martin, M. Kiledjian$ & L. Tong.$ (2012). Dxo1 is a new type of eukaryotic enzyme with both decapping and 5'-3' exoribonuclease activities. Nature Struct. Mol. Biol. 19, 1011-1017. (*--equal first authors, $--co-corresponding authors) Reprint(PDF)
X. Jiao,* J.H. Chang,* T. Kilic, L. Tong$ & M. Kiledjian.$ (2013). A mammalian pre-mRNA 5' end capping quality control mechanism and an unexpected link of capping to pre-mRNA processing. Mol. Cell, 50, 104-115. (*--equal first authors, $--co-corresponding authors) (Preview by J.S. Mugridge & J.D. Gross, 2-4) Reprint(PDF)
A.R. Jurado, D. Tan, X. Jiao, M. Kiledjian & L. Tong. (2014). Structure and function of pre-mRNA 5'-end capping quality control and 3'-end processing. Biochem. 53, 1882-1898. Reprint(PDF)
V.Y.F. Wang, X. Jiao, M. Kiledjian & L. Tong. (2015). Structural and biochemical studies of the distinct activity profiles of Rai1 proteins. Nucl. Acids Res. 43, 6596-6606. Reprint(PDF)
X. Jiao, S. Doamekpor, J.G. Bird, B.E. Nickels, L. Tong, R.P. Hart & M. Kiledjian. (2017). 5' end nicotinamide adenine dinucleotide cap in human cells promotes RNA decay through DXO-mediated deNADding. Cell, 168, 1015-1027. Reprint(PDF)
J.-S. Yun,* J.-H. Yoon,* Y.J. Choi, Y.J. Son, S.H. Kim, L. Tong$ & J.H. Chang.$ (2018). Molecular mechanism for the inhibition of DXO by adenosine 3',5'-bisphosphate. Biochem. Biophys. Res. Commun. 504, 89-95. (*-equal first authors, $-co-corresponding authors)
A. Kwasnik,*$ V.Y.F. Wang,* M. Krzyszton, A. Gozdek, M. Zakrzewska-Placzek, K. Stepniak, J. Poznanski, L. Tong$ & J. Kufel.$ (2019). Arabidopsis DXO1 links RNA turnover and chloroplast function independently of its enzymatic activity. Nucl. Acids Res. 47, 4751-4764. (*-equal first authors, $-co-corresponding authors)
E. Grudzien-Nogalska,* Y. Wu,* X. Jiao, H. Cui, M.K. Mateyak, R.P. Hart, L. Tong$ & M. Kiledjian.$ (2019). Structural and mechanistic basis of mammalian Nudt12 deNADding. Nat. Chem. Biol. 15, 575-582. (*-equal first authors, $-co-corresponding authors)
S.K. Doamekpor,* A. Gozdek,* A. Kwasnik, J. Kufel$ & L. Tong.$ (2020). A novel 5'-hydroxyl dinucleotide hydrolase activity for the DXO/Rai1 family of enzymes. Nucl. Acids Res. 48, 349-358. (Epub 11/28/19) (*-equal first authors, $-co-corresponding authors)
S.K. Doamekpor,* E. Grudzien-Nogalska,* A. Mlynarska-Cieslak, J. Kowalska, M. Kiledjian$ & L. Tong.$ (2020). DXO/Rai1 enzymes remove 5'-end FAD and dephospho-CoA caps on RNAs. Nucl. Acids Res. 48, 6136-6148. (*-equal first authors, $-co-corresponding authors)
S. Sharma, E. Grudzien-Nogalska,* K. Hamilton,* X. Jiao, J. Yang, L. Tong & M. Kiledjian. (2020). Mammalian Nudix proteins cleave nucleotide metabolite caps on RNAs. Nucl. Acids Res. 48, 6788-6798. (*-equal second authors)
S. Sharma,* J. Yang,* S.K. Doamekpor, E. Grudzien-Nogalska, L. Tong & M. Kiledjian. (2022). Identification of a novel deFADding activity in human, yeast and bacterial 5' to 3' exoribonucleases. Nucl. Acids Res. 50, 8807-8817. (*-equal first authors)
S.K. Doamekpor,* S. Sharma,* M. Kiledjian$ & L. Tong.$ (2022). Recent insights into noncanonical 5' capping and decapping of RNA. J. Biol. Chem. 298, 102171. (*-equal first authors, $-co-corresponding authors)

Funding for this project

NIH R01GM090059 (2011-2016)
NIH R35GM118093 (2016-)