## 2 Results
### 3222401L13 Rik Interacts with Npus3 to Control Gene Expression  
Among the most significantly downregulated genes identified from the RNA sequencing data was the transcription factor Neuronal PAS Domain Protein 3 (Npas3) (Table S3). We confirmed this finding by qPCR in primary mouse and human iPSC-derived astrocytes (Figure 6A). Npas3 is known as an important transcription factor in astrocytes that mediates the expression of genes involved in brain development and synapse function [37]. This study also demonstrated that an astrocyte-specific knockout of Npas3, similar to our data, causes deficits in synaptic density and complexity, along with behavioral abnormalities in mice. Additionally, Npas3 target genes were found to be significantly enriched in genes associated with schizophrenia, autism, and intellectual disability [38]. Based on these findings, we hypothesized that the effects observed after the knockdown (KD) of 3222401L13 Rik might, at least in part, be mediated by the deregulation of Npas3. IncRNAs are known to bind to DNA and form triple helices, acting as scaffolds to facilitate interactions between proteins, RNA, and DNA [39]. We employed the Triplex Domain Finder tool [39] to assess how many downregulated genes could potentially bind _3222401L13 Rik_ via their promoter region. This analysis revealed that 46% (352 out of 765 transcripts) exhibited _3222401L13 Rik_-binding regions (Figure 6B) (Table S5) that were localized toward the 3\({}^{\prime}\) end of _3222401L13 Rik_ (Figure 6C), including the potential binding site for the _Npas3_ promoter (Figure 6C), further supporting our biochemical data, suggesting that _3222401L13 Rik_ could directly bind to the promoter of _Npas3_. It is known that _Npas3_ expression is regulated by autoregulation, where the Npas3 protein binds to its own gene promoter to drive its expression [38]. Therefore, we hypothesized that _3222401L13 Rik_ might interact with both the _Npas3_ promoter and Npas3 itself to regulate its expression. Indeed,  
Figure 5: The synaptic support functions of _3222401L13 Rik_ are conserved in human astrocytes. (**A**) KD of _ENSG00000272070_ in human iPSC-derived astrocytes (unpaired _t_-test; *_p_ \(<\) 0.05). (**B**) Expression levels of interferon response genes after the KD of _ENSG00000272070_ in human iPSC-derived astrocytes (unpaired _t_-test; ***_p_ \(<\) 0.001; ns = not significant). (**C**) Expression levels of genes associated with synaptic support after the KD of _ENSG00000272070_ in human iPSC-derived astrocytes (unpaired _t_-test; *_p_ \(<\) 0.05; **_p_ \(<\) 0.01). (**D**) qPCR showing the expression of the glutamate transporters _GLT-1_ and _GLAST_ after the KD of _ENSG00000272070_ in human iPSC-derived astrocytes (unpaired _t_-test; *_p_ \(<\) 0.05; **_p_ \(<\) 0.01). (**E**) Glutamate uptake after the KD of _ENSG00000272070_ (unpaired _t_-test; ***_p_ \(<\) 0.001). (**F**) Increase in intracellular Ca2+ levels in response to ATP stimulation after the KD of _ENSG00000272070_ (unpaired _t_-test; **_p_ \(<\) 0.01). Data are depicted as mean \(\pm\) standard error. NC: negative control. KD: knockdown. we observed a direct interaction between 3222401L13 Rik and Npas3, as demonstrated by RNA immunoprecipitation followed by qPCR (Figure 6D). Figure 6: The overexpression of the interaction partner Npas3 can rescue molecular and functional changes induced by the loss of 3222401L13 Rik. (**A**) qPCR showing the expression levels of _Npas3/Npas3_ in mouse (left panel) and human iPSC-derived (right panel) astrocytes after the KD of 3222401L13 Rik_ (unpaired \(t\)-test; \({}^{\star}p<0.05\), \({}^{\star}\ast\ast\ast\ast\)\(p<0.0001\)). (**B**) Venn diagram showing the proportion of downregulated genes (352 out of 765) containing a promoter region that can bind 3222401L13 Rik. The Triplex Domain Finder tool [39] was used to identify promoter regions of the downregulated genes that have a binding motif for 3222401L13 Rik.