Céline Verheggen

Cajal body-specific small nuclear RNAs: a novel class of 2′-O-methylation and pseudouridylation guide RNAs

Cajal (coiled) bodies are conserved subnuclear organelles that are present in the nucleoplasm of both animal and plant cells. Although Cajal bodies were first described nearly 100 years ago, their function has remained largely speculative. Here, we describe a novel class of human small nuclear RNAs that localize specifically to Cajal bodies. The small Cajal body‐ specific RNAs (scaRNAs) are predicted or have already been demonstrated to function as guide RNAs in site‐specific synthesis of 2′‐O‐ribose‐methylated nucleotides and pseudouridines in the RNA polymerase II‐transcribed U1, U2, U4 and U5 spliceosomal small nuclear RNAs (snRNAs). Our results provide strong support for the idea that the Cajal body, this mysterious nuclear organelle, provides the cellular locale for post‐transcriptional modification of spliceosomal snRNAs.

Mammalian and yeast U3 snoRNPs are matured in specific and related nuclear compartments

Nucleolar localization of vertebrate box C/D snoRNA involves transit through Cajal bodies, but the significance of this event is unkown. To define better the function of this compartment, we analyzed here the maturation pathway of mammalian U3. We show that 3′‐extended U3 precursors possess a mono‐methylated cap, and are not associated with fibrillarin and hNop58. Importantly, these precursors are detected at both their transcription sites and in Cajal bodies. In addition, mature U3, the core box C/D proteins and the human homolog of the methyltransferase responsible for U3 cap tri‐methylation, hTgs1, are all present in Cajal bodies. In yeast, U3 follows a similar maturation pathway, and equivalent 3′‐extended precursors are enriched in the nucleolus and in the nucleolar body, a nucleolar domain that concentrates Tgs1p under certain growth conditions. Thus, spatial organization of U3 maturation appears to be conserved across evolution, and involves specialized and related nuclear compartments, the nucleolus/nucleolar body in yeast and Cajal bodies in higher eukaryotes. These are likely places for snoRNP assembly, 3′ end maturation and cap modification.

Hypermethylation of the Cap Structure of Both Yeast snRNAs and snoRNAs Requires a Conserved Methyltransferase that Is Localized to the Nucleolus

The m(7)G caps of most spliceosomal snRNAs and certain snoRNAs are converted posttranscriptionally to 2,2,7-trimethylguanosine (m(3)G) cap structures. Here, we show that yeast Tgs1p, an evolutionarily conserved protein carrying a signature of S-AdoMet methyltransferase, is essential for hypermethylation of the m(7)G caps of both snRNAs and snoRNAs. Deletion of the yeast TGS1 gene abolishes the conversion of the m(7)G to m(3)G caps and produces a cold-sensitive splicing defect that correlates with the retention of U1 snRNA in the nucleolus. Consistently, Tgs1p is also localized in the nucleolus. Our results suggest a trafficking pathway in which yeast snRNAs and snoRNAs cycle through the nucleolus to undergo m(7)G cap hypermethylation.

The Hsp90 chaperone controls the biogenesis of L7Ae RNPs through conserved machinery

RNA-binding proteins of the L7Ae family are at the heart of many essential ribonucleoproteins (RNPs), including box C/D and H/ACA small nucleolar RNPs, U4 small nuclear RNP, telomerase, and messenger RNPs coding for selenoproteins. In this study, we show that Nufip and its yeast homologue Rsa1 are key components of the machinery that assembles these RNPs. We observed that Rsa1 and Nufip bind several L7Ae proteins and tether them to other core proteins in the immature particles. Surprisingly, Rsa1 and Nufip also link assembling RNPs with the AAA + adenosine triphosphatases hRvb1 and hRvb2 and with the Hsp90 chaperone through two conserved adaptors, Tah1/hSpagh and Pih1. Inhibition of Hsp90 in human cells prevents the accumulation of U3, U4, and telomerase RNAs and decreases the levels of newly synthesized hNop58, hNHP2, 15.5K, and SBP2. Thus, Hsp90 may control the folding of these proteins during the formation of new RNPs. This suggests that Hsp90 functions as a master regulator of cell proliferation by allowing simultaneous control of cell signaling and cell growth.

HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II.

RNA polymerases are key multisubunit cellular enzymes. Microscopy studies indicated that RNA polymerase I assembles near its promoter. However, the mechanism by which RNA polymerase II is assembled from its 12 subunits remains unclear. We show here that RNA polymerase II subunits Rpb1 and Rpb3 accumulate in the cytoplasm when assembly is prevented and that nuclear import of Rpb1 requires the presence of all subunits. Using MS-based quantitative proteomics, we characterized assembly intermediates. These included a cytoplasmic complex containing subunits Rpb1 and Rpb8 associated with the HSP90 cochaperone hSpagh (RPAP3) and the R2TP/Prefoldin-like complex. Remarkably, HSP90 activity stabilized incompletely assembled Rpb1 in the cytoplasm. Our data indicate that RNA polymerase II is built in the cytoplasm and reveal quality-control mechanisms that link HSP90 to the nuclear import of fully assembled enzymes. hSpagh also bound the free RPA194 subunit of RNA polymerase I, suggesting a general role in assembling RNA polymerases.

A common sequence motif determines the Cajal body‐specific localization of box H/ACA scaRNAs

Post‐transcriptional synthesis of 2′‐O‐methylated nucleotides and pseudouridines in Sm spliceosomal small nuclear RNAs takes place in the nucleoplasmic Cajal bodies and it is directed by guide RNAs (scaRNAs) that are structurally and functionally indistinguishable from small nucleolar RNAs (snoRNAs) directing rRNA modification in the nucleolus. The scaRNAs are synthesized in the nucleoplasm and specifically targeted to Cajal bodies. Here, mutational analysis of the human U85 box C/D‐H/ACA scaRNA, followed by in situ localization, demonstrates that box H/ACA scaRNAs share a common Cajal body‐specific localization signal, the CAB box. Two copies of the evolutionarily conserved CAB consensus (UGAG) are located in the terminal loops of the 5′ and 3′ hairpins of the box H/ACA domains of mammalian, Drosophila and plant scaRNAs. Upon alteration of the CAB boxes, mutant scaRNAs accumulate in the nucleolus. In turn, authentic snoRNAs can be targeted into Cajal bodies by addition of exogenous CAB box motifs. Our results indicate that scaRNAs represent an ancient group of small nuclear RNAs which are localized to Cajal bodies by an evolutionarily conserved mechanism.

Box C/D small nucleolar RNA trafficking involves small nucleolar RNP proteins, nucleolar factors and a novel nuclear domain

Nucleolar localization of box C/D small nucleolar (sno) RNAs requires the box C/D motif and, in vertebrates, involves transit through Cajal bodies (CB). We report that in yeast, overexpression of a box C/D reporter leads to a block in the localization pathway with snoRNA accumulation in a specific sub‐nucleolar structure, the nucleolar body (NB). The human survival of motor neuron protein (SMN), a marker of gems/CB, specifically localizes to the NB when expressed in yeast, supporting similarities between these structures. Box C/D snoRNA accumulation in the NB was decreased by mutation of Srp40 and increased by mutation of Nsr1p, two related nucleolar proteins that are homologous to human Nopp140 and nucleolin, respectively. Box C/D snoRNAs also failed to accumulate in the NB, and became delocalized to the nucleoplasm, upon depletion of any of the core snoRNP proteins, Nop1p/fibrillarin, Snu13p, Nop56p and Nop5p/Nop58p. We conclude that snoRNP assembly occurs either in the nucleoplasm, or during transit of snoRNAs through the NB, followed by routing of the complete snoRNP to functional sites of ribosome synthesis.

Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments

Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments

The human cap-binding complex is functionally connected to the nuclear RNA exosome

Nuclear processing and quality control of eukaryotic RNA is mediated by the RNA exosome, which is regulated by accessory factors. However, the mechanism of exosome recruitment to its ribonucleoprotein (RNP) targets remains poorly understood. Here we report a physical link between the human exosome and the cap-binding complex (CBC). The CBC associates with the ARS2 protein to form CBC–ARS2 (CBCA) and then further connects, together with the ZC3H18 protein, to the nuclear exosome targeting (NEXT) complex, thus forming CBC–NEXT (CBCN). RNA immunoprecipitation using CBCN factors as well as the analysis of combinatorial depletion of CBCN and exosome components underscore the functional relevance of CBC-exosome bridging at the level of target RNA. Specifically, CBCA suppresses read-through products of several RNA families by promoting their transcriptional termination. We suggest that the RNP 5′ cap links transcription termination to exosomal RNA degradation through CBCN.

Interaction between the small-nuclear-RNA cap hypermethylase and the spinal muscular atrophy protein, survival of motor neuron.

The biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) requires the cytoplasmic assembly of the Sm‐core complex, followed by the hypermethylation of the small nuclear RNA (snRNA) 5′ cap. Both the Sm‐core complex and the snRNA trimethylguanosine cap are required for the efficient nuclear import of snRNPs. Here, we show that trimethylguanosine synthase 1 (TGS1), the human homologue of the yeast snRNA cap hypermethylase, interacts directly with the survival of motor neuron (SMN) protein. Both proteins are similarly distributed, localizing in the cytoplasm and in nuclear Cajal bodies. The interaction between TGS1 and SMN is disrupted by a mutation in SMN that mimics the predominant isoform of the protein that is expressed in patients with the neurodegenerative disease, spinal muscular atrophy. These data indicate that, in addition to its function in cytoplasmic Sm‐core assembly, the SMN protein also functions in the recruitment of the snRNA cap hypermethylase.