Measuring Proximity-Mediated Function of mRNA Regulatory Proteins by Engineered Tethering.

A powerful approach for studying the functional consequences of site-specific RNA-protein interactions is to artificially tether a protein to a messenger (or noncoding) RNA through a selective, high-affinity interaction. We share a strategy for evaluating the contribution of protein positioning within an mRNA on gene expression. We introduced an RNA hairpin recognition site for the MS2 coat protein into the untranslated regions or coding sequence of mRNAs expressing a luminescent reporter protein, NanoLuc. Effector proteins fused to the MS2 coat protein could thus be targeted to distinct regions across the mRNA. We illustrate this approach using ZFP36L2, which recruits the CCR4-NOT complex for poly(A) tail deadenylation. Tethering ZFP36L2 to the 3'-UTR decreased NanoLuc expression, as expected, given the known interaction of this adapter protein with adenine uridine-rich elements (AREs). Intriguingly, ZFP36L2 also decreased NanoLuc expression when bound within the coding sequence, revealing that ZFP36L2-and potentially many other mRNA regulatory proteins-can function when targeted to diverse locations within an mRNA. This multi-target tethering strategy enables exploration of the interplay between mRNA-protein proximity and gene expression.

Sequence and tissue targeting specificity of ZFP36L2 reveals Elavl2 as a novel target with co-regulation potential.

Zinc finger protein 36 like 2 (ZFP36L2) is an RNA-binding protein that destabilizes transcripts containing adenine-uridine rich elements (AREs). The overlap between ZFP36L2 targets in different tissues is minimal, suggesting that ZFP36L2-targeting is highly tissue specific. We developed a novel Zfp36l2-lacking mouse model (L2-fKO) to identify factors governing this tissue specificity. We found 549 upregulated genes in the L2-fKO spleen by RNA-seq. These upregulated genes were enriched in ARE motifs in the 3'UTRs, which suggests that they are ZFP36L2 targets, however the precise sequence requirement for targeting was not evident from motif analysis alone. We therefore used gel-shift mobility assays on 12 novel putative targets and established that ZFP36L2 requires a 7-mer (UAUUUAU) motif to bind. We observed a statistically significant enrichment of 7-mer ARE motifs in upregulated genes and determined that ZFP36L2 targets are enriched for multiple 7-mer motifs. Elavl2 mRNA, which has three 7-mer (UAUUUAU) motifs, was also upregulated in L2-fKO spleens. Overexpression of ZFP36L2, but not a ZFP36L2(C176S) mutant, reduced Elavl2 mRNA expression, suggesting a direct negative effect. Additionally, a reporter assay demonstrated that the ZFP36L2 effect on Elavl2 decay is dependent on the Elavl2-3'UTR and requires the 7-mer AREs. Our data indicate that Elavl2 mRNA is a novel target of ZFP36L2, specific to the spleen. Likely, ZFP36L2 combined with other RNA binding proteins, such as ELAVL2, governs tissue specificity.

Inhibition of Isoleucyl-tRNA Synthetase by the Hybrid Antibiotic Thiomarinol.

Hybrid antibiotics are an emerging antimicrobial strategy to overcome antibiotic resistance. The natural product thiomarinol A is a hybrid of two antibiotics: holothin, a dithiolopyrrolone (DTP), and marinolic acid, a close analogue of the drug mupirocin that is used to treat methicillin-resistant Staphylococcus aureus (MRSA). DTPs disrupt metal homeostasis by chelating metal ions in cells, whereas mupirocin targets the essential enzyme isoleucyl-tRNA synthetase (IleRS). Thiomarinol A is over 100-fold more potent than mupirocin against mupirocin-sensitive MRSA; however, its mode of action has been unknown. We show that thiomarinol A targets IleRS. A knockdown of the IleRS-encoding gene, ileS, exhibited sensitivity to a synthetic analogue of thiomarinol A in a chemical genomics screen. Thiomarinol A inhibits MRSA IleRS with a picomolar Ki and binds to IleRS with low femtomolar affinity, 1600 times more tightly than mupirocin. We find that thiomarinol A remains effective against high-level mupirocin-resistant MRSA and provide evidence to support a dual mode of action for thiomarinol A that may include both IleRS inhibition and metal chelation. We demonstrate that MRSA develops resistance to thiomarinol A to a substantially lesser degree than mupirocin and the potent activity of thiomarinol A requires hybridity between DTP and mupirocin. Our findings identify a mode of action of a natural hybrid antibiotic and demonstrate the potential of hybrid antibiotics to combat antibiotic resistance.

Targeting the Oncogenic Long Non-coding RNA SLNCR1 by Blocking Its Sequence-Specific Binding to the Androgen Receptor.

Long non-coding RNAs (lncRNAs) are critical regulators of numerous physiological processes and diseases, especially cancers. However, development of lncRNA-based therapies is limited because the mechanisms of many lncRNAs are obscure, and interactions with functional partners, including proteins, remain uncharacterized. The lncRNA SLNCR1 binds to and regulates the androgen receptor (AR) to mediate melanoma invasion and proliferation in an androgen-independent manner. Here, we use biochemical analyses coupled with selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) RNA structure probing to show that the N-terminal domain of AR binds a pyrimidine-rich motif in an unstructured region of SLNCR1. This motif is predictive of AR binding, as we identify an AR-binding motif in lncRNA HOXA11-AS-203. Oligonucleotides that bind either the AR N-terminal domain or the AR RNA motif block the SLNCR1-AR interaction and reduce SLNCR1-mediated melanoma invasion. Delivery of oligos that block SLNCR1-AR interaction thus represent a plausible therapeutic strategy.