Atomic structure of human Argonaute-2 protein bound to a microRNA 鈥榞uide鈥� could lead to better understanding of RNA interference mechanisms
Cold Spring Harbor, NY — In a study published in the journal Cell on May 24, 黑料吃瓜资源 (CSHL) scientists describe the three-dimensional atomic structure of a human protein bound to a piece of RNA that 鈥済uides鈥� the protein鈥檚 ability to silence genes. The protein, Argonaute-2, is a key player in RNA interference (RNAi), a powerful cellular phenomenon that has important roles in diverse biological processes, including an organism鈥檚 development.
Detailed knowledge of the structure of human Argonaute-2 and the way it interacts with its RNA guides will greatly improve our understanding of its biological mechanism of action,鈥� says CSHL Professor and HHMI Investigator Leemor Joshua-Tor, Ph.D., the study鈥檚 leader. 鈥淪uch precise structural information of the human Argonaute bound to an important RNA guide could potentially aid both basic research to understand the function of genes and also advance the development of RNAi as a therapeutic strategy in clinical settings.鈥�
Upon the activation of a gene within a cell, the gene鈥檚 DNA is copied into a messenger RNA (mRNA) 鈥渢ranscript.鈥� The instructions encoded within this transcript are then used as a blueprint by the cell鈥檚 protein synthesis machinery to generate a working protein. The gene is 鈥渟ilenced鈥� or prevented from giving rise to the protein, however, when an Argonaute-2 protein that is bound to a small piece of 鈥済uide鈥� RNA鈥攅ither a short-interfering RNA or a microRNA鈥攊ntercepts the mRNA molecule. The guide RNA, whose nucleotide sequence matches that of the target mRNA, acts as a homing device that helps the Argonaute-2 protein zero in on the mRNA target.
A few years ago, Joshua-Tor collaborated with CSHL Professor and HHMI Investigator Gregory Hannon, Ph.D., who is also a co-author in this study, to show that Argonaute proteins, which are made up of different domains or parts, act like a pair of molecular scissors that slice up target mRNAs, thus preventing proteins from being made and enforcing the silencing of their genes. The discovery of the Argonautes鈥� 鈥渟licer鈥� activity stemmed in part from solving the crystal structure of an Argonaute protein from Pyrococcus furiosus, an archebacterium that thrives in extremely high temperatures.
鈥淏ut we still know nothing about the biological functions or mechanisms of action of archebacterial Argonautes,鈥� says Joshua-Tor. 鈥淲e therefore next focused on solving the structures of Argonautes from higher organisms such as mammals, in which Argonaute functions and target recognition are well documented.鈥�
Joshua-Tor鈥檚 team and other research groups subsequently determined the atomic structures of individual parts of Argonaute proteins from higher organisms. While these studies revealed several important details鈥攆or example, the interaction between two parts of the Argonaute protein, called the PAZ and Mid domains, with the two ends of guide RNAs鈥擩oshua-Tor鈥檚 goal was to solve the structure of the entire human Argonaute protein in complex with a single human guide RNA.
Overcoming a complicated series of technical challenges, her team has achieved this goal by analyzing the structure of a full-length human Argonaute-2 protein bound to a small RNA called miR-20a, which is known to play a role in cancer development. Although Argonautes from higher organisms diverged from their archebacterial cousins more than three billion years ago, the team鈥檚 analysis shows a remarkable similarity between the two structures, especially in the regions that are important for target recognition and slicing activity.
鈥淥ur structure shows that the guide RNA, which is anchored at both ends by the PAZ and Mid domains, kinks and twists its way through the structure of the entire protein, making several points of contact within each domain and with the linker loops that join them,鈥� explains Joshua-Tor. 鈥淭he guide RNA thus acts like a backbone that rigidly locks together the otherwise flexible Argonaute protein and gives it stability.鈥�
The researchers speculate that the path threaded through the Argonaute by the guide RNAs could have evolved to maximize mutual stability, in turn making the protein-RNA complexes long-lived. This long life is critical for many biological processes that are mediated by Argonautes. 鈥淭his is also the kind of information that might help us to design better synthetic guide RNAs for therapeutic use,鈥� explains Joshua-Tor. 鈥淚t will also be useful to researchers who are trying to find more precise ways of blocking Argonaute activity.鈥�
Written by: Hema Bashyam, Science Writer | [email protected] | 516-367-8455
Funding
The work was supported by the Louis Morin Charitable Trust and by a grant (GM062534) from the. Leemor Joshua-Tor and Greg Hannon are Howard Hughes Medical Institute Investigators.
Citation
鈥淭he structure of human Argonaute-2 in complex with miR-20a鈥� appeared online in Cell on May 24. The full citation is: Elad Elkayam, Claus-D. Kuhn, Ante Tocilj, Astrid D. Haase, Emily M. Greene, Gregory J. Hannon, and Leemor Joshua-Tor. The paper can be downloaded at using the doi 10.1016/j.cell.2012.05.017.