NAR’s Breakthrough Articles present high-impact studies answering long-standing questions in the field of nucleic acids research and/or opening up new areas and mechanistic hypotheses for investigation. These articles are chosen by the Editors on the recommendation of Editorial Board Members and Referees. Articles are accompanied by a brief synopsis explaining the findings of the paper and where they fit in the broader context of nucleic acids research. They represent the very best papers published at NAR.
The CRISPR system is an adaptive prokaryotic viral defense system that involves a range of complex nucleoprotein machines and targeting RNA molecules that target and degrade invading viral nucleic acid. This field has progressed quickly and many mechanistic questions have been answered. One exception is the process of spacer capture and processing, coupled with integration into the host genome, which underlies the basis of adaptive immunity. This study presents a biochemical study of the spacer capture and integration process for the model subtype I-A system of S. solfataricus. Key observations include: specific integration in this system requires long leader sequences and ATP hydrolysis – breaking the prevailing paradigm; integration of spacers is accompanied by prespacer processing in a PAM-dependent manner; cas1-2 binds and protects prespacers, allowing processing by nucleases including Cas4, resulting in generation of substrates suitable for integration.
Reconstitution of the CstF complex unveils a regulatory role for CstF-50 in recognition of 3′-end processing signals
This article describe the reconstitution of cleavage stimulation factor (CstF) and physical and structural analysis for its RNA recognition behavior and role in alternative mRNA processing. It demonstrates that hexameric assembly of CstF creates a high affinity platform to target G/U-rich sequences longer than 10nt, also demonstrating CstF-77 is key in boosting the weak activity of its RNA-binding component. The authors describe a potential role for CstF-50 in the recognition of 3’-end processing signals by restriction of dynamic flexibility in the CstF complex rather than direct contacts with the RNA. A crystal structure of a CstF-50/CstF-77 complex provides a molecular basis for the recruitment of CstF-50 by CstF-77. They conclude that the CstF complex serves as the platform to read the two downstream elements (G/U- and U-rich) simultaneously, using CstF-64 as the primary binding module to increase affinity, while CstF-50 confers the ability to engage both signals efficiently.
Designer tRNAs for efficient incorporation of non-canonical amino acids by the pyrrolysine system in mammalian cells
The study describes the generation of two suppressor tRNAs that boost the efficiency of the pyrrolysine system for the genetic incorporation of non-canonical amino acids (ncAAs) into proteins in mammalian cells. The tRNAs extend the repertoire of tools applicable to study processes in the live cell. The archaeal (Pyl)/tRNAPyl pair is commonly used for the incorporation of ncAAs into proteins but the system suffers of low ncAA incorporation rate in cells. By screening rationally designed tRNA sets, the authors selected two tRNA variants giving higher amber suppression yields. Compared to the tRNAPyl, the engineered tRNAs feature higher intracellular concentrations and bear distinct posttranscriptional modifications. They demonstrate incorporation of AAs designed for bioorthogonal chemistry into a relevant biological target in live cells. These results open the way to the development of small intramolecular sensors for the study of protein dynamics in the natural cellular environment.