Blanc SVSE 8 - Blanc - SVSE 8 - Biochimie, biologie moléculaire et structurale

AluACA RNAs, a novel class of human box H/ACA noncoding RNAs – AluACA

AluACA RNAs, a novel class of human box H/ACA noncoding RNAs

Our group has a long-standing interest in understanding of the functional complexity of box C/D and H/ACA small nucleolar and Cajal body-specific RNPs (snoRNPs and scaRNPs). During studying a group of human C/D and H/ACA RNPs which are associated with WDR79, we discovered a new class of human H/ACA RNPs, called the AluACA RNPs. The AluACA RNAs are processed from intronic Alu repetitive sequences.

We hoped that investigation of the biogenesis, trafficking and nuclear localization of AluACA RNPs would provide us with significant new insights into the biology of human WDR79-associated RNPs.

WDR79-associated small RNPs play important roles in the cell. They participate in site-specific pseudouridylation (box H/ACAs scaRNPs) and 2’-O-ribose methylation (box C/D scaRNPs) of spliceosomal snRNAs, catalyze telomere synthesis (telomerase). The function of the recently discovered WDR79-associated AluACA RNPs remained fully unknown. In this project, we wanted to get new insights into biogenesis, intracellular trafficking and function of AluACA RNPs and other WDR79-associated small RNPs. We systematically investigated the processing and assembly of intron-encoded AluACA RNA into metabolically stable H/ACA RNPs. To identify associated proteins and RNAs, we attempted to purify human AluACA RNP overexpressed in HeLa cells. Box H/ACA scaRNA, AluACA RNAs and telomerase RNA share a common WDR79-binding element the CAB box (consensus ugAG) that is missing from box C/D scaRNAs. Therefore, to understand the intranuclear trafficking of box C/D scaRNPs, we wanted to dissect their WDR79 binding elements. Finally, we wanted to test the capacity of ALuACA RNPs to control RNAPII activity as it has been reported for the related rodent B2 RNAs.

Intranuclear localization of wild-type and mutant WDR79-associated AluACA RNAs and box C/D RNAs was performed by fluorescent in situ hybridization microscopy. In vivo processing and accumulation of wild-type and a series of mutant and chimeric AluACA RNAs was monitored by Northern blot analysis and RNase A/T1 protection analyses by using sequence-specific antisense probes. Assembly of wild-type and mutant AluACA RNAs and box C/D scaRNAs into small RNPs was assayed by co-immunoprecipitation experiments. Genome-wide protein and RNA association with chromatin was determined by chromatin immunoprecipitation (ChIP) and chromatin isolation by RNA purification (ChIRP) followed by deep sequencing and bioinformatics analyses.

We have shown that human WDR79-associated RNPs fall into four distinct groups: box H/ACA scaRNPs, box C/D scaRNPs, AluACA RNPs and the telomerase RNP. We showed that AluACA RNAs carry aberrantly short or long 5' hairpins which are not expected to support RNA accumulation and RNP assembly. Systematic mutational analysis demonstrated that the 3’-hairpins of human AluACA RNAs and the human telomerase RNA (hTR) share a common RNA element, called the BIO box, that supports RNA 3’ end processing and H/ACA RNP assembly. The BIO box likely supports specific binding of the Nhp2 H/ACA RNP protein that binds in a non-specific manner to canonical H/ACA RNAs. We demonstrated that overaccumulation of AluACA is toxic to the cell. This finding, although excluded the possibility of overexpressing AluACA RNAs for purification, strongly supported the biological importance of AluACA RNPs. Mouse Alu related B2 RNAs have been reported to interact with RNAPII. Therefore, we investigated human nuclear RNAs interacting with RNAPII. While we failed to detect AluACA RNAs, we found the 7SK snRNA specifically associated with a fraction of RNAPII hyperphosphorylated at Ser5 and Ser7. We showed that the 7SK snRNP promotes expression of RNAPII-specific snRNA genes as an integral component of the snRNA gene-specific transcription factor, the little elongation complex. The 7SK is the first snRNP that functions as a canonical transcription factor. WDR79 targets both box H/ACA and C/D scaRNPs into the Cajal body. Earlier we showed that WDR79 binds to the CAB box (ugAG) of H/ACA scaRNAs. Now, we have demonstrated that box C/D scaRNAs are targeted into the Cajal body by GU-dominated dinucleotide repeat sequences through binding WDR79.

Our work has already provided new insights into the biology of human small nuclear RNPs. Our most significant achievements are:
(i) Demonstration that AluACA RNAs and human telomerase RNA share a common RNA structural element (BIO box) supporting RNP assembly.
(ii) Demonstration that 7SK snRNP functions as a genuine transcription factor.
(iii) Dissection of the Cajal body targeting elements of box C/D scaRNAs.

The BIO box was assumed to be a telomerase-specific RNA element that promotes 3’ end processing and accumulation of hTR. Our preliminary results indicate that the BIO box functions as an Nhp2 docking site that tethers Nhp2 to the terminal stem-loop of the 3’-hairpins of hTR and AluACA RNAs with high specificity and efficiency. In collaboration with Drs Lebaron S. and Leulliot N (Laboratoire de Cristallographie et RMN Biologiques, Université Paris Descartes,Paris), we currently investigate the association of Nhp2 with AluACA RNAs and hTR. Thus, our results will also facilitate the understanding of the biogenesis of telomerase RNP. Our finding that besides controlling the nuclear activity of P-TEFb, the 7SK snRNP also functions in RNAPII-specific snRNA gene expression, demonstrates that 7SK is a multifunctional regulatory snRNP and opens up new exciting areas to investigate in the future.

1. Marnef, A., Richard, P., Pinzón, N. and Kiss, T. (2014) Targeting of vertebrate intron-encoded box C/D 2’-O-methylation guide RNAs into the Cajal body. Nucleic Acids Res., 42, 6616-6629.
(http://nar.oxfordjournals.org/content/42/10/6616.long)
2. Marnef, A., Jády, B.E. and Kiss, T. (2015) Human polypyrimidine tract-binding protein interacts with mitochondrial tRNAThr in the cytosol. Nucleic Acids Res., (in press) pii: gkv1355
3. Egloff, S., Vitali, P., Raffel, R., Murphy, S. and Kiss, T. (2016) The 7SK snRNP associates with the little elongation complex to promote snRNA gene expression. Genes & Dev., (submitted)

Eukaryotic genome sequencing projects led to the astonishing discovery that the number of protein-coding genes shows a surprisingly moderate increase during evolution, although the genome size is rapidly growing in higher eukaryotes. This finding led to the recently emerging view that in addition to the protein-coding genes, the eukaryotic genomes contain another, thus far hidden layer of genetic information. Indeed, during that past decade it became apparent that the eukaryotic genome encodes a tremendous number of non-protein-coding or non-coding RNAs (ncRNAs) which function as regulatory RNAs in all aspects of gene expression and thereby, largely contribute to the biological complexity of eukaryotic organisms. Box H/ACA RNAs represent an abundant, evolutionarily conserved and functionally diverse group of ncRNAs. The H/ACA RNAs function in pseudouridylation of various classes of cellular RNAs, nucleolytic processing of rRNAs, synthesis of telomeric DNA and they serve as substrates for microRNA processing. All H/ACA RNAs associate with four H/ACA core ribonucleoproteins, dyskerin, Nhp2, Nop10 and Gar1, and they accumulate either in the nucleolus or in the nucleoplasmic Cajal bodies. The Cajal body-specific H/ACA RNAs also associate with Wdr79. In our laboratory, recent characterization of human Wdr79-associated RNAs identified more than 400 novel putative H/ACA RNAs. Besides several canonical H/ACA RNAs, we identified 348 novel human H/ACA RNAs which are encoded by genomic Alu repetitive elements located within introns of protein-coding genes. We have demonstrated that the newly discovered Alu-derived H/ACA RNAs, termed AluACA RNAs, are synthesized and processed from pre-mRNA intronic Alu sequences in an H and ACA box-dependent manner. The mature AluACA RNPs associate with the four H/ACA core proteins and Wdr79, but unexpectedly, they co-localize with spliceosomal small nuclear (sn)RNAs in the nucleoplasm. The Alu elements are the most abundant repetitive elements in the human genome with largely unknown function. Originated from the 7SL signal recognition particle RNA gene, Alu elements were propagated by retrotransposition in the genomes of primates. Thus, the newly discovered AluACA RNAs represent a novel, abundant, human- or maybe primate-specific subclass of H/ACA RNAs. Here, I propose a research program to dissect the biogenesis, subnuclear trafficking and the cellular function of this fascinating group of human ncRNPs.

Project coordination

Tamas Kiss (Organisme de recherche)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

Help of the ANR 194,930 euros
Beginning and duration of the scientific project: June 2013 - 42 Months

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