5 research outputs found
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hnRNPK recognition of the B motif of Xist and other biological RNAs
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.
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Structural and Functional Effects of Cu Metalloprotein-Driven Silver Nanoparticle Dissolution
Interactions of a model Cu-metalloprotein, azurin, with
10ā100
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 CuĀ(II) and formation
of AgĀ(I) azurin species under aerobic conditions implicates CuĀ(II)
azurin as a catalyst of NP oxidative dissolution. Consistent with
NP oxidation potentials, largest concentrations of AgĀ(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.
CuĀ(II)
azurin displacement upon reaction with NPs was significantly greater
than when reacted with AgĀ(I)Ā(<i>aq</i>) alone. Regardless
of NP size, dialysis experiments show minimal reactivity between azurin
and the AgĀ(I)Ā(<i>aq</i>) 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
Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation
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