Biology subjects

Ojha, S.

Publications and source records attributed to Ojha, S..

5 recordsLinked to original sources

bioRxiv2020

Non-apoptotic function of caspase-3 in morphogenesis of epithelial tubes of Drosophila renal system

Cells trigger apoptosis to eliminate themselves from the system, when tissue needs to be sculptured or they detect any abnormality within them, thus preventing irreparable damage to the host. Drosophila Malpighian tubules express apoptotic proteins, without succumbing to cell death. Here we present evidence to show apoptosis independent role of executioner caspase, Drice, for precise architecture and function of Malpighian tubules. Drice is required for precise cytoskeleton organization and convergent extension, failing which the morphology, size, cellular number and arrangement gets affected. Acquisition of star shape of stellate cells in adult Malpighian tubules requires Drice. We demonstrate that Drice regulates expression of Rho1GTPase and localization of polarity proteins. Our study shows a probable mechanism by which Drice governs tubulogenesis via Rho1GTPase mediated coordinated organization of actin cytoskeleton and membrane stablisation. Furthermore, defective morphology of tubules leads to abnormal osmoregulation and excretory functions. Collectively our findings suggest a possible non-apoptotic function of caspase-3 in the fine tuning of cell mobility during tubule development and our results will add to the growing understanding of diverse roles of caspases during its evolution in metazoans.

developmental biology↗

bioRxiv2020

A Novel Stress Response Pathway Regulates Ribosome Biogenesis

Production of ribosomes is an energy-intensive process owing to the intricacy of these massive macromolecular machines. Each human ribosome contains 80 ribosomal proteins and four non-coding RNAs. Accurate assembly requires precise regulation of protein and RNA subunits. In response to stress, the integrated stress response (ISR) rapidly inhibits global translation. How rRNA is coordinately regulated with the rapid inhibition of ribosomal protein synthesis is not known. Here we show that stress specifically inhibits the first step of rRNA processing. Unprocessed rRNA is stored within the nucleolus, and, when stress resolves, it re-enters the ribosome biogenesis pathway. Retention of unprocessed rRNA within the nucleolus aids in the maintenance of this organelle. This response is independent of the ISR or inhibition of cellular translation but represents an independent stress-response pathway that we term Ribosome Biogenesis Stress Response (RiBiSR). Failure to coordinately regulate ribosomal protein translation and rRNA production results in nucleolar fragmentation. Our study unveils a novel stress response pathway that aims at conserving energy, preserving the nucleolus, and prevents further stress by regulation of rRNA processing.

biochemistry↗

bioRxiv2020

Implementation of PAR-CLIP to characterize RNA-protein interactions in prokaryotes at nucleotide resolution

The identification of RNAs that are recognized by RNA-binding proteins (RNA-BPs) using techniques such as "Crosslinking and Immunoprecipitation" (CLIP) has revolutionized the genome-wide discovery of RNA-BP RNA targets. Among the different versions of CLIP that have been developed, the use of photoactivable nucleoside analogs incorporated into cellular RNA has resulted in high efficiency photoactivable ribonucleoside-enhanced CLIP (PAR-CLIP). Nonetheless, PAR-CLIP has not yet been applied in prokaryotes. To determine if PAR-CLIP can be used in prokaryotes, we determined suitable conditions for the incorporation of 4-thiouridine (4SU), a photoactivable nucleoside, into E. coli RNA, and for the isolation of crosslinked RNA. Applying this technique to Hfq, a well-characterized regulator of small RNA (sRNA)-messenger RNA (mRNA) interactions, we showed that PAR-CLIP identified most of the known sRNA targets of Hfq, as well as functionally relevant sites of Hfq-mRNA interactions at nucleotide resolution. Based on our findings, PAR-CLIP represents an improved method to identify both the RNAs and the specific regulatory sites that are recognized by RNA-BPs in prokaryotes.

microbiology↗

bioRxiv2020

Dual-level autoregulation of the E. coli DeaD RNA helicase via mRNA stability and Rho-dependent transcription termination

DEAD-box proteins (DBPs) are RNA remodeling factors associated with RNA helicase activity that are found in nearly all organisms. Despite extensive studies on the mechanisms used by DBPs to regulate RNA function, very little is known about how DBPs themselves are regulated. In this work, we have analyzed the expression and regulation of DeaD/CsdA, the largest of the DBPs in Escherichia coli (E. coli). We show that deaD transcription initiates 838 nts upstream of the start of the coding region. We have also found that DeaD is autoregulated through a negative feedback mechanism that operates both at the level of mRNA stability and Rho-dependent transcription termination, and this regulation is dependent upon the 5 untranslated region (5 UTR). These findings suggest that DeaD might be regulating the conformation of its own mRNA through its RNA helicase activity to facilitate ribonuclease and Rho access to its 5UTR.

microbiology↗

bioRxiv2019

Development of PAR-CLIP to analyze RNA-protein interactions in prokaryotes

The ability to identify RNAs that are recognized by RNA-binding proteins (RNA-BPs) using techniques such as "Crosslinking and Immunoprecipitation" (CLIP) has revolutionized the genome-wide discovery of RNA targets. Among the different versions of CLIP developed, the incorporation of photoactivable nucleoside analogs into cellular RNA has proven to be especially valuable, allowing for high efficiency photoactivable ribonucleoside-enhanced CLIP (PAR-CLIP). Although PAR-CLIP has become an established technique for use in eukaryotes, it has not yet been applied in prokaryotes. To determine if PAR-CLIP can be used in prokaryotes, we first investigated whether 4-thiouridine (4SU), a photoactivable nucleoside, can be incorporated into E. coli RNA. After determining 4SU incorporation into RNA, we developed suitable conditions for crosslinking of proteins in E. coli cells and for the isolation of crosslinked RNA. Applying this technique to Hfq, a well-characterized regulator of small RNA (sRNA) - messenger RNA (mRNA) interactions, we showed that PAR-CLIP identified most of the known sRNA targets of Hfq. Based on our results, PAR-CLIP represents an improved method to identify the RNAs recognized by RNA-BPs in prokaryotes.

microbiology↗