A dual reporter approach to quantify defects in messenger RNA processing.
Splicing and nuclear export are vital components of eukaryotic gene expression. Defects in splicing due to cis mutations are known to cause a number of human diseases. Here we present a dual reporter system that can be used to look at splicing or export deficiencies resulting from an insufficiency in components of the cotranscriptional machinery. The constructs use a bidirectional promoter to coexpress a test reporter and a control reporter. In the splicing construct, maximal expression of the test reporter is dependent on efficient splicing and splicing-related nuclear export, whereas the control reporter is an intronless complementary DNA expression cassette. The dual reporters allow a robust ratiometric output that is independent of cell number or transfection efficiency. Therefore, our construct is internally controlled and amenable to high-throughput analysis. As a counterscreen, we have a nonsplicing control construct in which neither reporter bears an intron. We demonstrate the sensitivity of our construct to defects in nuclear export by depleting UAP56 and NXF1, essential components of the cotranscriptional machinery.
Banerjee
Ayan
A
Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.
Sammarco
Mimi C
MC
Ditch
Scott
S
Grabczyk
Ed
E
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays; D006801:Humans; D008969:Molecular Sequence Data; D029543:Nucleocytoplasmic Transport Proteins / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism
C122111:NXF1 protein, human; D029543:Nucleocytoplasmic Transport Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C487886:DDX39B protein, human; D053487:DEAD-box RNA Helicases