We suggest that nElavl regulates the protein interacting partners of this critical enzyme by maintaining a balance between the isoforms selleck screening library of the Gls gene. Taken together, we establish nElavl proteins as regulators of neuron-specific AS, determine an nElavl-RNA map associated with alternative splicing and uncover a new nElavl-regulated biological pathway, namely the glutamate synthesis pathway. By investigating other nElavl targets our data set also offers the possibility of identifying other interesting functions of these neuronal proteins. A 17.7 kb targeting

vector (see Supplemental Experimental Procedures) was selected in SV-129 ES cells, transferred into the germline of SV129/FVB mice, and the ACNF targeting cassette auto-excised in the male germ cells. All animal studies in this work were in accordance with the Code of Practice for the Housing and Care of Animals Used in Scientific Procedures, and was approved by the Rockefeller University Comparative Biosciences Center. Western Selleck Anti-cancer Compound Library blots were performed using 50 μg of cortex extract per lane. A pan anti-nElavl antibody (α-nElavl; paraneoplastic Hu antibody; RU IRB approved protocol 0148; patient

code NA-0018, a 63-year-old with small cell lung cancer and Hu encephalomyelopathy who had a pan-sensory neuropathy expired from prolonged status epilepticus) was used for IF. nElavl-RNA complexes in brain tissue were UV crosslinked and immunoprecipitated using specific human antisera. Isolated RNA molecules were reverse-transcribed, PCR amplified and sequenced on an Illumina GAIIx at the Rockefeller University Genomics Resource Center (see Supplemental Information). Three and one-half micrograms of total RNA from Elavl3−/−;Elavl4−/− and littermate WT P0 mice cortical tissue was reverse transcribed

and sense target DNA was prepared as described in “GeneChip Whole-Transcript next (WT) Sense Target Labeling Assay” protocol from Affymetrix. Labeled Target DNA was hybridized to GeneChip Mouse Exon 1.0 ST Array and to custom made Exon Junction Array (Affymetrix) at the Rockefeller University Genomics Resource Center. RT-PCR was used to validate alternative splicing changes as described (Licatalosi et al., 2008; Ule et al., 2005b). P0 cortex was dissected and immediately frozen in −80°C. RNA was isolated using Trizol plus RNA purification kit (Invitrogen). RNA was reverse transcribed using superscript III reverse transcriptase (Invitrogen). Abundance of RNA isoforms were determined by semiquantitative RT-PCR or where indicated by quantitative PCR, respectively. The number of PCR cycles used was in the linear range of product amplification. Rabbit anti-glutaminase antibody was courtesy of Norman Curthoys, Colorado State University. Cortex was dissected out at P0 and immediately frozen at −80°C.