No abstract

An increasing number of genetic variants have been implicated in autism spectrum disorders (ASD), and the functional study of such variants will be critical for the elucidation of autism pathophysiology. Here, we report a de novo balanced translocation disruption of TRPC6, a cation channel, in a non-syndromic autistic individual. Using multiple models, such as dental pulp cells, iPSC-derived neuronal cells and mouse models, we demonstrate that TRPC6 reduction or haploinsufficiency leads to altered neuronal development, morphology, and function. The observed neuronal phenotypes could then be rescued by TRPC6 complementation and by treatment with IGF1 or hyperforin, a TRPC6-specific agonist, suggesting that ASD individuals with alterations in this pathway might benefit from these drugs. We also demonstrate that MeCP2 levels affect TRPC6 expression. Mutations in MeCP2 cause Rett syndrome, revealing common pathways among ASDs. Genetic sequencing of TRPC6 in 1041 ASD individuals and 2872 controls revealed significantly more nonsynonymous mutations in the ASD population, and identified loss-of-function mutations with incomplete penetrance in two patients. Taken together, these findings suggest that TRPC6 is a novel predisposing gene for ASD that may act in a multiple-hit model. This is the first study to use iPSC-derived human neurons to model non-syndromic ASD and illustrate the potential of modeling genetically complex sporadic diseases using such cells.

RNA polymerase II (Pol II) termination is triggered by sequences present in the nascent transcript. Termination of pre-mRNA transcription is coupled to recognition of cis-acting sequences that direct cleavage and polyadenylation of the pre-mRNA. Termination of nonpolyadenylated [non-poly(A)] Pol II transcripts in Saccharomyces cerevisiae requires the RNA-binding proteins Nrd1 and Nab3. We have used a mutational strategy to characterize non-poly(A) termination elements downstream of the SNR13 and SNR47 snoRNA genes. This approach detected two common RNA sequence motifs, GUA[AG] and UCUU. The first motif corresponds to the known Nrd1-binding site, which we have verified here by gel mobility shift assays. We also show that Nab3 protein binds specifically to RNA containing the UCUU motif. Taken together, our data suggest that Nrd1 and Nab3 binding sites play a significant role in defining non-poly(A) terminators. As is the case with poly(A) terminators, there is no strong consensus for non-poly(A) terminators, and the arrangement of Nrd1p and Nab3p binding sites varies considerably. In addition, the organization of these sequences is not strongly conserved among even closely related yeasts. This indicates a large degree of genetic variability. Despite this variability, we were able to use a computational model to show that the binding sites for Nrd1 and Nab3 can identify genes for which transcription termination is mediated by these proteins.RNA polymerases synthesize discrete transcripts by initiating and terminating transcription in response to specific sequence elements. To initiate transcription, DNA sequences direct the binding of transcription initiation factors and polymerase to appropriate sites upstream of genes. Termination signals, providing for the creation of functional 3Ј ends, may reside either in the DNA template or, alternatively, are found in the nascent RNA transcript.Eucaryotic RNA polymerases have evolved distinct mechanisms for termination. RNA polymerase III (Pol III) requires no protein factors but terminates efficiently after transcribing four to six consecutive U residues, presumably due to instability of the RNA-DNA hybrid in the enzyme active site (1, 7). RNA Pol I terminates in response to a protein factor, Reb1, which blocks further elongation by binding to a DNA sequence downstream of the termination site (28). The Reb1 site is situated in such a way that the paused polymerase contains an inherently unstable U-rich RNA-DNA hybrid in the active site.The RNA Pol II termination mechanism is more complex than those employed by the other eucaryotic RNA polymerases, requiring a large multiprotein complex that recognizes the poly(A) signal in the nascent transcript (4,17,20,21,27,43). Deletion or mutation of the poly(A) signal results in a failure to terminate messages at the appropriate site (9, 19). This observation suggests that processing of the nascent message is coupled to termination, but the mechanism of this coupling remains uncertain. In Saccharomyces cerevisiae, mutations in severa...