In Vivo Tissue-Specific Knockdown of circRNAs Using shRNAs in Drosophila melanogaster.

Studying circular RNAs' function in vivo has been challenging due to the lack of generic tools to manipulate their levels without affecting their linear counterparts. This is particularly challenging as the back-splice junction is the only sequence not shared between the linear and circular version. In this chapter, we describe a method to study circRNA function in vivo targeting shRNAs against the desired back-splice junction to achieve knockdown with tissue-specific resolution in flies.

Alternative polyadenylation factor CPSF6 regulates temperature compensation of the mammalian circadian clock.

A defining property of circadian clocks is temperature compensation, characterized by the resilience of their near 24-hour free-running periods against changes in environmental temperature within the physiological range. While temperature compensation is evolutionary conserved across different taxa of life and has been studied within many model organisms, its molecular underpinnings remain elusive. Posttranscriptional regulations such as temperature-sensitive alternative splicing or phosphorylation have been described as underlying reactions.

Organismal landscape of clock cells and circadian gene expression in .

Background: Circadian rhythms time physiological and behavioral processes to 24-hour cycles. It is generally assumed that most cells contain self-sustained circadian clocks that drive circadian rhythms in gene expression that ultimately generating circadian rhythms in physiology. While those clocks supposedly act cell autonomously, current work suggests that in some of them can be adjusted by the brain circadian pacemaker through neuropeptides, like the Pigment Dispersing Factor (PDF).

The chromatin factor ROW cooperates with BEAF-32 in regulating long-range inducible genes.

Insulator proteins located at the boundaries of topological associated domains (TAD) are involved in higher-order chromatin organization and transcription regulation. However, it is still not clear how long-range contacts contribute to transcriptional regulation. Here, we show that relative-of-WOC (ROW) is essential for the long-range transcription regulation mediated by the boundary element-associated factor of 32kD (BEAF-32).

Artificially stimulating retrotransposon activity increases mortality and accelerates a subset of aging phenotypes in .

Transposable elements (TEs) are mobile sequences of DNA that can become transcriptionally active as an animal ages. Whether TE activity is simply a by-product of heterochromatin breakdown or can contribute toward the aging process is not known. Here, we place the TE under the control of the UAS GAL4 system to model TE activation during aging.

Best practice standards for circular RNA research.

Circular RNAs (circRNAs) are formed in all domains of life and via different mechanisms. There has been an explosion in the number of circRNA papers in recent years; however, as a relatively young field, circRNA biology has an urgent need for common experimental standards for isolating, analyzing, expressing and depleting circRNAs. Here we propose a set of guidelines for circRNA studies based on the authors' experience.

circMbl functions in cis and in trans to regulate gene expression and physiology in a tissue-specific fashion.

Muscleblind (mbl) is an essential muscle and neuronal splicing regulator. Mbl hosts multiple circular RNAs (circRNAs), including circMbl, which is conserved from flies to humans. Here, we show that mbl-derived circRNAs are key regulators of MBL by cis- and trans-acting mechanisms.

SARS-CoV-2 Nsp14 mediates the effects of viral infection on the host cell transcriptome.

Viral infection involves complex set of events orchestrated by multiple viral proteins. To identify functions of SARS-CoV-2 proteins, we performed transcriptomic analyses of cells expressing individual viral proteins. Expression of Nsp14, a protein involved in viral RNA replication, provoked a dramatic remodeling of the transcriptome that strongly resembled that observed following SARS-CoV-2 infection.

SARS-CoV-2 Nsp14 mediates the effects of viral infection on the host cell transcriptome.

Viral infection involves complex set of events orchestrated by multiple viral proteins. To identify functions of SARS-CoV-2 proteins, we performed transcriptomic analyses of cells expressing individual viral proteins. Expression of Nsp14, a protein involved in viral RNA replication, provoked a dramatic remodeling of the transcriptome that strongly resembled that observed following SARS-CoV-2 infection.

Parallel evolution of a splicing program controlling neuronal excitability in flies and mammals.

Alternative splicing increases neuronal transcriptomic complexity throughout animal phylogeny. To delve into the mechanisms controlling the assembly and evolution of this regulatory layer, we characterized the neuronal microexon program in and compared it with that of mammals. In nonvertebrate bilaterians, this splicing program is restricted to neurons by the posttranscriptional processing of the (eMIC) domain in .

SRCP: a comprehensive pipeline for accurate annotation and quantification of circRNAs.

Here we describe a new integrative approach for accurate annotation and quantification of circRNAs named Short Read circRNA Pipeline (SRCP). Our strategy involves two steps: annotation of validated circRNAs followed by a quantification step. We show that SRCP is more sensitive than other individual pipelines and allows for more comprehensive quantification of a larger number of differentially expressed circRNAs.

Using Drosophila to uncover molecular and physiological functions of circRNAs.

Circular RNAs (circRNAs) are a class of covalently closed RNA molecules generated by backsplicing. circRNAs are expressed in a tissue-specific manner, accumulate with age in neural tissues, and are highly stable. In many cases, circRNAs are generated at the expense of a linear transcript as back-splicing competes with linear splicing.

Extensive tissue-specific expression variation and novel regulators underlying circadian behavior.

Natural genetic variation affects circadian rhythms across the evolutionary tree, but the underlying molecular mechanisms are poorly understood. We investigated population-level, molecular circadian clock variation by generating >700 tissue-specific transcriptomes of ( ) and 141 Genetic Reference Panel (DGRP) lines. This comprehensive circadian gene expression atlas contains >1700 cycling genes including previously unknown central circadian clock components and tissue-specific regulators.

An in vivo strategy for knockdown of circular RNAs.

Exonic circular RNAs (circRNAs) are highly abundant 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.

Host-derived circular RNAs display proviral activities in Hepatitis C virus-infected cells.

Viruses subvert macromolecular pathways in infected host cells to aid in viral gene amplification or to counteract innate immune responses. Roles for host-encoded, noncoding RNAs, including microRNAs, have been found to provide pro- and anti-viral functions. Recently, circular RNAs (circRNAs), that are generated by a nuclear back-splicing mechanism of pre-mRNAs, have been implicated to have roles in DNA virus-infected cells.

A Parkinson's disease CircRNAs Resource reveals a link between circSLC8A1 and oxidative stress.

Circular RNAs (circRNAs) are brain-abundant RNAs of mostly unknown functions. To seek their roles in Parkinson's disease (PD), we generated an RNA sequencing resource of several brain region tissues from dozens of PD and control donors. In the healthy substantia nigra (SN), circRNAs accumulate in an age-dependent manner, but in the PD SN this correlation is lost and the total number of circRNAs reduced.

A lncRNA survey finds increases in neuroprotective LINC-PINT in Parkinson's disease substantia nigra.

Recent reports highlight regulatory functions of long noncoding RNAs (lncRNAs) in neurodegeneration and aging, but biomedical implications remain limited. Here, we report an rRNA-depletion-based long RNA-Sequencing Resource of 65 substantia nigra, amygdala, and medial temporal gyrus samples from Parkinson's disease (PD) and matched control brains. Using a lncRNA-focused analysis approach to identify functionally important transcripts, we discovered and prioritized many lncRNAs dysregulated in PD.

Thermosensitive alternative splicing senses and mediates temperature adaptation in .

Circadian rhythms are generated by the cyclic transcription, translation, and degradation of clock gene products, including (), but how the circadian clock senses and adapts to temperature changes is not completely understood. Here, we show that temperature dramatically changes the splicing pattern of in . We found that at 18°C, TIM levels are low because of the induction of two cold-specific isoforms: and .

PSI controls circadian period and the phase of circadian behavior under temperature cycle via splicing.

The circadian pacemaker consists of transcriptional feedback loops subjected to post-transcriptional and post-translational regulation. While post-translational regulatory mechanisms have been studied in detail, much less is known about circadian post-transcriptional control. Thus, we targeted 364 RNA binding and RNA associated proteins with RNA interference.

Past, present, and future of circRNAs.

Exonic circular RNAs (circRNAs) are covalently closed RNA molecules generated by a process named back-splicing. circRNAs are highly abundant in eukaryotes, and many of them are evolutionary conserved. In metazoans, circular RNAs are expressed in a tissue-specific manner, are highly stable, and accumulate with age in neural tissues.

NEAT1 is overexpressed in Parkinson's disease substantia nigra and confers drug-inducible neuroprotection from oxidative stress.

Recent reports attribute numerous regulatory functions to the nuclear paraspeckle-forming long noncoding RNA, nuclear enriched assembly transcript 1 (NEAT1), but the implications of its involvement in Parkinson's disease (PD) remain controversial. To address this issue, we assessed NEAT1 expression levels and cell type patterns in the substantia nigra (SN) from 53 donors with and without PD, as well as in interference tissue culture tests followed by multiple in-house and web-available models of PD. PCR quantification identified elevated levels of NEAT1 expression in the PD SN compared with control brains, an elevation that was reproducible across a multitude of disease models.

Craving for Introns.

Parenteau et al. (2019) and Morgan et al. (2019) showed that a subset of introns can work as non-coding RNAs that trap the spliceosome and decrease global splicing upon nutrient depletion in yeast, providing a new example of the functionality of introns, molecules that were previously assumed to be useless.

High-accuracy determination of internal circadian time from a single blood sample.

Background: The circadian clock is a fundamental and pervasive biological program that coordinates 24-hour rhythms in physiology, metabolism, and behavior, and it is essential to health. Whereas therapy adapted to time of day is increasingly reported to be highly successful, it needs to be personalized, since internal circadian time is different for each individual. In addition, internal time is not a stable trait, but is influenced by many factors, including genetic predisposition, age, sex, environmental light levels, and season.