RESUMO
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 in vitro 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. To address this issue, we developed a series of di- and trinucleotide capping reagents and in vitro transcription conditions to provide straightforward access to unconventionally capped RNAs with improved 5'-end homogeneity. We show that because of the transcription start site flexibility of T7 polymerase, R1ppApG-type structures (where R1 is either nicotinamide riboside or riboflavin) are efficiently incorporated into RNA during transcription from dsDNA templates containing both φ 6.5 and φ 2.5 promoters and enable high capping efficiencies (â¼90%). Moreover, uridine-initiated RNAs are accessible by transcription from templates containing the φ 6.5 promoter performed in the presence of R2ppUpG-type initiating nucleotides (where R2 is a sugar or phosphate moiety). We successfully employed this strategy to obtain several nucleotide-sugar-capped and uncapped RNAs. The capping reagents developed herein provide easy access to chemical probes to elucidate the biological roles of non-canonical RNA 5' capping.
RESUMO
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.
Assuntos
Exorribonucleases/metabolismo , NAD/genética , Capuzes de RNA/metabolismo , RNA Mitocondrial/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Exorribonucleases/genética , Mitocôndrias/genética , Capuzes de RNA/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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. We demonstrate that Nudt16 is a novel deNADding enzyme in mammalian cells. Additionally, we identified seven Nudix proteins-Nudt2, Nudt7, Nudt8, Nudt12, Nudt15, Nudt16 and Nudt19, to possess deCoAping activity in vitro. Moreover, our screening revealed that both mammalian Nudt2 and Nudt16 hydrolyze FAD-capped RNAs in vitro with Nudt16 regulating levels of FAD-capped RNAs in cells. All decapping activities identified hydrolyze the metabolite cap substrate within the diphosphate linkage. Crystal structure of human Nudt16 in complex with FAD at 2.7 Å resolution provide molecular insights into the binding and metal-coordinated hydrolysis of FAD by Nudt16. In summary, our study identifies novel cellular deNADding and deFADding enzymes and establishes a foundation for the selective functionality of the Nudix decapping enzymes on non-canonical metabolite caps.
Assuntos
Flavina-Adenina Dinucleotídeo/química , Pirofosfatases/genética , Pirofosfatases/ultraestrutura , Capuzes de RNA/genética , Coenzima A/química , Coenzima A/genética , Cristalografia por Raios X , Flavina-Adenina Dinucleotídeo/genética , Humanos , NAD/química , NAD/ultraestrutura , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Conformação Proteica , Pirofosfatases/química , Pirofosfatases/classificação , Capuzes de RNA/química , Capuzes de RNA/ultraestrutura , Nudix HidrolasesRESUMO
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 â¼200 nucleotides) in human cells and that these RNAs are stabilized in the absence of DXO.
Assuntos
Coenzima A/metabolismo , Exorribonucleases/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Kluyveromyces/enzimologia , Proteínas Nucleares/metabolismo , Capuzes de RNA/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Animais , Exorribonucleases/química , Exorribonucleases/genética , Flavina-Adenina Dinucleotídeo/análise , Células HEK293 , Humanos , Técnicas In Vitro , Camundongos , Modelos Moleculares , NAD/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Capuzes de RNA/análise , Especificidade por Substrato , Transcrição GênicaRESUMO
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 Mg2+ ions at 1.6 Å resolution provides insights into the molecular basis of the deNADding activity in 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 nutrient or environmental stress manifests changes in NAD-capped RNA levels that are selectively responsive to Nudt12 or DXO, respectively, indicating an association of deNADding to cellular metabolism.
Assuntos
NAD/metabolismo , Pirofosfatases/metabolismo , RNA Mensageiro/metabolismo , Humanos , NAD/química , Pirofosfatases/química , Pirofosfatases/genética , RNA Mensageiro/químicaRESUMO
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 ~15% 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 transcriptional outputs, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Capuzes de RNA/genética , RNA Mitocondrial/genética , Transcrição Gênica , Citoplasma/genética , Citoplasma/metabolismo , Humanos , Mitocôndrias/genética , NAD/genética , Oxirredução , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Sítio de Iniciação de TranscriçãoRESUMO
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.
Assuntos
Adenina/farmacologia , NAD/antagonistas & inibidores , Niacinamida/farmacologia , Capuzes de RNA/antagonistas & inibidores , Adenina/análogos & derivados , Adenina/química , Conformação Molecular , NAD/metabolismo , Niacinamida/análogos & derivados , Niacinamida/química , Capuzes de RNA/metabolismoRESUMO
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 Escherichia coli, Saccharomyces cerevisiae, 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. We further show that NAD-capped RNA levels in human cells respond to changes in cellular NAD concentrations, indicating that NAD capping provides a mechanism for human cells to directly sense and respond to alterations in NAD metabolism. Our findings establish NAD-capQ as a versatile, rapid, and accessible methodology to detect and quantitate 5'-NAD caps on endogenous RNA in any organism.
Assuntos
Colorimetria , NAD/química , Capuzes de RNA/química , Capuzes de RNA/genética , RNA/química , RNA/genética , Alelos , Linhagem Celular , Colorimetria/métodos , Humanos , Espaço Intracelular , Espectrometria de Massas , Mutação , NAD/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genéticaRESUMO
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. Furthermore, the components of the general decapping machinery can also be recruited by mRNA-specific regulatory proteins to activate decapping. The Nudix hydrolase, Dcp2, identified as a first decapping enzyme, cleaves capped mRNA and initiates 5'-3' degradation. Extensive studies on Dcp2 led to broad understanding of its activity and the regulation of transcript specific decapping and decay. Interestingly, seven additional Nudix proteins possess intrinsic decapping activity in vitro and at least two, Nudt16 and Nudt3, are decapping enzymes that regulate mRNA stability in cells. Furthermore, a new class of decapping proteins within the DXO family preferentially function on incompletely capped mRNAs. Importantly, it is now evident that each of the characterized decapping enzymes predominantly modulates only a subset of mRNAs, suggesting the existence of multiple decapping enzymes functioning in distinct cellular pathways. WIREs RNA 2017, 8:e1379. doi: 10.1002/wrna.1379 For further resources related to this article, please visit the WIREs website.
Assuntos
Endorribonucleases/metabolismo , Estabilidade de RNA/fisiologia , Animais , HumanosRESUMO
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. Here we report Nudt3 is a Nudix protein that possesses mRNA decapping activity in cells and is a modulator of MCF-7 breast cancer cell migration. Reduction of Nudt3 protein levels in MCF-7 cells promotes increased cell migration and corresponding enhanced filopodia extensions. Importantly, this phenotype was reversed by complementation with wild type, but not catalytically inactive Nudt3 protein indicating Nudt3 decapping activity normally functions to control cell migration. Genome-wide analysis of Nudt3 compromised cells identified elevated levels of transcripts involved in cell motility including integrin ß6, lipocalin-2, and fibronectin. The observed increase in mRNA abundance was dependent on Nudt3 decapping activity where integrin ß6 and lipocalin-2 were modulated directly through mRNA stability, while fibronectin was indirectly controlled. Moreover, increased cell migration observed in Nudt3 knockdown cells was mediated through the extracellular integrin ß6 and fibronectin protein nexus. We conclude that Nudt3 is an mRNA decapping enzyme that orchestrates expression of a subset of mRNAs to modulate cell migration and further substantiates the existence of multiple decapping enzymes functioning in distinct cellular pathways in mammals.
Assuntos
Hidrolases Anidrido Ácido/fisiologia , Movimento Celular/fisiologia , Hidrolases Anidrido Ácido/genética , Regulação para Baixo , Endorribonucleases , Fibronectinas/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Cadeias beta de Integrinas/metabolismo , Células MCF-7RESUMO
Downregulation of CPEB1, a sequence-specific RNA-binding protein, in a mouse mammary epithelial cell line (CID-9) causes epithelial-to-mesenchymal transition (EMT), based on several criteria. First, CPEB1 knockdown decreases protein levels of E-cadherin and ß-catenin but increases those of vimentin and Twist1. Second, the motility of CPEB1-depleted cells is increased. Third, CID-9 cells normally form growth-arrested, polarized and three-dimensional acini upon culture in extracellular matrix, but CPEB1-deficient CID-9 cells form nonpolarized proliferating colonies lacking a central cavity. CPEB1 downregulates Twist1 expression by binding to its mRNA, shortening its poly(A) tract and repressing its translation. CID-9 cultures contain both myoepithelial and luminal epithelial cells. CPEB1 increases during CID-9 cell differentiation, is predominantly expressed in myoepithelial cells, and its knockdown prevents expression of the myoepithelial marker p63. CPEB1 is present in proliferating subpopulations of pure luminal epithelial cells (SCp2) and myoepithelial cells (SCg6), but its depletion increases Twist1 only in SCg6 cells and fails to downregulate E-cadherin in SCp2 cells. We propose that myoepithelial cells prevent EMT by influencing the polarity and proliferation of luminal epithelial cells in a mechanism that requires translational silencing of myoepithelial Twist1 by CPEB1.
Assuntos
Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Animais , Caderinas/biossíntese , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Regulação para Baixo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Proteína 1 Relacionada a Twist/biossíntese , Proteína 1 Relacionada a Twist/genética , Fatores de Poliadenilação e Clivagem de mRNA/deficiência , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
The translational efficiency and stability of synthetic mRNA in both cultured cells and whole animals can be improved by incorporation of modified cap structures at the 5'-end. mRNAs are synthesized in vitro by a phage RNA polymerase transcribing a plasmid containing the mRNA sequence in the presence of all four NTPs plus a cap dinucleotide. Modifications in the cap dinucleotide at the 2'- or 3'-positions of m(7)Guo, or modifications in the polyphosphate chain, can improve both translational efficiency and stability of the mRNA, thereby increasing the amount and duration of protein expression. In the context of RNA-based immunotherapy, the latter is especially important for antigen production and presentation by dendritic cells. Protocols are presented for synthesis of modified mRNAs, their introduction into cells and whole animals, and measurement of their translational efficiency and stability.
Assuntos
Biossíntese de Proteínas , Análogos de Capuz de RNA/química , Estabilidade de RNA , RNA Mensageiro , Transfecção/métodos , Animais , Humanos , RNA Mensageiro/síntese química , RNA Mensageiro/química , RNA Mensageiro/genéticaRESUMO
Decapping is an essential step in multiple pathways of mRNA degradation. Previously, we synthesized mRNAs containing caps that were resistant to decapping, both to dissect the various pathways for mRNA degradation and to stabilize mRNA for more sustained protein expression. mRNAs containing an α-ß CH(2) group are resistant to in vitro cleavage by the decapping enzyme hDcp2 but poorly translated. mRNAs containing an S substitution at the ß-phosphate are well translated but only partially resistant to hDcp2. We now describe seven new cap analogs substituted at the ß-phosphate with BH(3) or Se, or substituted at either the α-ß or ß-γ O with NH. The analogs differ in affinity for eIF4E and efficiency of in vitro incorporation into mRNA by T7 RNA polymerase. Luciferase mRNAs capped with these analogs differ in resistance to hDcp2 hydrolysis in vitro, translational efficiency in rabbit reticulocyte lysate and in HeLa cells, and stability in HeLa cells. Whereas mRNAs capped with m(2)(7,2'-O)Gpp(S)pG were previously found to have the most favorable properties of translational efficiency and stability in mammalian cells, mRNAs capped with m(7)Gpp(BH3)pm(7)G are translated with the same efficiency but are more stable. Interestingly, some mRNAs exhibit a lag of up to 60 min before undergoing first-order decay (t(1/2) â 25 min). Only mRNAs that are efficiently capped, resistant to decapping in vitro, and actively translated have long lag phases.
Assuntos
Ácidos Bóricos/metabolismo , Compostos de Nitrogênio/metabolismo , Polifosfatos/metabolismo , Biossíntese de Proteínas , Estabilidade de RNA , RNA Mensageiro/análise , Selênio/metabolismo , Animais , RNA Polimerases Dirigidas por DNA/metabolismo , Endorribonucleases/metabolismo , Células HeLa , Humanos , Camundongos , Estrutura Molecular , Polifosfatos/química , Capuzes de RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Coelhos , Reticulócitos/química , Estereoisomerismo , Especificidade por Substrato , Proteínas Virais/metabolismoRESUMO
Analogs of the mRNA cap are widely employed to study processes involved in mRNA metabolism as well as being useful in biotechnology and medicinal applications. Here we describe synthesis of six dinucleotide cap analogs bearing a single phosphorothioate modification at either the alpha, beta, or gamma position of the 5',5'-triphosphate chain. Three of them were also modified with methyl groups at the 2'-O position of 7-methylguanosine to produce anti-reverse cap analogs (ARCAs). Due to the presence of stereogenic P centers in the phosphorothioate moieties, each analog was obtained as a mixture of two diastereomers, D1 and D2. The mixtures were resolved by RP HPLC, providing 12 different compounds. Fluorescence quenching experiments were employed to determine the association constant (K(AS)) for complexes of the new analogs with eIF4E. We found that phosphorothioate modifications generally stabilized the complex between eIF4E and the cap analog. The most strongly bound phosphorothioate analog (the D1 isomer of the beta-substituted analog m(7)Gpp(S)pG) was characterized by a K(AS) that was more than fourfold higher than that of its unmodified counterpart (m(7)GpppG). All analogs modified in the gamma position were resistant to hydrolysis by the scavenger decapping pyrophosphatase DcpS from both human and Caenorhabditis elegans sources. The absolute configurations of the diastereomers D1 and D2 of analogs modified at the alpha position (i.e., m(7)Gppp(S)G and m(2) (7,2'-O )Gppp(S)G) were established as S(P) and R(P) , respectively, using enzymatic digestion and correlation with the S(P) and R(P) diastereomers of guanosine 5'-O-(1-thiodiphosphate) (GDPalphaS). The analogs resistant to DcpS act as potent inhibitors of in vitro protein synthesis in rabbit reticulocyte lysates.
Assuntos
Proteínas de Caenorhabditis elegans/química , Endorribonucleases/química , Fator de Iniciação 4E em Eucariotos/química , Fosfatos/química , Oligonucleotídeos Fosforotioatos/química , Pirofosfatases/química , Análogos de Capuz de RNA/química , Animais , Guanosina/análogos & derivados , Guanosina/química , Humanos , Hidrólise , Estrutura Molecular , Oligonucleotídeos Fosforotioatos/síntese química , Oligonucleotídeos Fosforotioatos/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Análogos de Capuz de RNA/síntese química , Análogos de Capuz de RNA/farmacologiaRESUMO
Studies conducted since the 1970s have revealed that the production of milk proteins in the mammary gland under the influence of lactogenic hormones (insulin, prolactin, and glucocorticoids) is regulated at multiple levels. Whereas earlier studies concentrated on transcriptional regulation and stabilization of milk protein mRNAs, more recent studies have revealed that translation of milk protein mRNAs is also dependent on lactogenic hormones. A general stimulation of translation in mammary epithelial cells is caused by amino acids (as signaling molecules) or by phosphorylation of the translational regulator 4E-BP1 in a synergistic response to signals from insulin and prolactin. However, a selective enhancement of milk protein mRNA translation is caused by cytoplasmic polyadenylation of mRNA, again in a synergistic response to these two hormones. Preliminary evidence indicates that the latter effect depends on the existence of a cytoplasmic polyadenylation element (CPE) in milk protein mRNAs and phosphorylation of its binding protein, CPEB. Experiments in whole animals, organ explants, and cell culture have shown that the poly(A) length of milk protein mRNAs changes as a function of the lactation cycle. Interestingly, cytoplasmic polyadenylation is likely to be responsible for the selective hormone-dependent enhancement of both translation and stability of milk protein mRNAs.
Assuntos
Proteínas do Leite/metabolismo , Biossíntese de Proteínas/genética , Aminoácidos/metabolismo , Animais , Hormônios/metabolismo , Humanos , Proteínas do Leite/genética , Poliadenilação , RNA Mensageiro/genéticaRESUMO
Synthetic capped RNA transcripts produced by in vitro transcription in the presence of m(7)Gp(3)G have found a wide application in studying such processes as mRNA translation, pre-mRNA splicing, mRNA turnover, and intracellular transport of mRNA and snRNA. However, because of the presence of a 3'-OH on both m(7)Guo and Guo moieties of the cap structure, one-third to one-half of the mRNAs contain a cap incorporated in the reverse orientation. The reverse cap structures bind poorly to eIF4E, the cap binding protein, and reduce overall translational efficiency. We therefore replaced the conventional m(7)Gp(3)G cap by "anti-reverse" cap analogs (ARCAs) in which the 3'-OH of m(7)Guo moiety was substituted by 3'-deoxy or 3'-O-methyl groups, leading to m(7)3'dGp(3)G or m(2)(7,3'-O) Gp(3)G, respectively. The class of ARCAs was extended to analogs possessing an O-methyl group or deoxy group at C2' of m(7)Guo. We have also developed a series of ARCAs containing tetra- and pentaphosphates. mRNAs capped with various ARCAs were translated 1.1- to 2.6-fold more efficiently than their counterparts capped with m(7)Gp(3)G in both in vitro and in vivo systems. In a separate series, a methylene group was introduced between the alpha- and beta-, or beta- and gamma-phosphate moieties, leading to m(2)(7,3'-O)Gpp(CH2)pG and m(2)(7,3'-O)Gp(CH2)ppG. These analogs are resistant to cleavage by the decapping enzymes Dcp1/Dcp2 and DcpS, respectively. mRNA transcripts capped with m(2)(7,3'-O)Gpp(CH2)pG were more stable when introduced into cultured mammalian cells. In this chapter, we describe the synthesis of representative ARCAs and their biophysical and biochemical characterization, with emphasis on practical applications in mRNA translation.
Assuntos
Biossíntese de Proteínas , Análogos de Capuz de RNA/síntese química , Estabilidade de RNA , Animais , Sistema Livre de Células/metabolismo , Células Cultivadas , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Espectroscopia de Ressonância Magnética/métodos , Metano/análogos & derivados , Metano/química , Modelos Biológicos , Conformação de Ácido Nucleico , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Análogos de Capuz de RNA/química , Análogos de Capuz de RNA/farmacologia , Estabilidade de RNA/efeitos dos fármacos , Especificidade por Substrato , Transcrição GênicaRESUMO
Capped RNAs synthesized by in vitro transcription have found wide utility for studying mRNA function and metabolism and for producing proteins of interest. We characterize here a recently synthesized series of cap analogs with improved properties that contain a sulfur substitution for a nonbridging oxygen in either the alpha-, beta-, or gamma-phosphate moieties, m(2) (7,2'-O )Gppp(S)G, m(2) (7,2'-O )Gpp(S)pG, and m(2) (7,2'-O )Gp(S)ppG, respectively. The new compounds were also modified at the 2'-O position of the m(7)Guo to make them anti-reverse cap analogs (ARCAs), i.e., they are incorporated exclusively in the correct orientation during in vitro transcription. Each of the S-ARCAs exists in two diastereoisomeric forms (D1 and D2) that can be resolved by reverse-phase HPLC. A major in vivo pathway for mRNA degradation is initiated by removal of the cap by the pyrophosphatase Dcp1/Dcp2, which cleaves between the alpha- and beta-phosphates. Oligonucleotides capped with m(2) (7,2'-O )Gpp(S)pG (D2) were completely resistant to hydrolysis by recombinant human Dcp2 in vitro, whereas those capped with m(2) (7,2'-O )Gpp(S)pG (D1) and both isomers of m(2) (7,2'-O )Gppp(S)G were partially resistant. Luciferase mRNA capped with m(2) (7,2'-O )Gpp(S)pG (D2) had a t (1/2) of 257 min in cultured HC11 mammary epithelial cells compared with 86 min for m(7)Gp(3)G-capped mRNA. Luciferase mRNAs capped with m(2) (7,2'-O )Gpp(S)pG (D1) and m(2) (7,2'-O )Gpp(S)pG (D2) were translated 2.8-fold and 5.1-fold, respectively, more efficiently in HC11 cells than those capped with m(7)Gp(3)G. The greater yield of protein due to combining higher translational efficiency with longer t (1/2) of mRNA should benefit applications that utilize RNA transfection such as protein production, anti-cancer immunization, and gene therapy.