No description available.

N6-methyladenosine (m6A) is the most ubiquitous post-transcriptional RNA modification and has an important role in determining the fate of mRNA transcripts. Among the key proteins of the m6A pathway are methyltransferases (METTL family enzymes), demethylases (FTO, ALKBH family enzymes), and m6A binding proteins (e.g., YTHDF family) which recognize RNA sequences depending on the amount and localization of m6A in target transcripts and subsequently influence the fate of mRNA transcripts. The role of methyltransferases and demethylases is to provide a dynamic balance of m6A levels and possibly to convey mechanisms of specificity for these so-called epitranscriptomic marks, which are not yet fully understood. The main objective of this work was to determine the relative changes in the expression of key m6A pathway proteins during early postnatal development and adulthood in the rat brain. We found that the level of expression of key m6A pathway proteins decreases from birth to adulthood, with the exception of a transient increase between postnatal days 10 and 18. During this period, we also found significant changes in the expression of respiratory chain complexes. However, further research is needed to provide evidence of a mechanistic link between the m6A pathway and brain energy homeostasis during...

Le contrôle de l’expression des gènes est un processus crucial pour le développement, la reproduction ou les mécanismes d’acclimatation aux stress environnementaux et met en jeu des voies de régulation post-transcriptionnelles agissant sur les ARN messagers (ARNm). Ces molécules portent des modifications chimiques dont l’une des plus abondantes est la N6-méthyladénosine ou m6A. Cette modification permet notamment d’attirer des protéines spécifiques qualifiées de « lecteurs » qui, chez les mammifères, agissent principalement pour favoriser la dégradation et/ou la traduction des ARNm. Mes travaux de thèse ont eu pour objectif de caractériser les fonctions d’un de ces lecteurs, nommé ECT2, chez la plante modèle Arabidopsis thaliana. Dans un premier temps, sa fonction de liaison aux ARNm méthylés ainsi que son rôle dans le développement de la plante ont été démontrés. Au niveau moléculaire, une approche de protéomique a permis d’identifier de nombreux partenaires d’ECT2 dont la majorité est impliquée dans le métabolisme des ARNm parmi lesquels des facteurs inhibiteurs de traduction. Les résultats d’une analyse de translatomique permettent de proposer un modèle où ECT2 jouerait un rôle de répresseur de la traduction d’ARNm en coopération avec ses partenaires LARP1 et DCP5, deux facteurs évolutivement conservés qui agissent dans le contrôle de la traduction des ARNm. Enfin, j’ai également découvert que la protéine ECT2 est dynamiquement modifiée via des phosphorylations en réponse à un stress thermique, ce qui semble notamment affecter sa capacité à reconnaitre les résidus m6A. Ces travaux suggèrent pour la première fois que l’activité d’un lecteur peut être régulée par des phosphorylations en réponse à des variations environnementales. / Control of gene expression is a crucial process for development, reproduction or acclimation to environmental stresses and involves post-transcriptional regulatory pathways acting on messenger RNAs (mRNAs). These molecules carry chemical modifications of which N6-methyladenosine (m6A) is one of the most abundant. This modification allows notably the recruitment of specific proteins qualified as “readers” which, in mammals, mostly act to promote decay and/or translation of mRNAs. My thesis aimed to characterize the functions of one of these readers, named ECT2, in the model plant Arabidopsis thaliana. First, its binding function to methylated mRNAs and its role in plant development was demonstrated. At the molecular level, a proteomic approach identified numerous ECT2’s protein partners, mainly involved in mRNA metabolism including translation inhibition factors. Results obtained from a translatome analysis suggest a model where ECT2 could play a repressive role on the translation of methylated mRNAs cooperatively with its partners LARP1 and DCP5, two evolutionarily conserved factors acting in translational control of mRNAs. Finally, I also discovered that ECT2 is dynamically modified with phosphorylations in response to heat stress affecting especially its ability to recognize m6A residues. These works suggests for the first time that the activity of an m6A reader could be regulated by phosphorylations in response to environmental changes.

In the yeast Saccharomyces cerevisiae, the IME4 methyltransferase, interacts genetically with methyl binding protein, Pho92, to affect the expression of PHO regulon target genes. Cells mutant in IME4 or PHO92 show increases in the RNA abundance of PHO regulon target genes. The increase in the RNA abundance of the PHO regulon target genes is not additive in the cells double mutant in IME4 and PHO92. Hence, Ime4 and Pho92 interact in a single pathway in PHO regulon. Surprisingly, cells overexpressing IME4 and MUM2 shows increase in some PHO regulon target genes, indicating that IME4 affects the PHO regulon target genes through multiple mechanisms in different conditions. A promoter swap experiment revealed that one of the PHO regulon mRNAs that codes for phosphatase, PHO5, is a direct target of Ime4. Further experiments are required to examine whether the same is true for all PHO regulon mRNAs.

The extent and nature of physio-chemical changes that take place in detached leaves after harvest and during storage have been reviewed and discussed by Osborne (1962) and Rogers (1955). These changes include loss of moisture (Wittwer et al., 1962), chlorophyll degredation (Person et al., 1957), Protein loss (Thimann and Manmahan, 1960), and result in subsequent appearance of the visual manifestations of senescence of plant tissues. As lettuce, like most leafy vegetables, deteriorates rapidly and steadily after harvest. Loss of quality is inevitable and can only be minimized by rapid handling and with the best possible storage conditions (Pratt et al., 1954). In recent years, abundant work has been done to delay senescence by the use of various chemicals. Among the investigated chemicals, kinetin (6-furfurylarninopurine) and its related c ompounds show some promise. Van Overbeek et al. (1941) reported a potent new growthpromoting factor (kinetin) in coconut milk. This chemical is active in causing many of the growth reactions of c oconut milk at exceedingly small dosages. Subsequently several arninopurine compounds were synthesized. One of which is SD 4901 (Verdan), N6-benzyladenine, an experimental senescence inhibitor, was developed by Shell Development Company, Modesto, California in 1960. Many reports showed that this chemical is capable of delaying senescence of plant tissues on the basis of restoring protein molecules and respiration inhibition. On the other hand, others have shown stimulation of respiration and delaying of senescence. Paucity of scientific literature on the stability of those chemicals on leafy vegetables gave impetus to a study of the comparative influence of pre- and post-harvest applications of 6-furfurylaminopurine and N6-benzyladenine as related to successive harvest times. Such studies may have considerable economic bearing upon storing and shipping leafy vegetables to distant markets. This thesis presents effects of different concentrations (5, 10, and 20 ppm.) of pre- and post-harvest applications of 6-furfurylaminopurine and N6-benzyladenine as related to three successive harvest times (at one week intervals) on chlorophyll content, moisture content, total nitrogen, insoluble and soluble nitrogen, oxygen uptake, 0 and co2 production during storage (at 40 Fo and 85 percent RH) of "Great Lakes" variety of lettuce.

tRNA molecules contain more than 80 chemically unique nucleotide base modifications that contribute to the chemical and physical diversity of RNAs as well as add to the overall fitness of the cell. For instance, base modifications have been shown to play a critical role in tRNA molecules by improving the fidelity and efficiency of translation. Most of this work has been carried out extensively in Gram-negative bacteria, however, the role of modified bases in tRNAs as they relate to thermostability, structure, and transcriptional regulation in Gram-positive bacteria, such as Bacillus subtilis and Bacillus anthracis, are not well characterized. Infections by Gram-positive bacteria that have become more resistant to established drug regiments are on the rise, making Gram-positive bacteria a serious threat to public safety. My thesis work examined what role partial base modification of the tyrosyl-anticodon stem-loops (ASLTyr ) of B. subtilis and B. anthracis have on thermostability, structure, and transcriptional regulation. The ASLTyr molecules have three modified residues which include Queuine (Q34), 2-thiomethyl-N6-dimethylallyl (ms2i6A37), and pseudouridine (Y39). Differential Scanning Calorimetry (DSC) and UV melting were employed to examine the thermodynamic effects of partial modification on ASLTyr stability. The DSC and UV data indicated that the Y39 and i6A37 modifications improved the molecular stability of the ASL. To examine the effects of partial base modification on ASLTyr structure, NMR spectroscopy was employed. The NMR data indicated that the unmodified and [Y39]-ASLTyr form a protonated C-A+ Watson-Crick-like base pair instead of the canonical bifurcated C-A+ interaction. Additionally, the loop regions of the unmodified and [Y39]-ASLTyr molecules were well ordered. Interestingly, the [i6A37]- and [i6A37; Y39]- ASLTyr molecules did not form a protonated C-A+ base pair and the bases of the loop region were not well ordered. The NMR data also suggested that the unmodified and partially modified molecules do not adopt the canonical U-turn structure. The structures of the unmodified, [Y39]-, and [i6A37;Y39]-ASLTyr molecules did not depend on the presence of Mg2+, but the structure of the [i6A37]-ASLTyr molecule did depend on the presence of multivalent cations. Finally, to determine the repercussions that partial modification has on physiology and tRNA mediated transcriptional regulation in B. anthracis, antibiotic sensitivity tests, growth curves, and quantitative real-time polymerase chain reaction (qRT-PCR) were employed. Strains deficient in ms2 showed comparable growth to the parent strain when cultured in defined media, but Q deficient strains did not. The loss of ms2i6A37 conferred resistance to spectinomycin and ciprofloxacin, whereas the loss of Q34 resulted in sensitivity to erythromycin. Changes in the ratio full-length to truncated transcripts of the tyrS1 and tyrS2 genes were used to monitor tRNA mediated transcriptional regulation. The qRT-PCR data suggested that tyrS1 and tyrS2 are T-box regulated and that the loss of ms2i6A37 and Q34 might affect the interaction of the tRNATyr molecule with the specifier sequence, which is located in the 5’-untranscribed region (UTR) of the messenger RNA (mRNA).

A new series of N6,5'-bis-ureido-5'-amino-5'-deoxyadenosine derivatives was prepared and evaluated for anticancer activities using the NCI 60 panel of human cancers. Certain of the derivatives showed promising activities (low micromolar GI50's) against several of the representative cancers. These included cell lines from the following general cell types in the NCI 60: Leukemia, Breast, Central Nervous System, Non-Small Cell Lung, Ovarian, Prostate, Renal, and Colon cancers. Select compounds were also screened for their affinities for protein kinases. The synthesis of the compounds was straightforward and involved N6 acylation with arylisocyanates, preceded by activation and nucleophilic substitution of the 5'-position to give the desired 5'-azido-5'-deoxyadenosine derivatives. Reduction of the 5'-azido function with either H2/Pd-C, or Ph3P/H2O, gave the desired 5'-amino-5' deoxyadenosine products. Acylation of the 5'-amino group with N-methyl 4-nitrophenylcarbamate gave the N6,5'-bis-ureido-5'-amino-5' deoxyadenosine products. Yields ranged from good (50–75%) to excellent (75–95%) for all synthetic transformations.

Recently the Peterson lab discovered a promising anticancer adenosine derivative-- 2´,3´-bis-O-tert-butyldimethylsilyl-5´-deoxy-5´-[N-(methylcarbamoyl)amino]-N6-(N-phenylcarbamoyl)adenosine. This compound showed selective toxicity against human colon cancer cells in vitro with LC50's = 6--10 µM. It was hypothesized that the lead compound exerted its cytotoxic effects by interacting with a protein kinase. A systematic Structure Activity Relationship (SAR) was undertaken in an attempt to increase the kinase-binding affinity of the lead compound. Many regions of the lead compound were examined: the N6-phenyl urea moiety, the 5´-N-methyl urea group, the 2´,3´-bis-O-TBS groups, the nucleobase, and the ribose sugar. Results of these studies produced some promising new derivatives. In particular, one analogue exhibited potent cancer cell growth inhibition with an average GI50 of 0.58 μM (NCI-60). In addition, another compound showed selective toxicity for the non-small cell adenocarcinoma cell line NCI-H522 with an LC50 of 10 nM. Efficient methods for the preparation of a wide variety of N6-aryl and -alkyl substituted derivatives were developed. One versatile route involved the installation of an N6-ethoxy carbonyl and subsequent displacement with an alkly- or arylamine. Synthetic routes for the preparation of of a variety of 2´,3´-bis-O-acylated analogues were also developed. Nucleoside mono-, di-, and triphosphate bioisosteres in which the phosphoester or phosphoanhydride have been replaced by an unnatural functional group have been extensively investigated. A simple and efficient method was developed for the preparation of carbamoyl analogues of nucleoside mono-, di-, and triphosphate surrogates. This method uses a modified version of the Kočovský reaction to install mono-, di-, and triphosphate mimics in good to excellent yields (ave = 75%).

Följande studie behandlar hur den kommunala och regionala platsmarknadsföringen i norra Sverige fungerar och om det finns en koppling mellan dem. För att undersöka detta har Umeå- och Örnsköldsvik kommun valts ut för att företräda både kommunerna i sig och som representanter för N6-initiativet. Initiativet består av de fem största städerna i norra Sverige som tillsammans vill ändra bilden av norra Sverige. Metoden består av intervjuer med tjänstepersoner inom kommunerna, samt inom N6-initiativets verksamhet. Det kombineras med en innehållsanalys av de berörda kommunernas webbplatser, samt av initiativets hemsida och tillhörande informationsvideo. Resultatet består av en sammanställning av intervjuer och innehållsanalyser, samt en koppling till tidigare forskning som presenterats i studien. Det som framgår av undersökningen är att både Umeå och Örnsköldsvik kommun använder sig av olika strategier för att marknadsföra sig, däremot finns det flera likheter mellan kommunernas val av marknadsföring, såsom kultur och idrott. Det framgår även hur de aktivt anstränger sig för att behålla platsens identitet och unikhet i arbetet med platsmarknadsföring, vilket ofta kan influeras av trender. Ett genomgående tema under intervjuerna är konkurrens, där slutsatsen är att konkurrensen inte färgat N6-initiativets samarbeten. Däremot används initiativet inte direkt i kommunernas arbete, trots att det bidragit med flertalet positiva aspekter.

[ES] La N6-metiladenosina (m6A) es una modificación generalizada en los ARN celulares de diferentes organismos que puede afectar muchos procesos y vías celulares. En las plantas, ocurre mediante un complejo de metilación que contiene varias proteínas: MTA, MTB, FIP37, VIR y HAKAI. Esta modificación es eliminada por desmetilasas de la familia AlkB, mientras que los miembros de la familia ETC son las proteínas mejor descritas que reconocen y procesan los ARN m6A-modificados. Estudios de epitransciptómica viral han revelado un papel igualmente importante de m6A durante la infección por virus; sin embargo, no existe una función pro- o antiviral de m6A generalizada. El laboratorio donde se ha llevado a cabo este trabajo ha sido pionero en el estudio del efecto de m6A en la interacción planta-virus, utilizando como virus modelo el AMV. El AMV pertenece a la familia Bromoviridae, y su genoma está formado por tres (+)ssARN. Los ARN1/2 codifican las subunidades de replicasa (P1 y P2), mientras que el ARN3 codifica la proteína de movimiento (MP) y sirve como molde para la síntesis del sgARN4, que codifica la proteína de cubierta (CP). Al comienzo de esta tesis, nuestro laboratorio ya había informado sobre: la presencia de supuestos motivos m6A en el 3'UTR/RNA3, una región crítica para la replicación de AMV, la primera m6A-desmetilasa de Arabidopsis (ALKBH9B), la relevancia funcional de ALKBH9B para mantener niveles adecuados de m6A/A para la correcta replicación de AMV, la capacidad de la CP de AMV para interactuar con ALKBH9B, posiblemente para usurpar la actividad de ALKBH9B, y la capacidad de las proteínas de Arabidopsis ECT2/3/5 para interactuar con el ARNv de AMV que contienen m6A. Dada la relevancia funcional de m6A en la biología de AMV, en esta tesis se decidió profundizar en el conocimiento de las implicaciones del mecanismo de regulación de m6A en el ciclo infeccioso viral de AMV. Para ello, se decidió: profundizar en la comprensión funcional de la m6A-desmetilasa ALKBH9B, evaluar la función in vivo de los supuestos dos sitios m6A presentes en el 3'UTR/ARN3, y explorar una posible implicación de algunas m6A metiltransferasas en la infección causada por AMV. El mapeo de los subdominios funcionales de atALKBH9B determinó la presencia de IDRs en la región N-terminal, dentro del dominio interno similar a AlkB y en la región C-terminal. Alrededor del 78% del RBD identificado en ALKBH9B está contenido en el IDR C-terminal. Debido a que las IDRs se localizan con frecuencia en proteínas que se someten a LLPS, un proceso que probablemente contribuye a la formación y estabilidad de los gránulos de ARN, es posible que las IDR y la RBD de ALKBH9B puedan actuar de manera cooperativa para promover la formación de gránulos de ARN. El análisis de los putativos motivos DRACH localizados en el bucle de hpB y en el tallo inferior de hpE del 3'UTR/ARN3 de AMV demostró que son sitios críticos involucrados en la replicación in vivo de AMV. La identidad de los residuos 2012A, 2013A y 2014A en el bucle hpB parece ser un requisito estructural clave para la replicación y/o acumulación de AMV. Con respecto a hpE, nuestros resultados determinaron que el supuesto residuo de m6A (1902A), así como el apareamiento de bases del tallo inferior de hpE, también son requisitos esenciales para la síntesis in vivo de ARNs de cadena positiva en AMV. Hasta donde sabemos, esta es la primera evidencia en AMV que muestra que el bucle de hpB y el tallo inferior de hpE están involucrados en la replicación/acumulación viral y la síntesis de ARNs de cadena positiva, respectivamente. Finalmente, en cuanto al estudio de la influencia de las m6A-metiltransferasas en el ciclo de infección viral de AMV, no se determinó un efecto proviral y/o antiviral en el complejo m6A-ARNm metiltransferasa conformado por atMTA:atMTB, ni en el putativo complejo m6A- ARNr metiltransferasa conformado por atMETTL5-like:atTRMT112-like sobre la biología de AMV. / [CA] La N6-metiladenosina (m6A) és una modificació generalitzada en els ARN cellulars de diferents organismes que pot afectar molts processos i vies cellulars. En les plantes, ocorre mitjançant un complex de metilació que conté diverses proteïnes: MTA, MTB, FIP37, VIR i HAKAI. Aquesta modificació és eliminada per desmetilasas de la família AlkB, mentre que els membres de la família ETC són les proteïnes més ben descrites que reconeixen i processen els ARN m6A-modificats. Estudis de epitransciptómica viral han revelat un paper igualment important de m6A durant la infecció per virus; no obstant això, no existeix una funció pro- o antiviral de m6A generalitzada. El laboratori on s'ha dut a terme aquest treball ha sigut pioner en l'estudi de l'efecte de m6A en la interacció planta-virus, utilitzant com a virus model el AMV. El AMV pertany a la família Bromoviridae, i el seu genoma està format per tres (+) ssARN. Els ARN1/2 codifiquen les subunitats de replicasa (P1 i P2), mentre que l'ARN3 codifica la MP i serveix com a motle per a la síntesi del sgARN4, que codifica la CP. Al començament d'aquesta tesi, el nostre laboratori ja havia informat sobre: la presència de suposats motius m6A en el 3'UTR/RNA3, una regió crítica per a la replicació de AMV, la primera m6A-desmetilasa de Arabidopsis (ALKBH9B), la rellevància funcional d'ALKBH9B per a mantindre nivells adequats de m6A/A per a la correcta replicació de AMV, la capacitat de la CP de AMV per a interactuar amb ALKBH9B, possiblement per a usurpar l'activitat d'ALKBH9B, i la capacitat de les proteïnes de Arabidopsis ECT2/3/5 per a interactuar amb el ARNv de AMV que contenen m6A. Donada la rellevància funcional de m6A en la biologia de AMV, en aquesta tesi es va decidir aprofundir en el coneixement de les implicacions del mecanisme de regulació de m6A en el cicle infecciós viral de AMV. Per a això, es va decidir: aprofundir en la comprensió funcional de la m6A-desmetilasa ALKBH9B, avaluar la funció in vivo dels supòsits dos llocs m6A presents en el 3'UTR/ARN3, i explorar una possible implicació d'algunes m6A metiltransferasas en la infecció causada per AMV. El mapatge dels subdominis funcionals de atALKBH9B va determinar la presència de IDRs a la regió N-terminal, dins del domini intern similar a AlkB i a la regió C-terminal. Al voltant del 78% del RBD identificat en ALKBH9B està contingut en el IDR C-terminal. Pel fet que les IDRs es localitzen amb freqüència en proteïnes que se sotmeten a LLPS, un procés que probablement contribueix a la formació i estabilitat dels grànuls d'ARN, és possible que les IDR i la RBD d'ALKBH9B puguen actuar de manera cooperativa per a promoure la formació de grànuls d'ARN. L'anàlisi dels putatius motius DRACH localitzats en el bucle de hpB i en la tija inferior de hpE del 3'UTR/ARN3 de AMV va demostrar que són llocs crítics involucrats en la replicació in vivo de AMV. La identitat dels residus 2012A, 2013A i 2014A en el bucle hpB sembla ser un requisit estructural clau per a la replicació i/o acumulació de AMV. Respecte a hpE, els nostres resultats van determinar que el suposat residu de m6A (1902A), així com l'aparellament de bases de la tija inferior de hpE, també són requisits essencials per a la síntesi in vivo de ARNs de cadena positiva en AMV. Fins on sabem, aquesta és la primera evidència en AMV que mostra que el bucle de hpB i la tija inferior de hpE estan involucrats en la replicació/acumulació viral i la síntesi de ARNs de cadena positiva, respectivament. Finalment, quant a l'estudi de la influència de les m6A-metiltransferasas en el cicle d'infecció viral de AMV, no es va determinar un efecte proviral i/o antiviral en el complex m6A-ARNm metiltransferasa conformat per atMTA:atMTB, ni en el putatiu complex m6A-ARNr metiltransferasa conformat per atMETTL5-like:atTRMT112-like sobre la biologia de AMV. / [EN] N6-methyladenosine (m6A) is a widespread modification on cellular RNAs of different organisms that can impact many cellular processes and pathways. In plants, m6A-methylation is mainly installed by a methylation complex containing several proteins: MTA, MTB, FIP37, VIR, and HAKAI. This modification is removed by demethylases of the AlkB family, and members of the ECT family are the best described proteins that recognize and process m6A-modified RNAs. Studies of viral epitransciptomics have revealed an equally important role of m6A during virus infection; however, there is no global pro- or antiviral role of m6A that can be generalized. The laboratory where this work was carried out has been a pioneer in the study of the effect of m6A on plant-viruses, using AMV as a model-virus. AMV belongs to the Bromoviridae family and, as the rest of the members of this family, its genome consists of three (+)ssRNAs. RNA1 and RNA2 encode the replicase subunits (P1 and P2), whereas RNA 3 encodes the MP and serves as a template for the synthesis of sgRNA 4, which encodes CP. At the beginning of this thesis, our laboratory had already reported on: the presence of putative m6A-motifs in the 3'UTR RNA3, a critical region for AMV replication, the first Arabidopsis m6A-demethylase (ALKBH9B), the functional relevance of ALKBH9B to maintain adequate m6A/A levels for correct AMV replication, the ability of AMV-CP to interact with ALKBH9B, possibly to usurp ALKBH9B activity, and the capability of Arabidopsis ECT2/3/5 to interact with m6A-containing AMV vRNAs. Given the functional relevance of m6A on the biology of AMV, in this thesis it was decided to deepen the knowledge of the implications of the m6A regulation mechanism on the viral infectious cycle of AMV. For this, it was decided: deepen the functional understanding of the m6A-demethylase ALKBH9B, evaluate the in vivo function of the putative two m6A-sites present in the 3'UTR-RNA 3, and explore a possible involvement of some m6A-methyltransferases in infection caused by AMV. We mapped functional subdomains in the atALKBH9B m6A-demethylase required for its binding to the vRNA and to the CP of AMV. Remarkably, it was observed the presence of IDRs in the N-terminal region, within the internal domain like AlkB and in the C-terminal region. About 78% of the RBD identified in ALKBH9B is contained in the C-terminal IDR. In this context, it has been proposed that the capability to specifically target different RNAs in RBPs containing IDRs is due to conformational flexibility as well as the establishment of extended conserved electrostatic interfaces with RNAs. Additionally, due that IDRs are frequently localized in proteins that undergo LLPS, a process that likely contributes to the formation and stability of RNA granules, it's possible that the IDRs and the RBD of ALKBH9B could act cooperatively to promote RNA granule formation. The analysis of the putative DRACH-motifs located in the hpB loop and the lower-stem of hpE in the 3'UTR RNA 3 present hot sites involved in AMV replication in vivo. The identity of residues 2012A, 2013A and 2014A in the hpB loop appears to be a key structural requirement for AMV replication and/or accumulation. Regarding hpE, our results determined that the putative m6A-residue 1902A, as well as the base pairing of the lower-stem of hpE, are also essential requirements for the in vivo plus-strand synthesis in AMV. To our knowledge, this is the first evidence in AMV to show that the hpB loop and the lower-stem of hpE are involved in viral replication/accumulation and plus-strand synthesis, respectively. Finally, regarding the study of the influence of m6A-methyltransferases on the viral infection cycle of AMV, a non-proviral and/or antiviral effect was determined in the m6A-mRNA methyltransferase complex made up of atMTA:atMTB, nor of the putative m6A-rRNA methyltransferase complex made up of atMETTL5-like:atTRMT112-like on the biology of AMV. / Alvarado Marchena, LF. (2022). Insights into the Molecular Mechanisms of the N6-Methyladenosine (m6A) Methylation Machinery in the Regulation of the Infection Cycle of RNA Plant Viruses [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/185122 / TESIS