Distinct domain organization and diversity of 2'-5'-oligoadenylate synthetases.

The 2'-5'-oligoadenylate synthetases (OAS) are important components of the innate immune system that recognize viral double-stranded RNA (dsRNA). Upon dsRNA binding, OAS generate 2'-5'-linked oligoadenylates (2-5A) that activate ribonuclease L (RNase L), halting viral replication. The OAS/RNase L pathway is thus an important antiviral pathway and viruses have devised strategies to circumvent OAS activation. OAS enzymes are divided into four classes according to size: small (OAS1), medium (OAS2), and large (OAS3) that consist of one, two, and three OAS domains, respectively, and the OAS-like protein (OASL) that consists of one OAS domain and tandem domains similar to ubiquitin. Early investigation of the OAS enzymes hinted at the recognition of dsRNA by OAS, but due to size differences amongst OAS family members combined with the lack of structural information on full-length OAS2 and OAS3, the regulation of OAS catalytic activity by dsRNA was not well understood. However, the recent biophysical studies of OAS have highlighted overall structure and domain organization. In this review, we present a detailed examination of the OAS literature and summarized the investigation on 2'-5'-oligoadenylate synthetases.

Phase separation and clustering of an ABC transporter in Mycobacterium tuberculosis.

Phase separation drives numerous cellular processes, ranging from the formation of membrane-less organelles to the cooperative assembly of signaling proteins. Features such as multivalency and intrinsic disorder that enable condensate formation are found not only in cytosolic and nuclear proteins, but also in membrane-associated proteins. The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected hosts, has a cytoplasmic regulatory module consisting of 2 phosphothreonine-binding Forkhead-associated domains joined by an intrinsically disordered linker with multiple phospho-acceptor threonines. Here we demonstrate that the regulatory modules of Rv1747 and its homolog in Mycobacterium smegmatis form liquid-like condensates as a function of concentration and phosphorylation. The serine/threonine kinases and sole phosphatase of Mtb tune phosphorylation-enhanced phase separation and differentially colocalize with the resulting condensates. The Rv1747 regulatory module also phase-separates on supported lipid bilayers and forms dynamic foci when expressed heterologously in live yeast and M. smegmatis cells. Consistent with these observations, single-molecule localization microscopy reveals that the endogenous Mtb transporter forms higher-order clusters within the Mycobacterium membrane. Collectively, these data suggest a key role for phase separation in the function of these mycobacterial ABC transporters and their regulation via intracellular signaling.