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RNAs are often studied in nonnative sequence contexts to facilitate structural studies. However, seemingly innocuous changes to an RNA sequence may perturb the native structure and generate inaccurate or ambiguous structural models. To facilitate the investigation of native RNA secondary structure by selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE), we engineered an approach that couples minimal enzymatic steps to RNA chemical probing and mutational profiling (MaP) reverse transcription (RT) methods—a process we call template switching and mutational profiling (Switch-MaP). In Switch-MaP, RT templates and additional library sequences are added postprobing through ligation and template switching, capturing reactivities for every nucleotide. For a candidate SAM-I riboswitch, we compared RNA structure models generated by the Switch-MaP approach to those of traditional primer-based MaP, including RNAs with or without appended structure cassettes. Primer-based MaP masked reactivity data in the 5′ and 3′ ends of the RNA, producing ambiguous ensembles inconsistent with the conserved SAM-I riboswitch secondary structure. Structure cassettes enabled unambiguous modeling of an aptamer-only construct but introduced nonnative interactions in the full-length riboswitch. In contrast, Switch-MaP provided reactivity data for all nucleotides in each RNA and enabled unambiguous modeling of secondary structure, consistent with the conserved SAM-I fold. Switch-MaP is a straightforward alternative approach to primer-based and cassette-based chemical probing methods that precludes primer masking and the formation of alternative secondary structures due to nonnative sequence elements. 

SreA is one of seven candidate S-adenosyl methionine (SAM) class I riboswitches identified in Listeria monocytogenes, a saprophyte and opportunistic foodborne pathogen. SreA precedes genes encoding a methionine ATP-binding cassette (ABC) transporter, which imports methionine, and is presumed to regulate transcription of its downstream genes in a SAM-dependent manner. The proposed role of SreA in controlling the transcription of genes encoding an ABC transporter complex may have important implications for how the bacteria senses and responds to the availability of the metabolite SAM in the diverse environments in which L. monocytogenes persists. Here we validate SreA as a functional SAM-I riboswitch through ligand binding studies, structure characterization, and transcription termination assays. We determined that SreA has both a similar structure and SAM binding properties to other well characterized SAM-I riboswitches. Despite apparent structural similarities to previously described SAM-I riboswitches, SreA induces transcription termination in response to comparatively lower (nM) ligand concentrations. Furthermore, SreA is a leaky riboswitch that permits some transcription of the downstream even in the presence of mM SAM suggesting that L. monocytogenes may “dampen” the expression of genes for methionine import, but likely does not turn them “OFF”. 

Coyotes (Canis latrans) exist throughout North America and increasingly thrive in dense urban spaces; they also cause controversies when they eat small pets or seem to pose a threat. Based on fieldwork in Los Angeles, and an archive of over 400 conversations collected from the online application Nextdoor (2015–2019), we theorize the emergence of what we call the cloud coyote. Cloud coyotes are not representations but lively actors in coyote politics animated by discussion, debate, and a settler logic of property relations in places like Los Angeles. They do this by performing a threat and justifying a response that includes various attempts at extermination, containment, and assimilation, all of which—even supposedly humane alternatives—further sediment forms of settler colonialism in urban Los Angeles. We diagnose this process, show how it works, and argue that anticolonial practices—in both Los Angeles and its cloudy territories like Nextdoor—are needed to escape from perpetuating its violence. 

Although not canonically polyadenylated, the long noncoding RNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) is stabilized by a highly conserved 76-nt triple helix structure on its 3′ end. The entire MALAT1 transcript is over 8000 nt long in humans. The strongest structural conservation signal in MALAT1 (as measured by covariation of base pairs) is in the triple helix structure. Primary sequence analysis of covariation alone does not reveal the degree of structural conservation of the entire full-length transcript, however. Furthermore, RNA structure is often context dependent; RNA binding proteins that are differentially expressed in different cell types may alter structure. We investigate here the in-cell and cell-free structures of the full-length human and green monkey (Chlorocebus sabaeus) MALAT1 transcripts in multiple tissue-derived cell lines using SHAPE chemical probing. Our data reveal levels of uniform structural conservation in different cell lines, in cells and cell-free, and even between species, despite significant differences in primary sequence. The uniformity of the structural conservation across the entire transcript suggests that, despite seeing covariation signals only in the triple helix junction of the lncRNA, the rest of the transcript's structure is remarkably conserved, at least in primates and across multiple cell types and conditions. 

Abstract Contained within a galaxy cluster, the Intracluster Medium is comprised of hot, X-ray emitting material between member galaxies. The nearest (∼16.1 Mpc) large galaxy cluster is Virgo. Virgo covers an approximately 12° diameter field, making it challenging to observe in its entirety. The HaloSat satellite utilized a 7° radius field of view to obtain the first full cluster observations since 1994. We then fit the cluster spectra with a plasma emission model of fixed metallicity. Our best fit temperature is kT  = 1.39 ± 0.11 keV. We report a cluster luminosity of 5.8 ± 0.7 × 10 43  erg s −1 and an emission measure of 5.8 ± 0.6 × 10 66  cm −3 . 

Pd-catalyzed C–H arylation of heteorarenes is an important and widely studied synthetic transformation; however, the regioselectivity is often substrate-controlled. Here, we report catalyst-controlled regioselectivity in the Pd-catalyzed oxidative coupling of N-(phenylsulfonyl)indoles and aryl boronic acids using O2 as the oxidant. Both C2- and C3-arylated indoles are obtained in good yield with >10:1 selectivity. A switch from C2 to C3 regioselectivity is achieved by including 4,5-diazafluoren-9-one or 2,2'-bipyrimidine as an ancillary ligand to a "ligand-free" Pd(OTs)2 catalyst system. Density functional theory calculations indicate that the switch in selectivity arises from a change in the mechanism, from a C2-selective oxidative-Heck pathway to a C3-selective C–H activation/reductive elimination pathway.