ABSTRACT
Mutations in Als2 gene cause several autosomal recessive forms of motor neuron diseases including Juvenile Amyotrophic Lateral Sclerosis (JALS), Juvenile Primary Lateral Sclerosis (PLSJ) and Infantile-onset Ascending Hereditary Spastic Paralysis (IAHSP). To find novel protein-protein interactions of Als2 protein we performed a yeast two hybrid screen and fished out the Ubiquitously Expressed Transcript (UXT) protein. UXT is a novel gene encoding for an α-class prefoldin type chaperone which acts as a co-activator for various transcriptional factors such as Nf-κB and AR. The interaction between Als2 and UXT was confirmed by co-immunoprecipitation. Co-localization between endogenous Als2 and UXT was mainly found in the cytoplasm of neuronal Neuro2a cells with immunofluorescence microscopy. Cell cycle arrest of Neuro2a cells showed that Als2 and Uxt transcriptional levels are synchronously changing. Our results suggest that Als2 is a binding partner of Uxt and Als2/Uxt interaction could be important for the activation of Nf-κB pathway. These results provides basis for future research to investigate the role of Nf-κB pathway in the development of motor neuron diseases.
Subject(s)
Guanine Nucleotide Exchange Factors/metabolism , Molecular Chaperones/metabolism , Neoplasm Proteins/metabolism , Animals , Cell Cycle/genetics , Cell Cycle Proteins , Cell Line , Guanine Nucleotide Exchange Factors/genetics , HEK293 Cells , Humans , Mice , Molecular Chaperones/genetics , NF-kappa B/metabolism , Neoplasm Proteins/genetics , Two-Hybrid System TechniquesABSTRACT
We describe a novel Drosophila gene, dtl (Drosophila Tat-like), which encodes a 60-kDa protein with RNA binding activity and a methyltransferase (MTase) domain. Dtl has an essential role in Drosophila development. The homologs of DTL recently described include PIMT (peroxisome proliferator-activated receptor-interacting protein with a methyltransferase domain), an RNA-binding protein that interacts with and enhances the nuclear receptor coactivator function, and TGS1, the methyltransferase involved in the formation of the 2,2,7-trimethylguanosine (m3G) cap of non-coding small RNAs. DTL is expressed throughout all of the developmental stages of Drosophila. The dtl mRNA has two ORFs (uORF and dORF). The product of dORF is the 60-kDa PIMT/TGS1 homolog protein that is translated from an internal AUG located 538 bp downstream from the 5' end of the message. This product of dtl is responsible for the formation of the m3G cap of small RNAs of Drosophila. Trimethylguanosine synthase activity is essential in Drosophila. The deletion in the dORF or point mutation in the putative MTase active site results in a reduced pool of m3G cap-containing RNAs and lethality in the early pupa stage. The 5' region of the dtl message also has the coding capacity (uORF) for a 178 amino acid protein. For complete rescue of the lethal phenotype of dtl mutants, the presence of the entire dtl transcription unit is required. Transgenes that carry mutations within the uORF restore the MTase activity but result in only partial rescue of the lethal phenotype. Interestingly, two transgenes bearing a mutation in uORF or dORF in trans can result in complete rescue.
Subject(s)
Drosophila Proteins/physiology , Drosophila melanogaster/embryology , Gene Expression Regulation, Developmental , Protein D-Aspartate-L-Isoaspartate Methyltransferase/chemistry , RNA-Binding Proteins/physiology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/chemistry , Blotting, Northern , Blotting, Western , Cell Nucleus/metabolism , DNA, Complementary/metabolism , Drosophila Proteins/chemistry , Escherichia coli/metabolism , Gene Deletion , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Methyltransferases/metabolism , Models, Genetic , Molecular Sequence Data , Mutagenesis , Mutation , Open Reading Frames , Phenotype , Plasmids/metabolism , Point Mutation , Protein Biosynthesis , Protein Structure, Tertiary , RNA/metabolism , RNA-Binding Proteins/chemistry , Sequence Homology, Amino Acid , TransfectionABSTRACT
A protein family including the recently identified PIMT/Tgs1 (PRIP-interacting protein with methyltransferase domain/trimethylguanosine synthase) was identified by searching databases for homologues of a newly identified Drosophila protein with RNA-binding activity and methyltransferase domain. Antibodies raised against a short peptide of the mammalian homologue show a 90-kDa isoform expressed specifically in rat brain and testis and a 55-kDa form expressed ubiquitously. In HeLa cells, the larger isoform of the protein is nuclear and associated with a 600-kDa complex, while the smaller isoform is mainly cytoplasmic and co-localizes to the tubulin network. Inhibition of PIMT/Tgs1 expression by siRNA in HeLa cells resulted in an increase in the percentage of cells in G2/M phases. In yeast two-hybrid and in vitro GST pull down experiments, the conserved C-terminal region of PIMT/Tgs1 interacted with the WD domain containing EED/WAIT-1 that acts as a polycomb-type repressor in the nucleus and also binds to integrins in the cytoplasm. Our experiments, together with earlier data, indicate that isoforms of the PIMT/Tgs1 protein with an RNA methyltransferase domain function both in the nucleus and in the cytoplasm and associate with both elements of the cytoskeletal network and nuclear factors known to be involved in gene regulation.
Subject(s)
Cell Nucleus/metabolism , Cytoplasm/metabolism , Methyltransferases/chemistry , Protein D-Aspartate-L-Isoaspartate Methyltransferase/chemistry , Amino Acid Sequence , Animals , Blotting, Western , Brain/metabolism , Databases as Topic , Down-Regulation , Drosophila , G2 Phase , Glutathione Transferase/metabolism , HeLa Cells , Humans , Immunoblotting , Male , Methyltransferases/metabolism , Mitosis , Models, Genetic , Molecular Sequence Data , Precipitin Tests , Protein Binding , Protein Isoforms , Protein Structure, Tertiary , RNA Interference , RNA, Small Interfering/metabolism , Rats , Sequence Homology, Amino Acid , Testis/metabolism , Tissue Distribution , Tubulin/metabolism , Two-Hybrid System TechniquesABSTRACT
We have isolated a novel Drosophila (d) gene coding for two distinct proteins via alternative splicing: a homologue of the yeast adaptor protein ADA2, dADA2a, and a subunit of RNA polymerase II (Pol II), dRPB4. Moreover, we have identified another gene in the Drosophila genome encoding a second ADA2 homologue (dADA2b). The two dADA2 homologues, as well as many putative ADA2 homologues from different species, all contain, in addition to the ZZ and SANT domains, several evolutionarily conserved domains. The dada2a/rpb4 and dada2b genes are differentially expressed at various stages of Drosophila development. Both dADA2a and dADA2b interacted with the GCN5 histone acetyltransferase (HAT) in a yeast two-hybrid assay, and dADA2b, but not dADA2a, also interacted with Drosophila ADA3. Both dADA2s further potentiate transcriptional activation in insect and mammalian cells. Antibodies raised either against dADA2a or dADA2b both immunoprecipitated GCN5 as well as several Drosophila TATA binding protein-associated factors (TAFs). Moreover, following glycerol gradient sedimentation or chromatographic purification combined with gel filtration of Drosophila nuclear extracts, dADA2a and dGCN5 were detected in fractions with an apparent molecular mass of about 0.8 MDa whereas dADA2b was found in fractions corresponding to masses of at least 2 MDa, together with GCN5 and several Drosophila TAFs. Furthermore, in vivo the two dADA2 proteins showed different localizations on polytene X chromosomes. These results, taken together, suggest that the two Drosophila ADA2 homologues are present in distinct GCN5-containing HAT complexes.
