Quantitative Analysis of RNA‐mediated Protein–Protein Interactions in Living Cells by FRET

Abstract

Specific assembly of ribonucleoprotein complexes is essential in controlling various cellular functions including gene regulation. Diverse scaffolds containing proteins or nucleic acids could play key roles in stabilizing specific ribonucleoprotein complexes by enhancing protein-protein or RNA-protein interactions. One such example is the assembly of active RNA polymerase II transcription elongation complex originating from HIV-1 long terminal repeat promoter that involves HIV-1-encoded Tat protein and viral mRNA structure, trans-activation responsive RNA, and human CyclinT1 which is a subunit of the positive transcription elongation factor complex b. By using genetically encoded fluorescent proteins fused with Tat and human CyclinT1, here we demonstrate that human CyclinT1 was diffused throughout the nucleus and specific interactions between Tat and human CyclinT1 altered the localization of human CyclinT1 to specific nuclear foci. We also found that trans-activation responsive RNA enhanced protein-protein interactions between human CyclinT1 and Tat in living cells. Our results highlights the importance of trans-activation responsive RNA as a scaffold for stable and high affinity assembly of two protein partners to form a regulatory switch essential in HIV-1 gene regulation. RNA-mediated assembly of ribonucleoprotein complexes could be a general mechanism for stable ribonucleoprotein complex formation and a key step in regulating other cellular processes and viral replication. Furthermore, our results suggest that Tat interactions with human CyclinT1 change the nuclear location of positive transcription elongation factor complex b to modulate positive transcription elongation factor complex b function and transcription of cellular genes.