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Triple-negative breast cancer (TNBC), the deadliest breast cancer subtype, lacks broadly applicable targeted therapies. Induction of "viral mimicry" by activation of viral double-stranded RNA (dsRNA) sensors has potential therapeutic applications for TNBC and other cancers. Suppressors of dsRNA sensing prevent sensing of endogenous dsRNAs and resulting autoimmunity. Depletion of the suppressor of dsRNA sensing ADAR1 causes activation of dsRNA sensors and cell death in many cancer cell lines. These ADAR1-dependent cells are generally also dependent on the dsRNA-binding protein PACT, which is highly expressed and essential in many TNBC cell lines. While PACT is known as an activator of the dsRNA sensor PKR, overexpression of PACT had no effect on activation of PKR in multiple TNBC cell lines. Conversely, depletion of PACT in PACT-dependent cell lines caused robust activation of PKR and cell death, in addition to induction of integrated stress response genes and NF-κB targets. These phenotypes were entirely dependent on PKR. Rescue experiments revealed that PACT dimerization and dsRNA binding are required to suppress PKR activation. While depletion of PACT alone in ADAR1/PACT-independent cell lines had no effect on PKR activation, combined depletion of both PACT and ADAR1 in those cell lines caused robust PKR activation and cell death, supporting a partially redundant role for ADAR1 and PACT in suppression of dsRNA sensing. Taken together, these findings support a vital role for PACT in suppressing PKR activation and highlight the therapeutic potential of targeting PACT to treat TNBC.

The family of RNase P endoribonucleases comprises diverse enzyme architectures ranging from complex ribonucleoprotein assemblies to single polypeptides as small as ∼23 kDa termed homologs of Aquifex RNase P (HARPs). The HARPs of two hyperthermophilic bacteria (Aquifex aeolicus and Thermodesulfatator indicus) and one thermophilic archaeon (Methanothermobacter thermautotrophicus) restore (although at reduced growth rate) the viability of Escherichia coli cells with a lethal knockdown of its endogenous RNA-based RNase P. Potential causes for retarded growth were analyzed by RNA-seq, northern blot, and primer extension. This revealed inefficient processing by HARPs in the E. coli host, particularly for precursor tRNAs (pre-tRNAs) with acceptor stems extended by one or more G-C base pairs, and also for the non-tRNA substrate pre-4.5S RNA. Yet, E. coli pre-tRNAs fortuitously carrying an A residue immediately upstream of the (canonical or aberrant) cleavage site were processed more efficiently, particularly by the two bacterial HARPs. Follow-up in vitro processing assays using RNase P model substrates confirmed that an A residue immediately upstream of the cleavage site increases efficiency and accuracy in reactions catalyzed by HARPs. In the case of A. aeolicus that entirely relies on its HARP for RNase P activity, pre-tRNAs apparently coevolved with the enzyme, as 38 of the 44 tRNA transcripts carry an A residue and none a stable G-C or C-G bp immediately upstream of the native cleavage site, two attributes that are clearly favorable for accurate and efficient catalysis by this class of protein-only RNase P.

The anti-vascular endothelial growth factor (VEGF) aptamer, t44.27, is a 27-mer RNA that functions as the active component of pegaptanib, an antiangiogenic medicine for neovascular age-related macular degeneration. The t44.27 aptamer is extensively 2'-modified and tightly binds to the heparin-binding domain (HDB) of VEGF₁₆₅ in a Ca²⁺-dependent manner. However, the molecular mechanism by which the aptamer selectively recognizes VEGF HDB to antagonize its function is poorly understood. We found that t44.27 binds to VEGF HBD in a two-step manner using a different region in the molecule: a transient interaction using a structured region of t44.27 followed by a tight complex formation with a larger interaction surface. Ca²⁺ binding stabilizes t44.27 base-pair formation suitable for the initial transient interaction. Meanwhile, the tight complex formation was essential for t44.27 to exert a competitive inhibition of heparin binding to VEGF HBD. These results provide structural insight into how the RNA aptamer specifically interacts with its target molecule to inhibit its activity.

During eukaryotic translation initiation, the small (40S) ribosomal subunit is recruited to the 5' cap and subsequently scans the 5' untranslated region (5' UTR) of mRNA in search of the start codon. The molecular mechanism of mRNA scanning remains unclear, particularly the requirement for and identity of a translocase. Here, using GFP reporters in Saccharomyces cerevisiae, we show that order-of-magnitude variations in the length of unstructured 5' UTRs have only modest effects on protein synthesis, whereas structured 5' UTRs strongly inhibit translation. Thus, when not hindered by secondary structure, mRNA scanning is not rate limiting. Loss-of-function mutations in eIF4A, Ded1, and Slh1 reveal that these translational helicases are dispensable for mRNA scanning. Our data suggest that one-dimensional diffusion predominately enables 40S movement along the 5' UTR during mRNA scanning.

Double-stranded RNA plays a key role in various biological processes. The discovery of RNA interference, a gene-silencing mechanism, revolutionized the study of gene function. dsRNA has since been used in novel therapeutics and as an agricultural biocontrol alternative to chemical pesticides. Microbial production typically involves expression systems with convergent T7 promoters. However, convergent transcription from DNA-dependent RNA polymerases can lead to transcriptional interference. In this study, we designed multiple plasmid DNA constructs to investigate the effect of convergent and divergent T7 RNA polymerase production of dsRNA via in vitro transcription and in vivo in Escherichia coli, prior to dsRNA yield quantification and analysis of product quality. We demonstrate that higher yields of larger dsRNA are typically obtained using convergent promoters during in vivo production. A typical fold increase of 2.1 was obtained for dsRNAs >400 bp. However, production of smaller dsRNAs (<250 bp) by divergent promoters resulted in increased yields (2.2-fold). Furthermore, our data demonstrate that in vitro transcription production of dsRNA using divergent T7 promoters results in significantly higher yields of dsRNA, with a maximum fold increase of 6.46. Finally, independent of size, we demonstrate that dsRNA synthesized from DNA templates with multiple transcriptional terminators, compared to run-off transcription, improved the quality and purity of dsRNA due to decreased formation of dsRNA multimers or aggregates. This study demonstrates that optimal production of dsRNAs is not limited to a single method and can be optimized depending on the size of dsRNA, application, yield, and quality required.

Adaptation of adenosine-to-inosine (A-to-I) RNA editing has only been validated for a few nonsynonymous (recoding) sites, where the editable state confers higher fitness than the uneditable or fully edited state. However, adaptation of noncoding RNA editing is unexplored, limiting our understanding of the significance of post-transcriptional modifications. In this study, we report extensive A-to-I editing in Drosophila lncRNA roX1, a key component of the dosage compensation pathway. Despite dramatically higher roX1 expressions in males compared to females, editing levels show minimum gender specificity, indicating nonselective, promiscuous editing. Cross-sample comparison reveals no correlation between roX1 editing levels and the extent of dosage compensation. Furthermore, Adar mutant male flies do not display abnormal dosage compensation of X chromosomal genes. These findings suggest that rampant RNA editing in roX1 is unlikely to play functional roles in dosage compensation and does not appear to offer a selective advantage over either a genomic G or an uneditable allele. Our results align with the molecular error hypothesis that the adaptive changes represent only a small fraction of the genome. Nevertheless, we do not exclude the possibility that, despite a global nonadaptive signal, individual editing sites in roX1 may still be adaptive, contingent on supporting experimental evidence.

The heterotrimeric GTPase eukaryotic translation initiation factor 2 (eIF2) delivers the initiator Met-tRNA_i to the ribosomal translation preinitiation complex (PIC). eIF2β has three lysine-rich repeats (K-boxes), important for binding to the GTPase-activating protein eIF5, the guanine nucleotide exchange factor eIF2B, and the regulator eIF5-mimic protein (5MP). Here, we combine X-ray crystallography with NMR to understand the molecular basis and dynamics of these interactions. The crystal structure of yeast eIF5-CTD in complex with eIF2β K-box 3 reveals an extended binding site on eIF2β, far beyond the K-box. We show that eIF2β contains three distinct binding sites, centered on each of the K-boxes, and that human eIF5, eIF2Bε, and 5MP1 can bind to all three sites. Our results reveal how eIF2B speeds up the dissociation of eIF5 from eIF2-GDP to promote nucleotide exchange; and how 5MP1 can destabilize eIF5 binding to eIF2 and the PIC, to promote stringent start codon selection. All these affinities are increased by CK2 phosphomimetic mutations, highlighting the role of CK2 in both remodeling and stabilizing the translation apparatus.

Long noncoding RNAs perform integral roles in eukaryotic life cycles, particularly the 7SK snRNA, which is responsible for RNA polymerase II transcription modulation and progression when interacting with P-TEFb, and the 7SL RNA involved in signal recognition particle mediation of cotranslation activities of endoplasmic reticulum bound proteins. These RNAs retain important secondary structures that interact with proteins involved in transcription and translation regulation. RNA-protein interactions involving the RNA stem-loops have been previously characterized using chemical probing techniques, cryo-electron microscopy, and nuclear magnetic resonance. However, complete three-dimensional structures of the full-length 7SK and 7SL have not been resolved, limiting our understanding of these RNAs' tertiary landscapes and mechanisms. Our study bridges this gap in knowledge by using small-angle X-ray scattering and coarse-grained computational modeling of previously determined secondary structures through SimRNA to produce full-sequence, three-dimensional atomistic models of both 7SK and 7SL RNAs. We used size exclusion chromatography connected with light scattering and circular dichroism spectroscopy to verify RNA size, and compare previously identified secondary structures in solution. We additionally used all-atom, structure-based potential simulations to generate optimized models within our calculated SAXS envelopes. 7SK's total morphology is thus presented as a highly versatile structure whose well-defined stem-loops interact with each other in three-dimensional space. 7SL RNA is presented as a tightly wound and somewhat rigid structure, with significant base-pairing features in its Alu-domain whereupon it forms likely scaffolds for signal recognition peptide formation.

RNA-binding proteins shape biology through their widespread functions in RNA biochemistry. Their function requires the recognition of specific RNA motifs for targeted binding. These RNA-binding elements can be composed of both unmodified and chemically modified RNAs, of which over 170 chemical modifications have been identified in biology. Unmodified RNA sequence preferences for RNA-binding proteins have been widely studied, with numerous methods available to identify their preferred sequence motifs. However, only a few techniques can detect preferred RNA modifications, and no current method can comprehensively screen the vast array of hundreds of natural RNA modifications. Prior work demonstrated that λ-dynamics is an accurate in silico method to predict RNA base binding preferences of an RNA-binding antibody. This work extends that effort by using λ-dynamics to predict unmodified and modified RNA-binding preferences of human Pumilio, a prototypical RNA-binding protein. A library of RNA modifications was screened at eight nucleotide positions along the RNA to identify modifications predicted to affect Pumilio binding. Computed binding affinities were compared with experimental data to reveal high predictive accuracy. In silico force field accuracies were also evaluated between CHARMM36 and Amber RNA force fields to determine the best parameter set to use in binding calculations. This work demonstrates that λ-dynamics can predict RNA interactions to a bona fide RNA-binding protein without the requirements of chemical reagents or new methods to experimentally test binding at the bench. Advancing in silico methods like λ-dynamics will unlock new frontiers in understanding how RNA modifications shape RNA biochemistry.