Stabilization and analysis of intron lariats in vivo.
Methods
; 37(4): 368-75, 2005 Dec.
Article
em En
| MEDLINE
| ID: mdl-16314266
ABSTRACT
The analysis of lariats produced in vivo during pre-mRNA splicing is a powerful tool for elucidation of regulatory mechanisms and identification of natural recursive splicing events. Nevertheless, this analysis is technically challenging because lariats normally have short half-lives. With appropriate controls, RT-PCR amplification and sequencing of the region spanning the 2'-5' phosophodiester bond at the branch junction can be a sensitive and versatile method for lariat analysis. This approach can be facilitated and enhanced by reducing the activity of debranching enzyme (DBR) in order to stabilize lariats. We have generated a set of plasmids for dsRNA-mediated knockdown of DBR under diverse conditions in transgenic Drosophila and in cultured cells. We describe the use of these plasmids and protocols for lariat analysis. We have generated transgenic Drosophila strains carrying a GAL4-regulated RNAi construct that allows selective knockdown of DBR in specific tissues or developmental stages, using the large collection of available GAL4 expression lines. These strains should prove useful for detailed developmental analyses of alternative and recursive splicing and for genetic analyses of splicing factors. Similar approaches should be readily adaptable to other organisms.
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Base de dados:
MEDLINE
Assunto principal:
RNA Nucleotidiltransferases
/
RNA de Cadeia Dupla
/
RNA Mensageiro
/
Íntrons
/
Splicing de RNA
Idioma:
En
Ano de publicação:
2005
Tipo de documento:
Article