Mechanism of efficient double-strand break repair by a long non-coding RNA.

Mechanistic studies in DNA repair have focused on roles of multi-protein DNA complexes, so how long non-coding RNAs (lncRNAs) regulate DNA repair is less well understood. Yet, lncRNA LINP1 is over-expressed in multiple cancers and confers resistance to ionizing radiation and chemotherapeutic drugs. Here, we unveil structural and mechanistic insights into LINP1's ability to facilitate non-homologous end joining (NHEJ). We characterized LINP1 structure and flexibility and analyzed interactions with the NHEJ factor Ku70/Ku80 (Ku) and Ku complexes that direct NHEJ. LINP1 self-assembles into phase-separated condensates via RNA-RNA interactions that reorganize to form filamentous Ku-containing aggregates. Structured motifs in LINP1 bind Ku, promoting Ku multimerization and stabilization of the initial synaptic event for NHEJ. Significantly, LINP1 acts as an effective proxy for PAXX. Collective results reveal how lncRNA effectively replaces a DNA repair protein for efficient NHEJ with implications for development of resistance to cancer therapy.

Complexity and ultrastructure of infectious extracellular vesicles from cells infected by non-enveloped virus.

Enteroviruses support cell-to-cell viral transmission prior to their canonical lytic spread of virus. Poliovirus (PV), a prototype for human pathogenic positive-sense RNA enteroviruses, and picornaviruses in general, transport multiple virions en bloc via infectious extracellular vesicles, 100~1000 nm in diameter, secreted from host cells. Using biochemical and biophysical methods we identify multiple components in secreted microvesicles, including mature PV virions; positive-sense genomic and negative-sense replicative, template viral RNA; essential viral replication proteins; and cellular proteins. Using cryo-electron tomography, we visualize the near-native three-dimensional architecture of secreted infectious microvesicles containing both virions and a unique morphological component that we describe as a mat-like structure. While the composition of these mat-like structures is not yet known, based on our biochemical data they are expected to be comprised of unencapsidated RNA and proteins. In addition to infectious microvesicles, CD9-positive exosomes released from PV-infected cells are also infectious and transport virions. Thus, our data show that, prior to cell lysis, non-enveloped viruses are secreted within infectious vesicles that also transport viral unencapsidated RNAs, viral and host proteins. Understanding the structure and function of these infectious particles helps elucidate the mechanism by which extracellular vesicles contribute to the spread of non-enveloped virus infection.

In situ structure and assembly of the multidrug efflux pump AcrAB-TolC.

Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug efflux pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled efflux pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with efflux pump inhibitors.