Differential Conformational Dynamics in Two Type-A RNA-Binding Domains of TRBP Protein Drive Its Versatile Double-Stranded RNA Recognition and Binding

Core Concepts

The two type-A double-stranded RNA-binding domains (dsRBDs) of TRBP protein exhibit differential conformational dynamics, which enables them to recognize and bind to a variety of double-stranded RNA structures.

Abstract

The study investigates the intrinsic and RNA-induced conformational dynamics of the two type-A dsRBDs (dsRBD1 and dsRBD2) of the TRBP protein, a key player in the RNA interference (RNAi) pathway.
The key highlights are:

TRBP-dsRBD2 exhibits a more rigid and constrained conformational space compared to the highly dynamic TRBP-dsRBD1, as observed through NMR relaxation experiments and molecular dynamics simulations.

TRBP-dsRBD2 binds tightly to a short 12 bp double-stranded RNA (D12 RNA) with a Kd of ~1.2 μM, while TRBP-dsRBD1 shows weaker binding affinity.

In the presence of D12 RNA, TRBP-dsRBD2 undergoes enhanced conformational exchange on the microsecond-millisecond timescale, particularly in the RNA-binding regions and the surrounding areas. However, the amplitude of these motions is significantly lower compared to TRBP-dsRBD1.

The authors propose a dynamics-driven model where the two tandem dsRBDs of TRBP work synergistically - the flexible dsRBD1 recognizes diverse RNA structures, while the more rigid dsRBD2 binds tightly. The RNA-induced conformational dynamics in both domains may enable them to diffuse along the RNA, assisting associated proteins like Dicer in RNA processing.

Overall, the study highlights the crucial role of conformational dynamics in dictating the versatility of double-stranded RNA recognition and binding by the TRBP protein.

Stats

TRBP-dsRBD2 binds to the 12 bp D12 RNA with a Kd of ~1.2 μM.
The core average R1 rate of TRBP-dsRBD2 is 1.43 ± 0.05 s-1.
The core average R2 rate of TRBP-dsRBD2 is 10.92 ± 0.37 s-1.
The core average [1H]-15N NOE of TRBP-dsRBD2 is 0.73 ± 0.03.

Quotes

"Exploring the intricacies of RNA-protein interactions by delving into dynamics-based measurements not only adds valuable insights into the mechanics of RNA-protein interactions but also underscores the significance of conformational dynamics in dictating the functional outcome in such tightly regulated biological processes."
"The experimental challenge lies in the meticulous design of RNA sequences that maintain duplex stability amid the presence of bulges and internal loops of varying lengths and sequences. The task is further complicated by managing RNA length judiciously, as longer sequences can introduce line-broadening in NMR spectroscopy."

Key Insights Distilled From

Differential conformational dynamics in two type-A RNA-binding domains drive the double-stranded RNA recognition and binding

by Parvez,F., S... at www.biorxiv.org 08-06-2023

https://www.biorxiv.org/content/10.1101/2023.08.06.552166v3

Deeper Inquiries

How do the conformational dynamics of TRBP-dsRBD1 and TRBP-dsRBD2 enable them to cooperatively recognize and bind to diverse RNA structures during the RNAi pathway?

The conformational dynamics of TRBP-dsRBD1 and TRBP-dsRBD2 play a crucial role in their ability to recognize and bind to diverse RNA structures during the RNAi pathway. TRBP-dsRBD1 exhibits high conformational plasticity, allowing it to interact with a wide range of structurally and sequentially diverse dsRNAs. This flexibility enables TRBP-dsRBD1 to recognize various RNA structures, including those with bulges and internal loops. On the other hand, TRBP-dsRBD2 is characterized by a more rigid structure with conserved RNA-binding regions, leading to strong and stable interactions with RNA molecules.
During their interaction with RNA, both dsRBDs undergo conformational changes on fast (picoseconds to nanoseconds) and moderate (microseconds to milliseconds) timescales. These dynamic changes enable the dsRBDs to diffuse along the length of the RNA, facilitating precise interactions with different RNA structures. TRBP-dsRBD1's flexibility allows it to adapt to the structural diversity of RNA molecules, while TRBP-dsRBD2's rigidity ensures stable binding to RNA. Together, the complementary conformational dynamics of TRBP-dsRBD1 and TRBP-dsRBD2 enable them to cooperatively recognize and bind to diverse RNA structures, contributing to the efficiency of the RNAi pathway.

What are the potential implications of the differential conformational dynamics between the two dsRBDs on the overall function of the TRBP protein in regulating gene expression?

The differential conformational dynamics between TRBP-dsRBD1 and TRBP-dsRBD2 have significant implications for the overall function of the TRBP protein in regulating gene expression. TRBP is a key player in the RNAi pathway, where it binds to various pre-miRNAs and siRNAs, each with different sequences and structures. The distinct conformational dynamics of TRBP-dsRBD1 and TRBP-dsRBD2 allow them to interact with different RNA substrates in a coordinated manner.
TRBP-dsRBD1's high conformational plasticity enables it to recognize a wide range of RNA structures, contributing to the versatility of TRBP in binding diverse RNA molecules. On the other hand, TRBP-dsRBD2's rigid structure and strong RNA-binding affinity ensure stable interactions with specific RNA targets. The cooperative action of these two dsRBDs, with their differential dynamics, allows TRBP to efficiently process and regulate gene expression through the RNAi pathway.
Overall, the differential conformational dynamics between TRBP-dsRBD1 and TRBP-dsRBD2 enhance the adaptability of TRBP in recognizing and binding to various RNA structures, ultimately influencing the precision and effectiveness of gene regulation mechanisms in the cell.

How can the insights from this study on the role of protein dynamics in RNA recognition be extended to understand the mechanisms of other RNA-binding proteins involved in various cellular processes?

The insights gained from this study on the role of protein dynamics in RNA recognition can be extended to understand the mechanisms of other RNA-binding proteins involved in various cellular processes. By studying the conformational dynamics of RNA-binding proteins, researchers can uncover how these proteins interact with RNA molecules and regulate essential cellular functions.
Understanding the conformational dynamics of RNA-binding proteins can provide insights into how they recognize specific RNA structures, modulate RNA processing, and participate in diverse cellular processes such as RNA splicing, transport, and translation. By investigating the dynamic behavior of RNA-binding proteins, researchers can elucidate the mechanisms underlying RNA-protein interactions and their functional outcomes.
Moreover, the knowledge gained from studying protein dynamics in RNA recognition can be applied to design novel therapeutic strategies targeting RNA-binding proteins in diseases where RNA processing is dysregulated. By leveraging the insights from this study, researchers can advance our understanding of the intricate interplay between protein dynamics and RNA recognition in various cellular processes, paving the way for new discoveries in RNA biology and therapeutics.