HtrA2, a trimeric proapoptotic serine protease is involved in several diseases including cancer and neurodegenerative disorders. Its unique ability to mediate apoptosis via multiple pathways makes it an important therapeutic target. In HtrA2, C-terminal PDZ domain upon substrate binding regulates its functions through coordinated conformational changes the mechanism of which is yet to be elucidated. Although allostery has been found in some of its homologs, it has not been characterized in HtrA2 so far. Here, with an in silico and biochemical approach we have shown that allostery does regulate HtrA2 activity. Our studies identified a novel non-canonical selective binding pocket in HtrA2 which initiates signal propagation to the distal active site through a complex allosteric mechanism. This non-classical binding pocket is unique among HtrA family proteins and thus unfolds a novel mechanism of regulation of HtrA2 activity and hence apoptosis.

Circular RNAs (circRNAs) are a large family of noncoding RNAs that have emerged as novel regulators of gene expression. However, little is known about the function of circRNAs in pancreatic β-cells. Here, transcriptomic analysis of mice pancreatic islet RNA-sequencing data identified 77 differentially expressed circRNAs between mice fed with a normal diet and a high-fat diet. Surprisingly, multiple circRNAs were derived from the intron 2 of the preproinsulin 2 (Ins2) gene and are termed as circular intronic (ci)-Ins2. The expression of ci-Ins2 transcripts in mouse pancreatic islets, and βTC6 cells were confirmed by reverse transcription PCR, DNA sequencing, and RNase R treatment experiments. The level of ci-Ins2 was altered in βTC6 cells upon exposure to elevated levels of palmitate and glucose. Computational analysis predicted the interaction of several RNA-binding proteins with ci-Ins2 and their flanking region, suggesting their role in the ci-Ins2 function or biogenesis. Additionally, bioinformatics analysis predicted the association of several microRNAs with ci-Ins2. Gene ontology and pathway analysis of genes targeted by miRNAs associated with ci-Ins2 suggested the regulation of several key biological processes. Together, our findings indicate that differential expression of circRNAs, especially ci-Ins2 transcripts, may regulate β-cell function and may play a critical role in the development of diabetes.

Advancement in the RNA sequencing techniques has discovered hundreds of thousands of circular RNAs (circRNAs) in humans. However, the physiological function of most of the identified circRNAs remains unexplored. Recent studies have established that spliceosomal machinery and RNA-binding proteins modulate circRNA biogenesis. Furthermore, circRNAs have been implicated in regulating crucial cellular processes by interacting with various proteins and microRNAs. However, there are several challenges in understanding the mechanism of circRNA biogenesis, transport, and their interaction with cellular factors to regulate cellular events because of their low abundance and sequence similarity with linear RNA. Addressing these challenges requires systematic studies that directly visualize the circRNAs in cells at single-molecule resolution along with the molecular regulators. In this review, we present the design, benefits, and weaknesses of RNA imaging techniques such as single-molecule RNA fluorescence in situ hybridization and BaseScope in fixed cells and fluorescent RNA aptamers in live-cell imaging of circRNAs. Furthermore, we propose the potential use of molecular beacons, multiply labeled tetravalent RNA imaging probes, and Cas-derived systems to visualize circRNAs.

Biomimetic matrices offer a great advantage to understand several biological processes including regeneration. The study involves the development of a hybrid biomimetic scaffold and the uniqueness lies in the use of mucin, as a constituent protein. Through this study, the role of the protein in bone regeneration is deciphered through its development as a 3D model. As a first step towards understanding the protein, the interactions of mucin and collagen are determined by in silico studies considering that collagen is the most abundant protein in the bone microenvironment. Both proteins are reported to be involved in bone biology though the exact role of mucin is a topic of investigation. The in silico studies of collagen–mucin suggest to have a proper affinity toward each other, forming a strong basis for 3D scaffold development. The developed 3D scaffold is a double network system comprising of mucin and collagen and vinyl end functionalized polyethylene glycol. In situ deposition of mineral crystals has been performed enzymatically. Biological evaluation of these mineral deposited scaffolds is done in terms of their bone regeneration potential and a comparison of the two systems with and without mineral deposition is presented.