Investigating the NRAS 5' UTR as a Target for Small Molecules

Neuroblastoma RAS (NRAS) is an oncogene that is deregulated and highly mutated in cancers including melanomas and acute myeloid leukemias. Constitutively activated NRAS induces the MAPK and AKT signaling pathways and leads to uncontrolled proliferation and cell growth, making it an attractive target for small molecule inhibition. Like all RAS-family proteins, it has proven difficult to identify small molecules that directly inhibit the protein. An alternative approach would involve targeting the NRAS mRNA. The 5′ untranslated region (5′ UTR) of the NRAS mRNA is reported to contain a G-quadruplex (G4) that regulates translation of NRAS mRNA. Stabilizing the G4 structure with small molecules could reduce NRAS protein expression in cancer cells by impacting translation. Here we report a novel class of small molecule that binds to the G4 structure located in the 5′ UTR of the NRAS mRNA. We used a small molecule microarray (SMM) screen to identify molecules that selectively bind to the NRAS-G4. Biophysical studies demonstrated that compound 18 binds reversibly to the NRAS-G4 structure with submicromolar affinity. A Luciferase based reporter assay indicated that 18 inhibits the translation of NRAS via stabilizing the NRAS-G4 in vitro but showed only moderate effects on the NRAS levels in cellulo. Rapid Amplification of cDNA Ends (RACE), RT-PCR analysis on 14 different NRAS-expressing cell lines, coupled with analysis of publicly available CAGE seq experiments, revealed that predominant NRAS transcript does not possess the G4 structure. Further analysis of published rG4 and G4 sequencing data indicated the presence of G4 structure in the promoter region of NRAS gene (DNA) but not in the mRNA. Thus, although many NRAS transcripts lack a G4 in many cell lines the broader concept of targeting folded regions within 5' UTRs to control translation remains a highly attractive strategy and this work represents an intriguing example of transcript heterogeneity impacting targetability.

Single-Channel Region-Based Speller for Controlling Home Appliances

The brain-computer interface (BCI) system uses electroencephalography (EEG) signals for correspondence between the human and the outside world. This BCI communication system does not require any muscle action; hence, it can be controlled with the help of brain activities only. Therefore, this kind of system is helpful for patients, who are completely paralyzed or suffering from diseases like ALS (Amyotrophic Lateral Sclerosis), and spinal cord injury, etc., but having a normal functioning brain. A region-based P300 speller system for controlling home electronic appliances is proposed in this article. With the help of the proposed system, users can control and use appliances like an electronic door, fan, light, system, etc., without carrying out any physical movement. The experiments are conducted for five, ten, and fifteen trails for each subject. Among all classifiers, the ANN classifier provides the best off-line experiment accuracy of the order of 80% for fifteen flashes. Moreover, for the control translation, the Arduino module is also designed which is low cost and low power-based and physically controlled a device.

NSUN6, an RNA methyltransferase of 5-mC controls glioblastoma response to Temozolomide (TMZ) via NELFB and RPS6KB2 interaction.

Nop2/Sun RNA methyltransferase (NSUN6) is an RNA 5 - methyl cytosine (5mC) transferase with little information known of its function in cancer and response to cancer therapy. Here, we show that NSUN6 methylates both large and small RNA in glioblastoma and controls glioblastoma response to temozolomide with or without influence of the MGMT promoter status, with high NSUN6 expression conferring survival benefit to glioblastoma patients and in other cancers. Mechanistically, our results show that NSUN6 controls response to TMZ therapy via 5mC mediated regulation of NELFB and RPS6BK2. Taken together, we present evidence that show that NSUN6 mediated 5mC deposition regulates transcriptional pause (by accumulation of NELFB and the general transcription factor complexes (POLR2A, TBP, TFIIA, TFIIE) on the preinitiation complex at TATA binding site to control translation machinery in glioblastoma response to alkylating agents. Our findings open a new frontier into controlling of transcriptional regulation by RNA methyltransferase and 5mC.

Precise tuning of bacterial translation initiation by non-equilibrium 5’-UTR unfolding observed in single mRNAs

Noncoding, structured 5’- untranslated regions (5’-UTRs) of messenger RNAs (mRNAs) control translation efficiency by forming structures that can either recruit or repel the ribosome. Here we exploit a bacterial, preQ1-sensing translational riboswitch to probe how binding of a small ligand controls binding of the bacterial ribosome to the Shine-Dalgarno (SD) sequence. Combining single-molecule fluorescence microscopy with mutational analyses, we find that the stability of 30S ribosomal subunit binding is inversely correlated with the free energy needed to unfold the 5’-UTR during mRNA accommodation from the standby site to the binding cleft. Ligand binding stabilizes 5’-UTR structure to both antagonize 30S recruitment and accelerate 30S dissociation. Depletion of small ribosomal subunit protein S1, known to resolve structured 5’-UTRs, further increases the energetic penalty for mRNA accommodation. The resulting model of rapid standby site exploration followed by gated non-equilibrium unfolding of the 5’-UTR during accommodation provides a mechanistic understanding of translation efficiency.

Conserved Upstream Open Reading Frame Nascent Peptides That Control Translation

Cells utilize transcriptional and posttranscriptional mechanisms to alter gene expression in response to environmental cues. Gene-specific controls, including changing the translation of specific messenger RNAs (mRNAs), provide a rapid means to respond precisely to different conditions. Upstream open reading frames (uORFs) are known to control the translation of mRNAs. Recent studies in bacteria and eukaryotes have revealed the functions of evolutionarily conserved uORF-encoded peptides. Some of these uORF-encoded nascent peptides enable responses to specific metabolites to modulate the translation of their mRNAs by stalling ribosomes and through ribosome stalling may also modulate the level of their mRNAs. In this review, we highlight several examples of conserved uORF nascent peptides that stall ribosomes to regulate gene expression in response to specific metabolites in bacteria, fungi, mammals, and plants.

Single-Channel Region-Based Speller for Controlling Home Appliances

The brain-computer interface (BCI) system uses electroencephalography (EEG) signals for correspondence between the human and the outside world. This BCI communication system does not require any muscle action; hence, it can be controlled with the help of brain activities only. Therefore, this kind of system is helpful for patients, who are completely paralyzed or suffering from diseases like ALS (Amyotrophic Lateral Sclerosis), and spinal cord injury, etc., but having a normal functioning brain. A region-based P300 speller system for controlling home electronic appliances is proposed in this article. With the help of the proposed system, users can control and use appliances like an electronic door, fan, light, system, etc., without carrying out any physical movement. The experiments are conducted for five, ten, and fifteen trails for each subject. Among all classifiers, the ANN classifier provides the best off-line experiment accuracy of the order of 80% for fifteen flashes. Moreover, for the control translation, the Arduino module is also designed which is low cost and low power-based and physically controlled a device.

The Role of the RNA-RNA Interactome in the Hepatitis C Virus Life Cycle

RNA virus genomes are multifunctional entities endowed with conserved structural elements that control translation, replication and encapsidation, among other processes. The preservation of these structural RNA elements constraints the genomic sequence variability. The hepatitis C virus (HCV) genome is a positive, single-stranded RNA molecule with numerous conserved structural elements that manage different steps during the infection cycle. Their function is ensured by the association of protein factors, but also by the establishment of complex, active, long-range RNA-RNA interaction networks-the so-called HCV RNA interactome. This review describes the RNA genome functions mediated via RNA-RNA contacts, and revisits some canonical ideas regarding the role of functional high-order structures during the HCV infective cycle. By outlining the roles of long-range RNA-RNA interactions from translation to virion budding, and the functional domains involved, this work provides an overview of the HCV genome as a dynamic device that manages the course of viral infection.

Quantitative Principles ofcis-translational control by general mRNA sequence features in eukaryotes

ABSTRACTBACKGROUNDGeneral translationalcis-elements are present in the mRNAs of all genes and affect the recruitment, assembly, and progress of preinitiation complexes and the ribosome under many physiological states. These elements are: mRNA folding, upstream open reading frames, specific nucleotides flanking the initiating AUG codon, protein coding sequence length, and codon usage. The quantitative contributions of these sequence features and how and why they coordinate together to control translation rates are not well understood.RESULTSHere we show that these sequence features specify 42%–81% of the variance in translation rates inS.cerevisiae, S.pombe, Arabidopsis thaliana, M.musculus, andH.Sapiens. We establish that control by RNA secondary structure is chiefly mediated by highly folded 25–60 nucleotide segments within mRNA 5’ regions; that changes in tri-nucleotide frequencies between highly and poorly translated 5’ regions are correlated between all species; and that control by distinct biochemical processes is extensively correlated as is regulation by a single process acting in different parts of the same mRNA.CONCLUSIONSOur work shows that the general features control a much larger fraction of the variance in translation rates than previously realized. We provide a more detailed and accurate understanding of the aspects of RNA structure that direct translation in diverse eukaryotes. In addition, we note that the strongly correlated regulation between and withincis-control features will cause more even densities of translational complexes along each mRNA and therefore more efficient use of the translation machinery by the cell.

Proteostatic Signaling & Control of Protein Synthesis

The tremendous diversity and complexity of proteins invariably results in protein misfolding, to which cells have evolved numerous mechanisms of mitigating. Degrading misfolded proteins is perhaps the most intuitive strategy, but also critical to managing proteostasis are the elaborate mechanisms of translational control. Attenuated rates of translation ameliorate protein misfolding by downregulating the flux of new protein and conserving ATP. Loss of translational control, particularly in neurons, constitutes a major proteostatic dysfunction capable of causing or exacerbating neurodegeneration, while interventions aimed at downregulating protein synthesis are generally neuroprotective. In this review, I examine the critical neuronal signaling networks employed to control translation with an emphasis on current research. This includes the Unfolded Protein Response (UPR), the mitochondrial UPR (mtUPR), mTORC1 signaling, and stress granule formation.

Proteostatic Signaling & Control of Protein Synthesis

The tremendous diversity and complexity of proteins invariably results in protein misfolding, to which cells have evolved numerous mechanisms of mitigating. Degrading misfolded proteins is perhaps the most intuitive strategy, but also critical to managing proteostasis are the elaborate mechanisms of translational control. Attenuated rates of translation ameliorate protein misfolding by downregulating the flux of new protein and conserving ATP. Loss of translational control, particularly in neurons, constitutes a major proteostatic dysfunction capable of causing or exacerbating neurodegeneration, while interventions aimed at downregulating protein synthesis are generally neuroprotective. In this review, I examine the critical neuronal signaling networks employed to control translation with an emphasis on current research. This includes the Unfolded Protein Response (UPR), the mitochondrial UPR (mtUPR), mTORC1 signaling, and stress granule formation.