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AGING 2021, Vol. 13, No. 2
Research Paper

CPEB3 regulates neuron-specific alternative splicing and involves
neurogenesis gene expression
Wenrui Qu1, Hongjuan Jin2, Bing-Peng Chen3, Jun Liu1,*, Rui Li1,*, Wenlai Guo1,*, Heng Tian1,*
1

Department of Hand Surgery, The Second Hospital of Jilin University, Changchun, Jilin Province, China
Department of Plastic and Reconstructive Surgery, The First Hospital of Jilin University, Changchun, China
3
Orthopedic Medical Center, The Second Hospital of Jilin University, Changchun, Jilin Province, China*Equal contribution
2

Correspondence to: Jun Liu, Rui Li, Wenlai Guo, Heng Tian; email: ambitiousor@edu.cn, l_rui@jlu.edu.cn,
guowl16@mails.jlu.edu.cn, th608774@jlu.edu.cn
Keywords: neuron, RNA-binding protein, CPEB3, alternative splicing, neurogenesis
Received: June 27, 2020
Accepted: October 27, 2020
Published: December 9, 2020
Copyright: © 2020 Qu et al. This is an open access article distributed under the terms of the Creative Commons Attribution
License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original
author and source are credited.

ABSTRACTIn the mammalian brain, alternative pre-mRNA splicing is a fundamental mechanism that modifies neuronal
function dynamically where secretion of different splice variants regulates neurogenesis, development,
pathfinding, maintenance, migration, and synaptogenesis. Sequence-specific RNA-Binding Protein CPEB3 hasdistinctive isoform-distinct biochemical interactions and neuronal development assembly roles. Nonetheless,the mechanisms moderating splice isoform options remain unclear. To establish the modulatory trend of
CPEB3, we cloned and excessively expressed CPEB3 in HT22 cells. We used RNA-seq to analyze CPEB3-regulated
alternative splicing on control and CPEB3-overexpressing cells. Consequently, we used iRIP-seq to identify
CPEB-binding targets. We additionally validated CPEB3-modulated genes using RT-qPCR. CPEB3 overexpression
had insignificant effects on gene expression in HT22 cells. Notably, CPEB3 partially modulated differential gene
splicing enhanced in the modulation of neural development, neuron cycle, neurotrophin, synapse, and specificdevelopment pathway, implying an alternative splicing regulatory mechanism associated with neurogenesis.
Moreover, qRT-PCR verified the CPEB3-modulated transcription of neurogenesis genes LCN2 and NAV2,
synaptogenesis gene CYLD, as well as neural development gene JADE1. Herein, we established that CPEB3 is a
critical modulator of alternative splicing in neurogenesis, which remarkably enhances the current
understanding of the CPEB3 mediated alternative pre-mRNA splicing.

INTRODUCTION
The mammalian brain is a uniquely complex but a wellstructured system, constituting various types of neuronalcells which form specific synaptic links with otherneurons [1]. It is particularly essential to comprehend how
neurogenesis, development, pathfinding, maintenance,
migration, and synaptogenesis are modulated with such
precision. Recently, alternative splitting decisions were
thought to control key neuronal developmental phases,
like neurogenesis, synaptic connectivity, plasticity, andremodeling [2–5]. Furthermore, a growing number of

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neurological illnesses (e.g., autism, depression,
Parkinson's disease) correlated with known or suspected
splicing deficiencies, suggests the potential significant
function of neural alternative splicing events in several
biological processes [6–8]. Recent advances have been
made toward understanding the contributions ofalternative splicing that modulate isoform switching
during neurogenesis [9].
Neurogenesis is typified by extensive alterations in thetranscriptomes, as well as proteomes of the differentiating
cells, required for the optimal transformation of neuralAGINGprecursor or stem cells to mature neurons [2, 10]. Various
gene modulatory pathways moderate the phases ofneuronal growth consisting of neuronal relocation,neuronal plasticity, synaptic formations, dendritic andaxonal outgrowth [2, 8]. The dynamic modulation of AS
in the nervous system is essential for moderating proteinprotein interactions, transcription systems, and neuronal
growth [2].
Diverse mechanisms of spatio-temporal gene regulation
have proved essential for the moderation of nervous
system design, including control of mRNA synthesis via
RNA-binding proteins (RBPs) [11]. Among these,
cytoplasmic polyadenylation element-binding protein(CPEB) family is a crucial RNA-binding protein that has
been noted in developing, synaptic plasticity and cellularsenescence [12, 13]. CPEB confines the strength ofglutamatergic synapses through regulating the translationof several plasticity-related proteins (PRPs) RNAs inneurons [14–16]. This RNA-binding protein controls thecytoplasmatic polyadenylation and translation of target
mRNAs at synapses via a self-sustaining, functional
prion-like shape [17]. This prion-like process shifts CPEB
monomers from their original conformation into a
differential, self-propagating shape, in which CPEB
establishes continuously active build-ups [18–20]. In thevertebrates, four members (CPEB1, CPEB2, CPEB3, and
CPEB4) have been identified, all expressed in the brain[21], and share the structure and sequence-specificidentity in the RNA-binding domain [22]. CPEB1
achieves the translation of mRNA in target mRNA andsubsequent binding of the cap-binding factor. Differently,
CPEB2-4 has distinct U-rich loop motifs, suggestingtargets unique to those subgroups [23]. Functionally,
CPEB3 modulates the translation of several PRP RNAs,
including NMDA receptor subunit 1 (NR1), AMPA-typeglutamate receptor subunits GluA2, and GluA1,
postsynaptic density protein 95 (PSD95), and thecytoskeletal protein actin [16, 24]. Particularly, CPEB3
allows a pathway for maintaining protein synthesischanges long after the initiating synaptic stimulus, acritical component, and a long-elusive piece in the puzzleof long-term memory maintenance [25]. Elevated calcium
influx resulting from NMDA stimuli activates calpain-2
and then cleaves the CPEB3 repression motif to achieve
non-polyadenylation activation of CPEB3-RNA targets[15]. Maintenance of translational control by CPEB3
guides the required protein modifications required for theestablishment and maintenance of neuronal development
and synaptosome [23]. These findings support theimportance of CPEB3 in its regulatory function in neurons.
As such, the RNA-binding protein CPEB3 mediates thetranslational activity of several identified mRNA targets
in neurons [26, 27]. Several mechanisms modulate
CPEB3-related translation in neuronal development,
migration, and synaptogenesis [14, 28]. Nevertheless,www.aging-us.com

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whether CPEB3 modulates differential splicing of premRNAs in the neuron cells remains unclear.
Here, we first cloned and excessively expressed CPEB3 in
HT22 cells obtained from neurons in the hippocampus ofmice. To establish the potential role of CPEB3 inmodulating gene expression or alternative splicing(AS) that might be related to neurogenesis andneuronal development, we obtained CPEB3-regulatedtranscriptomes in mouse HT22 cells via RNA-seq. We
then described the features of CPEB3 related differential
gene expression and alternative splicing based oncomparative analysis. Moreover, iRIP-seq was used to
identify CPEB-binding targets in differential gene
expression analysis and alternative splicing events. Thesefindings indicate that CPEB3 modulates neurogenesis andneuronal development. Herein, we revealed that the RNAbinding protein CPEB3 is a crucial modulator ofdifferential splicing in the central nervous system.

RESULTS
Profiling CPEB3-regulated gene expression
To determine the molecular mechanism of CPEB3mediated gene expression, we examined the model of
CPEB3 overexpression (CPEB3-OE) in HT22 cells.
Relative to the control group, the efficacy of CPEB3OE was remarkably higher (Figure 1A). At the sametime, the CPEB3-OE group had distinctly elevatedprotein levels (Figure 1B). To study the CPEB3moderated transcriptional modulation, we prepared
cDNA libraries of the control, as well as the CPEB3-OE
cells. The sequencing of the six RNA-seq libraries was
accomplished on the Illumina HiSeq4000 platform togenerate 150nt paired-end reads per sample. The
sequencing data were reviewed to validate theirreliability (Supplementary Table 1). In comparing gene
expression patterns across samples, the expression
values (fragments/kilobase of exon model per million
fragments mapped (FPKM)) were computed. Effective
excessive expression of CPEB3 was moreover verified
in a comparative RNA-seq assessment (Figure 1C). We
utilized the FPKM values to compute an association
matrix as per Pearson’s correlation coefficients.
Consequently, the diagonal of the heat map revealed thePearson association linking CEPB3-OE and control
cells (Figure 1D). The biological replicates were highly
associated.
CPEB3 resulted in some transcriptional difference
The RNA-seq data acquired from the CEPB3-OE andfrom control cells was used to explore CPEB3-regulated
genes at the transcriptional level. We employed thecriteria of an absolute fold change ≥2 and FDR ≤0.05

AGINGwith the edgeR package to determine the differentiallyexpressed genes (DEGs) modulated by CPEB3 at thetranscriptional level (Supplementary Table 2).
Consequently, 31 up modulated and 23 down modulated
genes related with CPEB3 were identified. We
constructed a volcano plot to show the remarkably
expressed genes correlated with CPEB3-OE (Figure1E). A heat map estimation of the DEG expression
trends in the RNA-seq samples showed highconsistency with the CPEB3-regulated transcription inboth datasets (Figure 1F).

It was possible that the activated expression of theneurogenesis genes was by CPEB3 excessive
expression. Therefore, we conducted a qPCR analysis tovalidate the RNA-seq results. To examine thepossibility, we assessed the levels of Lipocalin-2 (LCN2)
and serum amyloid A3 (SAA3) mRNA expression.
These genes were highly expressed in neurogenesis,
neuronal development, and neuroinflammation. We
demonstrated a significant increase in LCN2 and SAA3
expression, highly consistent with the sequencing data
(Figure 1G).

Figure 1. CPEB3 overexpression has little effect on gene expression in HT22 cells. (A) CPEB3 expression quantified by qRT-PCR.
Error bars represent mean ± SEM. ***p < 0.001. (B) Western blotting analysis of CPEB3 expression. (C) CPEB3 expression quantified by RNA
sequencing data. FPKM values were calculated as that has been explained in Materials and Methods. Error bars represent mean ± SEM. ***p
< 0.001. (D) The heat map shows the hierarchically clustered Pearson correlation matrix resulted from comparing the transcript expression
values for control and CPEB3 overexpression samples. (E) Identification of CPEB3 regulated genes. Up-regulated genes are labeled in red,
whereas down-regulated are labeled in blue in the volcano plot. (F) Hierarchical clustering of DEGs in control and CPEB3 overexpression
samples. FPKM values are log2-transformed and then median-centered by each gene. (G) Validation of gene expression of DEGs using qRTPCR. RNA-seq quantification is shown at left, and RT-qPCR validation is shown at right.

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CPEB3 overexpression modulates pre-mRNA
alternative splicing of neurons and mediates
neurogenesis-related genes
One primary aim of this study constituted gaining
insights into the function of CPEB3 on alternative
splicing regulation. To assess the CPEB3-dependent
AS effects in HT22 cells, we evaluated the data quality
using an analysis of differential splicing. A total of
93M ± 6.9M uniquely mapped reads were retrieved
from CPEB3-OE and control HT22 cells, in which
about 47.62%~ 48.83% were junction reads(Supplementary Table 1). Analysis of the annotation of
the reference genome identified 170,257 novel splice
junctions using the Tophat2 pipeline), 141,720
annotated splice junctions and 68.03% annotatedexons (187,900 out of 284,564 annotated exons)(Supplementary Table 3). Subsequently, we employed
the ABLas software [29] to assess the AS events fromthe RNA-seq dataset to determine the global variationsin the AS profiles in response to CPEB3-OE. We
detected 167,859 novel ASEs, excluding intron
retention (IR) and 35,128 known ASEs in the model

gene named in the reference genome (Supplementary
Table 3). Using a stringent cutoff of p-value ≤0.05,
changed AS ratio ≥ 0.2, we identified high-confidence
RASEs, which gave rise to 497 RASEs(Supplementary Table 4). Complete RASEs included
82 known intron-retention (IR) RASEs and 415 non-IR(NIR) RASEs. The NIR RASEs constituted 104
alternative 3’ splice site (A3SS), 136 alternative 5’
splice site (A5SS), 72 exons skipping (ES), andcassette exon (CE, 43) (Figure 2A). The data indicatedthat CPEB3 modulates ASEs in HT22 cells globally.
In the biological process terms of GO assessments, theup modulated genes A large number of genes withalternative splicing in the CPEB3 overexpressed cells
were enriched in cell proliferation and transcription,
including cell division, mitosis, regulation oftranscription, RNA splicing, transcription (Figure 2B).
Enriched KEGG pathways included VEGF, RIG-I-likereceptor, and Toll-like receptor signaling pathways(Figure 2C, Supplementary Table 5). These findings
posit that CPEB3 potentially plays a critical role inneurodevelopment and immunity.

Figure 2. RNA-seq data reveals CPEB3 regulates gene alternative splicing in HT22 cells. (A) Classification of CPEB3 overexpression
regulated alternatively spliced events. (B) The top 10 enriched GO biological processes of the CPEB3-regulated alternatively spliced genes. (C)The top 10 enriched KEGG pathways of the CPEB3-regulated alternatively spliced genes.

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Validation of CPEB3-regulated AS events in HT22cells
To confirm the differential splicing effects determinedusing the RNA-Sequencing data, we evaluated potential
differential splicing events using qPCR. We designed
the PCR primer pairs for multiplexing the long andshort splicing isoforms (Supplementary Table 6). The
differential splicing events verified by the qPCR data
were consistent with the RNA-Seq data, located intumor necrosis factor receptor-associated factor 6(TRAF6), JADE1, and neuron navigator 2 (NAV2). We
found a significant increase in TRAF6 and JADE1.
There were distinct reductions in NAV in the CPEB-OE
cell lines. All the results were consistent with the RNAseq analysis. Most of these genes are involved inneuronal development, neuron progenitors, or neuroinflammation directly or indirectly involved inneurological disorders (Figure 3). The findings
validated the CPEB3-modulated ASEs determined by
ABLas assessments of RNA-seq.
CPEB3-mRNA interaction map in HT22 cells
To establish the correlation of CPEB3-mRNA
interaction in HT22 cells, we constructed atranscriptome-wide binding profile of CPEB3 using theiRIP-seq approach [30]. iRIP captures both direct andindirect RNA–RBP interactions. The flag-tagged
CPEB3 was employed for immunoprecipitation, andtwo separate iRIP replicates were conducted. FlagCPEB3 protein was identified through western blottingof both total cell lysate and Flag IP samples, but not in
the IgG control (Figure 4A). We sequenced the cDNA
libraries from anti-Flag IP and the complete cell lysate
control on an Illumina X-ten platform. The heat map
revealed the hierarchically classified Pearson
association matrix generated from comparing thetranscript abundance between anti-flag and IgG
immunoprecipitated samples (Figure 4B). A plot of theassayed levels of each gene was reflected using the
FPKM in each pair of the samples, which suggested thattranscripts were enriched in the IP samples (Figure 4C).
Reads distribution across reference genome revealed theFlag-CPEB3 reads were statistically enhanced in genesites consisting of the 5’UTR, noncoding (NC) exons,and introns, when contrasted with the input control
reads (Figure 4D). Peak distribution across different
genomic regions revealed the IP group was enhanced ingene regions consisting of the 5’UTR, 3’UTR, CDS,
and introns (Figure 4E). The peaks were identified
using the ABLIRC algorithm. Peaks from the two setsof experiments overlapped well (Figure 4F). It further
indicated that these genes interacting with CPEB3 werehighly enhanced for modulation of transcription, protein
phosphorylation, and regulation of translation (Gowww.aging-us.com

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biological process terms, Figure 4G, left panel).
Enriched KEGG cascades constituted MARK signalingcascade, focal adhesion, neurotrophin signaling cascade,Wnt signaling axis, and the cell cycle (Figure 4G, rightpanel, Supplementary Table 7). The CPEB3-boundgenes were enriched in transcription, neurodevelopment,
and neurogenesis genes.
Functional analysis and validation of the CPEB3bound genes in HT22 cells
We used the ABLIRC tool to elucidate the CPEB3bound genes from the iRIP-seq reads. Consequently, 76of the CPEB3-bound genes (17691) overlapped with the
CPEB3-regulated alternative splicing genes (371)(Figure 5A), indicating that CPEB3 could modulatedifferential splicing processes via direct binding to
RNA targets. We overlapped the CPEB3-bound genes
with the RASG. Go enrichment to determine the
CPEB3 binding related differential splicing modulation.
This further revealed that the CPEB3-modulated genes
are involved in synaptic formation (Figure 5B).
Enriched KEGG pathways were enriched for metabolic
pathways, pyruvate metabolism, and Toll-like receptor
signaling pathway (Figure 5C). We found that the GCrich motifs were highly enhanced in the AS overlapped
CPEB bound peak (Figure 5D), suggesting that the first
GC in the motif were the critical sites for CPEB3
binding to its targets. We performed qPCR analyses tovalidate the CPEB3-bound genes overlapping withRASG detected in this study. Lcn2 was markedly
enriched in the anti-CPEB3 immunoprecipitatecontrasted with the input. The association of Lcn2
mRNA to CPEB3 was besides confirmed by the qPCR
assays (Figure 5E). Notably, both the RNA-seq, as wellas the iRIP-seq peaks posited that Lcn2 is a direct targetof CPEB3 protein in HT22 cells. Similarly, NAV2 wasdistinctly lower in the anti-CPEB3 immunoprecipitatecontrasted with the input, in agreement with RNA-seq
data (Figure 5G). Moreover, CPEB3 led a significant
increase of cylindromatosis (CYLD) in the CPEB3-OE
IP arm, which was confirmed by the qPCR assay(Figure 5F). This result indicated that CPEB3-mediatedalternative splicing of CYLD is involved insynaptogenesis. Collectively, we concluded that CPEB3
is preferentially bound to neurogenesis andsynaptogenesis genes.

DISCUSSION
The RNA-binding protein CPEB3 mediates thetranslational activity of several identified mRNA targets
in neurons. However, the regulatory mechanism throughwhich CPEB3 mediates translation remains unknown.
To our best knowledge, we first established that CPEB3
broadly modulates the differential splicing of genes that

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Figure 3. Validation of CPEB3 regulated AS events. (A–C) IGV-sashimi plot showed an alternative 3’ splicing sites (A), an exon skipping(B), and a cassette exon (C) events in three different genes. Reads variations on the alternative exon were plotted in the left panel with thetranscripts shown below. The schematic diagrams depict the structures of ASEs, AS1 (purple line), and AS2 (green line). The constitutive exon
sequences are denoted by black boxes, intron sequences by a horizontal line (right panel, top), while alternative exon by the red box. RNAseq quantification and RT-qPCR validation of ASEs are shown at the bottom of the right panel. Error bars represent mean ± SEM. *p < 0.05.

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participate in neurogenesis in HT22 cells. Herein, we
utilized RNA-seq and iRIP-seq strategy to elucidate the
CPEB3-RNA interactions in neuron cells globally. It
involved 31 up modulated genes and 23 down
modulated genes, posting that CPEB3 has an
insignificant impact on gene expression in HT22 cells.
Interestingly, we found that the overexpression of the
CPEB3 resulted in elevated LCN2 mRNA levels in
HT22 cells, which suggested that CPEB3 modulates the
splicing trend of LCN2 pre-mRNA. LCN2 is analternative mechanism for the delivery and absorptionof physiological iron [31]. Recently, LCN2 wasreported to possess various roles in cell irontransportation and homeostasis [32, 33]. Within thebrain, the iron-binding potential of LCN2 was proved tobe significant in regulating hippocampal neuronaldendritic spine density and morphology [34].
Importantly, LCN2 is also a critical modulator ofneurogenesis that regulates the neural stem cells (NSCs)
maintenance, self-renewal, proliferation, differentiation,and hippocampal plasticity and function [35]. Moreover,the iRIP-seq analysis showed that CPEB3 preferentiallybinds LCN2. Similarly, SAA3, an acute-phase proteinwith a cytokine-like feature, was elevated in CPEB-OE
cell lines. SAA3 is a crucial modulator in neuronalsurvival and death during inflammation [36]. Thesefindings contribute to the existing knowledge on themechanisms that modulate neurogenesis and neuronaldevelopment.
Evaluation of RNA-seq data obtained in the present
study showed that CPEB3 globally modulates AS in

Figure 4. RIP-seq analysis of CPEB3. (A) Western blotting analysis of immunoprecipitated CPEB3 from HT22 cells. (B) The heat map shows
the hierarchically clustered Pearson correlation matrix resulted from comparing the transcript abundance between anti-flag and IgG
immunoprecipitated samples. (C) Scatter plot of transcript abundance across the reference genome in paired samples. (D) Reads distribution
across the reference genome. Error bars represent mean ± SEM. *p < 0.05. (E) Peak distribution across different genomic regions. (F) Venn
diagram of peaks in two replicates. (G) The top 10 enriched GO biological processes(left) and KEGG pathways(right) of the CPEB3-boundgenes.

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Figure 5. Integrated analysis of CPEB3-bound genes and the regulated alternative splicing events (RASE) in response to
CPEB3 overexpression. (A) The overlap of CPEB3-bound peaks with CPEB3-regulated alternative splicing events. (B)Top 10 GO biologicalprocess in which the overlapped genes in A were enriched. (C) Top 10 KEGG pathways in which the overlapped genes in A were enriched. (D)The CG motifs over-represented in CPEB3 peaks overlapped with RASEs. The motifs were searched by running HOMER pipeline. (E–G) The
reads density landscape of CPEB3-binding peaks on DEGs and RASEs (left). RIP-qPCR validation of the CPEB3 binding a peak was showed(right). The asterisk (*) indicates *P < 0.05,** P < 0.01.

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HT22 cells. Particularly, CPEB3 binding is linked toincreased RASE numbers. In the functional assessments,
these changes in gene expression impacted cell division,transcription regulation, mitosis, and multiple signaling
pathways. Specifically, CPEB3 correlated with the RIGI-like receptor, Toll-like receptor, and VEGF signalingcascade. Recent mounting evidence posited that VEGF
supports neuronal progenitors as they proliferate,migrate, and differentiate [37]. Our findings provide
comprehensive insights on the differential splicing
modulation mechanism of CPEB3 as neuronaldevelopment and neurogenesis.
In particular, the broad significance of AS eventsoccurring involves the modulation of transcription. For
instance, the genes constitute TRAF6, JADE1, andNAV2. TRAF6 has significantly been reported tomodulate neuronal fate [38]. Previous studies suggested
that JADE1 links cell development, cell cycles, DNAtranscription, and replication hence participating in
epigenetic processes [39]. Jade1 is also subject toposttranscriptional regulation, resulting in varioustranscripts, which mediate a regulated expression during
cell cycle progression [40]. The CPEB3-OE HT22 cells
exhibit high expression levels of NAV2, which play arole in nervous system development, including neurite
outgrowth, axonal elongation, neuronal proliferation,and migration [41, 42]. Notably, CPEB3 had similareffects on NAV2 through the iRIP-seq analysis.
Overall, the iRIP-seq approach identifies the intact
RNA biomolecules bound by RBPs. Herein, we utilized
the ABLIRC algorithm to effectively determine binding
peaks and motifs of CPEB3 protein from iRIP-seq
results. iRIP-seq elucidated some indirect CPEB3
binding regions in the 5’ UTR sites, and likely in the
introns and NC-exon regions. CPEB3 enrichedregulation of binding genes in the regulation oftranslation, regulation of transcription, MAPK signalingcascade, focal adhesion, neurotrophin signaling cascade,Wnt signaling cascade, and the cell cycle. Therefore,
our study expands current knowledge of the primaryrole of CPEB3 in the regulation of neurogenesis.
Overall, 35,128 alternative splicing events wereidentified, and 447 RASEs were detected. Among them,
76 of the pre-mRNAs were modulated by the directbinding of CPEB3 in HT22 cells. Furthermore, weestablished that differential splicing modulation of
CPEB3 is global, which makes RNA-binding protein
versatile in the regulation of various alternative splice
events. When CPEB3-bound peaks overlapped withRASG events, synaptic vesicle fused to presynaptic
membrane, pyruvate metabolism, and the toll-likereceptor. The CPEB-binding genes in these pathways
included LCN2, CYLD, and NAV2. LCN2 and NAV2

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have been well-established to contribute to neurogenesis
and neuronal development. Our results regarding
CPEB3-modulated differential splicing of LCN2, as
well as NAV2, revealed a more multiplex network inneurogenesis. CYLD, a deubiquitinase distinct for K63linkage polyubiquitins, is highly expressed in the brain.
CYLD has been reported as one of the abundant
proteins in the affinity-purified PSD fraction [43]. In
addition, CYLD modulates dendritic growth andpostsynaptic differentiation in mouse hippocampal
neurons [44]. These results revealed that CPEB3mediated alternative splicing of CYLD is potentiallyinvolved in synaptogenesis.

CONCLUSION
Herein, for the first time, we established that CPEB3
broadly modulates the differential splicing of genes thatparticipate in neurogenesis in HT22 cells. The directmodulation of the gene expression enhanced inneuroinflammation and synaptogenesis has also been
demonstrated. More studies should be conducted to
explore the biological role of CPEB3-modulated neurite
outgrowth, axonal elongation, synapse formation, andneuroinflammation. Hence, we provide new insightsthat CPEB3 potentially participate in AS events,
necessary for the discovery of novel biological roles of
CPEB3 in the process of neurogenesis and neuraldevelopment.

MATERIALS AND METHODS
Cloning and plasmid construction
We employed CE Design V1.04 (Vazyme, Nanjing,
China) to design the primer pairs utilized for Hot Fusion.
Every primer includes gene-distinct fragment sequence
and a 17-30 bp pIRES-hrGFP-1a vector sequence.
F-primer: agcccgggcggatccgaattcATGCAGGATGATTTACTGATG R-primer: gtcatccttgtagtcctcgagGCTCC
AGCGGAACGGGAC Using EcoRI and XhoI (NEB),
we digested the pIRES-hrGFP-1a vector at 37° C for
2h~3h. Then, the vector digested by the enzyme gelelectrophoresed on 1.0% agarose gel, followed bypurification employing the Qiagen column kit (Qiagen.
Inc, Valencia, CA). Using Trizol reagent (15596, Life
Technologies, USA), we extracted total RNA from the
HT22 cells. Subsequently, we reverse-transcribed the
RNA to cDNA using an oligo-dT primer. After that, theinsert fragment using PCR, we detected the insert
fragment. Ligation of the vector that was linearizedusing digestion with EcoRI and XhoI (NEB) and the
PCR insert was accomplished using ClonExpress® II
One Step Cloning Kit (Vazyme). Through chemicaltransformation, we introduced the plasmids into the

AGINGEscherichia coli strain. Subsequently, the cells weregrown on LB agar plates added gL/ml ampicillin in an
overnight incubation at 37° C. We screened the colonies
through colony PCR (28 cycles) using universal primers(positioned on the backbone vector). The inserts
sequence were Sanger sequenced for verification.
Assessment of CPEB3 overexpression
We utilized the GAPDH (glyceraldehyde-3-phosphate
dehydrogenase) gene as our standard control to examine
the impacts of excessive expression of CPEB3. cDNA
generation was accomplished through standard
procedures, and RT-qPCR using the Bestar SYBRGreen RT-PCR Master Mix (2220, DBI Bioscience,
Shanghai, China) run on the Bio-Rad S1000 machine.
Using the 2-AACT approach, we normalized theconcentrations of our assay transcripts to the GAPDHmRNA levels [45]. Comparisons were accomplishedwith the Student's paired t-test in the GraphPad Prism
software (San Diego, CA, USA).

RNA-Seq raw data cleaning and alignment
Firstly, we dropped the raw reads with more than 2-N
bases. After that, we implemented an adaptor and lowquality base trimming from raw sequenced reads in
FASTX-Toolkit (Version 0.0.13). Subsequently, we
dropped short reads below 16nt. After that, mapping of
the trimmed clean reads to the GRch38 genome using
tophat2, allowing four mismatches, was performed [46].
We applied the uniquely mapped reads for genereads number counting and FPKM calculation(fragments per kilobase of transcript per million
fragments mapped) [47].
RNA-Seq and differentially expressed genes (DEG)
evaluation
The R Bioconductor package edgeR was used to screenout the DEGs [48]. We set a false discovery rate <0.05
and fold change>2 or < 0.5 as the cutoff values for
identifying DEGs.

RNA extraction and sequencing

RNA-Seq and differential splicing assessment

Before RNA extraction, the HT22 cells were made intoa fine powder via grinding. We utilized the TRIZOL
reagent for the RNA isolations. After that, we
performed two phenol-chloroform treatments to remove
contaminating biomolecules and salts from the RNA
isolates, followed by the addition of RQ1 DNase(Promega, Madison, WI, USA) to digest any
contaminating DNA. We checked the quality andquantity levels of the purified RNA by assessing theirabsorbance at 260/280nm (A260/A280) on a Smartspec
Plus (BioRad, Hercules, CA, USA). Moreover, we gelelectrophoresed the purified RNA on a 1.5% agarose
gel to check its integrity.

The differential splicing events (ASEs) and regulationrelated differential splicing events (RASEs) between
these samples were identified and quantified via the
ABLas pipeline, as outlined previously [49]. In short,the identification of ten kinds of ASEs was as per the
splice junction reads, i.e., exon skipping (ES),alternative 5' splice site (A5SS), alternative 3'splice site
(A3SS), intron retention (IR), mutually exclusive exons(MXE), mutually exclusive 5'UTRs (5pMXE), mutually
exclusive 3'UTRs (3pMXE), cassette exon, A3SS&ES,
and A5SS&ES.

For each sample, we used 1ng of the RNA in preparing
the RNA-seq library using the VAHTS Stranded
mRNA-seq Library Prep Kit (NR604, Vazyme,
Nanjing, China). Subsequently, we purified thepolyadenylated mRNAs, and then fragmented them,followed by the generation of double-strand cDNA. We
then ligated the ds-cDNAs to the VAHTS RNA
Adapters (N803/N804, Vazyme, Nanjing, China)
following the step of end repair + A tailing. The
purified ligation products (size 200-500 bps) werecleaved using the heat-labile UDG. We then amplified
the resulting single-strand cDNAs, which were purified,quantified, and kept at -80° C, waiting for sequencing.
For high-throughput sequencing, we performed library
preparation as outlined by the manufacturer andconducted on an Illumina HiSeq X Ten system togenerate 150-nt paired-end products.

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To examine RBP modulated ASE, we performed thestudent's t-test to inspect the importance of the ratio
alteration of AS events. Those events, which wereremarkable at P-value cutoff correlated with a false
discovery rate cutoff of 5%, were regarded to be RBP
modulated ASEs.
Reverse transcription qPCR verification of DEGs
and AS events
To establish the validity of the RNA-seq data in HT22
cells, we conducted qRT-PCR of the selected DEGs.
We used the remaining total RNA from RNA-seq
library preparation for the RT-qPCR. First, we
generated cDNA via reverse transcription using an MMLV Reverse Transcriptase (R021, Vazyme, Nanjing,
China). After that, we performed qPCR using the SYBRGreen PCR Reagents Kit (QR0100, Yeasen, Shanghai,
China) on Step One Real Time PCR System. The PCR
conditions constituted denaturing at 95° C for 10min,

AGING40 cycles involving denaturing at 95° C for 15s,
annealing at 60° C for 1min, and extension at 60° C for
1min. Each sample was amplified in triplicates. We
used the GAPDH gene as the standard to normalize theRNA expression levels of all the genes.

secondary antibody (anti-mouse or anti-rabbit 1:10,000)(Abcam) was measured using the enhanced
chemiluminescence (ECL) reagent (Bio-Rad, 170506).

Meanwhile, the qRT-PCR assay was also used for ASE
verification. The primers for identifying ASEs are
itemized in Additional file 1. To identify alternative
isoforms, we utilized a boundary-spanning primer forthe sequence comprising of the junction of theconstitutive exon, alternative exon, and an opposing
primer in a constitutive exon. The boundary-spanning
primer of alternative exon was developed as per themodel exon to identify model splicing or altered exon to
identify differential splicing.

The CPEB3-bound RNAs were extracted from theimmunoprecipitation of anti-Flag using the TRIzol
reagent (15596-026, Invitrogen, NY, USA). cDNA
libraries were built using the KAPA RNA Hyper Prep
Kit (KAPA, KK8541) as per the protocol outlined by
the manufacturer. High-throughput sequencing of thecDNA libraries was accomplished on an Illumina Xten
platform for 150 bp paired-end sequencing.

The Primers we used herein are presented in the
Additional file 1.

After aligning the reads onto the genome using TopHat
2 [46], we only utilized the uniquely mapped reads inthe subsequent analysis. We used the ABLIRC
approach to establish the binding sites of CPEB3 on the
genome [29]. The reads with at least 1bp overlap weregrouped as peaks. For every gene, we appliedcomputational simulation in randomly generating reads
with similar numbers and lengths as reads in peaks. We
further mapped the outputting reads to the same genes;
hence, we generated random max peak height from theoverlapping reads. We repeated the whole process 500
times. We selected all the reported peaks with heights
higher relative to the random max peaks (p-value <0.05). The IP and input samples were assessed using thesimulation separately, with the IP peaks that had
overlaps with Input peaks removed. Finally, the target
genes of IP were established using the peaks and thebinding motifs of IP protein were named using the
HOMER software (Heinz, Benner et al. 2010).

Co-Immunoprecipitation
Firstly, we lysed the HT22 in ice-chilled lysis buffer(IxPBS, 0.5% sodium deoxycholate, 0.1% SDS, 0.5%NP40) with RNase repressor (Takara, 2313) and aprotease suppressor (Solarbio, 329-98-6) on ice for5min. Subsequently, the mixture was vibrated
vigorously, followed by centrifugation at 13,000 x g at
4° C for 20min to eliminate the cell debris. We
incubated the supernatant with DynaBeads protein A/G(Thermo, 26162) conjugated with anti-flag antibody(Sigma, F1804) or normal IgG at 4° C for the overnight.
The beads were rinsed with Low-salt Wash buffer,
High-salt Wash buffer, and 1X PNK Buffer,respectively. We resuspended the beads in the elution
buffer, followed by dividing into two classes, one for
RNA extraction from CPEB3-RNA complexes andanother for the western blotting assay for CPEB3.
Western blot
We resuspended the samples in 40µl elution Buffer(50mM Tris-Cl (PH=8.0), 10mM EDTA (PH=8.0),
1%SDS, followed by incubation at 70° C for 20min at
1,400 rpm. After that, the samples were centrifuged at
13,200xg for a short time period. We then moved thesupernatant to a fresh EP tube while on the magnetic
separator. After the complexes were eluted by boilingfor about 10min in boiling water containing 1X SDS
sample buffer, the proteins were resolved on 10% SDSPAGE, with TBST buffer (20 mM Tris-buffered saline
and 0.1% Tween-20) added 5% non-fat milk powder for
1h at RT. We incubated the membranes with theprimary antibody: Flag antibody (1:2,000, Sigma,
F7425), actin (1:2000, CUSABIO), followed by HRPconjugated secondary antibody. The conjugatedwww.aging-us.com

2340

iRIP-Seq library preparation and sequencing

Data analyses

Functional enrichment estimations
To categorize the functional groups of peak associatedgenes (target genes), Gene Ontology (GO) terms and
KEGG pathways were elucidated using the KOBAS 2.0
server [50]. We utilized the Hypergeometric test andBenjamini-Hochberg FDR controlling protocol todescribe the enrichment of each term.
Availability of data and materials
The data generated and discussed in this publication areavailable under GEO Series accession (GSE153168).

Abbreviations
RBP: RNA-binding proteins; CPEB: cytoplasmic
polyadenylation element-binding protein; PRP:
plasticity-related protein; AS: alternative splicing;AGINGPSD95: postsynaptic density protein 95; FPKM:
fragments/kilobase of exon model per million fragments
mapped; LCN2: Lipocalin-2; SAA3: serum amyloid A3;
DEGs: differentially expressed genes; A3SS: alternative
3’ splice site; A5SS: alternative 5’ splice site; ES: exonsskipping; CE: cassette exon; CYLD: cylindromatosis;
NAV2: neuron navigator 2; TRAF6: tumor necrosis
factor receptor-associated factor 6.

5.

Jia Y, Mu JC, Ackerman SL. Mutation of a U2 snRNA
gene causes global disruption of alternative splicing
and neurodegeneration. Cell. 2012; 148:296–308.
https://doi.org/10.1016/j.cell.2011.11.057
PMID:22265417

6.

Le François B, Zhang L, Mahajan GJ, Stockmeier CA,
Friedman E, Albert PR. A novel alternative splicing
mechanism that enhances human 5-HT1A receptor
RNA stability is altered in major depression. J Neurosci.
2018; 38:8200–10.
https://doi.org/10.1523/JNEUROSCI.0902-18.2018
PMID:30093565

7.

Stamova BS, Tian Y, Nordahl CW, Shen MD,
Rogers S, Amaral DG, Sharp FR. Evidence fordifferential alternative splicing in blood of young
boys with autism spectrum disorders. Mol Autism.
2013; 4:30.
https://doi.org/10.1186/2040-2392-4-30
PMID:24007566

8.

Mills JD, Janitz M. Alternative splicing of mRNA in themolecular pathology of neurodegenerative diseases.
Neurobiol Aging. 2012; 33:1012.e11–24.
https://doi.org/10.1016/j.neurobiolaging.2011.10.030
PMID:22118946

9.

Raj B, O’Hanlon D, Vessey JP, Pan Q, Ray D, Buckley NJ,
Miller FD, Blencowe BJ. Cross-regulation between an
alternative splicing activator and a transcription
repressor controls neurogenesis. Mol Cell. 2011;
43:843–50.
https://doi.org/10.1016/j.molcel.2011.08.014
PMID:21884984

AUTHOR CONTRIBUTIONSQW, JH, BC, and RL designed and supervised all
experiments. JL and HT carried out all experiments.
QW executed the data analysis and drafted themanuscript. JL, RL, and HT revised the manuscript. All
authors reviewed the final manuscript.

CONFLICTS OF INTEREST
The authors declare that they have no conflicts ofinterest.

FUNDING
This work was also supported by the National Natural
Science Foundation of China (NSFC, No. 81901365).
This work was also supported by Jilin Science andTechnology Agency funds in China (No. 20180101118JC,
20180520115JH, and 20190103076JH) and Departmentof Finance of Jilin province (2019SRCJ005 and2020SCZT051).

REFERENCES
1.

Norris AD, Calarco JA. Emerging roles of alternative
pre-mRNA splicing regulation in neuronal development
and function. Front Neurosci. 2012; 6:122.
https://doi.org/10.3389/fnins.2012.00122
PMID:22936897

2.

Vuong CK, Black DL, Zheng S. The neurogenetics ofalternative splicing. Nat Rev Neurosci. 2016; 17:265–81.
https://doi.org/10.1038/nrn.2016.27 PMID:27094079

3.

Traunmüller L, Gomez AM, Nguyen TM, Scheiffele P.
Control of neuronal synapse specification by a highly
dedicated alternative splicing program. Science. 2016;
352:982–86.
https://doi.org/10.1126/science.aaf2397
PMID:27174676

4.

Iijima T, Wu K, Witte H, Hanno-Iijima Y, Glatter T,
Richard S, Scheiffele P. SAM68 regulates neuronal
activity-dependent alternative splicing of neurexin-1.
Cell. 2011; 147:1601–14.
https://doi.org/10.1016/j.cell.2011.11.028
PMID:22196734

www.aging-us.com

2341

10. Furlanis E, Scheiffele P. Regulation of neuronaldifferentiation, function, and plasticity by alternative
splicing. Annu Rev Cell Dev Biol. 2018; 34:451–69.
https://doi.org/10.1146/annurev-cellbio-100617062826 PMID:30028642
11. Conlon EG, Manley JL. RNA-binding proteins inneurodegeneration: mechanisms in aggregate. Genes
Dev. 2017; 31:1509–28.
https://doi.org/10.1101/gad.304055.117
PMID:28912172
12. Bender CL, Yang Q, Sun L, Liu SJ. NH125 reduces thelevel of CPEB3, an RNA binding protein, to promote
synaptic GluA2 expression. Neuropharmacology. 2016;
101:531–37.
https://doi.org/10.1016/j.neuropharm.2015.03.017
PMID:25842244
13. Bender CL, Sun X, Farooq M, Yang Q, Davison C,
Maroteaux M, Huang YS, Ishikawa Y, Liu SJ. Emotional
stress induces structural plasticity in bergmann glial
cells via an AC5-CPEB3-GluA1 pathway. J Neurosci.
2020; 40:3374–84.

AGINGhttps://doi.org/10.1523/JNEUROSCI.0013-19.2020
PMID:32229518
14. Peng SC, Lai YT, Huang HY, Huang HD, Huang YS. A
novel role of CPEB3 in regulating EGFR genetranscription via association with Stat5b in neurons.
Nucleic Acids Res. 2010; 38:7446–57.
https://doi.org/10.1093/nar/gkq634 PMID:20639532
15. Wang CF, Huang YS. Calpain 2 activated through Nmethyl-D-aspartic acid receptor signaling cleaves
CPEB3 and abrogates CPEB3-repressed translation inneurons. Mol Cell Biol. 2012; 32:3321–32.
https://doi.org/10.1128/MCB.00296-12
PMID:22711986
16. Chao HW, Tsai LY, Lu YL, Lin PY, Huang WH, Chou HJ, Lu
WH, Lin HC, Lee PT, Huang YS. Deletion of CPEB3
enhances hippocampus-dependent memory via
increasing expressions of PSD95 and NMDA receptors.
J Neurosci. 2013; 33:17008–22.
https://doi.org/10.1523/JNEUROSCI.3043-13.2013
PMID:24155305
17. Rayman JB, Kandel ER. Erratum: Functional Prions in
the Brain. Cold Spring Harb Perspect Biol. 2017;
9:a033597.
https://doi.org/10.1101/cshperspect.a033597
PMID:28765158
18. Si K, Choi YB, White-Grindley E, Majumdar A, Kandel
ER. Aplysia CPEB can form prion-like multimers insensory neurons that contribute to long-term
facilitation. Cell. 2010; 140:421–35.
https://doi.org/10.1016/j.cell.2010.01.008
PMID:20144764
19. Si K, Lindquist S, Kandel ER. A neuronal isoform of theaplysia CPEB has prion-like properties. Cell. 2003;
115:879–91.
https://doi.org/10.1016/s0092-8674(03)01020-1
PMID:14697205
20. Fiumara F, Rajasethupathy P, Antonov I, Kosmidis S,
Sossin WS, Kandel ER. MicroRNA-22 gates long-termheterosynaptic plasticity in aplysia through presynaptic
regulation of CPEB and downstream targets. Cell Rep.
2015; 11:1866–75.
https://doi.org/10.1016/j.celrep.2015.05.034
PMID:26095361
21. Theis M, Si K, Kandel ER. Two previously undescribed
members of the mouse CPEB family of genes andtheir inducible expression in the principal cell layersof the hippocampus. Proc Natl Acad Sci USA. 2003;
100:9602–07.
https://doi.org/10.1073/pnas.1133424100
PMID:12871996
22. Huang YS, Kan MC, Lin CL, Richter JD. CPEB3 and CPEB4
in neurons: analysis of RNA-binding specificity andwww.aging-us.com

2342

translational control of AMPA receptor GluR2 mRNA.
EMBO J. 2006; 25:4865–76.
https://doi.org/10.1038/sj.emboj.7601322
PMID:17024188
23. Qu WR, Sun QH, Liu QQ, Jin HJ, Cui RJ, Yang W, Song B,
Li BJ. Role of CPEB3 protein in learning and memory:
new insights from synaptic plasticity. Aging (Albany
NY). 2020; 12:15169–82.
https://doi.org/10.18632/aging.103404
PMID:32619199
24. Stephan JS, Fioriti L, Lamba N, Colnaghi L, Karl K,
Derkatch IL, Kandel ER. The CPEB3 protein is afunctional prion that interacts with the actin
cytoskeleton. Cell Rep. 2015; 11:1772–85.
https://doi.org/10.1016/j.celrep.2015.04.060
PMID:26074072
25. Si K, Kandel ER. The role of functional prion-likeproteins in the persistence of memory. Cold Spring
Harb Perspect Biol. 2016; 8:a021774.
https://doi.org/10.1101/cshperspect.a021774
PMID:27037416
26. Ford L, Asok A, Tripp AD, Parro C, Fitzpatrick M, de Solis
CA, Shafiian N, Fioriti L, Soni RK, Kandel ER. The low
complexity motif of cytoplasmic polyadenylation
element binding protein 3 (CPEB3) is critical for the
trafficking of its targets in neurons. bioRxiv. 2020.
[Epub ahead of print].
https://doi.org/10.1101/2020.05.16.100032
27. Ford L, Ling E, Kandel ER, Fioriti L. CPEB3 inhibitstranslation of mRNA targets by localizing them to P
bodies. Proc Natl Acad Sci USA. 2019; 116:18078–87.
https://doi.org/10.1073/pnas.1815275116
PMID:31416913
28. Hosoda N, Funakoshi Y, Hirasawa M, Yamagishi R,
Asano Y, Miyagawa R, Ogami K, Tsujimoto M, Hoshino
S. Anti-proliferative protein tob negatively regulates
CPEB3 target by recruiting Caf1 deadenylase. EMBO J.
2011; 30:1311–23.
https://doi.org/10.1038/emboj.2011.37
PMID:21336257
29. Xia H, Chen D, Wu Q, Wu G, Zhou Y, Zhang Y, Zhang L.
CELF1 preferentially binds to exon-intron boundaryand regulates alternative splicing in HeLa cells. Biochim
Biophys Acta Gene Regul Mech. 2017; 1860:911–21.
https://doi.org/10.1016/j.bbagrm.2017.07.004
PMID:28733224
30. Tu Y, Wu X, Yu F, Dang J, Wei Y, Yu H, Liao W, Zhang Y,
Wang J. Tristetraprolin-RNA interaction map reveals anovel TTP-RelB regulatory network for innate immunity
gene expression. Mol Immunol. 2020; 121:59–71.
https://doi.org/10.1016/j.molimm.2020.02.004
PMID:32163759

AGING

31. Richardson DR. 24p3 and its receptor: dawn of a newiron age? Cell. 2005; 123:1175–77.
https://doi.org/10.1016/j.cell.2005.12.008
PMID:16377555

induced by CK1α contributes to cell cycle progression.
Cell Cycle. 2016; 15:1034–45.
https://doi.org/10.1080/15384101.2016.1152429
PMID:26919559

32. Devireddy LR, Gazin C, Zhu X, Green MR. A cell-surface
receptor for lipocalin 24p3 selectively mediates
apoptosis and iron uptake. Cell. 2005; 123:1293–305.
https://doi.org/10.1016/j.cell.2005.10.027
PMID:16377569

41. McNeill EM, Klöckner-Bormann M, Roesler EC, TaltonLE, Moechars D, Clagett-Dame M. Nav2 hypomorphic
mutant mice are ataxic and exhibit abnormalities incerebellar development. Dev Biol. 2011; 353:331–43.
https://doi.org/10.1016/j.ydbio.2011.03.008
PMID:21419114

33. Bao G, Clifton M, Hoette TM, Mori K, Deng SX, Qiu A,
Viltard M, Williams D, Paragas N, Leete T, Kulkarni R, Li
X, Lee B, et al. Iron traffics in circulation bound to asiderocalin (Ngal)-catechol complex. Nat Chem Biol.
2010; 6:602–09.
https://doi.org/10.1038/nchembio.402
PMID:20581821
34. Mucha M, Skrzypiec AE, Schiavon E, Attwood BK,
Kucerova E, Pawlak R. Lipocalin-2 controls neuronal
excitability and anxiety by regulating dendritic spine
formation and maturation. Proc Natl Acad Sci USA.
2011; 108:18436–41.
https://doi.org/10.1073/pnas.1107936108
PMID:21969573
35. Ferreira AC, Santos T, Sampaio-Marques B, Novais A,
Mesquita SD, Ludovico P, Bernardino L, Correia-Neves
M, Sousa N, Palha JA, Sousa JC, Marques F. Lipocalin-2
regulates adult neurogenesis and contextual
discriminative behaviours. Mol Psychiatry. 2018;
23:1031–39.
https://doi.org/10.1038/mp.2017.95 PMID:28485407
36. Liu J, Wang D, Li SQ, Yu Y, Ye RD. Suppression of LPSinduced tau hyperphosphorylation by serum amyloid
A. J Neuroinflammation. 2016; 13:28.
https://doi.org/10.1186/s12974-016-0493-y
PMID:26838764
37. Rosenstein JM, Krum JM, Ruhrberg C. VEGF in the
nervous system. Organogenesis. 2010; 6:107–14.
https://doi.org/10.4161/org.6.2.11687
PMID:20885857
38. Kisiswa L, Fernández-Suárez D, Sergaki MC, Ibáñez CF.
RIP2 gates TRAF6 interaction with death receptor
p75NTR to regulate cerebellar granule neuron survival.
Cell Rep. 2018; 24:1013–24.
https://doi.org/10.1016/j.celrep.2018.06.098
PMID:30044969
39. Siriwardana NS, Meyer R, Havasi A, Dominguez I,
Panchenko MV. Cell cycle-dependent chromatin
shuttling of HBO1-JADE1 histone acetyl transferase(HAT) complex. Cell Cycle. 2014; 13:1885–901.
https://doi.org/10.4161/cc.28759 PMID:24739512
40. Borgal L, Rinschen MM, Dafinger C, Liebrecht VI, Abken
H, Benzing T, Schermer B. jade-1S phosphorylationwww.aging-us.com

2343

42. Pook C, Ahrens JM, Clagett-Dame M. Expression
pattern of Nav2 in the murine CNS with development.
Gene Expr Patterns. 2020; 35:119099.
https://doi.org/10.1016/j.gep.2020.119099
PMID:32081718
43. Thein S, Pham A, Bayer KU, Tao-Cheng JH, Dosemeci A.
IKK regulates the deubiquitinase CYLD at thepostsynaptic density. Biochem Biophys Res Commun.
2014; 450:550–54.
https://doi.org/10.1016/j.bbrc.2014.06.019
PMID:24928390
44. Li J, Sekine-Aizawa Y, Ebrahimi S, Tanaka S, Okabe S.
Tumor
suppressor
protein
CYLD
regulates
morphogenesis of dendrites and spines. Eur J Neurosci.
2019; 50:2722–39.
https://doi.org/10.1111/ejn.14421
PMID:31001844
45. Livak KJ, Schmittgen TD. Analysis of relative gene
expression data using real-time quantitative PCR andthe 2(-Delta Delta C(T)) method. Methods. 2001;
25:402–08.
https://doi.org/10.1006/meth.2001.1262
PMID:11846609
46. Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R,
Salzberg SL. TopHat2: accurate alignment oftranscriptomes in the presence of insertions, deletions
and gene fusions. Genome Biol. 2013; 14:R36.
https://doi.org/10.1186/gb-2013-14-4-r36
PMID:23618408
47. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan
G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L.
Transcript assembly and quantification by RNA-seq
reveals unannotated transcripts and isoform switching
during cell differentiation. Nat Biotechnol. 2010;
28:511–15.
https://doi.org/10.1038/nbt.1621 PMID:20436464
48. Robinson MD, McCarthy DJ, Smyth GK. edgeR: abioconductor package for differential expression
analysis of digital gene expression data. Bioinformatics.
2010; 26:139–40.
https://doi.org/10.1093/bioinformatics/btp616
PMID:19910308

AGING

49. Jin L, Li G, Yu D, Huang W, Cheng C, Liao S, Wu Q,
Zhang Y. Transcriptome analysis reveals the complexityof alternative splicing regulation in the fungus
verticillium dahliae. BMC Genomics. 2017; 18:130.
https://doi.org/10.1186/s12864-017-3507-y
PMID:28166730

combinations of lineage-determining transcription
factors prime cis-regulatory elements required for
macrophage and B cell identities. Mol Cell. 2010;
38:576–89.
https://doi.org/10.1016/j.molcel.2010.05.004
PMID:20513432

50. Heinz S, Benner C, Spann N, Bertolino E, Lin YC, Laslo P,
Cheng JX, Murre C, Singh H, Glass CK. Simple

www.aging-us.com

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SUPPLEMENTARY MATERIALSSupplementary Tables
Please browse full text version to see the data of Supplementary Tables 1, 2, 5 and 7.

Supplementary Table 1. Summary of RNA-seq reads used in the analysis.
Supplementary Table 2. CPEB3-OE versus Ctrl DEGs.

Supplementary Table 3. Alternative splicing events.
Known AS events#Sample3pMXE 5pMXE A3SS A3SS&ES A5SS A5SS&ES

ES

IntronR MXE

cassetteExon

Total Detected junction AS/100SJ

CPEB3_1st

416

1373

3565

323

4734

416

2019

2009

179

1158

16192

1883088.6

CPEB3_2nd

388

1359

3420

296

4500

398

1949

1978

186

1032

15506

178425

8.69

CPEB3_3rd

440

1385

3634

293

4825

410

2074

1963

203

1191

16418

199214

8.24

Ctrl_1st

380

1291

3283

280

4443

376

1930

1979

165

1048

15175

178945

8.48

Ctrl_2nd

381

1276

3341

267

4542

384

1834

1883

170

1011

15089

172304

8.76

Ctrl_3rd

365

1119

3334

275

4109

361

1829

1890

171

966

14419

169493

8.51

Total

991

3102

8138

843

10127

972

3885

4632

406

2032

35128

31197711.26

3pMXE 5pMXE

A3SS

ES

IntronR

MXE

Novel AS events
SampleA3SS&ES A5SS A5SS&ES

cassetteExon Total Detected junction AS/100SJ

Ctrl_1st

735

1859

4468

510

5566

632

2264

7407

273

959

24673

59714

41.32

Ctrl_2nd

728

1857

4313

488

5613

593

1962

6803

244

964

23565

57352

41.09

SRSF6_1st

796

1933

4765

548

6353

678

2085

7436

287

1115

25996

71257

36.48

SRSF6_2nd

724

1955

5178

585

6504

657

2361

6885

325

1188

26362

77410

34.06

Total

1982

4606

12609

1539

15249

1769

5379

15388

790

2684

61995

16785936.93#Sample

3pMXE

5pMXE

A3SS

A3SS&ES

A5SS

A5SS&ESES

IntronR

MXE

cassetteExon

Total

Detected
AS/100SJ
junction

Ctrl_1st

1037

2443

7283

821

8777

1040

4495

8522

546

2528

37492

200907

18.66

Ctrl_2nd

1040

2424

7194

815

8932

1032

4169

7955

520

2555

36636

199202

18.39

SRSF6_1st

1111

2549

7705

884

9876

1108

4408

8592

574

2767

39574

213797

18.51

SRSF6_2nd

1053

2573

8200

920

10070

1099

4778

8045

605

2871

40214

219556

18.32

Total

2461

5485

16857

2013

20314

2389

8571

17014

1269

5026

81399

32611524.96

SampleType 3pMXE 5pMXE A3SS A3SS&ES A5SS A5SS&ES
CPEB3_vs_Ctrl Up
4
13
60
2
71
3
CPEB3_vs_Ctrl Down
9
13
44
2
65
3

ES
39
33

All AS events

Supplementary Table 4. CPEB3-OE versus Ctrl differential ASs (RAS).
IntronR MXE cassetteExon Total
48
6
22
268
34
5
21
229

Supplementary Table 5. GO enrichment and KEGG pathway of RAS.

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Supplementary Table 6. DEG_Primer sequence.
Gene
DEG-Cpeb3-F
DEG-Cpeb3-R
DEG-Saa3-F
DEG-Saa3-R
DEG-Lcn2-F
DEG-Lcn2-R

Sequence
GCTGATTGGAGAACGCAACAAACTGGCAATGATAATGGACCGTGATGACTTTAGCAGCCCAGGCGAGCCTACTCTGACATGAAGATTTCAGAAGTCTTTCGGAAACTCAGGTACAGAGCTACAA

AS_Primer sequence
Gene
Traf6-M/A-F
Traf6-M-R
Traf6-A-R
Cyld-M/A-R
Cyld-M-F
Cyld-A-F
Nrp2-M/A-R
Nrp2-M-F
Nrp2-A-F
Nav2-M/A-R
Nav2-M-F
Nav2-A-F
Jade1-M/A-R
Jade1-M-F
Jade1- A-F
Naa16-M/A-R
Naa16-M-F
Naa16-A-F
Dnm1l-M/A-F
Dnm1l-M-R
Dnm1l-A-R
H2-T24-M/A-R
H2-T24-M-F
H2-T24-A-F

Sequence
AAGCCAGAAGAAGGTGTCAGAGTCAGTCGATCTGCTCAGGAGCCTTCCTATCTGCTCAGGAGGCCTGAACTCATTGTGACAGTCGCGCCTGAGGCACTTTGAAGTGGCTCCTGGCACTTTGAATGATGGAGTGATCGGTCTTCTGATTTTAAAGGGGGCACCCTGCTTTTGCAGGGGGCACCCTGGTGAGGAAGGCAATGAAGAATGTCAATGAGTTGCGCAGCTGAAAAACTGGTTGCGCAGCTTGTCGTCAGAATCCTCACTGCTTGCGCAGCAGGCTGCCTG
CCTCCCACCGGCTGCCTGTAGACGAGAGGCTTCAGCTT
AACTTCTTACCTGGGGCGAA
TTATTGAAACCTGGGGCGAA
GCTGGCATAATTGGAATTGGTTTGATGGCAAGGTCCCATCTG
ACGGGACAAGGTCCCATCTG
CCTGACCTGGCAGTTGAATGTGAAGAGGCTCCCATTTCAGTTCTCCTTACCCCATTTCAG

RIP_Primer sequence
Gene
Cyld-F
Cyld-R
Nav2-1-F
Nav2-1-R
Nav2-2-F
Nav2-2-R
Nrp2-F
Nrp2-R

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Sequence
ACGTGGGCAGTCCTGTGAAGACACCGTCCTCTCCGCTAAACTCCCAGGTCCTACCGCAA
GGGTGGAGAGGTGACAGGTT
CTCTGTCACCACCGCCGTG
CTCGAACACCAAGCACCTGAG
ACTTCACAACCCAAGGCTCTG
AAACAACAGGGCCTGTCTCT

2346

AGING

Lcn2-F
Lcn2-R
Baiap2-F
Baiap2-R
Dnm1l-F
Dnm1l-R
Naa16-F
Naa16-R
Jade1-1-F
Jade1-1-R
Jade1-2-F
Jade1-2-R

GTCTTAACCGCTGAGCCATC
CACCTGTCTTAACACCACCAATACAGTGTGAGGACGCTGACAACAACCCAAGAACAAACCAAC
TTGTCCCGTGATCCAGCTGAACAAAGAAGAAACAGGCTAGGGAGCAGGTCTCACTATGTAGC
CTTGAAAGGCTGAGGCAGAAGA
TTGTTCCTGTCGCCTCGG
GGGAAACAGGCTGGAGACCGTGGGCAGGTGCTGGTTG
CGGGCAGCGACAAGGAAC

Supplementary Table 7. GO enrichment and KEGG pathway of CPEB3 binding genes.
Additional file 1. Primers sets, related to the experimental procedures. qRT-PCR primers for gene expression
qualification.
Gene
GAPDH
CPEB3

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PrimerSequence (5'-3')Forward
ReverseForward
ReverseCGGAGTCAACGGATTTGGTCGTATAGCCTTCTCCATGGTGGTGAAGACGATACGGACCCAGAGCTGAA
CCTGGCACTCATCACACATC

2347

Related Figures
Figure 1.A
Figure 1.A

AGING