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Heterogeneous nuclear ribonuclear protein K (hnRNPK) is an abundant RNA-binding protein crucial for a wide variety of biological processes. While its binding preference for multi-cytosine-patch (C-patch) containing RNA is well documented, examination of binding to known cellular targets that contain C-patches reveals an unexpected breadth of binding affinities. Analysis of in-cell crosslinking data reinforces the notion that simple C-patch preference is not fully predictive of hnRNPK localization within transcripts. The individual RNA-binding domains of hnRNPK work together to interact with RNA tightly, with the KH3 domain being neither necessary nor sufficient for binding. Rather, the RG/RGG domain is implicated in providing essential contributions to RNA-binding, but not DNA-binding, affinity. hnRNPK is essential for X chromosome inactivation, where it interacts with Xist RNA specifically through the Xist B-repeat region. We use this interaction with an RNA motif derived from this B-repeat region to determine the RNA-structure dependence of C-patch recognition. While the location preferences of hnRNPK for C-patches are conformationally restricted within the hairpin, these structural constraints are relieved in the absence of RNA secondary structure. Together, these results illustrate how this multi-domain protein's ability to accommodate and yet discriminate between diverse cellular RNAs allows for its broad cellular functions.

Poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2, respectively), upon binding damaged DNA, become activated to add long chains of poly(ADP-ribose) (PAR) to themselves and other nuclear proteins. This activation is an essential part of the DNA damage response. The PAR modifications recruit the DNA repair machinery to sites of DNA damage and result in base excision and single-strand break repair, homologous recombination, nucleotide excision repair, and alternative nonhomologous end joining. More recently, both PARP1 and PARP2 have been shown to bind to or be activated by RNA, a property that could interfere with the function of PARP1 and PARP2 in the response to DNA damage or lead to necrosis by depletion of cellular NAD+. We have quantitatively evaluated the in vitro binding of a variety of RNAs to PARP1 and PARP2 and queried the ability of these RNAs to switch on enzymatic activity. We find that while both proteins bind RNAs without specificity toward sequence or structure, their interaction with RNA does not lead to auto-PARylation. Thus, although PARP1 and PARP2 are promiscuous with respect to activation by DNA, they both demonstrate exquisite selectivity against activation by RNA.

The Xist lncRNA mediates X chromosome inactivation (XCI). Here we show that Spen, an Xist-binding repressor protein essential for XCI , binds to ancient retroviral RNA, performing a surveillance role to recruit chromatin silencing machinery to these parasitic loci. Spen loss activates a subset of endogenous retroviral (ERV) elements in mouse embryonic stem cells, with gain of chromatin accessibility, active histone modifications, and ERV RNA transcription. Spen binds directly to ERV RNAs that show structural similarity to the A-repeat of Xist, a region critical for Xist-mediated gene silencing. ERV RNA and Xist A-repeat bind the RRM domains of Spen in a competitive manner. Insertion of an ERV into an A-repeat deficient Xist rescues binding of Xist RNA to Spen and results in strictly local gene silencing in cis. These results suggest that Xist may coopt transposable element RNA-protein interactions to repurpose powerful antiviral chromatin silencing machinery for sex chromosome dosage compensation., eLife digest The genetic material inside cells is often packaged into thread-like structures called chromosomes. In humans, mice and other mammals, a pair of sex chromosomes determines the genetic or chromosomal sex of each individual. Those who inherit two “X” chromosomes are said to be chromosomally female, while chromosomal males have one “X” and one “Y” chromosome. This means females have twice as many copies of genes on the X chromosome as a male does, which turns out to be double the number that the body needs. To solve this problem, mammals have developed a strategy known as dosage compensation. The second X chromosome in females becomes “silent”: its DNA remains unchanged, but none of the genes are active. A long noncoding RNA molecule called Xist is responsible for switching off the extra X genes in female cells. It does this by coating the entirety of the second X chromosome. Normally, RNA molecules transmit the coded instructions in genes to the cellular machinery that manufactures proteins. “Noncoding” RNAs like Xist, however, are RNAs that have taken on different jobs inside the cell. Researchers believe that the ancestral Xist gene may have once encoded a protein but changed over time to produce only a noncoding RNA. Carter, Xu et al. therefore set out to find out how exactly this might have happened, and also how Xist might have acquired its ability to switch genes off. Initial experiments used mouse cells grown in the laboratory, in which a protein called Spen was deleted. Spen is known to help Xist silence the X chromosome. In female cells lacking Spen, the second X chromosome remained active. Other chromosomes in male and female cells also had stretches of DNA that became active upon Spen’s removal. These DNA sequences, termed endogenous retroviruses, were remnants of ancestral viral infections. In other words, Spen normally acted as an antiviral defense. Analysis of genetic sequences showed that Spen recognized endogenous retrovirus sequences resembling a key region in Xist, a region which was needed for Xist to work properly. Inserting fragments of endogenous retroviruses into a defective version of Xist lacking this region also partially restored its ability to inactivate genes, suggesting that X chromosome silencing might work by hijacking cellular defenses against viruses. That is, female cells essentially ‘pretend’ there is a viral infection on the second X chromosome by coating it with Xist (which mimics endogenous retroviruses), thus directing Spen to shut it down. This research is an important step towards understanding how female cells carry out dosage compensation in mammals. More broadly, it sheds new light on how ancient viruses may have shaped the evolution of noncoding RNAs in the human genome.

Dosage compensation between the sexes has emerged independently multiple times during evolution, often harnessing long noncoding RNAs (lncRNAs) to alter gene expression on the sex chromosomes. In eutherian mammals, X chromosome inactivation (XCI) in females proceeds via the lncRNAXist, which coats one of the two X chromosomes and recruits repressive proteins to epigenetically silence gene expressionin cis1,2. HowXistevolved new functional RNA domains to recruit ancient, pleiotropic protein partners is of great interest. Here we show that Spen, anXist-binding repressor protein essential for XCI3-7, binds to ancient retroviral RNA, performing a surveillance role to recruit chromatin silencing machinery to these parasitic loci.Speninactivation leads to de-repression of a subset of endogenous retroviral (ERV) elements in embryonic stem cells, with gain of chromatin accessibility, active histone modifications, andERVRNA transcription. Spen binds directly toERVRNAs that show structural similarity to the A-repeat ofXist, a region critical forXist-mediated gene silencing8-9.ERVRNA andXistA-repeat bind the RRM3 domain of Spen in a competitive manner. Insertion of an ERV into an A-repeat deficientXistrescues binding ofXistRNA to Spen and results in local gene silencingin cis. These results suggest that insertion of an ERV element into proto-Xistmay have been a critical evolutionary event, which allowedXistto coopt transposable element RNA-protein interactions to repurpose powerful antiviral chromatin silencing machinery for sex chromosome dosage compensation.

Engineered silver nanoparticles (AgNPs) undergo profound chemical transformations in the environment. Upon exposure to biological systems, a protein corona forms to coat the NPs, altering NP bioavailability, toxicity and environmental fate. Although recent studies have explored the importance of the engineered properties of NPs in the formation of protein coronas, less is known about the impact of environmental conditions. In this work, protein corona populations are compared across AgNPs of varied sizes and surface coatings, as well as in aqueous solutions with additives that mimic environmentally relevant conditions. Bioinformatic analysis of the protein corona populations reveals that, in low buffer concentrations, the formation of the protein corona is dominated by electrostatic interactions between the proteins and NPs. The influence of electrostatics is attenuated by addition of solution additives such as sodium chloride and cysteine. Protein enrichment on the NPs is compared across 6 samples with varied AgNP engineered properties and solution conditions. Clustered results reveal that engineered surface coatings strongly mediate protein corona formation. However, under conditions that mimic varied biological and environmental systems, the protein affinities are similar. Indeed, the AgNP protein coronas characterized under environmentally relevant conditions share 70% of the protein corona population. Results provide insight into the relative importance of engineered NP properties and uncontrollable environmental variables, such as solution reaction conditions, in the formation of protein coronas.

Interactions of a model Cu-metalloprotein, azurin, with 10-100 nm silver nanoparticles (NPs) were examined to elucidate the role of oxidative dissolution and protein interaction on the biological reactivity of NPs. Although minimal protein and NP structural changes were observed upon interaction, displacement of Cu(II) and formation of Ag(I) azurin species under aerobic conditions implicates Cu(II) azurin as a catalyst of NP oxidative dissolution. Consistent with NP oxidation potentials, largest concentrations of Ag(I) azurin species were recorded in reaction with 10 nm NPs (50%). Apo-protein was also observed under anaerobic reaction with NPs of all sizes and upon aerobic reaction with larger NPs (20 nm), where NP oxidation is slowed. Cu(II) azurin displacement upon reaction with NPs was significantly greater than when reacted with Ag(I)(aq) alone. Regardless of NP size, dialysis experiments show minimal reactivity between azurin and the Ag(I)(aq) species formed as a result of NP oxidative dissolution, indicating Cu displacement from azurin occurs at the NP surface. Mechanisms of azurin-silver NP interaction are proposed. Results demonstrate that NP interactions not only impact protein structure and function, but also NP reactivity, with implications for targeting, uptake, and cytotoxicity.