Protocol to study the direct binding of proteins to RNA:DNA hybrids or RNA-DNA chimeras in living cells using cross-linking immunoprecipitation.

RNA-binding proteins (RBPs) are involved in many biological processes. The direct interaction between protein and RNA can be studied using cross-linking immunoprecipitation (CLIP) techniques in living cells. Here, we present a protocol to characterize the direct binding of proteins to RNA:DNA hybrids or RNA-DNA chimeras in living cells using CLIP.

Transcriptional activation of candidate paused genes after partial hepatectomy.

Partial hepatectomy is a model of acute liver injury that is known to induce a strong reprogrammation of gene expression. Transcriptional induction of Immediate Early Genes is extremely fast and this would be due to the release of RNA Polymerase II poised for elongation at 'paused' genes. Using bioinformatic analysis, we identified 23 genes sharing features of paused genes before hepatectomy, and with predicted quick and strong expression induction after.

Post-transcriptional gene regulation: From mechanisms to RNA chemistry and therapeutics.

A better understanding of the RNA biology and chemistry is necessary to then develop new RNA therapeutic strategies. This review is the synthesis of a series of conferences that took place during the 6th international course on post-transcriptional gene regulation at Institut Curie. This year, the course made a special focus on RNA chemistry.

53BP1 interacts with the RNA primer from Okazaki fragments to support their processing during unperturbed DNA replication.

RNA-binding proteins (RBPs) are found at replication forks, but their direct interaction with DNA-embedded RNA species remains unexplored. Here, we report that p53-binding protein 1 (53BP1), involved in the DNA damage and replication stress response, is an RBP that directly interacts with Okazaki fragments in the absence of external stress. The recruitment of 53BP1 to nascent DNA shows susceptibility to in situ ribonuclease A treatment and is dependent on PRIM1, which synthesizes the RNA primer of Okazaki fragments.

At the crossroads of RNA biology, genome integrity and cancer.

This article is the synthesis of the scientific presentations that took place during two international courses at Institute Curie, one on post-transcriptional gene regulation and the other on genome instability and human disease, that were joined together in their 2021 edition. This joined course brought together the knowledge on RNA metabolism and the maintenance of genome stability.

An alternative D. melanogaster 7SK snRNP.

Background: The 7SK small nuclear RNA (snRNA) found in most metazoans is a key regulator of P-TEFb which in turn regulates RNA polymerase II elongation. Although its primary sequence varies in protostomes, its secondary structure and function are conserved across evolutionary distant taxa.

Results: Here, we describe a novel ncRNA sharing many features characteristic of 7SK RNAs, in D.

Functional Interaction between HEXIM and Hedgehog Signaling during Drosophila Wing Development.

Studying the dynamic of gene regulatory networks is essential in order to understand the specific signals and factors that govern cell proliferation and differentiation during development. This also has direct implication in human health and cancer biology. The general transcriptional elongation regulator P-TEFb regulates the transcriptional status of many developmental genes.

The Drosophila 7SK snRNP and the essential role of dHEXIM in development.

Regulation of the positive transcription elongation factor, P-TEFb, plays a major role in controlling mammalian transcription and this is accomplished in part by controlled release of P-TEFb from the 7SK snRNP that sequesters the kinase in an inactive state. We demonstrate here that a similar P-TEFb control system exists in Drosophila. We show that an RNA previously suggested to be a 7SK homolog is, in fact, associated with P-TEFb, through the action of a homolog of the human HEXIM1/2 proteins (dHEXIM).

Maturation by LctT is required for biosynthesis of full-length lantibiotic lacticin 481.

In lantibiotic lacticin 481 biosynthesis, LctT cleaves the precursor peptide and exports mature lantibiotic. Matrix-assisted laser desorption ionization-time of flight mass spectrometry revealed that a truncated form of lacticin 481 is produced in the absence of LctT or after cleavage site inactivation. Production of truncated lacticin 481 is 4-fold less efficient, and its specific activity is about 10-fold lower.

3'-box-dependent processing of human pre-U1 snRNA requires a combination of RNA and protein co-factors.

Using an in vitro system we have recently shown that the 3' ends of human pre-snRNAs synthesized by RNA polymerase II are produced by RNA processing directed by the snRNA gene-specific 3' box. Towards a complete characterization of this processing reaction we have further investigated the in vitro requirements for proper 3' end formation of pre-U1 snRNA. Here we show that the 5' cap plays a stimulatory role and processing requires creatine phosphate.

The 3' ends of human pre-snRNAs are produced by RNA polymerase II CTD-dependent RNA processing.

Proper 3' end formation of the human pre-snRNAs synthesized by pol II requires the cis-acting 3' box, although the precise function of this element has proved difficult to determine. In vivo, 3' end formation is tightly linked to transcription. However, we have now been able to obtain transcription-independent 3' box-dependent processing in vitro.

The C-terminal domain of pol II and a DRB-sensitive kinase are required for 3' processing of U2 snRNA.

The human snRNA genes transcribed by RNA polymerase II (e.g. U1 and U2) have a characteristic TATA-less promoter containing an essential proximal sequence element.

The LcnC homologue cannot replace LctT in lacticin 481 export.

Lacticin 481 is produced by Lactococcus lactis subsp. lactis and belongs to subgroup AII of the lanthionine-containing bacteriocins. The putative homodimeric LctT involved in lacticin 481 production shares significant similarities with the 'LcnC' protein encoded by 'lcnC', located on the chromosome of the lactic acid bacterium, L.