The interaction between RPAP3 and TRBP reveals a possible involvement of the HSP90/R2TP chaperone complex in the...

Area of Science:

• Molecular Biology
• RNA Biology
• Biochemistry

Background:

• MicroRNAs (miRNAs) are crucial regulators of gene expression, silencing messenger RNAs (mRNAs) via the RNA-induced silencing complex (RISC).
• RISC assembly involves Dicer processing pre-miRNAs and transfer to AGO proteins, with TRBP or PACT assisting Dicer.
• The R2TP complex, an HSP90 co-chaperone, is implicated in ribonucleoprotein particle assembly.

Purpose of the Study:

• To investigate the interaction between the R2TP complex component RPAP3 and proteins involved in miRNA biogenesis.
• To elucidate the structural basis of the RPAP3-TRBP interaction.
• To explore the functional consequences of this interaction on miRNA pathway regulation.

Main Methods:

• Co-immunoprecipitation assays to detect protein-protein interactions.
• X-ray crystallography to determine the structure of the RPAP3-TRBP complex.
• HSP90 inhibition assays to assess protein sensitivity.
• Assessment of miRNA activity in the presence and absence of RPAP3.

Main Results:

• RPAP3 binds TRBP, but not PACT, with specific domain interactions identified (RPAP3-TPR1 and TRBP-dsRBD3).
• The crystal structure revealed key residues mediating the RPAP3-TRBP interaction.
• TRBP binding to RPAP3 is mutually exclusive with TRBP binding to Dicer.
• AGO(1/2), TRBP, and Dicer are sensitive to HSP90 inhibition; TRBP sensitivity increases without RPAP3.
• RPAP3 appears to inhibit miRNA activity, suggesting a regulatory role.

Conclusions:

• RPAP3 directly interacts with TRBP, influencing its association with Dicer.
• The R2TP chaperone complex, via RPAP3, may regulate miRNA pathway activity.
• RPAP3 might sequester TRBP, thereby modulating miRNA-mediated gene silencing.