Abstract
DBP5, also known as DDX19, GLE1 and inositol hexakisphosphate (IP6) function in messenger RNA (mRNA) export at the cytoplasmic surface of the nuclear pore complex in eukaryotic cells. DBP5 is a DEAD-box RNA helicase, and its activity is stimulated by interactions with GLE1 and IP6. In addition, these three factors also have unique role(s). To investigate how these factors influenced the cytoplasmic mRNA expression and cell phenotype change, we performed RNA microarray analysis to detect the effect and function of DBP5, GLE1 and IP6 on the cytoplasmic mRNA expression. The expression of some cytoplasmic mRNA subsets (e.g. cell cycle, DNA replication) was commonly suppressed by the knock-down of DBP5, GLE1 and IPPK (IP6 synthetic enzyme). The GLE1 knock-down selectively reduced the cytoplasmic mRNA expression required for mitotic progression, results in an abnormal spindle phenotype and caused the delay of mitotic process. Meanwhile, G1/S cell cycle arrest was observed in DBP5 and IPPK knock-down cells. Several factors that function in immune response were also down-regulated in DBP5 or IPPK knock-down cells. Thereby, IFNβ-1 mRNA transcription evoked by poly(I:C) treatment was suppressed. These results imply that DBP5, GLE1 and IP6 have a conserved and individual function in the cytoplasmic mRNA expression. Variations in phenotype are due to the difference in each function of DBP5, GLE1 and IPPK in intracellular mRNA metabolism.
Highlights
To validate that the effect of DBP5 siRNA was specific to its corresponding messenger RNA (mRNA), we performed rescue analysis by combining the knock-down of DBP5 and transfection of the rescue plasmid expressing siRNA-resistant DBP5 (Fig 1E) because we observed the phenotype from single siRNA against DBP5 (Fig 1A and 1C)
Each value is the mean with standard deviation (SD) of three independent experiments
DBP5, GLE1 and IPPK play a role in mRNA export, and are conserved from S. cerevisiae to human
Summary
GLE1 deleterious mutation was found in amyotrophic lateral sclerosis (ALS) patients [31] This mutant GLE1 did not inhibit the mRNA export but has a tendency to form stress granules. DBP5, GLE1 and IP6 function in mRNA export in an integrated manner, these three factors reported to have multiple roles. We were interested in the finding that GLE1 showed a relation with neurodegenerative diseases but DBP5 and IP6 were not related to them This prompted us to identify the exact effect on the cytoplasmic mRNA expression from each factor. We recovered the cytoplasmic RNA, and analyzed the array data and cell phenotypes to determine whether DBP5, GLE1 and IP6 have a general and unique role in the cytoplasmic mRNA expression. Results imply that DBP5, GLE1 and IP6 function as mRNA export regulators as well as exerting unique functions through regulating the unique target mRNA expression in the cytoplasm
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