Biallelic NUDT2 variants defective in mRNA decapping cause a neurodevelopmental disease.

Dysfunctional RNA processing caused by genetic defects in RNA processing enzymes has a profound impact on the nervous system, resulting in neurodevelopmental conditions. We characterized a recessive neurological disorder in 18 children and young adults from 10 independent families typified by intellectual disability, motor developmental delay and gait disturbance. In some patients peripheral neuropathy, corpus callosum abnormalities and progressive basal ganglia deposits were present.

NADcapPro and circNC: methods for accurate profiling of NAD and non-canonical RNA caps in eukaryotes.

Accurate identification of NAD-capped RNAs is essential for delineating their generation and biological function. Previous transcriptome-wide methods used to classify NAD-capped RNAs in eukaryotes contain inherent limitations that have hindered the accurate identification of NAD caps from eukaryotic RNAs. In this study, we introduce two orthogonal methods to identify NAD-capped RNAs more precisely.

Preparation of RNAs with non-canonical 5' ends using novel di- and trinucleotide reagents for co-transcriptional capping.

Many eukaryotic and some bacterial RNAs are modified at the 5' end by the addition of cap structures. In addition to the classic 7-methylguanosine 5' cap in eukaryotic mRNA, several non-canonical caps have recently been identified, including NAD-linked, FAD-linked, and UDP-glucose-linked RNAs. However, studies of the biochemical properties of these caps are impaired by the limited access to transcribed RNA probes of high quality, as the typical capping efficiencies with NAD or FAD dinucleotides achieved in the presence of T7 polymerase rarely exceed 50%, and pyrimidine derivatives are not incorporated because of promoter sequence limitations.

Identification of a novel deFADding activity in human, yeast and bacterial 5' to 3' exoribonucleases.

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.

Recent insights into noncanonical 5' capping and decapping of RNA.

The 5' N-methylguanosine cap is a critical modification for mRNAs and many other RNAs in eukaryotic cells. Recent studies have uncovered an RNA 5' capping quality surveillance mechanism, with DXO/Rai1 decapping enzymes removing incomplete caps and enabling the degradation of the RNAs, in a process we also refer to as "no-cap decay." It has also been discovered recently that RNAs in eukaryotes, bacteria, and archaea can have noncanonical caps (NCCs), which are mostly derived from metabolites and cofactors such as NAD, FAD, dephospho-CoA, UDP-glucose, UDP-N-acetylglucosamine, and dinucleotide polyphosphates.

Xrn1 is a deNADding enzyme modulating mitochondrial NAD-capped RNA.

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.

mRNA-Decapping Associated DcpS Enzyme Controls Critical Steps of Neuronal Development.

Homozygous mutations in the gene encoding the scavenger mRNA-decapping enzyme, DcpS, have been shown to underlie developmental delay and intellectual disability. Intellectual disability is associated with both abnormal neocortical development and mRNA metabolism. However, the role of DcpS and its scavenger decapping activity in neuronal development is unknown.

SOD1 regulates ribosome biogenesis in KRAS mutant non-small cell lung cancer.

SOD1 is known as the major cytoplasmic superoxide dismutase and an anticancer target. However, the role of SOD1 in cancer is not fully understood. Herein we describe the generation of an inducible Sod1 knockout in KRAS-driven NSCLC mouse model.

InsP is a small-molecule regulator of NUDT3-mediated mRNA decapping and processing-body dynamics.

Regulation of enzymatic 5' decapping of messenger RNA (mRNA), which normally commits transcripts to their destruction, has the capacity to dynamically reshape the transcriptome. For example, protection from 5' decapping promotes accumulation of mRNAs into processing (P) bodies-membraneless, biomolecular condensates. Such compartmentalization of mRNAs temporarily removes them from the translatable pool; these repressed transcripts are stabilized and stored until P-body dissolution permits transcript reentry into the cytosol.

Mammalian Nudix proteins cleave nucleotide metabolite caps on RNAs.

We recently reported the presence of nicotinamide adenine dinucleotide (NAD)-capped RNAs in mammalian cells and a role for DXO and the Nudix hydrolase Nudt12 in decapping NAD-capped RNAs (deNADding) in cells. Analysis of 5'caps has revealed that in addition to NAD, mammalian RNAs also contain other metabolite caps including flavin adenine dinucleotide (FAD) and dephosphoCoA (dpCoA). In the present study we systematically screened all mammalian Nudix proteins for their potential deNADing, FAD cap decapping (deFADding) and dpCoA cap decapping (deCoAping) activity.

DXO/Rai1 enzymes remove 5'-end FAD and dephospho-CoA caps on RNAs.

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.

Structural and mechanistic basis of mammalian Nudt12 RNA deNADding.

We recently demonstrated that mammalian cells harbor nicotinamide adenine dinucleotide (NAD)-capped messenger RNAs that are hydrolyzed by the DXO deNADding enzyme. Here, we report that the Nudix protein Nudt12 is a second mammalian deNADding enzyme structurally and mechanistically distinct from DXO and targeting different RNAs. The crystal structure of mouse Nudt12 in complex with the deNADding product AMP and three Mg ions at 1.

Nicotinamide-Containing Di- and Trinucleotides as Chemical Tools for Studies of NAD-Capped RNAs.

We report the chemical synthesis of a set of nicotinamide adenine dinucleotide (NAD) cap analogues containing chemical modifications that reduce their susceptibility to NAD-RNA-degrading enzymes. These analogues can be incorporated into transcripts in a similar way as NAD. Biochemical characterization of RNAs carrying these caps with DXO, NudC, and Nudt12 enzymes led to the identification of compounds that can be instrumental in unraveling so far unaddressed biological aspects of NAD-RNAs.

"NAD-capQ" detection and quantitation of NAD caps.

RNA 5' cap structures comprising the metabolic effector nicotinamide adenine dinucleotide (NAD) have been identified in diverse organisms. Here we report a simple, two-step procedure to detect and quantitate NAD-capped RNA, termed "NAD-capQ." By use of NAD-capQ we quantitate NAD-capped RNA levels in , , and human cells, and we measure increases in NAD-capped RNA levels in cells from all three organisms harboring disruptions in their respective "deNADding" enzymes.

CapZyme-Seq Comprehensively Defines Promoter-Sequence Determinants for RNA 5' Capping with NAD.

Nucleoside-containing metabolites such as NAD can be incorporated as 5' caps on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report CapZyme-seq, a high-throughput-sequencing method that employs NCIN-decapping enzymes NudC and Rai1 to detect and quantify NCIN-capped RNA. By combining CapZyme-seq with multiplexed transcriptomics, we determine efficiencies of NAD capping by Escherichia coli RNAP for ∼16,000 promoter sequences.

Eukaryotic RNA 5'-End NAD Capping and DeNADding.

A hallmark of eukaryotic mRNAs has long been the 5'-end mG cap. This paradigm was recently amended by recent reports that Saccharomyces cerevisiae and mammalian cells also contain mRNAs carrying a novel nicotinamide adenine dinucleotide (NAD) cap at their 5'-end. The presence of an NAD cap on mRNA uncovers a previously unknown mechanism for controlling gene expression through nucleotide metabolite-directed mRNA turnover.

5' End Nicotinamide Adenine Dinucleotide Cap in Human Cells Promotes RNA Decay through DXO-Mediated deNADding.

Eukaryotic mRNAs generally possess a 5' end N7 methyl guanosine (mG) cap that promotes their translation and stability. However, mammalian mRNAs can also carry a 5' end nicotinamide adenine dinucleotide (NAD) cap that, in contrast to the mG cap, does not support translation but instead promotes mRNA decay. The mammalian and fungal noncanonical DXO/Rai1 decapping enzymes efficiently remove NAD caps, and cocrystal structures of DXO/Rai1 with 3'-NADP illuminate the molecular mechanism for how the "deNADding" reaction produces NAD and 5' phosphate RNA.

Reversible methylation of mA in the 5' cap controls mRNA stability.

Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5' end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N,2'-O-dimethyladenosine (mA), is a reversible modification that influences cellular mRNA fate.

New insights into decapping enzymes and selective mRNA decay.

Removal of the 5' end cap is a critical determinant controlling mRNA stability and efficient gene expression. Removal of the cap is exquisitely controlled by multiple direct and indirect regulators that influence association with the cap and the catalytic step. A subset of these factors directly stimulate activity of the decapping enzyme, while others influence remodeling of factors bound to mRNA and indirectly stimulate decapping.

A long noncoding RNA associated with susceptibility to celiac disease.

Recent studies have implicated long noncoding RNAs (lncRNAs) as regulators of many important biological processes. Here we report on the identification and characterization of a lncRNA, lnc13, that harbors a celiac disease-associated haplotype block and represses expression of certain inflammatory genes under homeostatic conditions. Lnc13 regulates gene expression by binding to hnRNPD, a member of a family of ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs).

Nudt3 is an mRNA decapping enzyme that modulates cell migration.

Removal of the 5'-end 7-methylguanosine cap structure is a critical step in the highly regulated process of mRNA decay. The Nudix hydrolase, Dcp2, was identified as a first decapping enzyme and subsequently shown to preferentially modulate stability of only a subset of mRNAs. This observation led to the hypothesis that mammalian cells possess multiple decapping enzymes that may function in distinct pathways.

Thalamic WNT3 Secretion Spatiotemporally Regulates the Neocortical Ribosome Signature and mRNA Translation to Specify Neocortical Cell Subtypes.

Unlabelled: Neocortical development requires tightly controlled spatiotemporal gene expression. However, the mechanisms regulating ribosomal complexes and the timed specificity of neocortical mRNA translation are poorly understood. We show that active mRNA translation complexes (polysomes) contain ribosomal protein subsets that undergo dynamic spatiotemporal rearrangements during mouse neocortical development.

Structural and biochemical studies of the distinct activity profiles of Rai1 enzymes.

Recent studies showed that Rai1 and its homologs are a crucial component of the mRNA 5'-end capping quality control mechanism. They can possess RNA 5'-end pyrophosphohydrolase (PPH), decapping, and 5'-3' exonuclease (toward 5' monophosphate RNA) activities, which help to degrade mRNAs with incomplete 5'-end capping. A single active site in the enzyme supports these apparently distinct activities.

DcpS is a transcript-specific modulator of RNA in mammalian cells.

The scavenger decapping enzyme DcpS is a multifunctional protein initially identified by its property to hydrolyze the resulting cap structure following 3' end mRNA decay. In Saccharomyces cerevisiae, the DcpS homolog Dcs1 is an obligate cofactor for the 5'-3' exoribonuclease Xrn1 while the Caenorhabditis elegans homolog Dcs-1, facilitates Xrn1 mediated microRNA turnover. In both cases, this function is independent of the decapping activity.