FMRP Regulates the Nuclear Export of Adam9 and Psen1 mRNAs: Secondary Analysis of an N6-Methyladenosine Dataset

Fragile X mental retardation protein (FMRP) binds to and regulates the translation of amyloid-β protein precursor (App) mRNA, but the detailed mechanism remains to be determined. Differential methylation of App mRNA could underlie FMRP binding, message localization and translation efficiency. We sought to determine the role of FMRP and N6-methyladeonsine (m6A) on nuclear export of App mRNA. We utilized the m6A dataset by Hsu and colleagues to identify m6A sites in App mRNA and to determine if the abundance of message in the cytoplasm relative to the nucleus is altered in Fmr1 knockout mouse brain cortex. Given that processing of APP to Aβ and soluble APP alpha (sAPPα) contributes to disease phenotypes, we also investigated whether Fmr1KO associates with nuclear export of the mRNAs for APP protein processing enzymes, including β-site amyloid cleaving enzyme (Bace1), A disintegrin and metalloproteinases (Adams), and presenilins (Psen). Fmr1KO did not alter the nuclear/cytoplasmic abundance of App mRNA. Of 36 validated FMRP targets, 35 messages contained m6A peaks but only Agap2 mRNA was selectively enriched in Fmr1KO nucleus. The abundance of the APP processing enzymes Adam9 and Psen1 mRNA, which code for a minor alpha-secretase and gamma-secretase, respectively, were selectively enriched in wild type cytoplasm.

www.nature.com/scientificreports www.nature.com/scientificreports/  Table 5 18 ), RPKM values were extracted for nuclear and cytoplasmic fractions isolated from cortices of WT and Fmr1 KO mice (postnatal day 11) and the mean expression level was plotted as response variable versus mouse genotype as predictor. Error bars represent standard error of the mean (SEM). Asterisks indicate statistical differences between nuclear and cytoplasmic compartments computed by 2-way ANOVA with post-hoc Bonferroni multiple comparison tests (p < 0.050). Screened FMRP targets were previously reviewed 9 . Targets are presented in alphabetical order. See Figure 2 for the remaining targets.
www.nature.com/scientificreports www.nature.com/scientificreports/  Table 5 18 ), RPKM values were extracted for nuclear and cytoplasmic fractions isolated from cortices of WT and Fmr1 KO mice (postnatal day 11) and the mean expression level was plotted as response variable versus mouse genotype as predictor. Error bars represent standard error of the mean (SEM). Asterisks indicate statistical differences between nuclear and cytoplasmic compartments computed by 2-way ANOVA with post-hoc Bonferroni multiple comparison tests (p < 0.050). Screened FMRP targets were previously reviewed 9 . Targets are presented in alphabetical order. See Figure 1 for the remaining targets.
www.nature.com/scientificreports www.nature.com/scientificreports/ Hsu and colleagues recently combined photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) with m 6 A immunoprecipitation (m 6 A-IP) to determine if FMRP binds directly to m 6 A methylation modifications on messenger RNA (mRNA) 18 . They demonstrated that FMRP binds directly to m 6 A sites in mRNAs, FMRP deletion increases nuclear m 6 A-mRNA levels, and the abundance of FMRP mRNA targets in the cytoplasm relative to the nucleus decreases in Fmr1 KO mice 18 . These results strongly suggest that FMRP functions in the nuclear export of m 6 A-modified FMRP-target mRNAs.
The mRNA coding for amyloid-β precursor protein (APP) is an FMRP target. FMRP binds to a guanine-rich sequence in the coding region of both the mouse (App) and human (APP) variants of App mRNA and inhibits protein synthesis 19,20 . APP is the parent protein that is processed by secretases to produce amyloid-β (Aβ), which is the most prevalent protein found in the senile plaques of Alzheimer's disease, as well soluble APP alpha (sAPPα), which is elevated in autism [21][22][23] . APP is dysregulated in Fmr1 KO mice through a metabotropic glutamate receptor 5 (mGluR 5 )-dependent pathway, whereby activation of mGluR 5 rapidly displaces FMRP from the coding region of App mRNA and thus increases translation of APP 24 . The detailed mechanism through which FMRP represses translation of APP remains to be determined.
We hypothesize that FMRP regulates localization, and hence protein synthesis of App mRNA through an m 6 A-dependent pathway. Furthermore, differential methylation of App mRNA, and not variations in FMRP levels or activity, could explain cases of autism spectrum disorder that do not accompany FMRP aberrations. Thus, cross-talk between FMRP and m 6 A-App mRNA could have implications for FXS, Alzheimer's disease, and autism. Here, we utilized the Supplementary Information provided by Hsu and colleagues to identify m 6 A sites in App mRNA and to determine if the abundance of message in the cytoplasm relative to the nucleus is altered in Fmr1 knockout (KO) mouse brain cortex. Given that processing of APP may also contribute to disease-associated differences in the APP metabolites Aβ and sAPPα, we also investigated whether Fmr1 KO associates with nuclear export of the mRNAs for APP processing enzymes, including β-site amyloid cleaving enzyme 1 (Bace1) and A disintegrin and metalloproteinases (Adam) 9, 10, and 17.

Results
The relative abundance of App mRNA in the cytoplasm versus the nucleus, based on RNAseq in cortical tissue from wild type (WT) and Fmr1 KO C57BL/6 J mice (postnatal day 11), indicated significantly increased abundance of App mRNA in the cytoplasm that did not change in response to Fmr1 knockdown (Fig. 1, Supplementary Table S1). The reported data were in reads per kilobase per million (RPKM), which normalizes the RNAseq data for both sequencing depth and the length of the gene (Hsu Supplementary Information  www.nature.com/scientificreports www.nature.com/scientificreports/ Methylation profiling of mouse cortical tissue identified multiple m 6 A sites in App mRNA (Table 1). There appears to be two highly reproducible m 6 A sites at 84-211 and 1222-1390, with additional sites at 536-600 and 878-1017. The first m 6 A site encompasses the ATG start codon at position 150, and the other three sites are within the coding region of App mRNA (NM_001198823). The 878-1017 methylation site is immediately downstream of a near canonical G-quartet sequence in the coding region of App mRNA (position 825-846; X59379) 19 . The average Log2 enrichment was high for two of the four sites including the 84-211 site encompassing the start codon of App mRNA and the 1222-1390 site in the coding region ( Table 2).
The majority of validated FMRP targets contained m 6 A sites, but Fmr1 KO did not alter the abundance of these messages in the cytoplasm relative to the nucleus. Using the FMRP target list prepared by Sethna and colleagues 9 , we found that 35 out of 36 known FMRP target mRNAs (all but Sapap3/4 mRNA) contained m 6 A peaks (Figs. 1 and 2). In comparison, of the 24,661 screened mRNAs in the dataset, 12% did not contain any m 6 A peaks. The 35 m 6 A-containing FMRP target mRNAs can be grouped based on nuclear and cytoplasmic localization. Twelve mRNAs including App mRNA had statistically significantly more message in the cytoplasm than the nucleus in both WT and Fmr1 KO cortex. Five mRNAs had statistically significant more message in the nucleus compared to the cytoplasm in both WT and Fmr1 KO cortex. Eleven mRNAs did not differ between cytoplasm and nuclear localization in WT or Fmr1 KO cortex. Four mRNAs (Dlg4, Mapk1, Pk3cb, and Pten) exhibited significantly increased cytoplasmic levels selectively in WT. Finally, one mRNA (Rps6kb1) exhibited significantly increased nuclear levels selectively in WT. Fmr1 mRNA levels were low in Fmr1 KO cytoplasm. A single message (ArfGAP with GTPase domain; Agap2) was significantly enriched in the nucleus of Fmr1 KO , suggesting that loss of FMRP reduced nuclear export. Only five mRNAs (Arc, Eef1a1, Fmr1, Gabrd, Shank3) exhibited genotype-specific differences by 2-way ANOVA (Supplementary Table S1).
The mRNAs for APP processing enzymes contained altered nuclear/cytoplasmic abundance as a function of Fmr1 KO status. For Adam9 and Psen1, WT mRNA levels were significantly increased in the cyptoplasm versus  Table 5 18 ), RPKM values were extracted for nuclear and cytoplasmic fractions isolated from cortices of WT and Fmr1 KO mice (postnatal day 11) and the mean expression level was plotted as response variable versus mouse genotype as predictor. Error bars represent standard error of the mean (SEM). Asterisks indicate statistical differences between nuclear and cytoplasmic compartments computed by 2-way ANOVA with post-hoc Bonferroni multiple comparison tests (p < 0.050).
www.nature.com/scientificreports www.nature.com/scientificreports/ nucleus (Fig. 3, Supplementary Table S2). Fmr1 KO reduced this difference to non-significant levels. Adam10 mRNA levels did not differ by genotype or location. Levels of Adam17 mRNA were significantly lower in the cytoplasm compared to the nucleus for both WT and Fmr1 KO animals. Levels of Bace1 and Psen2 mRNAs were significantly higher in cytoplasm than nucleus, but genotype did not exert a significant effect. Given the effects of Fmr1 KO on Adam9 and Psen1 mRNA localization, we examined methylation profiling, which identified five m 6 A sites in both Adam9 (Tables 3 & 4

Discussion
Methylation at m 6 A is the most abundant post-transcriptional mRNA modification in polyadenylated mRNAs and long non-coding RNAs in higher eukaryotes 25 . Recent findings indicate that FMRP target mRNAs contain an increased number of m 6 A peaks, mostly enriched in the coding regions of genes 26 , and that FMRP functions as an m 6 A reader protein that modulates neuron differentiation and mRNA stability through m 6 A-dependent mRNA mechanisms 12,[26][27][28][29] . Out of 842 FMRP mRNA targets identified by Darnell and colleagues 11 , 95% had m 6 A modifications in mouse brain cerebellum and 96% in cortex 26 . App mRNA is a validated FMRP target 19 .
App gene expression is negatively regulated by cytosine methylation [30][31][32] , but little is known regarding methylation-dependent regulation of App mRNA other than that small nuclear ribonucleoprotein (SNRP) splicing factors regulate alternative splicing through a methylation-dependent mechanism 17,33 . To our knowledge, nuclear-cytoplasmic transport of App mRNA has not been reported 34 . Hsu and colleagues performed m 6 A-Seq in cytoplasmic and nuclear samples from P11 cortical tissue isolated from WT and Fmr1 KO C57BL/6 J mice, and provided the normalized dataset as Supplementary Information to their manuscript 18 . Based on their global analysis of the dataset, they propose that FMRP is an m 6 A reader protein that binds directly to m 6 A sites in mRNA and functions in the export of those messages to the cytoplasm. This is an important phenomenon that could underlie FXS pathogenesis; thus, we wanted to determine if cross-talk between FMRP and m 6 A methylation affects the nuclear export of App mRNA.
We found that App mRNA contains four m 6 A sites and is more abundant in the cytoplasm relative to the nucleus. Fmr1 KO did not alter the abundance of App mRNA in the cytoplasm or the nucleus suggesting that crosstalk between FMRP and m 6 A sites does not regulate nuclear-cytoplasmic transport of this message. It is not surprising that App mRNA levels were similar between WT and Fmr1 KO samples as we previously demonstrated that App mRNA is a stable message and altered protein levels are not dependent on message decay 19 .
It is of interest that there is high enrichment of m 6 A in App mRNA in the region encompassing the start codon but not at the near canonical G-quartet region. RNAs that contain m 6 A can bind eukaryotic initiation factor 3 (eIF3) without having a 5'-cap. This may facilitate additional cap-independent mRNA translation during cell stress 35 . In addition, the App m 6 A region that crosses the start codon also includes a nexus with an overlapping  www.nature.com/scientificreports www.nature.com/scientificreports/ interleukin-1 acute box, an iron response element and a target for microRNA-346, all of which may participate in neuronal iron (Fe) homeostasis 36 . The guanine-rich sequence in the coding region of App mRNA functions as a binding site for FMRP and heterogeneous nuclear ribonucleoprotein C (hnRNP C), which compete for binding and inversely regulate APP protein synthesis 20 . FMRP represses translation by recruiting App mRNA to processing bodies whereas hnRNP C promotes translation by displacing FMRP 20 . It remains to be determined if m 6 A modification regulates App mRNA nuclear export through hnRNP C or other RBP, which may vary as a function of development and disease. PAR-CLIP previously identified three FMRP binding sites in APP mRNA (Ascano Supplementary Fig. 7: site 1: 888-948 in the coding region, site 2 in the coding region: 2169-2228, site 3 in the 3'-UTR: 3337-3396) 37 . Site 1 overlaps with the guanine-rich site previously identified in mouse. The other two sites were not identified as m 6 A peaks in the Hsu dataset 18 . Overall, the findings indicate that FMRP does not regulate nuclear-cytoplasmic transport of App mRNA through an m 6 A-dependent pathway.
We further asked if the nuclear/cytoplasmic transport of other known FMRP targets or APP secretases were regulated by FMRP/m 6 A crosstalk. Of 36 validated FMRP targets 9 , 35 messages contained m 6 A peaks. Several FMRP target mRNAs (Dlg4, Mapk1, Pik3cb, Pten and Rps6kb1) exhibited significantly altered nuclear/cytoplasmic distribution in WT samples, but there were trends for the same phenomenon in the Fmr1 KO , suggesting that FMRP/m 6 A crosstalk does not play a prominent role in nuclear transport of these messages. Only Agap2 mRNA was selectively enriched in Fmr1 KO nucleus suggesting that loss of FMRP reduced its nuclear export. Agap2 mRNA codes for phosphoinositide-3 kinase enhancer (PIKE) protein, which is an important regulator of group 1 mGluR-dependent phosphoinositide-3 kinase (PI3K) activity 38,39 . The gene for Agap2 is highly enriched in key pathways involved in amyloid-beta formation, the regulation of cardiocyte differentiation, and in actin cytoskeleton reorganization 40 . The Agap2 promoter is hypermethylated in Alzheimer's disease 41 . Agap2 mRNA was not included in the Edupuganti pulsed-SILAC translation dataset 29 , suggesting that FMRP regulates nuclear export but not protein synthesis. Of the 36 validated FMRP mRNA targets reviewed by Sethna and colleagues 9 , only 5 are present in the Edupuganti dataset (EEF1A1, FMR1, GSK3B, MAPK1, SOD1).  Table 6. Psen1 mRNA Mean Log 2 Enrichment ± SEM based on Table 5 data. a Instances are appearances for WT-nucleus/WT-cytoplasm/KO-nucleus/KO-cytoplasm, maximum 2 appearances per group. b Mean values and SEM are calculated as if "missing" sites had a Log2 enrichment of zero. UTR = untranslated region. www.nature.com/scientificreports www.nature.com/scientificreports/ Adam9 mRNA, which encodes for a minor α-secretase, as well as Psen1 mRNA, which codes for gamma secretase, were selectively reduced in the nucleus of WT samples but not Fmr1 knockouts, suggesting that FMRP may play a role in cytoplasmic transport of these secretase coding mRNAs (Fig. 4). This finding is unexpected in light of western blot data showing equal ADAM9 protein levels between WT and Fmr1 KO and lack of FMRP/Adam9 mRNA co-immunoprecipitation 42 even though Adam9 mRNA possess a near canonical G-quartet (DWGGN 0-2 DWGGN 0-1 DWGGN 0-1 DWGG) 7 at position 3756 in the 3'-UTR (TAGG_CT_GGAG_A_AAGG_ AAGG) (NM_001270996). Deletion of ADAM9 does not appreciably alter levels of α-secretase processing of APP 43 , but this may be due to compensatory upregulation of ADAM10 44 . An in-depth investigation of ADAM9 protein or mRNA levels in human subjects with APP-related disorders, such as Alzheimer's disease and autism spectrum disorder, has yet to be performed. It may be possible that ADAM9 disruption functions in some but not all APP-related disorders.
Overall, the main findings of this study were that FMRP/m 6 A crosstalk does not mediate the nuclear export of App mRNA nor export of the majority of other validated FMRP target mRNAs, but does affect the nuclear export of mRNAs for two APP secretases, Adam9 and Psen1. The function of m 6 A sites in Adam9 and Psen1 messages remains to be determined. Specifically, mRNA methylation has the potential to affect RNA folding, splicing, stability, sorting, transport, localization, storage, degradation and/or translation [14][15][16] . Disruption of ADAM9 function could play a role in some but not all APP-related disorders. Further investigation of ADAM9, AGAP2 and PSEN1 levels in human subjects with APP-related disorders could help in understanding Alzheimer's disease and autism spectrum disorders. It also remains to be determined how the binding and activity of other RBP are affected by m 6 A methylation and if m 6 A methylation is altered as a function of development and environment. The limitation of this study is the dataset is dependent on one time point, which precludes analysis as a function of development and disease severity. The strengths of the study are the large dataset, nuclear/cytoplasmic distribution data in quadruplicate, and utilization of the most widely used FXS model.

Methods
Dataset: We utilized the m 6 A dataset generated by Hsu and colleagues, which is available online at http://www. jbc.org/content/294/52/19889.long, to extract data regarding m 6 A modifications to App, Adam9 and Psen1 mRNAs (Hsu Supplementary Table 3 18 ) as well as FMRP target mRNA nuclear/cytoplasmic distributions (Hsu Supplementary Table 5 18 ). The Hsu dataset was generated by performing RNA isolation and m 6 A-Seq on nuclear and cytoplasmic fractions isolated from cortices of wild type (WT) and Fmr1 KO mice in the C57BL/6 J background (postnatal day 11). m6A-Seq data were available for 23,869 mRNAs and nuclear/cytoplasmic distribution data were available for 24,661 mRNAs. m6A-Seq was performed in duplicates and nuclear/cytoplasmic distribution in quadruplicate.
Analyses: Data were analyzed in accordance with STROBE guidelines (https://strobe-statement.org/index.php ?id=available-checklists). Means, standard deviations from the mean (SEM), and 2-way ANOVA with post-hoc Bonferroni multiple comparison tests were computed to describe the results. Statistical significance was defined as p < 0.050.

Data availability
All materials and data associated with the manuscript are or will be made available to readers by contacting the corresponding author.