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). In addition, a Drosophila La related protein (now called dLARP7) is shown to be the functional homolog of human LARP7. The Drosophila 7SK snRNP (d7SK snRNP) responded to treatment of cells with P-TEFb inhibitors and to nuclease treatment of cell lysates by releasing P-TEFb. Supporting a critical role for the d7SK snRNP in Drosophila development, dLARP7 and dHEXIM were found to be ubiquitously expressed throughout embryos and tissues at all stages. Importantly, knockdown of dHEXIM was embryonic lethal, and reduction of dHEXIM in specific tissues led to serious developmental defects. Our results suggest that regulation of P-TEFb by the d7SK snRNP is essential for the growth and differentiation of tissues required during Drosophila development.

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. This transcriptional dynamic, confirmed by RT-qPCR for Jun , Fos , Btg2, is very precocious. RNA Pol II CTD Ser2 hyperphosphorylation indicates a switch to productive elongation and release from transcriptional pause.

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. Conversely, depletion of FEN1, resulting in the accumulation of uncleaved RNA primers, increases 53BP1 levels at replication forks, suggesting that RNA primers contribute to the recruitment of 53BP1 at the lagging DNA strand. 53BP1 depletion induces an accumulation of S-phase poly(ADP-ribose), which constitutes a sensor of unligated Okazaki fragments. Collectively, our data indicate that 53BP1 is anchored at nascent DNA through its RNA-binding activity, highlighting the role of an RNA-protein interaction at replication forks.

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. melanogaster. We examined the structure of the corresponding gene and determined the expression profiles of the encoded RNA, called snRNA:7SK:94F, during development. It is probably produced from the transcription of a lncRNA which is processed into a mature snRNA. We also addressed its biological function and we show that, like dm7SK, this alternative 7SK interacts in vivo with the different partners of the P-TEFb complex, i.e. HEXIM, LARP7 and Cyclin T. This novel RNA is widely expressed across tissues. CONCLUSION: We propose that two distinct 7SK genes might contribute to the formation of the 7SK snRNP complex in D. melanogaster.

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. Our results also indicate that the pre-U1 processing activity is heat sensitive and that an RNA component is required. In addition, the exact sequence adjacent to the 3' box influences the position of the pre-U1 3' end produced in vitro. Interestingly, the processing extract active for 3'-box-dependent processing also contains an activity that converts the 3' end of RNA containing the U1 Sm protein binding site and the 3' terminal stem-loop into the mature form.

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. Its biological activity is controlled by an inhibitory complex composed of HEXIM and the 7SK snRNA. Here, we examine the function of HEXIM during Drosophila development. Our key finding is that HEXIM affects the Hedgehog signaling pathway. HEXIM knockdown flies display strong phenotypes and organ failures. In the wing imaginal disc, HEXIM knockdown initially induces ectopic expression of Hedgehog (Hh) and its transcriptional effector Cubitus interuptus (Ci). In turn, deregulated Hedgehog signaling provokes apoptosis, which is continuously compensated by apoptosis-induced cell proliferation. Thus, the HEXIM knockdown mutant phenotype does not result from the apoptotic ablation of imaginal disc; but rather from the failure of dividing cells to commit to a proper developmental program due to Hedgehog signaling defects. Furthermore, we show that ci is a genetic suppressor of hexim. Thus, HEXIM ensures the integrity of Hedgehog signaling in wing imaginal disc, by a yet unknown mechanism. To our knowledge, this is the first time that the physiological function of HEXIM has been addressed in such details in vivo.

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. lactis IL1403. LctT and 'LcnC' belong to the recently defined family of AMS (ABC transporter maturation and secretion) proteins. Inactivation of the 'lcnC' gene demonstrates that it is not responsible for the weak lacticin 481 production observed in a strain expressing only the precursor peptide LctA, and the modification enzyme LctM. This result indicates that the two AMS proteins, 'LcnC' and LctT, are not interchangeable in the machinery of processing/export of lacticin 481.

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.

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. This finally demonstrates that the 3' end of pre-snRNAs is produced by RNA processing rather than by termination of transcription. The phosphorylated form of the C-terminal domain (CTD) of pol II activates the processing event in vitro, consistent with our previous demonstration of the role of the CTD in pre-snRNA 3' end formation in vivo. In addition, we show that sequences upstream from the 3' box of the U2 snRNA gene influence 3' end formation both in vivo and 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. Formation of the 3' end of these non-polyadenylated RNAs requires a specialized 3' box element whose function is promoter specific. Here we show that truncation of the C-terminal domain (CTD) of RNA polymerase II and treatment of cells with CTD kinase inhibitors, including DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole), causes a dramatic reduction in proper 3' end formation of U2 transcripts. Activation of 3' box recognition by the phosphorylated CTD would be consistent with the role of phospho-CTD in mRNA processing. CTD kinase inhibitors, however, have little effect on initiation or elongation of transcription of the U2 genes, whereas elongation of transcription of the beta-actin gene is severely affected. This result highlights differences in transcription of snRNA and mRNA genes.