Biology subjects

Kiledjian, M.

Publications and source records attributed to Kiledjian, M..

8 recordsLinked to original sources

bioRxiv2024

Defective DcpS Decapping Manifests in Creatine Deficiency Syndrome and Neurological impairment

Biallelic mutations in the DCPS gene disrupting the decapping activity of the scavenger decapping protein DcpS, leads to neurodevelopmental deficiencies and intellectual disability. However, the molecular basis for the neurogenesis defects in these individuals remains unknown. Here we show that cells derived from individuals with a DCPS mutation harbor a creatine deficiency and a corresponding elevation of the creatine precursor, guanidinoacetate (GAA). The altered metabolite levels are a consequence of a reduction in both the mRNA and protein levels for the enzyme that converts GAA into creatine, guanidinoacetate methyltransferase. Importantly, the compromised neurogenesis and neurite outgrowth phenotypes observed during the differentiation of DcpS mutant patient derived induced pluripotent stem cell differentiation into neurons was reversed upon supplementation of creatine monohydrate. These findings suggest creatine deficiency as the major underlying factor for the neurogenetic defect detected in DcpS mutant cells and a potential driver of the neurological deficiencies in affected individuals.

cell biology↗

bioRxiv2024

Extracellular Exosomal RNAs are Glyco-Modified

Epitranscriptomic modifications play pivotal roles in regulating RNA function, encompassing base alterations and the addition of both canonical m7G and noncanonical nucleotide metabolite caps. Recently, the spectrum of modifications has extended to include glyco modification at the 5 cap or within the RNA. Despite this expansion, the functional implications of glyco modification on RNA remain elusive. Our study reveals that mammalian cells labeled with N-azidoacetylgalactosamine-tetraacylated (Ac4GalNAz) produce small noncoding and nonpolyadenylated glyco-modified RNA (glycoRNA), primarily localized within exosome vesicles. The resistance of glycoRNA-containing exosomes to RNase treatment suggests that Ac4GalNAz-derived glycoRNA constitutes intraluminal cargo, distinct from recently reported cell surface glycoRNAs. Furthermore, we demonstrate that exosome cargo can be transferred to naive cells, underscoring exosome-mediated intercellular communication of glycoRNAs. Inhibition of exosome biogenesis leads to the accumulation of intracellular glycoRNA while blocking glycan transfer to proteins concomitantly reduced the targeting of glycoRNA within exosomes. These findings highlight a correlation between protein and RNA glycosylation, suggesting that the accumulation of glycoRNAs within exosomes is a regulated process. Our results support a functional role for glyco-modification in mediating RNA targeting into exosomes, offering new insights for enhancing recent advances in exosome-directed diagnostics and therapeutic applications.

cell biology↗

bioRxiv2022

Identification of a novel deFADding activity in 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. 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 E. 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 evolutionarily conserved intrinsic property of 5-3 exoribonucleases.

molecular biology↗

bioRxiv2021

Xrn1 is a deNADding Enzyme Modulating Mitochondrial NAD Levels

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 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 in turn intramitochondrial NAD levels. Our findings uncover a functional role for mitochondrial NAD-capped RNAs as a reservoir to maintain overall NAD homeostasis. We propose NAD-capped RNAs function as a cistern for mitochondrial NAD with Xrn1 serving as a rheostat for NAD-capped RNAs.

molecular biology↗

bioRxiv2021

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. Here, we show that human neurons derived from patients with a DcpS mutation have compromised differentiation and neurite outgrowth. Moreover, in the developing mouse neocortex, DcpS is required for the radial migration, polarity, neurite outgrowth and identity of developing glutamatergic neurons. Collectively, these findings demonstrate that the scavenger mRNA decapping activity contributes to multiple pivotal roles in neural development, and further corroborate that mRNA metabolism and neocortical pathologies are associated with intellectual disability.

neuroscience↗

bioRxiv2018

Structural and biochemical studies define Nudt12 as a new class of RNA deNADding enzyme in mammalian cells

We recently demonstrated mammalian cells harbor NAD-capped mRNAs that are hydrolyzed by the DXO deNADding enzyme. Here we report the Nudix protein Nudt12 is a second mammalian deNADding enzyme structurally and mechanistically distinct from DXO and targets different RNAs. Crystal structure of mouse Nudt12 in complex with the deNADding product AMP and three Mg2+ ions at 1.6 [A] resolution provides exquisite insights into the molecular basis of the deNADding activity within the NAD pyrophosphate. Disruption of the Nudt12 gene stabilizes transfected NAD-capped RNA in cells and its endogenous NAD-capped mRNA targets are enriched in those encoding proteins involved in cellular energetics. Furthermore, exposure of cells to metabolic stress manifests changes in NAD-capped RNA levels indicating an association between NAD-capped RNAs and cellular metabolism. Lastly, we show that the bacterial RppH protein also possesses deNADding activity toward NAD-capped RNA but not free NAD, revealing a third class of deNADding enzymes.

molecular biology↗

bioRxiv2018

Mitochondrial RNA capping: highly efficient 5’-RNA capping with NAD+ and NADH by yeast and human mitochondrial RNA polymerase

Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non-canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~10% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD+ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial gene expression, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly.

biochemistry↗

bioRxiv2017

“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. The results define preferred transcription start-site (TSS) positions for NAD+ capping and define a consensus promoter sequence for NAD+ capping: HRRASWW (TSS underlined). By applying CapZyme-Seq to E. coli total cellular RNA, we establish that sequence determinants for NCIN capping in vivo match the NAD+-capping consensus defined in vitro, and we identify and quantify NCIN-capped small RNAs. Our findings define the promoter-sequence determinants for NCIN capping with NAD+ and provide a general method for analysis of NCIN capping in vitro and in vivo.

biochemistry↗