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Identification of metabolite caps including FAD on the 5' end of RNA has uncovered a previously unforeseen intersection between cellular metabolism and gene expression. To understand the function of FAD caps in cellular physiology, we characterised the proteins interacting with FAD caps in budding yeast. Here we demonstrate that highly conserved 5'-3' exoribonucleases, Xrn1 and Rat1, physically interact with the RNA 5' FAD cap and both possess FAD cap decapping (deFADding) activity and subsequently degrade the resulting RNA. Xrn1 deFADding activity was also evident in human cells indicating its evolutionary conservation. Furthermore, we report that the recently identified bacterial 5'-3' exoribonuclease RNase AM also possesses deFADding activity that can degrade FAD-capped RNAs in vitro and in Escherichia coli cells. To gain a molecular understanding of the deFADding reaction, an RNase AM crystal structure with three manganese ions coordinated by a sulfate molecule and the active site amino acids was generated that provided details underlying hydrolysis of the FAD cap. Our findings reveal a general propensity for 5'-3' exoribonucleases to hydrolyse and degrade RNAs with 5' end noncanonical caps in addition to their well characterized 5' monophosphate RNA substrates indicating an intrinsic property of 5'-3' exoribonucleases., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The existence of non-canonical nicotinamide adenine diphosphate (NAD) 5'-end capped RNAs is now well established. Nevertheless, the biological function of this nucleotide metabolite cap remains elusive. Here, we show that the yeast Saccharomyces cerevisiae cytoplasmic 5'-end exoribonuclease Xrn1 is also a NAD cap decapping (deNADding) enzyme that releases intact NAD and subsequently degrades the RNA. The significance of Xrn1 deNADding is evident in a deNADding deficient Xrn1 mutant that predominantly still retains its 5'-monophosphate exonuclease activity. This mutant reveals Xrn1 deNADding is necessary for normal growth on non-fermenting sugar and is involved in modulating mitochondrial NAD-capped RNA levels and may influence intramitochondrial NAD levels. Our findings uncover a contribution of mitochondrial NAD-capped RNAs in overall NAD regulation with the deNADding activity of Xrn1 fulfilling a central role., (© 2022. The Author(s).)

In eukaryotes, the DXO/Rai1 enzymes can eliminate most of the incomplete and non-canonical NAD caps through their decapping, deNADding and pyrophosphohydrolase activities. Here, we report that these enzymes can also remove FAD and dephospho-CoA (dpCoA) non-canonical caps from RNA, and we have named these activities deFADding and deCoAping. The crystal structures of mammalian DXO with 3'-FADP or CoA and fission yeast Rai1 with 3'-FADP provide elegant insight to these activities. FAD and CoA are accommodated in the DXO/Rai1 active site by adopting folded conformations. The flavin of FAD and the pantetheine group of CoA contact the same region at the bottom of the active site tunnel, which undergoes conformational changes to accommodate the different cap moieties. We have developed FAD-capQ to detect and quantify FAD-capped RNAs and determined that FAD caps are present on short RNAs (with less than ∼200 nucleotides) in human cells and that these RNAs are stabilized in the absence of DXO., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Recently, we reported that two homologous yeast proteins, Rai1 and Dxo1, function in a quality control mechanism to clear cells of incompletely 5' end-capped messenger RNAs (mRNAs). Here, we report that their mammalian homolog, Dom3Z (referred to as DXO), possesses pyrophosphohydrolase, decapping, and 5'-to-3' exoribonuclease activities. Surprisingly, we found that DXO preferentially degrades defectively capped pre-mRNAs in cells. Additional studies show that incompletely capped pre-mRNAs are inefficiently spliced at all introns, a fact that contrasts with current understanding, and are also poorly cleaved for polyadenylation. Crystal structures of DXO in complex with substrate mimic and products at a resolution of up to 1.5Å provide elegant insights into the catalytic mechanism and molecular basis for their three apparently distinct activities. Our data reveal a pre-mRNA 5' end capping quality control mechanism in mammalian cells, indicating DXO as the central player for this mechanism, and demonstrate an unexpected intimate link between proper 5' end capping and subsequent pre-mRNA processing., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Recent studies showed that Rai1 is a crucial component of the mRNA 5'-end-capping quality-control mechanism in yeast. The yeast genome encodes a weak homolog of Rai1, Ydr370C, but little is known about this protein. Here we report the crystal structures of Ydr370C from Kluyveromyces lactis and the first biochemical and functional studies on this protein. The overall structure of Ydr370C is similar to Rai1. Ydr370C has robust decapping activity on RNAs with unmethylated caps, but it has no detectable pyrophosphohydrolase activity. Unexpectedly, Ydr370C also possesses distributive, 5'-3' exoRNase activity, and we propose the name Dxo1 for this new eukaryotic enzyme with both decapping and exonuclease activities. Studies of yeast in which both Dxo1 and Rai1 are disrupted reveal that mRNAs with incomplete caps are produced even under normal growth conditions, in sharp contrast to current understanding of the capping process.

The scavenger decapping enzyme Dcs1 has been shown to facilitate the activity of the cytoplasmic 5'-3' exoribonuclease Xrn1 in eukaryotes. Dcs1 has also been shown to be required for growth in glycerol medium. We therefore wondered whether the capacity to activate RNA degradation could account for its requirement for growth on this carbon source. Indeed, a catalytic mutant of Xrn1 is also unable to grow in glycerol medium, and removal of the nuclear localization signal of Rat1, the nuclear homolog of Xrn1, restores glycerol growth. A cytoplasmic 5'-3' exoribonuclease activity is therefore essential for yeast growth on glycerol, suggesting that Xrn1 activation by Dcs1 is physiologically important. In fact, Xrn1 is essentially inactive in the absence of Dcs1 in vivo. We analyzed the role of Dcs1 in the control of exoribonuclease activity in vitro and propose that Dcs1 is a specific cofactor of Xrn1. Dcs1 does not stimulate the activity of other 5'-3' exoribonucleases, such as Rat1, in vitro. We demonstrate that Dcs1 improves the apparent affinity of Xrn1 for RNA and that Xrn1 and Dcs1 can form a complex in vitro. We examined the biological significance of this regulation by performing 2D protein gel analysis. We observed that a set of proteins showing decreased levels in a DCS deletion strain, some essential for respiration, are also systematically decreased in an XRN1 deletion mutant. Therefore, we propose that the activation of Xrn1 by Dcs1 is important for respiration.

The 5'-->3' exoribonucleases (XRNs) comprise a large family of conserved enzymes in eukaryotes with crucial functions in RNA metabolism and RNA interference. XRN2, or Rat1 in yeast, functions primarily in the nucleus and also has an important role in transcription termination by RNA polymerase II (refs 7-14). Rat1 exoribonuclease activity is stimulated by the protein Rai1 (refs 15, 16). Here we report the crystal structure at 2.2 A resolution of Schizosaccharomyces pombe Rat1 in complex with Rai1, as well as the structures of Rai1 and its murine homologue Dom3Z alone at 2.0 A resolution. The structures reveal the molecular mechanism for the activation of Rat1 by Rai1 and for the exclusive exoribonuclease activity of Rat1. Biochemical studies confirm these observations, and show that Rai1 allows Rat1 to degrade RNAs with stable secondary structure more effectively. There are large differences in the active site landscape of Rat1 compared to related and PIN (PilT N terminus) domain-containing nucleases. Unexpectedly, we identified a large pocket in Rai1 and Dom3Z that contains highly conserved residues, including three acidic side chains that coordinate a divalent cation. Mutagenesis and biochemical studies demonstrate that Rai1 possesses pyrophosphohydrolase activity towards 5' triphosphorylated RNA. Such an activity is important for messenger RNA degradation in bacteria, but this is, to our knowledge, the first demonstration of this activity in eukaryotes and suggests that Rai1/Dom3Z may have additional important functions in RNA metabolism.

The exosome is a 3' to 5' exoribonuclease central to many cellular processes, including mRNA decay. now present the biochemical reconstitution and crystal structure of the eukaryotic exosome. This remarkable achievement provides key insights into the composition and assembly of the human and yeast exosomes, revealing functions of individual subunits.

The scavenger decapping enzyme Dcs1 has been shown to facilitate the activity of the cytoplasmic 5'-3' exoribonuclease Xrn1 in eukaryotes. Dcs1 has also been shown to be required for growth in glycerol medium. We therefore wondered whether the capacity to activate RNA degradation could account for its requirement for growth on this carbon source. Indeed, a catalytic mutant of Xrn1 is also unable to grow in glycerol medium, and removal of the nuclear localization signal of Rat1, the nuclear homolog of Xrn1, restores glycerol growth. A cytoplasmic 5'-3' exoribonuclease activity is therefore essential for yeast growth on glycerol, suggesting that Xrn1 activation by Dcs1 is physiologically important. In fact, Xrn1 is essentially inactive in the absence of Dcs1 in vivo. We analyzed the role of Dcs1 in the control of exoribonuclease activity in vitro and propose that Dcs1 is a specific cofactor of Xrn1. Dcs1 does not stimulate the activity of other 5'-3' exoribonucleases, such as Rat1, in vitro. We demonstrate that Dcs1 improves the apparent affinity of Xrn1 for RNA and that Xrn1 and Dcs1 can form a complex in vitro. We examined the biological significance of this regulation by performing 2D protein gel analysis. We observed that a set of proteins showing decreased levels in a DCS deletion strain, some essential for respiration, are also systematically decreased in an XRN1 deletion mutant. Therefore, we propose that the activation of Xrn1 by Dcs1 is important for respiration. [ABSTRACT FROM AUTHOR]