Human NDE1 splicing and mammalian brain development

S Mosca,A Meneghello,C G Woods,E Buratti,M Raponi,D Baralle

doi:10.1038/srep43504

Abstract

Exploring genetic and molecular differences between humans and other close species may be the key to explain the uniqueness of our brain and the selective pressures under which it evolves. Recent discoveries unveiled the involvement of Nuclear distribution factor E-homolog 1 (NDE1) in human cerebral cortical neurogenesis and suggested a role in brain evolution; however the evolutionary changes involved have not been investigated. NDE1 has a different gene structure in human and mouse resulting in the production of diverse splicing isoforms. In particular, mouse uses the terminal exon 8 T, while Human uses terminal exon 9, which is absent in rodents. Through chimeric minigenes splicing assay we investigated the unique elements regulating NDE1 terminal exon choice. We found that selection of the terminal exon is regulated in a cell dependent manner and relies on gain/loss of splicing regulatory sequences across the exons. Our results show how evolutionary changes in cis as well as trans acting signals have played a fundamental role in determining NDE1 species specific splicing isoforms supporting the notion that alternative splicing plays a central role in human genome evolution, and possibly human cognitive predominance.

Highlights

Human Nuclear distribution factor E-homolog 1 (NDE1) expression was analysed by examining fetal human tissues
Using mouse endogenous NDE1 primers (Materials and Methods, 2.4) we obtained two bands: the higher band originated from use of an alternative 3′splice site in exon 8 T (Fig. 2B, lanes 1 to 8); and a more intense band corresponding to the main splicing isoform that includes the joining together of exons 7, 8, and 8 T (Fig. 2B, lanes 1 to 8)
In order to investigate the role of splicing factors in the human NDE1 splicing, we focused on polypyrimidine tract-binding proteins (PTB), neural PTB and heterogeneous nuclear ribonucleoprotein H, as these factors have consensus sequences in human exon 8 T (Fig. 4B) that are disrupted in the generated mutant M1 and

Summary

Results

Characterization of NDE1 splicing pattern: differences between human and mouse. Endogenous. Cells with human minigenes show three bands with different intensities that, when sequenced, were confirmed to represent the following splicing events: full transcript (7-8-8T-9), skipping of exon 8 T (7-8-9) and skipping of exon 8, ∆8 isoform (7–9) (Fig. 3A, lanes 1–5). In order to address the potential significance of these nucleotide differences, several nucleotides were substituted in the human NUDE1 minigene with the corresponding mouse sequence by site-directed mutagenesis (favouring common sequences between gallus and mouse) (Fig. 4) In this way one minigene for each mutation was generated which was transiently transfected into SK-N-SH cell lines for splicing analyses (Fig. 5A).

Discussion

Methods

Mouse minigene and Human minigene were transfected into

Additional Information