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.

Nonconventional chemical inhibitors of microRNA: therapeutic scope.

MicroRNAs (miRNAs) are a class of genomically encoded small RNA molecules (∼22nts in length), which regulate gene expression post transcriptionally. The term microRNA or miRNA was coined in 2001, and research in the past decade has shed light on their widespread occurrence, evolutionary conservation and tissue specific functions. It is estimated that they modulate the gene expression of approximately 60% of the mammalian genes by regulating the levels of target mRNAs to which they can bind on the basis of sequence complementarities. miRNAs are produced in a well coordinated series of steps from being transcribed in the nucleus to exerting their function in the cytoplasm. miRNAs are now implicated in diverse biological phenomena ranging from development to stress response which makes miRNAs one of the central regulatory molecules which modulate information flow along the central dogma of gene expression. More importantly, like any regulatory molecule, deregulation of miRNAs is causally associated with several diseases (mainly cancer) and is directly involved in a variety of pathophysiologies owing to their aberrant expression. Thus, modulation of miRNA levels is of prime therapeutic importance. Conventional methods of miRNA knockdown using chemically modified antisense-oligonucleotides have been explored extensively but face the challenges of modes of delivery, biostability and biodistribution. This calls for the development of more alternative and non-conventional methods to target miRNA. Small molecules targeting RNA chemical and structural space provide one such timely opportunity. In this article we first provide a brief overview of miRNA biogenesis and its disease associations. We then summarize the major developments in conventional oligonucleotide based approaches to miRNA knockdown and its status. We then focus on the more non-conventional methods like oligonucleotide enzymes and small molecules and provide an outlook on the future of such methods.

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.

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

BACKGROUND: The 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. RESULTS: Integration 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. CONCLUSIONS: Our 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.