TDP-43 mediates transcriptional repression (7,8) and acts on mRNA stability (9,10) and miRNA processing (11)

TDP-43 mediates transcriptional repression (7,8) and acts on mRNA stability (9,10) and miRNA processing (11). repeats within the SKAR pre-mRNA. SKAR is usually a component of the exon junction complex, which recruits S6K1, thereby facilitating the pioneer round of translation and promoting cell growth. Indeed, we found that expression of the alternatively spliced SKAR enhanced S6K1-dependent VTP-27999 signaling pathways and the translational yield of a splice-dependent reporter. Consistent with this, TDP-43 knockdown also increased translational yield and significantly increased cell size. This indicates a novel mechanism of deregulated translational control upon TDP-43 deficiency, which might contribute to pathogenesis of the protein aggregation diseases frontotemporal dementia and amyotrophic lateral sclerosis. == INTRODUCTION == TDP-43 [transactivation response (TAR) DNA binding protein of 43 kDa] is usually neuropathologically as well as genetically linked to frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) (14). Besides hyperphosphorylation, fragmentation and aggregation of TDP-43 in neurodegenerative disease, nuclear depletion of ERK2 TDP-43 is usually a hallmark of affected neurons (1). Thus, in addition to a harmful gain of misfunction, loss of (nuclear) TDP-43 function may contribute to disease pathogenesis. TDP-43 is usually a RNA binding protein (RBP) involved in various aspects of RNA metabolism (5,6). TDP-43 mediates transcriptional repression (7,8) and functions on mRNA stability (9,10) and miRNA processing (11). Pertaining to alternate splicing, TDP-43 mediates exon skipping of cystic fibrosis transmembrane conductance regulator (CFTR) exon 9 (12) and apolipoprotein A-II exon 3 (13) as well as exon inclusion of survival of motor neuron exon 7 (14). Other reported and validated TDP-43 target RNAs include cyclin-dependent kinase 6 (15), splicing component of 35 kDa (SC35) (16) and histone deacetylase 6 (HDAC6) (1721). In addition, recent screenings have identified many other novel target RNAs by use of RNA sequencing after crosslinking and immunoprecipitation (CLIP) with TDP-43 VTP-27999 antibodies (20,21); however, functional investigation is largely missing so far. To expand the knowledge about TDP-43 splice targets, we have used Affymetrix exon arrays to identify alternatively spliced transcripts upon TDP-43 knockdown. Thereby, we discovered exon 3 inclusion of S6 kinase 1 (S6K1) Aly/REF-like target (SKAR, also known as POLDIP3 or PDIP46) to be highly dependent on TDP-43, but not on FUS/TLS, another RNA-binding protein involved in FTD/ALS (2225). RNAi-mediated silencing of TDP-43 in non-neuronal and neuronal cell lines significantly reduced the main SKAR isoform, made up of all nine exons, and concomitantly VTP-27999 increased the SKAR isoform lacking exon 3. Retransfection experiments showed only subtle defects of the C-terminal glycine-rich domain name (GRD) as well as of disease-associated TDP-43 point mutations, but highlighted the involvement of the RNA acknowledgement motif (RRM) 1. TDP-43 specifically bound to the proximal intronic region downstream of exon 3 within the SKAR pre-mRNA. Mutagenesis of either a 5-GA-3 repeat or the consensus TDP-43 binding motif 5-UGUGUGU-3 (26) within this region largely abolished the binding of TDP-43 to the SKAR pre-mRNA and significantly reduced the splicing of SKAR minigene constructs that were generated as splicing reporters. Because SKAR, itself an RRM-containing protein, is usually a component of the exon junction complex (EJC) (27), we assessed the effects VTP-27999 on S6K1-dependent pioneer round of translation and cell growth. We found that the alternative SKAR isoform is usually significantly more active than SKAR . Furthermore, TDP-43 siRNA increased S6K1-dependent signaling and translational yield as well as cell size. Thus, loss of TDP-43 and producing option splicing of SKAR increases splicing-dependent global translation and may thereby contribute to disease pathogenesis by disturbing cellular protein homeostasis. == METHODS == == cDNA constructs == Wild-type and mutant Flag-TDP-43 constructs have been explained previously (17). SKAR and cDNA were PCR amplified from scrambled and siRNATDP-43treated HEK293E cells, respectively, and were subcloned into pCMV-Myc (Clontech) via BglII/NotI. Intron made up of SKAR DNA forin vitrotranscription/UV-crosslinking experiments.