Characterization of cytoplasmic polyadenylation element binding 2 protein expression and its RNA binding activity

Abstract

Cytoplasmic polyadenylation element binding (CPEB) proteins are translational regulators that are involved in the control of cellular senescence, synaptic plasticity, learning, and memory. We have previously found all four known CPEB family members to be transcribed in the mouse hippocampus. Aside from a brief description of CPEB2 in mouse brain, not much is known about its biological role. Hence, this study aims to investigate CPEB2 expression in mouse brain. With reverse transcription polymerase chain reaction (RT-PCR) of total mouse brain cDNA, we identified four distinct CPEB2 splice variants. Single-cell RT-PCR showed that CPEB2 is predominantly expressed in neurons of the juvenile and adult brain and that individual cells express different sets of splice variants. Staining of brain slices with a custom-made CPEB2 antibody revealed ubiquitous expression of the protein in many brain regions, including hippocampus, striatum, thalamus, cortex, and cerebellum. We also found differential expression of CPEB2 protein in excitatory, inhibitory, and dopaminergic neurons. In primary hippocampal cultures, the subcellular localization of CPEB2 in neurons and astrocytes resembled that of CPEB1. Electrophoretic mobility shift assay and RNA coimmunoprecipitation revealed CPEB2 interaction with β-catenin and Ca(2+) /calmodulin-dependent protein kinase II (both established CPEB1 targets), indicating an overlap in RNA binding specificity between CPEB1 and CPEB2. Furthermore, we identified ephrin receptor A4 as a putative novel target of CPEB2. In conclusion, our study identifies CPEB2 splice variants to be differentially expressed among individual cells and across cell types of the mouse hippocampus, and reveals overlapping binding specificity between CPEB2 and CPEB1.

The Alternative Splicing of Cytoplasmic Polyadenylation Element Binding Protein 2 Drives Anoikis Resistance and the Metastasis of Triple Negative Breast Cancer

“…In response to stress, CPEB2 dissociates from the target transcript and allows the polyadenylate tail to elongate and/or alternative polyadenylation sites to be used followed by subsequent translation of the mRNA by the ribosomal complex (29 -33). Two such mRNA transcripts reported to be regulated by CPEB2 are HIF1␣ and TWIST1 (25)(26)(27)(28), factors strongly associated with cancer progression and the EMT. As we show that CPEB2A sensitizes TNBC cells to anoikis, one can hypothesize that CPEB2A is the specific isoform of CPEB2 acting as an inhibitor of HIF1␣ and TWIST1 polyadenylation and translation, As we also observed a previously unknown antagonism between the two CPEB2 splice variants, CPEB2A and -B, we can further hypothesize that CPEB2B has the opposing function.…”

“…The identity of the CPEB2 isoforms was confirmed by gel extraction of the PCR fragments followed by DNA sequencing, which verified that CPEB2A excludes 90 nucleotides corresponding to exon 4 of the CPEB2B gene (nucleic acids 1944 -2034; data not shown and Refs. [25][26][27][28]. The CPEB2B isoform is a recently discovered one that was partially characterized in murine models earlier this year (25)(26)(27)(28).…”

“…[25][26][27][28]. The CPEB2B isoform is a recently discovered one that was partially characterized in murine models earlier this year (25)(26)(27)(28). These data demonstrate that aberrantly spliced CPEB2 pre-mRNA produces the splice variant of CPEB2, CPEB2B, which is strongly associated with AnR in TNBC.…”

Regulation of the Expression of Cytoplasmic Polyadenylation Element Binding Proteins for the Treatment of Cancer

“…The CPEB proteins are composed of 4 subtypes (CPEB1-4) where CPEB2-4 are closely related and CPEB1 is the most distant member of the family (12)(13)(14). All CPEB proteins have a similar structure with the C-terminal regions composed of two RNA recognition motifs (RRMs) and two zinc-fingercontaining sequence-specific RNA-binding proteins, as well as a regulatory N-terminal region (1,9,12).…”

“…CPEB-mediated effects on translation require at least two CPEs separated by less than 50 nucleotides in the target mRNA, which indicates the formation of CPEB-dimer (10). CPEBs can recruit the translational repression or cytoplasmic polyadenylation machineries to their target mRNAs and nucleate a complex of factors that regulates poly(A) elongation through a deadenylating enzyme (1,4,7,(11)(12)(13). …”