The NOTCH2NLA (also known as N2N or NOTCH2NL) gene, reported to consist of six exons by Duan et al. 2004, and of five exons by Fiddes et al. 2018 and Suzuki et al. 2018, localizes to the chromosomal band 1q21.1 and originates from the partial duplication of the NOTCH2 gene, which localizes to the chromosomal band 1p12 (Duan et al. 2004, Florio et al. 2018) or from duplication of the NOTCH2NLR gene (Fiddes et al. 2019). The 211 N-terminal amino acids correspond to the EGF repeats 1 to 6 of NOTCH2 (the EGF repeats 1 and 6 of NOTCH2 are incomplete in NOTCH2NLA) (Duan et al. 2004) and the 24 C-terminal amino acids are unique to NOTCH2NLA (Duan et al. 2004). The exon sequences of NOTCH2NLA are almost completely identical to corresponding exons of NOTCH2, while corresponding introns are more divergent (Duan et al. 2004). Fiddes et al. 2018 and Suzuki et al. 2018 reported that the duplicated segment of the NOTCH2 gene in NOTCH2NLA, NOTCH2NLB, NOTCH2NLC, and NOTCH2NLR includes the promoter region of NOTCH2 and exons 1-4 of NOTCH2 that encode EGF repeats 1-6. The duplicated segment does not include the NOTCH2 gene regions that encode the transmembrane and cytoplasmic domains (Fiddes et al. 2018). NOTCH2 introns 1-4 are also included in the duplicated segment (Suzuki et al. 2018). The fifth exon of NOTCH2NLA, NOTCH2NLB, NOTCH2NLC, and NOTCH2NLR is derived from the NOTCH2 intronic sequence (intron 4 of NOTCH2) and encodes a C-terminus of ~20 amino acids unique to NOTCH2NL proteins (Fiddes et al. 2018, Suzuki et al. 2018). Several polymorphic alleles (paratypes) of NOTCH2NLA, NOTCH2NLB and NOTCH2NLC genes exist in humans that mainly differ in their start and stop codons, thus producing polymorphic protein isoforms of different lengths (Fiddes et al. 2018). Occasionally, a protein product of an identical sequence can be produced by paratypes of two different NOTCH2NL genes (Fiddes et al. 2018). While all NOTCH2NL paratypes are transcribed, paratypes containing premature stop codons may not be translated (Fiddes et al. 2018). In addition to the specific combination of NOTCH2NL paratypes (called NOTCH2NL haplotype), individuals can also differ by the total copy number of NOTCH2NL genes, due to variability in the number of the copies of NOTCH2NLC (Fiddes et al. 2018).
NOTCH2NLA is widely expressed, with its expression pattern and levels largely overlapping with NOTCH2 (Duan et al. 2004). The highest NOTCH2NLA mRNA levels are seen in leukocytes and lymph nodes (Duan et al. 2004). NOTCH2NLA was identified as one of human-specific genes that are highly expressed in human cortical neuronal progenitor cells, both the apical (ventricular) radial glia (aRG) and the basal (outer) radial glia (bRG)(Florio et al. 2018). In the human fetal neocortical wall, the expression of NOTCH2NLA mRNA is restricted to the ventricular zone, one of the three germinal zones (regions that serve as primary sites for neurogenesis in the developing brain (Florio et al. 2018). Total NOTCH2NL mRNA levels in aRG, bRG, and differentiating neurons are within the same range as NOTCH2 mRNA levels (Florio et al. 2018). By single cell transcriptomic analysis of combined expression of NOTCH2NL genes expression in multiple regions of human fetal brains ranging in age from 11 to 21.5 gestational weeks, it was found that the expression pattern of NOTCH2NLs closely resembles the expression pattern of NOTCH2, as could be expected from their duplicated NOTCH2 promoter regions, with the highest levels in various radial glia (RG) populations, including bRG, as well as astrocytes and microglia (Fiddes et al. 2018, Fiddes et a. 2019). Detailed analysis of expression of different NOTCH2NL paratypes in human embryonic stem cells (hESCs) shows that transcripts of some paratypes are more abundant (Fiddes et al. 2018). NOTCH2 is highly expressed throughout human embryonic corticogenesis, reaching a peak at gestational week 9, while NOTCH2NLA and NOTCH2NLB are expressed at lower levels between gestational weeks 7 and 9 and their expression increases at later stages, in gestational week 21, including in the germinal zones of the cortical wall, such as the outer subventricular zone (oSVZ) (Suzuki et al. 2018). Overall, levels of NOTCH2NLA mRNA are lower than levels of NOTCH2NLB mRNA during human embryonic corticogenesis (Suzuki et al. 2018). By RNA in situ hybridization, NOTCH2 mRNA is expressed mostly along the apical part of the ventricular zone of the developing human brain, while mRNAs of NOTCH2NLs are detected throughout the ventricular zone (Suzuki et al. 2018). In an hESC-based model of human cortical neurogenesis, NOTCH2 is highly expressed in all the stages, while the expression level of NOTCH2NLs steadily increases until two months of differentiation.
NOTCH2NLA mRNA is spliced and polyadenylated (Duan et al. 2004).
While the predicted molecular mass of NOTCH2NLA is 26 kDa, its actual mass is closer to 36 kDa, which suggests that the EGF repeats of NOTCH2NLA may undergo glycosylation similar to EGF repeats of NOTCH2 (Duan et al. 2004).
NOTCH2NLA is distributed throughout the cell (Duan et al. 2004) and also secreted (Duan et al. 2004, Fiddes et al. 2018).
Electroporation of the human NOTCH2NLA gene into embryonic day 13.5 mouse embryos in utero under the control of a constitutive promoter in neocortical aRGs promotes proliferation of the basal progenitors in the mouse embryonic neocortex (Florio et al. 2018). Cortical organoids generated from hESCs in which CRISPR/Cas9 was used to homozygously delete NOTCH2NLA and NOTCH2NLB genes, and heterozygously delete the NOTCH2NLC gene, while NOTCH2 and NOTCH2NLR genes were unaffected, show early maturation and reduced size, along with the increased expression of neuronal differentiation genes such as BCL11B (CTIP2), DLX1, SEMA3A, UNC5D, and FGFR2, suggesting that the role of NOTCH2NLss is to delay differentiation of neuronal progenitors (Fiddes et al. 2018).
