rna-pull-down-procedure-to-identify-rna-targets-long-non-coding

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding ...

Aix-Marseille Université

Paraspeckles are nuclear bodies form around the long non-coding RNA, Neat1, and RNA-binding proteins. While their role is not fully understood, they are believed to control gene expression at a post-transcriptional level by means of the nuclear retention of mRNA containing in their 3'-UTR inverted repeats of Alu sequences (IRAlu). In this study, we found that, in pituitary cells, all components of paraspeckles including four major proteins and Neat1 displayed a circadian expression pattern. Furthermore the insertion of IRAlu at the 3'-UTR of the EGFP cDNA led to a rhythmic circadian nuclear retention of the egfp mRNA that was lost when paraspeckles were disrupted whereas insertion of a single antisense Alu had only a weak effect. Using real-time video-microscopy, these IRAlu were further shown to drive a circadian expression of EGFP protein. This study shows that paraspeckles, thanks to their circadian expression, control circadian gene expression at a post-transcriptional level.

Circadian RNA expression elicited by 3'-UTR IRAlu-paraspeckle associated elements.

Circadian clocks regulate rhythmic gene expression levels by means of mRNA oscillations that are mainly driven by post-transcriptional regulation. We identified a new post-transcriptional mechanism, which involves nuclear bodies called paraspeckles. Major components of paraspeckles including the long noncoding RNA Neat1, which is the structural component, and its major protein partners, as well as the number of paraspeckles, follow a circadian pattern in pituitary cells. Paraspeckles are known to retain within the nucleus RNAs containing inverted repeats of Alu sequences. We showed that a reporter gene in which these RNA duplex elements were inserted in the 3'-UTR region displayed a circadian expression. Moreover, circadian endogenous mRNA associated with paraspeckles lost their circadian pattern when paraspeckles were disrupted. This work not only highlights a new paraspeckle-based post-transcriptional mechanism involved in circadian gene expression but also provides the list of all mRNA associated with paraspeckles in the nucleus of pituitary cells.

Paraspeckles as rhythmic nuclear mRNA anchorages responsible for circadian gene expression.

You are what you eat; but when you eat also seems to be important for a healthy metabolism. In this issue of Cell Metabolism, Benegiamo et al. (2018) uncover a mechanism by which the RNA-binding protein NONO promotes the time-of-day-dependent expression of key metabolic genes at a post-transcriptional level in response to nutrition.

Mealtime Is NONO Speckled: Timing Hepatic Adaptation to Food.

The circadian clock drives daily rhythms of multiple physiological processes, allowing organisms to anticipate and adjust to periodic changes in environmental conditions. These physiological rhythms are associated with robust oscillations in the expression of at least 30% of expressed genes. While the ability for the endogenous timekeeping system to generate a 24-hr cycle is a cell-autonomous mechanism based on negative autoregulatory feedback loops of transcription and translation involving core-clock genes and their protein products, it is now increasingly evident that additional mechanisms also govern the circadian oscillations of clock-controlled genes. Such mechanisms can take place post-transcriptionally during the course of the RNA life cycle. It has been shown that many steps during RNA processing are regulated in a circadian manner, thus contributing to circadian gene expression. These steps include mRNA capping, alternative splicing, changes in splicing efficiency, and changes in RNA stability controlled by the tail length of polyadenylation or the use of alternative polyadenylation sites. RNA transport can also follow a circadian pattern, with a circadian nuclear retention driven by rhythmic expression within the nucleus of particular bodies (the paraspeckles) and circadian export to the cytoplasm driven by rhythmic proteins acting like cargo. Finally, RNA degradation may also follow a circadian pattern through the rhythmic involvement of miRNAs. In this review, we summarize the current knowledge of the post-transcriptional circadian mechanisms known to play a prominent role in shaping circadian gene expression in mammals. This article is categorized under: RNA Processing > Splicing Regulation/Alternative Splicing RNA Processing > RNA Editing and Modification RNA Export and Localization > Nuclear Export/Import.

Manon Torres

CNRS,
CRN2M-UMR7286,
Faculté de Médecine Nord,
CNRS, CRN2M-UMR7286, Faculté de Médecine Nord

Aix-Marseille Université

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Manon Torres

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}CNRS,CRN2M-UMR7286,Faculté de Médecine Nord,CNRS, CRN2M-UMR7286, Faculté de Médecine Nord

Aix-Marseille Université

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

CNRS,
CRN2M-UMR7286,
Faculté de Médecine Nord,
CNRS, CRN2M-UMR7286, Faculté de Médecine Nord