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ATP modulates siRNA interactions with an endogenous human Dicer complex.
Janice L Pellino1, Lukasz Jaskiewicz, Witold Filipowicz
1Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208-3500, USA.
RNA (New York, N.Y.)
|September 24, 2005
Summary
Human Dicer (hDcr) binds short interfering RNA (siRNA) in a complex, contrary to previous reports. This endogenous hDcr complex, distinct from purified hDcr, shows ATP-dependent siRNA binding, suggesting additional factors are involved in RNA interference.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Dicer enzyme is crucial for RNA interference (RNAi) by binding short interfering RNA (siRNA).
- Previous studies suggested human Dicer (hDcr) lacks direct siRNA binding activity, contrasting with its Drosophila counterpart.
- Understanding hDcr's role in siRNA binding is vital for elucidating RNAi mechanisms in humans.
Purpose of the Study:
- To investigate the siRNA-binding activity of endogenous hDcr in human cell extracts.
- To characterize the composition and properties of hDcr-containing complexes involved in siRNA binding.
- To compare the siRNA-binding characteristics of endogenous hDcr complexes with purified recombinant hDcr (rhDcr).
Main Methods:
- Native gel electrophoresis to analyze protein-RNA complexes.
- Antibody supershift assays to identify hDcr within complexes.
- Glycerol gradient sedimentation to determine complex size.
- Crosslinking experiments to assess direct hDcr-siRNA contact.
- Comparison of ATP dependence between endogenous and recombinant hDcr.
Main Results:
- An endogenous hDcr-containing complex (Complex D) with siRNA-binding activity was identified.
- Complex D, approximately 250 kDa, contains double-stranded siRNAs and requires specific siRNA structural features.
- Purified rhDcr alone also exhibits siRNA binding, but Complex D suggests the presence of additional protein factors.
- hDcr directly contacts siRNAs within Complex D, and its binding is ATP-dependent, unlike rhDcr alone.
- The ATP dependence of Complex D suggests regulatory factors influencing hDcr-siRNA interactions.
Conclusions:
- Endogenous hDcr participates in siRNA binding within a multi-protein complex in human cells.
- Complex D's properties suggest a role in the RNA-induced silencing complex (RISC) assembly pathway.
- The findings challenge previous notions of hDcr's lack of direct siRNA binding and highlight the importance of cellular context.