Biology subjects

Krishnamoorthy, A.

Publications and source records attributed to Krishnamoorthy, A..

3 recordsLinked to original sources

bioRxiv2024

TTF2 promotes replisome eviction from stalled forks in mitosis

Cells entering mitosis with incompletely replicated DNA face catastrophic chromosome segregation failure. During interphase, the replisome-associated E3 ubiquitin ligase TRAIP ubiquitylates barriers in front of the fork to allow replisome progression. In mitosis, TRAIP is reprogrammed from a trans-acting to a cis-acting ligase that can ubiquitylate the replisome itself. This enables the processing of unreplicated DNA by promoting replisome disassembly, fork breakage, and joining of the broken chromosome arms. Here, we describe a mechanism for this reprogramming: the ATPase TTF2 is recruited to the replisome, where its non-catalytic N-terminal domain tethers Cyclin B-CDK1-phosphorylated TRAIP to the leading strand DNA polymerase {varepsilon} in a geometry that allows replisome ubiquitylation. Thus, a phospho-regulated architectural switch alters replisome organization in mitosis to safeguard genome integrity before chromosome segregation.

biochemistry↗

bioRxiv2020

An in vivo strategy for knockdown of circular RNAs

Exonic circular RNAs (circRNAs) are highly abundant and evolutionarily conserved RNAs generated mostly from exons of protein-coding genes. Assaying the functions of circRNAs is not straightforward as common approaches for circRNA depletion tend to also alter the levels of mRNAs generated from the hosting gene. Here we describe a methodology for specific knockdown of circRNAs in vivo with tissue and cell resolution. We also describe an experimental and computational platform for determining the potential off-target effects as well as for verifying the obtained phenotypes. Briefly, we utilize miRNA-derived shRNAs targeted to the circRNA-specific back-splice junction to specifically downregulate the circRNA. We utilized this methodology to downregulate five circRNAs that are highly expressed in the fly nervous system. There were no effects on levels of the linear RNA or any RNA with complementarity to the expressed shRNA. Interestingly, downregulation of circCtrip resulted in developmental lethality that was recapitulated with a second shRNA. Moreover, we found that downregulation of individual circRNAs caused specific changes in the fly head transcriptome, suggesting roles for these circRNAs in the fly nervous system. Together, our results provide a methodological approach that enables the comprehensive study of circRNAs at the organismal and cell levels.

molecular biology↗

bioRxiv2018

An in vivo knockdown strategy reveals multiple functions for circMbl

Circular RNAs (circRNAs) are highly abundant and evolutionary conserved RNAs of mostly unknown functions. circRNAs are enriched in the brain and accumulate with age in flies, worms and mice. Despite their abundance, little is known about their functions, especially in the context of whole organisms. Here we report the development and use of shRNAs to knock down and study the function of circMbl, the most abundant circRNA in Drosophila. This circRNA is highly conserved through evolution and is generated from the locus of the essential splicing factor muscleblind (mbl). Briefly, we generated flies in which circMbl is reduced more than 90% without measurable off-target effects in the hosting gene as well as in other RNAs. These flies display specific defects that suggest roles of circMbl in muscle and neural tissues during development and in adult flies. More specifically, whole organism downregulation of circMbl leads to male developmental lethality, altered gene expression, behavioral defects, wing posture- and flight defects. Moreover, these phenotypes are recapitulated by a second shRNA targeting circMbl. Importantly, knockdown and overexpression of circMbl affect mostly the same genes but in the opposite direction. Last but not least, downregulation of circMbl in the fly central nervous system caused abnormal synaptic function. Together, our results demonstrate the functionality of circMbl at the organismal level likely by acting in multiple tissues. Moreover, here we provide the first proof of functionality of circRNAs in Drosophila as well as a methodological approach that enables the comprehensive study of circRNAs in vivo.\n\nSIGNIFICANCE STATEMENTCircular RNAs (circRNAs) are highly abundant and evolutionary conserved RNAs of mostly unknown functions. Here we report the development and use of a shRNA-based system to knockdown specific circRNAs in vivo. We generated flies in which circMbl, the most abundant circRNA is reduced more than 90% without measurable off-target effects. These flies display male developmental lethality, altered gene expression, behavioral defects, wing posture- and flight defects. These phenotypes are recapitulated by a second shRNA targeting circMbl. Moreover, downregulation of circMbl in the fly central nervous system caused abnormal synaptic function. Together, our results demonstrate the functionality of circMbl at the organismal level and provide a methodological approach that enables the comprehensive study of circRNAs in vivo.

molecular biology↗