Gopal Gunanathan Jayaraj

The Tuberculosis Drug Streptomycin as a Potential Cancer Therapeutic: Inhibition of miR‐21 Function by Directly Targeting Its Precursor

Dedicated to Professor Samir K. Brahmachari on the occasion of his 60th birthdayMicroRNAs (miRNAs) play crucial roles in regulating geneexpression in many cellular contexts. Deregulation ofmiRNAs has been implicated in a number of diseaseconditions and thus, small molecules that can modulatemature miRNA levels in cells can have immense therapeuticpotential. Aminoglycosides, mostly used as antibiotics, areknown specifically to bind to certain RNA secondarystructures. Herein, we report that one such aminoglycoside,streptomycin,candown-regulatethelevelsofmaturemiR-21,a miRNA with roles in a variety of cancers. We suggest thatstreptomycin down-regulates miR-21 by binding to pre-miRNA (its precursor) and blocking the function of theDicer enzyme, an essential step in miRNA maturation.MicroRNAs are a class of endogenous noncoding RNAsthat act post-transcriptionally to target mRNAs for transla-tional repression, cleavage, and destabilization.

Integrative transcriptome analysis suggest processing of a subset of long non-coding RNAs to small RNAs

BackgroundThe availability of sequencing technology has enabled understanding of transcriptomes through genome-wide approaches including RNA-sequencing. Contrary to the previous assumption that large tracts of the eukaryotic genomes are not transcriptionally active, recent evidence from transcriptome sequencing approaches have revealed pervasive transcription in many genomes of higher eukaryotes. Many of these loci encode transcripts that have no obvious protein-coding potential and are designated as non-coding RNA (ncRNA). Non-coding RNAs are classified empirically as small and long non-coding RNAs based on the size of the functional RNAs. Each of these classes is further classified into functional subclasses. Although microRNAs (miRNA), one of the major subclass of ncRNAs, have been extensively studied for their roles in regulation of gene expression and involvement in a large number of patho-physiological processes, the functions of a large proportion of long non-coding RNAs (lncRNA) still remains elusive. We hypothesized that some lncRNAs could potentially be processed to small RNA and thus could have a dual regulatory output.ResultsIntegration of large-scale independent experimental datasets in public domain revealed that certain well studied lncRNAs harbor small RNA clusters. Expression analysis of the small RNA clusters in different tissue and cell types reveal that they are differentially regulated suggesting a regulated biogenesis mechanism.ConclusionsOur analysis suggests existence of a potentially novel pathway for lncRNA processing into small RNAs. Expression analysis, further suggests that this pathway is regulated. We argue that this evidence supports our hypothesis, though limitations of the datasets and analysis cannot completely rule out alternate possibilities. Further in-depth experimental verification of the observation could potentially reveal a novel pathway for biogenesis.ReviewersThis article was reviewed by Dr Rory Johnson (nominated by Fyodor Kondrashov), Dr Raya Khanin (nominated by Dr Yuriy Gusev) and Prof Neil Smalheiser. For full reviews, please go to the Reviewer’s comment section.

Potential G-quadruplexes in the human long non-coding transcriptome

DNA G-quadruplexes are known as modulators of transcription. More recently G-quadruplexes, located in the untranslated regions of the mRNA of protein coding genes, have been described to negatively regulate gene expression at the post transcriptional/ translational levels. Here we describe the possibility of the existence of G-quadruplexes in non-coding RNA (ncRNA) and discuss their potential biological roles. Using an in house prediction tool (Quadfinder) we observe a significant occurrence and distribution of G-quadruplexes in ncRNA of various sizes. We also observe that most of non-coding RNAs harboring these potential quadruplex motifs peak at the sizes ranging from 200–300 bases. More importantly we report enrichment for single and dinucleotide loops indicating a degree of high stability of these G-quadruplexes and their potential functions in vivo. Subsequent in vitro analyses of a subset of these sequences were performed which support our predictions.

A molecular-beacon-based screen for small molecule inhibitors of miRNA maturation.

miRNAs are small non-coding RNAs that regulate about 60% of mammalian genes by modulating their transcript levels. Network scale studies of miRNA-mediated regulatory circuits demonstrate the central importance of this class of small RNA in the maintenance of biological robustness. More recently, several reports have described the deregulation of numerous miRNA to be causally associated with many diseases, including cancer. These studies have highlighted the potential for development of therapeutic modalities against miRNA. Previous screening protocols, for small molecules targeting miRNA function, are either costly or technically too complex to be applied in a high-throughput manner in standard chemical laboratories. We describe a simple in vitro screening method using a DNA-based molecular beacon that overcomes the limitations associated with earlier screens. We used this method to identify inhibitors of miR-27a function from a library of 14 aminoglycosides as a pilot study. Inhibitory molecules identified were further scrutinized to identify the validity of screen. With this proof of concept we illustrate the utility of a scalable molecular-beacon-based screening strategy for miRNA inhibitors.

The RNA Stem–Loop to G-Quadruplex Equilibrium Controls Mature MicroRNA Production inside the Cell

The biological role of the existence of overlapping structures in RNA is possible yet remains very unexplored. G-Rich tracts of RNA form G-quadruplexes, while GC-rich sequences prefer stem-loop structures. The equilibrium between alternate structures within RNA may occur and influence its functionality. We tested the equilibrium between G-quadruplex and stem-loop structure in RNA and its effect on biological processes using pre-miRNA as a model system. Dicer enzyme recognizes canonical stem-loop structures in pre-miRNA to produce mature miRNAs. Deviation from stem-loop leads to deregulated mature miRNA levels, providing readout of the existence of an alternate structure per se G-quadruplex-mediated structural interference in miRNA maturation. In vitro analysis using beacon and Dicer cleavage assays indicated that mature miRNA levels depend on relative amounts of K(+) and Mg(2+) ions, suggesting an ion-dependent structural shift. Further in cellulo studies with and without TmPyP4 (RNA G-quadruplex destabilizer) demonstrated that miRNA biogenesis is modulated by G-quadruplex to stem-loop equilibrium in a subset of pre-miRNAs. Our combined analysis thus provides evidence of the formation of noncanonical G-quadruplexes in competition with canonical stem-loop structure inside the cell and its effect on miRNA maturation in a comprehensive manner.

Classification of Chemical Chaperones Based on Their Effect on Protein Folding Landscapes

Various small molecules present in biological systems can assist protein folding in vitro and are known as chemical chaperones. De novo design of chemical chaperones with higher activity than currently known examples is desirable to ameliorate protein misfolding and aggregation in multiple contexts. However, this development has been hindered by limited knowledge of their activities. It is thought that chemical chaperones are typically poor solvents for a protein backbone and hence facilitate native structure formation. However, it is unknown if different chemical chaperones can act differently to modulate folding energy landscapes. Using a model slow folding protein, double-mutant Maltose-binding protein (DM-MBP), we show that a canonical chemical chaperone, trimethylamine-N-oxide (TMAO), accelerates refolding by decreasing the flexibility of the refolding intermediate (RI). Among a number of small molecules that chaperone DM-MBP folding, proline and serine stabilize the transition state (TS) enthalpically, while trehalose behaves like TMAO and increases the rate of barrier crossing through nonenthalpic processes. We propose a two-group classification of chemical chaperones based upon their thermodynamic effect on RI and TS, which is also supported by single molecule Förster resonance energy transfer (smFRET) studies. Interestingly, for a different test protein, the molecular mechanisms of the two groups of chaperones are not conserved. This provides a glimpse into the complexity of chemical chaperoning activity of osmolytes. Future work would allow us to engineer synergism between the two classes to design more efficient chemical chaperones to ameliorate protein misfolding and aggregation problems.

RNA G-quadruplexes: G-quadruplexes with "U" turns.

G-quadruplexes are non canonical secondary structures held together by Hoogsteen bonded planar guanine quartets formed in G-rich sequences in DNA and RNA. Considerable research over the past three decades has contributed to a great deal of understanding of these unusual structures in DNA. Various factors governing the stability of DNA quadruplexes coupled with their in vivo existence have been well documented. RNA has emerged as a key regulatory player in the functioning of the cell shifting the focus to RNA G-quadruplexes which were discovered recently. RNA G-quadruplexes demonstrate immense potential for in vivo existence and function due to their inherent chemistry. We have highlighted the major findings of the field and compared them to structural aspects of DNA quadruplexes. Further, the plausible functions of RNA G-quadruplexes such as translational suppression, splicing etc. are discussed in brief, suggesting scope for an extensive role of these structures in biological systems. As the field is growing, we endeavor to review the current knowledge and evaluate the various attributes of RNA G- quadruplex structure, stability, function and applications. We have also attempted to evaluate the physical and physiological role and relevance of these motifs.

Monitoring conformational heterogeneity of the lid of DnaK substrate-binding domain during its chaperone cycle.

DnaK or Hsp70 of Escherichia coli is a master regulator of the bacterial proteostasis network. Allosteric communication between the two functional domains of DnaK, the N‐terminal nucleotide‐binding domain (NBD) and the C‐terminal substrate‐ or peptide‐binding domain (SBD) regulate its activity. X‐ray crystallography and NMR studies have provided snapshots of distinct conformations of Hsp70 proteins in various physiological states; however, the conformational heterogeneity and dynamics of allostery‐driven Hsp70 activity remains underexplored. In this work, we employed single‐molecule Förster resonance energy transfer (sm‐FRET) measurements to capture distinct intradomain conformational states of a region within the DnaK‐SBD known as the lid. Our data conclusively demonstrate prominent conformational heterogeneity of the DnaK lid in ADP‐bound states; in contrast, the ATP‐bound open conformations are homogeneous. Interestingly, a nonhydrolysable ATP analogue, AMP‐PNP, imparts heterogeneity to the lid conformations mimicking the ADP‐bound state. The cochaperone DnaJ confers ADP‐like heterogeneous lid conformations to DnaK, although the presence of the cochaperone accelerates the substrate‐binding rate by a hitherto unknown mechanism. Irrespective of the presence of DnaJ, binding of a peptide substrate to the DnaK‐SBD leads to prominent lid closure. Lid closure is only partial upon binding to molten globule‐like authentic cellular substrates, probably to accommodate non‐native substrate proteins of varied structures.

Unique Structural Modulation of a Non-Native Substrate by Cochaperone DnaJ

The role of bacterial DnaJ protein as a cochaperone of DnaK is strongly appreciated. Although DnaJ unaccompanied by DnaK can bind unfolded as well as native substrate proteins, its role as an individual chaperone remains elusive. In this study, we demonstrate that DnaJ binds a model non-native substrate with a low nanomolar dissociation constant and, more importantly, modulates the structure of its non-native state. The structural modulation achieved by DnaJ is different compared to that achieved by the DnaK-DnaJ complex. The nature of structural modulation exerted by DnaJ is suggestive of a unique unfolding activity on the non-native substrate by the chaperone. Furthermore, we demonstrate that the zinc binding motif along with the C-terminal substrate binding domain of DnaJ is necessary and sufficient for binding and the subsequent binding-induced structural alterations of the non-native substrate. We hypothesize that this hitherto unknown structural alteration of non-native states by DnaJ might be important for its chaperoning activity by removing kinetic traps of the folding intermediates.

Differential strengths of molecular determinants guide environment specific mutational fates

Organisms maintain competitive fitness in the face of environmental challenges through molecular evolution. However, it remains largely unknown how different biophysical factors constrain molecular evolution in a given environment. Here, using deep mutational scanning, we quantified empirical fitness of >2000 single site mutants of the Gentamicin-resistant gene (GmR) in Escherichia coli, in a representative set of physical (non-native temperatures) and chemical (small molecule supplements) environments. From this, we could infer how different biophysical parameters of the mutations constrain molecular function in different environments. We find ligand binding, and protein stability to be the best predictors of mutants’ fitness, but their relative predictive power differs across environments. While protein folding emerges as the strongest predictor at minimal antibiotic concentration, ligand binding becomes a stronger predictor of mutant fitness at higher concentration. Remarkably, strengths of environment-specific selection pressures were largely predictable from the degree of mutational perturbation of protein folding and ligand binding. By identifying structural constraints that act as determinants of fitness, our study thus provides coarse mechanistic insights into the environment specific accessibility of mutational fates.