No abstract

A hallmark pathological feature of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord1. A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing2–4. Single nucleotide polymorphisms in UNC13A are among the strongest hits associated with FTD and ALS in human genome-wide association studies5,6, but how those variants increase risk for disease is unknown. Here we show that TDP-43 represses a cryptic exon-splicing event in UNC13A. Loss of TDP-43 from the nucleus in human brain, neuronal cell lines and motor neurons derived from induced pluripotent stem cells resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. The top variants associated with FTD or ALS risk in humans are located in the intron harbouring the cryptic exon, and we show that they increase UNC13A cryptic exon splicing in the face of TDP-43 dysfunction. Together, our data provide a direct functional link between one of the strongest genetic risk factors for FTD and ALS (UNC13A genetic variants), and loss of TDP-43 function.

Background: During energy starvation, cells utilize intracellular resources for survival; however, the resources used during glucose starvation are unknown. Results: In glucose-starved yeast, vacuolar hydrolysis and endocytosis promote survival whereas autophagy is dispensable. Conclusion: Vacuolar hydrolysis blocks autophagy and provides resources for survival during glucose starvation. Significance: This new survival mechanism could protect cells from starvation in many situations.

Dynamic ensembles hold great promise in advancing RNA-targeted drug discovery. Here, we subjected the transactivation response element (TAR) RNA from human immunodeficiency virus type-1 to experimental high-throughput screening against ~100,000 drug-like small molecules. Results were augmented with 170 known TAR-binding molecules and used to generate sub-libraries optimized for evaluating enrichment when virtually screening (VS) a dynamic ensemble of TAR determined by combining NMR spectroscopy data and molecular dynamics (MD) simulations. Ensemble-based VS scores molecules with an area under the receiver operator characteristic curve of ~0.85-0.94 and with ~40-75% of all hits falling within the top 2% of scored molecules. The enrichment decreased significantly for ensembles generated from the same MD simulations without input NMR data and for other control ensembles. The results demonstrate that experimentally determined RNA ensembles can significantly enrich libraries with true hits, and that the degree of enrichment is dependent on the accuracy of the ensemble.

No abstract

Diversification of RNA-targeted scaffolds offers great promise in the search for selective ligands of therapeutically relevant RNA such as HIV-1 TAR. We herein report the establishment of amiloride as a novel RNA-binding scaffold along with synthetic routes for combinatorial C(5)- and C(6)-diversification. Iterative modifications at the C(5)- and C(6)- positions yielded derivative 24, which demonstrated a 100-fold increase in activity over the parent dimethylamiloride in peptide displacement assays. NMR chemical shift mapping was performed using the 2D SOFAST- [1H-13C] HMQC NMR method, which allowed for facile and rapid evaluation of binding modes for all library members. Cheminformatic analysis revealed distinct differences between selective and non-selective ligands. In this study, we evolved dimethylamiloride from a weak TAR ligand to one of the tightest binding selective TAR ligands reported to date through a novel combination of synthetic methods and analytical techniques. We expect these methods to allow for rapid library expansion and tuning of the amiloride scaffold for a range of RNA targets and for SOFAST NMR to allow unprecedented evaluation of small molecule:RNA interactions.

Recent studies have shown that RNAs exist in dynamic equilibrium with short-lived low-abundance ‘excited states’ that form by reshuffling base pairs in and around non-canonical motifs. These conformational states are proposed to be rich in non-canonical motifs and to play roles in the folding and regulatory functions of non-coding RNAs but their structure proves difficult to characterize given their transient nature. Here, we describe an approach for determining sugar pucker conformation in RNA excited states through nuclear magnetic resonance measurements of C1΄ and C4΄ rotating frame spin relaxation (R1ρ) in uniformly 13C/15N labeled RNA samples. Application to HIV-1 TAR exposed slow modes of sugar repuckering dynamics at the μs and ms timescale accompanying transitions between non-helical (C2΄-endo) to helical (C3΄-endo) conformations during formation of two distinct excited states. In contrast, we did not obtain any evidence for slow sugar repuckering dynamics for nucleotides in a variety of structural contexts that do not undergo non-helical to helical transitions. Our results outline a route for significantly improving the conformational characterization of RNA excited states and suggest that slow modes of repuckering dynamics gated by transient changes in secondary structure are quite common in RNA.

Higher sensitivity of NMR spectrometers and novel isotopic labeling schemes have ushered the development of rapid data acquisition methodologies, improving the time resolution with which NMR data can be acquired. For nucleic acids, longitudinal relaxation optimization in conjunction with Ernst angle excitation (SOFAST-HMQC) for imino protons, in addition to rendering rapid pulsing, has been demonstrated to yield significant improvements in sensitivity per unit time. Extending such methodology to other spins offers a viable prospect to measure additional chemical shifts, thereby broadening their utilization for various applications. Here, we introduce the 2D [13C,1H] aromatic SOFAST-HMQC that results in overall sensitivity gain of 1.4–1.7 fold relative to the conventional HMQC and can also be extended to yield long-range heteronuclear chemical shifts such as the adenine imino nitrogens N1, N3, N7 and N9. The applications of these experiments range from monitoring real-time biochemical processes, drug/ligand screening, and to collecting data at very low sample concentration and/or in cases where isotopic enrichment cannot be achieved.