Cadherins

1. Characterisation and function of the protocadherin alpha gene and its products-how this relates to neurexin-3 structure, processing and function

2. Cadherins superfamily of genes which encode transmembrane glycoproteins (>100 different related genes) originally identified as calcium dependent cell adhesion molecules superfamily includes cadherins (type I and II) protocadherins desmogleins desmocollins plus many others involved in cell adhesion and cell-cell interactions classic cadherins have 5 extracelluar cadherin ectodomains the extracellular domains engage in homophilic interactions the cytoplasmic domain interacts with the cytoskeleton

3. Protocadherins subgroup of cadherins named due their expression in a variety of organisms expressed at high levels and in specific patterns in the brain 6 or 7 extracellular ectodomains cytoplasmic domain shows no similarity to classic cadherins the genomic structure of a subset of protocadherin genes is very different to the classic cadherins

4. Identification of 3 protocadherin clusters Wu and Maniatis 1999 (Cell97:779-790) identified 52 human cadherin-like genes on chromosome 5q31 BLAST searches of GenBank sequences cDNA cloning and sequencing organized into 3 tandem gene clusters covering 700kb extracellular domains of the encoded proteins contained 6 cadherin ectodomain repeats (EC1-6) similar to each other and other published protocadherins therefore designated as protocadherins genes (Pcdh)

6. carried out cloning and sequencing of PcdhcDNAs from a human brain cDNA library each cDNA is composed of one large variable region exon encoding the extracellular and the transmembrane domains and the 3 constant region exons ie: Pcdhα1 EC2 EC1 EC3 EC4 EC5 EC6 TM Cytoplasmic domain ~2400nt Extracellular domain

7. Pcdhα – 15 variable exons 15 different cDNA combinations Pcdhβ – 15 variable exons 15 different cDNA combinations Pcdhγ– 22 variable exons 22 different cDNA combinations Also observed cDNAs composed of only one variable region exon and a small portion of the following intronic sequence (also from independently derived cDNA libraries) Possibility of 2 types of mRNA for the 52 protocadherin genes in the 3 clusters It is thought that duplication and divergence of one gene cluster gave rise to the other 2 and sequence duplication and divergence of variable region exons within the clusters produced new variable region exons (evidence for gene conversion) Distinct patterns of cell-specific expression are observed in the brain Wu and Maniatis (1999) proposed 4 models that might explain the regulation of this cell-specific protocadherin gene expression

8. Proposed models for cell specific expression of individual protocadherins From Wu and Maniatis (1999)

9. multiple promoters and cis-alternative splicing is the correct model for protocadherin expression (Model 2) CSE = consensus sequence element present in the promoter region upstream of each variable exon From Yagi, Y. 2008. Development, Growth and Differentiation50:S130-S140.

10. Cell-specific expression studies Esumi et al. 2005 (Nature Genetics 37(2):171-176) Single cell analysis of Purkinje cells (large cerebellar neurons) from the brains of mice using multiple RT-PCR reactions Strategy a) Identified polymorphisms in the Pcdhαvariable region exons in 2 strains of mice (MSM and B6) – could determine transcript origin – MSM or B6 • crossed MSM and B6 mice to produce F1 mice (one B6 and one MSM Pcdhαcluster) • Single cell RT-PCR Single Purkinje cell Reverse transcription 1st round multiplex PCR 2nd round nested PCR and sequencing

11. Results 84 cells  43 gave a PcdhαPCR product  28 gave a product in all 3 replicates Conclusion Observed monoallelic and combinatorial expression in single cells Multiple Pcdhαpromoters work monoallelically and individually in a single cell This type of regulation could account for the combinations expressed in individual neurons and could have a role in the generation of neurons with a range of different adhesions and interactions

12. Neurexins family of proteins expressed in the nervous system thought to function as cell adhesion molecules during synaptogenesis and in intercellular signalling in mammals - 3 genes NRXN1, NRXN2 and NRXN3 Neurexin 1 - spans ~ 1.1Mb (23 exons on chr2) Neurexin 2 - spans ~ 0.12Mb (23 exons on chr11) Neurexin 3 - spans ~ 1.7Mb (almost 2% of chr14) (24 exons) however it is estimated that there may be over 1,000 neurexin isoforms expressed isoforms are generated through the use of 2 alternative promoters and alternative splicing at 5 different sites

13. From Rowen et al. 2002. Genomics79(4):587-597. 2 major isoforms – α-neurexin and β-neurexin α-neurexin – transcribed from a promoter upstream of exon 1 β-neurexin – transcribed from a promoter downstream of exon 17 β-neurexins are modified and truncated forms of the α-neurexins alternative splicing occurs at 5 different sites

14. Alternative splicing can potentially generate 1728 α-neurexin isoforms (8 x 3 x 2 x 2 x 18) (576 from observed transcript data) and 36 β-neurexin isoforms (2 x 18) (16 from observed transcript data) total of 1764 NRXN3 transcripts (592 from observed transcript data)

15. Drosophila and C. elegans only have one neurexin gene which do not encode β-neurexin transcripts and shows no apparent alternative splicing It is thought that neurexins have evolved from very simple genes in invertebrates to diversified genes in vertebrates with multiple promoters and extensive alternative splicing – consistent with their proposed role in synapse specification (Tabuchi and Sudhof 2002. Genomics 79(6):849-859) Invertebrates species do not have clustered protocadherin genes The protocadherins and neurexins are cell adhesion molecules expressed in the nervous system and both gene families have evolved to utilise alternative promoters and alternative splicing to generate the multiple isoforms necessary for vertebrate neuronal diversity