title: TDP-43 regulates β-adducin (Add2) transcript stability.
abstract: TDP-43 is an RNA-binding protein involved in several steps of mRNA metabolism including transcription, splicing and stability. It is also involved in ALS and FTD, neurodegenerative diseases characterized by TDP-43 nuclear depletion. We previously identified TDP-43 as a binder of the downstream element (DSE) of the β-Adducin (Add2) brain-specific polyadenylation site (A4 PAS), suggesting its involvement in pre-mRNA 3' end processing. Here, by using chimeric minigenes, we showed that TDP-43 depletion in HeLa and HEK293 cells resulted in down-regulation of both the chimeric and endogenous Add2 transcripts. Despite having confirmed TDP-43-DSE in vitro interaction, we demonstrated that the in vivo effect was not mediated by the TDP-43-DSE interaction. In fact, substitution of the Add2 DSE with viral E-SV40 and L-SV40 DSEs, which are not TDP-43 targets, still resulted in decreased Add2 mRNA levels after TDP-43 downregulation. In addition, we failed to show interaction between TDP-43 and key polyadenylation factors, such as CstF-64 and CPSF160 and excluded TDP-43 involvement in pre-mRNA cleavage and regulation of polyA tail length. These evidences allowed us to exclude the pre-hypothesized role of TDP43 in modulating 3' end processing of Add2 pre-mRNA. Finally, we showed that TDP-43 regulates Add2 gene expression levels by increasing Add2 mRNA stability. Considering that Add2 in brain participates in synapse assembly, synaptic plasticity and their stability, and its genetic inactivation in mice leads to LTP, LTD, learning and motor-coordination deficits, we hypothesize that a possible loss of Add2 function by TDP-43 depletion may contribute to ALS and FTD disease states.
authors.affiliation: a International Center for Genetic Engineering and Biotechnology (ICGEB) ; Trieste , Italy.
authors.forename: Luisa
authors.initials: L
authors.lastname: Costessi
authors.forename: Fabiola
authors.initials: F
authors.lastname: Porro
authors.forename: Alessandra
authors.initials: A
authors.lastname: Iaconcig
authors.forename: Andrés F
authors.initials: AF
authors.lastname: Muro
mesh_terms: D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D003598:Cytoskeletal Proteins / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D026723:Polyadenylation; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D039221:mRNA Cleavage and Polyadenylation Factors / Q000235:genetics; D000099291:Adducins
chemical_list: D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; C501062:ADD2 protein, human; D000099291:Adducins
keywords: 3′ end processing; 3′UTR; ActD; Add2; DSE; PAS; RBP; RNA binding protein; TDP-43; actinomycin D.; downstream element; mRNA stability; polyadenylation site