Cue in Srpk79DP1 mutant background did not extend the 50% survival

Cue in Srpk79DP1 mutant background did not extend the 50% survival PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19861958 time beyond the values found for the null mutant. Bruchpilot and overexpressed SRPK79D-PC and -PF but not -PB isoform co-localize at the presynaptic active zone Discussion A considerable number of proteins located at the presynaptic active zone of both vertebrates and invertebrates have been characterized in recent years. However, little is known about their assembly into the characteristic active zone structure. The identification of the Drosophila active zone protein Bruchpilot, a large protein with significant homology to the vertebrate active zone protein CAST/ERC, has spawned interest in the study of active zone development, structure and function by using the INK-128 custom synthesis highly accessible larval neuromuscular synapse of the genetic model organism Drosophila. Bruchpilot contains large coiled-coil regions similar to vertebrate proteins Piccolo and Bassoon that are Drosophila SRPK79D lacking in Drosophila, and thus may combine functions of several vertebrate active zone proteins. Here we have described the identification and characterization of a kinase with high homology to vertebrate SR protein kinases that is targeted to active zones and is required to prevent the accumulation of Bruchpilot in discrete spots within larval and adult nerves and the formation of large Bruchpilot containing electron-dense Vatalanib site agglomerates in neuronal axons. Since the transcripts of the gene located in chromosomal subdivision 79D have been controversial in successive flybase releases we first analyzed cDNAs by RTPCR. Srpk79D Transcripts Using the information provided by the Berkeley Drosophila Genome Project and flybase we confirmed by RTPCR and sequencing the structure of the RB transcript and of the transcript RC that had been annotated in an earlier release. We extended this information by the detection of an alternatively spliced exon of 159 bp. This newly identified exon is differentially included in both RB and RC transcripts generating the transcripts RE and RF, respectively. The 53 amino acids encoded by exon 7 are located in the non-conserved spacer region between the two highly conserved kinase sub-domains of the SRPK79D-PE and -PF isoforms. This spacer region was shown to play a role in the subcellular localization of yeast and human SRPKs. We find that the overexpressed SRPK79D-PC and PF but not the -PB isoform are targeted to synaptic active zones while the overexpressed -PB isoform accumulates in clearly defined perikaryal sub-regions. This demonstrates that in Drosophila the non-conserved N-terminal domain of SRPK79D isoforms that is generated by the use of alternative promotors contains important protein targeting information while the additional amino acids encoded by exon 7 Drosophila SRPK79D and 9 is retained as a large exon. Translation of this transcript would terminate in intron 6 and lead to a truncated protein of 695 amino acids which contains only the N-terminal half of the kinase domain and thus most likely would be nonfunctional. Our RTPCR experiments did not detect the RD transcript in adult flies. The RD transcript is based on the existence of a cDNA clone, which was generated in a high-throughput approach to produce cDNA clones for Drosophila melanogaster genes. We propose that this cloned cDNA was reverse-transcribed from an incompletely spliced RNA. This interpretation is supported by the fact that the amino acids of the hypothetical PD isoform encoded by intron 6 show n.Cue in Srpk79DP1 mutant background did not extend the 50% survival PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19861958 time beyond the values found for the null mutant. Bruchpilot and overexpressed SRPK79D-PC and -PF but not -PB isoform co-localize at the presynaptic active zone Discussion A considerable number of proteins located at the presynaptic active zone of both vertebrates and invertebrates have been characterized in recent years. However, little is known about their assembly into the characteristic active zone structure. The identification of the Drosophila active zone protein Bruchpilot, a large protein with significant homology to the vertebrate active zone protein CAST/ERC, has spawned interest in the study of active zone development, structure and function by using the highly accessible larval neuromuscular synapse of the genetic model organism Drosophila. Bruchpilot contains large coiled-coil regions similar to vertebrate proteins Piccolo and Bassoon that are Drosophila SRPK79D lacking in Drosophila, and thus may combine functions of several vertebrate active zone proteins. Here we have described the identification and characterization of a kinase with high homology to vertebrate SR protein kinases that is targeted to active zones and is required to prevent the accumulation of Bruchpilot in discrete spots within larval and adult nerves and the formation of large Bruchpilot containing electron-dense agglomerates in neuronal axons. Since the transcripts of the gene located in chromosomal subdivision 79D have been controversial in successive flybase releases we first analyzed cDNAs by RTPCR. Srpk79D Transcripts Using the information provided by the Berkeley Drosophila Genome Project and flybase we confirmed by RTPCR and sequencing the structure of the RB transcript and of the transcript RC that had been annotated in an earlier release. We extended this information by the detection of an alternatively spliced exon of 159 bp. This newly identified exon is differentially included in both RB and RC transcripts generating the transcripts RE and RF, respectively. The 53 amino acids encoded by exon 7 are located in the non-conserved spacer region between the two highly conserved kinase sub-domains of the SRPK79D-PE and -PF isoforms. This spacer region was shown to play a role in the subcellular localization of yeast and human SRPKs. We find that the overexpressed SRPK79D-PC and PF but not the -PB isoform are targeted to synaptic active zones while the overexpressed -PB isoform accumulates in clearly defined perikaryal sub-regions. This demonstrates that in Drosophila the non-conserved N-terminal domain of SRPK79D isoforms that is generated by the use of alternative promotors contains important protein targeting information while the additional amino acids encoded by exon 7 Drosophila SRPK79D and 9 is retained as a large exon. Translation of this transcript would terminate in intron 6 and lead to a truncated protein of 695 amino acids which contains only the N-terminal half of the kinase domain and thus most likely would be nonfunctional. Our RTPCR experiments did not detect the RD transcript in adult flies. The RD transcript is based on the existence of a cDNA clone, which was generated in a high-throughput approach to produce cDNA clones for Drosophila melanogaster genes. We propose that this cloned cDNA was reverse-transcribed from an incompletely spliced RNA. This interpretation is supported by the fact that the amino acids of the hypothetical PD isoform encoded by intron 6 show n.