| Full text | |
| Author(s): |
Basei, Fernanda L.
;
Moura, Livia A. R.
;
Kobarg, Jorg
Total Authors: 3
|
| Document type: | Journal article |
| Source: | JOVE-JOURNAL OF VISUALIZED EXPERIMENTS; v. N/A, n. 170, p. 13-pg., 2021-04-01. |
| Abstract | |
mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5'capping, poly-A addition and splicing. Besides constitutive splicing, alternative mRNA splicing allows the expression of multifunctional proteins from one gene. As interactome studies are generally the first analysis for new or unknown proteins, the association of the bait protein with splicing factors is an indication that it can participate in mRNA splicing process, but to determine in what context or what genes are regulated is an empirical process. A good starting point to evaluate this function is using the classical minigene tool. Here we present the adenoviral E1A minigene usage for evaluating the alternative splicing changes after different cellular stress stimuli. We evaluated the splicing of E1A minigene in HEK293 stably overexpressing Nek4 protein after different stressing treatments. The protocol includes E1A minigene transfection, cell treatment, RNA extraction and cDNA synthesis, followed by PCR and gel analysis and quantification of the E1A spliced variants. The use of this simple and well-established method combined with specific treatments is a reliable starting point to shed light on cellular processes or what genes can be regulated by mRNA splicing. (AU) | |
| FAPESP's process: | 17/03489-1 - From functional studies to searching for new inhibitors for cancer: exploring kinases that regulate the cell cycle of the human NEK family |
| Grantee: | Jörg Kobarg |
| Support Opportunities: | Research Projects - Thematic Grants |
| FAPESP's process: | 18/05350-3 - Study of the role of the interaction between mitofusin and Nek4 in the signaling between mitochondria and nucleus after cellular stress |
| Grantee: | Fernanda Luisa Basei |
| Support Opportunities: | Scholarships in Brazil - Post-Doctoral |