M6A/m-Seq of human B-lymphocyte cell lines from healthy controls and major depressive disorder patients
ABSTRACT: N6-Methyladenosine (m6A) and N6,2′-O-dimethyladenosine (m6Am) are abundant mRNA modifications that regulate transcript processing and translation. The role of both, here termed m6A/m, in the human stress response are currently unknown. Here, we provide m6A/m-Seq of immortalied cell lines derived from B lymphocytes from male healthy donors or male donors diagnosed with major depressive disorder (MDD), harvested 1 h after treatment with 100 nM cortisol or mock treatment. PolyA-RNA-fragments of each sample was processed both as m6A/m-sample (RNA immunoprecipiation RIP with an m6A and m6Am antibody) and RNA-input sample. Overall design: 3 biological replicates of cells from healthy male donors and 3 biological replicates of cells from male donors diagnosed with major depressive disorder each split in two treatment samples: mock treatment (ethanol only) or 100 nM cortisol in ethanol for 1 h creating 4 groups with each 3 biological replicates: healthy-mock: H_x_M, healthy-cortisol: H_x_C, MDD-mock: M_x_M and MDD-cortisol: M_x_C. Each samples was processed as m6A/m-immunoprecipitation-sample ("m6A") or as input RNA samples ("RNA"). Additionally, 1 RNA sample (equimolar mixed from all samples) was processed as IgG control.
Project description:mRNA modifications play important roles in regulating gene expression. One of the most abundant mRNA modifications is N6,2-O-dimethyladenosine (m6Am). Here, we demonstrate that m6Am is an evolutionarily conserved mRNA modification mediated by the Phosphorylated CTD Interacting Factor 1 (PCIF1), which catalyzes m6A methylation on 2-O-methylated adenine located at the 5' ends of mRNAs. Furthermore, PCIF1 catalyzes only 5' m6Am methylation of capped mRNAs but not internal m6A methylation in vitro and in vivo. To study the biological role of m6Am, we developed a robust methodology (m6Am-Exo-Seq) to map its transcriptome-wide distribution, which revealed no global crosstalk between m6Am and m6A under assayed conditions, suggesting that m6Am is functionally distinct from m6A. Importantly, we find that m6Am does not alter mRNA transcription or stability but negatively impacts cap-dependent translation of methylated mRNAs. Together, we identify the only human mRNA m6Am methyltransferase and demonstrate a mechanism of gene expression regulation through PCIF1-mediated m6Am mRNA methylation.
Project description:BACKGROUND:Studies testing the relationship between cortisol levels, depression, and antidepressant treatment response have yielded divergent results suggesting the possibility of moderators of a cortisol effect. Several studies indicate that age may moderate the relationship between cortisol and depression. In patients with Major Depressive Disorder (MDD), we studied the interactive effects of age and cortisol in association with MDD diagnostic status and mood and memory response to antidepressant treatment. METHODS:Serum cortisol levels in 66 unmedicated patients with MDD and 75 matched healthy controls (HC) were measured at baseline and retrospectively analyzed. Logistic regression was used to determine an association of age, cortisol and their interaction with MDD diagnosis in the pooled sample of MDD and HC participants. Thirty-four of the MDD participants (age range: 19-65 years; median: 36) underwent treatment with a selective serotonin reuptake inhibitor (SSRl) for 8 weeks. Clinician and self-ratings of depression symptoms, as well as tests of verbal and visual delayed recall were obtained at baseline and post treatment. Moderation analyses determined the effect of age on the relationship between baseline cortisol and treatment outcome. RESULTS:Cortisol, moderated by age, was associated with MDD diagnosis (p?<?.05), treatment-associated reduction of depression symptoms (p?<?.001) and improvement of delayed recall (p?<?.001). Modeling the Cortisol?×?Age interaction suggested that for participants below the median age of our sample, lower cortisol levels were associated with a lower rate of MDD diagnosis and higher antidepressant effects. On the contrary, in those above the median sample age, lower cortisol was associated with a higher rate of MDD and less improvement in depression symptoms and memory performance. CONCLUSIONS:Our results add to the body of literature suggesting that age might be an important factor in moderating the relationship between peripheral cortisol levels, depression, cognition, and prognosis. These results indicate that previous disparities in the literature linking peripheral cortisol levels with depression characteristics and treatment response may critically relate, at least in part, to the age of the participants studied.
Project description:FTO, the first RNA demethylase discovered, mediates the demethylation of internal N6-methyladenosine (m6A) and N6, 2-O-dimethyladenosine (m6Am) at the +1 position from the 5' cap in mRNA. Here we demonstrate that the cellular distribution of FTO is distinct among different cell lines, affecting the access of FTO to different RNA substrates. We find that FTO binds multiple RNA species, including mRNA, snRNA, and tRNA, and can demethylate internal m6A and cap m6Am in mRNA, internal m6A in U6 RNA, internal and cap m6Am in snRNAs, and N1-methyladenosine (m1A) in tRNA. FTO-mediated demethylation has a greater effect on the transcript levels of mRNAs possessing internal m6A than the ones with cap m6Am in the tested cells. We also show that FTO can directly repress translation by catalyzing m1A tRNA demethylation. Collectively, FTO-mediated RNA demethylation occurs to m6A and m6Am in mRNA and snRNA as well as m1A in tRNA.
Project description:BACKGROUND:Major Depressive Disorder (MDD) is the leading psychiatric disorder in low- and middle-income countries, and is to be the second leading cause of burden of disease by 2020. Cortisol plays a significant role in pathophysiology of MDD. Depression can alter serum cortisol level. However, the change in serum cortisol level and its association with depressive symptom severity and improvement among patients with MDD is not well studied. OBJECTIVE:To outline change in serum cortisol levels and its association with severity and improvement of depressive symptoms in newly diagnosed patients with MDD. METHOD:Hospital based longitudinal study was conducted among 34 newly diagnosed patients who met DSM-V criteria of MDD. Venous blood sample was performed twice; pre- and post- 8 weeks of treatment. Serum cortisol concentration was measured using an extracted radioimmunoassay. The 17-item Hamilton Depression Scale (HAM-D) was used to rate depression at baseline and after 8 weeks of treatment. Paired t-test was done to look the mean difference of serum cortisol level and HAM-D, before and after treatment. Pearson correlation was done to look the association between serum cortisol levels, HAM-D scores and, sociodemographic and clinical factors. Statistical significance was set at p<0.05. RESULTS:There is no significant difference in cortisol concentrations at baseline and end line (t (33) = 2.02, p = 0.052). However, there is significant difference in HAM-D total score (t (33) = 5.67, p<0.001). Baseline and end line serum cortisol levels were significantly correlated (r = .561, p = .001). Monthly family income is correlated with baseline HAM-D total score (r = -0.373, p = .030). There is no significant relationship between baseline serum cortisol level and HAM-D score. There is also no significant relationship between end line serum cortisol level and HAM-D score. CONCLUSIONS:The symptoms of MDD were reduced following treatment but there is no significant difference in serum cortisol levels. Baseline and end line serum cortisol levels were significantly correlated. We recommend further research based on large sample.
Project description:mRNAs are regulated by nucleotide modifications that influence their cellular fate. Two of the most abundant modified nucleotides are N6-methyladenosine (m6A), found within mRNAs, and N6,2'-O-dimethyladenosine (m6Am), which is found at the first transcribed nucleotide. Distinguishing these modifications in mapping studies has been difficult. Here, we identify and biochemically characterize PCIF1, the methyltransferase that generates m6Am. We find that PCIF1 binds and is dependent on the m7G cap. By depleting PCIF1, we generated transcriptome-wide maps that distinguish m6Am and m6A. We find that m6A and m6Am misannotations arise from mRNA isoforms with alternative transcription start sites (TSSs). These isoforms contain m6Am that maps to "internal" sites, increasing the likelihood of misannotation. We find that depleting PCIF1 does not substantially affect mRNA translation but is associated with reduced stability of a subset of m6Am-annotated mRNAs. The discovery of PCIF1 and our accurate mapping technique will facilitate future studies to characterize m6Am's function.
Project description:N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are abundant mRNA modifications that regulate transcript processing and translation. The role of both, here termed m6A/m, in the stress response in the adult brain in vivo is currently unknown. Here, we provide a detailed analysis of the stress epitranscriptome using m6A/m-seq, global and gene-specific m6A/m measurements. We show that stress exposure and glucocorticoids region and time specifically alter m6A/m and its regulatory network. We demonstrate that deletion of the methyltransferase Mettl3 or the demethylase Fto in adult neurons alters the m6A/m epitranscriptome, increases fear memory, and changes the transcriptome response to fear and synaptic plasticity. Moreover, we report that regulation of m6A/m is impaired in major depressive disorder patients following glucocorticoid stimulation. Our findings indicate that brain m6A/m represents a novel layer of complexity in gene expression regulation after stress and that dysregulation of the m6A/m response may contribute to the pathophysiology of stress-related psychiatric disorders.
Project description:N6-methyladenosine (m6A) is the most abundant modified base in eukaryotic mRNA and has been linked to diverse effects on mRNA fate and function. Current m6A mapping approaches rely on immunoprecipitation of m6A-containing RNA fragments to identify regions of transcripts that contain m6A. This approach localizes m6A residues to 100-200 nt-long regions of transcripts. The precise position of m6A in mRNAs cannot be identified on a transcriptome-wide level because there are no chemical methods to distinguish between m6A and adenosine. Here we show that anti-m6A antibodies can induce specific mutational signatures at m6A residues after ultraviolet light-induced antibody-RNA crosslinking and reverse transcription. Similarly, we find these antibodies induce mutational signatures at N6, 2’-O-dimethyladenosine (m6Am), a nucleotide found at the first encoded position of certain mRNAs. Using these mutational signatures, we map m6A and m6Am at single-nucleotide resolution in human and mouse mRNA and identify snoRNAs as a novel class of m6A-containing ncRNAs. UV-crosslinking and immunoprecipitation with m6A-specific antibodies was used to map m6A and m6Am in cellular RNA with single nucleotide resolution.
Project description:Various methyltransferases and demethylases catalyse methylation and demethylation of N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) but precise methylomes uniquely mediated by each methyltransferase/demethylase are still lacking. Here, we develop m6A-Crosslinking-Exonuclease-sequencing (m6ACE-seq) to map transcriptome-wide m6A and m6Am at quantitative single-base-resolution. This allows for the generation of a comprehensive atlas of distinct methylomes uniquely mediated by every individual known methyltransferase or demethylase. Our atlas reveals METTL16 to indirectly impact manifold methylation targets beyond its consensus target motif and highlights the importance of precision in mapping PCIF1-dependent m6Am. Rather than reverse RNA methylation, we find that both ALKBH5 and FTO instead maintain their regulated sites in an unmethylated steady-state. In FTO's absence, anomalous m6Am disrupts snRNA interaction with nuclear export machinery, potentially causing aberrant pre-mRNA splicing events.
Project description:FTO demethylates internal N 6-methyladenosine (m6A) and N 6,2'-O-dimethyladenosine (m6Am; at the cap +1 position) in mRNA, m6A and m6Am in snRNA, and N 1-methyladenosine (m1A) in tRNA in vivo, and in vitro evidence supports that it can also demethylate N 6-methyldeoxyadenosine (6mA), 3-methylthymine (3mT), and 3-methyluracil (m3U). However, it remains unclear how FTO variously recognizes and catalyzes these diverse substrates. Here we demonstrate-in vitro and in vivo-that FTO has extensive demethylation enzymatic activity on both internal m6A and cap m6Am Considering that 6mA, m6A, and m6Am all share the same nucleobase, we present a crystal structure of human FTO bound to 6mA-modified ssDNA, revealing the molecular basis of the catalytic demethylation of FTO toward multiple RNA substrates. We discovered that (i) N 6-methyladenine is the most favorable nucleobase substrate of FTO, (ii) FTO displays the same demethylation activity toward internal m6A and m6Am in the same RNA sequence, suggesting that the substrate specificity of FTO primarily results from the interaction of residues in the catalytic pocket with the nucleobase (rather than the ribose ring), and (iii) the sequence and the tertiary structure of RNA can affect the catalytic activity of FTO. Our findings provide a structural basis for understanding the catalytic mechanism through which FTO demethylates its multiple substrates and pave the way forward for the structure-guided design of selective chemicals for functional studies and potential therapeutic applications.
Project description:Depressed adults have shown blunted or elevated cortisol reactivity in response to various forms of psychosocial stress. However, there have been few studies of cortisol reactivity in children who had early onset depression or a history of depression during the preschool-school period. The present study utilized a laboratory stress paradigm and collected salivary cortisol from preschoolers at baseline (ages 3-5 years) and 24-month follow-up (ages 5-7 years). Repeated-measures multivariate analyses of variance (MANOVAs) were used to compare cortisol reactivity to mild stress between children with Major Depressive Disorder (MDD), elevated symptoms of depression (sub-syndromal MDD), and healthy controls. For healthy children, a quadratic cortisol reactivity curve was found at baseline (n=73), which appeared flatter under similar stressful situations at follow-up (n=14), which may reflect acclimation to the paradigm. In contrast, children with MDD (n=46) and sub-syndromal MDD (n=76) showed a peak cortisol response to the novelty of lab arrival and then reduced and blunted responses to stressors at baseline. These cortisol responses persisted at follow-up in children with a history of MDD (n=41) or sub-syndromal MDD (n=73). These results suggest that the hypothalamic-pituitary-adrenal (HPA) axis shows a blunted response to stress and failed to acclimate to familiar stressful situations in depressed and sub-syndromal depressed children.