The small nucle(ol)ar RNA cap trimethyltransferase is required for ribosome synthesis and intact nucleolar morphology

Nucleolar morphogenesis is a poorly defined process. Here we report that the Saccharomyces cerevisiae nucleolar trimethyl guanosine synthase I (Tgs1p), which specifically selects the m(7)G cap structure of snRNAs and snoRNAs for m(2,2,7)G conversion, is required not only for efficient pre-mRNA splicing but also for pre-rRNA processing and small ribosomal subunit synthesis. Mutational analysis indicates that the requirement for Tgs1p in pre-mRNA splicing, but not its involvement in ribosome synthesis, is dependent upon its function in cap trimethylation. In addition, we report that cells lacking Tgs1p showed a striking and unexpected loss of nucleolar structural organization. Tgs1p is not a core component of the snoRNP proteins; however, in vitro, the protein interacts with the KKD/E domain present at the carboxyl-terminal ends of several snoRNP proteins. Strains expressing versions of the snoRNPs lacking the KKD/E domain were also defective for nucleolar morphology and showed a loss of nucleolar compaction. We propose that the transient and functional interactions of Tgs1p with the abundant snoRNPs, through presumed interactions with the KKD/E domain of the snoRNP proteins, contribute substantially to the coalescence of nucleolar components. This conclusion is compatible with a model of self-organization for nucleolar assembly.     

CRM1 controls the composition of nucleoplasmic pre-snoRNA complexes to licence them for nucleolar transport

Transport of C/D snoRNPs to nucleoli involves nuclear export factors. In particular, CRM1 binds nascent snoRNPs, but its precise role remains unknown. We show here that both CRM1 and nucleocytoplasmic trafficking are required to transport snoRNPs to nucleoli, but the snoRNPs do not transit through the cytoplasm. Instead, CRM1 controls the composition of nucleoplasmic pre-snoRNP complexes. We observed that Tgs1 long form (Tgs1 LF), the long isoform of the cap hypermethylase, contains a leucine-rich nuclear export signal, shuttles in a CRM1-dependent manner, and binds to the nucleolar localization signal (NoLS) of the core snoRNP protein Nop58. In vitro data indicate that CRM1 binds Tgs1 LF and promotes its dissociation from Nop58 NoLS, and immunoprecipitation experiments from cells indicate that the association of Tgs1 LF with snoRNPs increases upon CRM1 inhibition. Thus, CRM1 appears to promote nucleolar transport of snoRNPs by removing Tgs1 LF from the Nop58 NoLS. Microarray/IP data show that this occurs on most snoRNPs, from both C/D and H/ACA families, and on the telomerase RNA. Hence, CRM1 provides a general molecular link between nuclear events and nucleocytoplasmic trafficking.     

Composition of yeast snRNPs and snoRNPs in the absence of trimethylguanosine caps reveals nuclear cap binding protein as a gained U1 component implicated in the cold-sensitivity of tgs1Δ cells

Small nuclear and nucleolar RNAs that program pre-mRNA splicing and rRNA processing have a signature 5'-trimethylguanosine (TMG) cap. Whereas the mechanism of TMG synthesis by Tgs1 methyltransferase has been elucidated, we know little about whether or how RNP biogenesis, structure and function are perturbed when TMG caps are missing. Here, we analyzed RNPs isolated by tandem-affinity purification from TGS1 and tgs1Δ yeast strains. The protein and U-RNA contents of total SmB-containing RNPs were similar. Finer analysis revealed stoichiometric association of the nuclear cap-binding protein (CBP) subunits Sto1 and Cbc2 with otherwise intact Mud1- and Nam8-containing U1 snRNPs from tgs1Δ cells. CBP was not comparably enriched in Lea1-containing U2 snRNPs from tgs1Δ cells. Moreover, CBP was not associated with mature Nop58-containing C/D snoRNPs or mature Cbf5- and Gar1-containing H/ACA snoRNPs from tgs1Δ cells. The protein composition and association of C/D snoRNPs with the small subunit (SSU) processosome were not grossly affected by absence of TMG caps, nor was the composition of H/ACA snoRNPs. The cold-sensitive (cs) growth defect of tgs1Δ yeast cells could be suppressed by mutating the cap-binding pocket of Cbc2, suggesting that ectopic CBP binding to the exposed U1 m(7)G cap in tgs1Δ cells (not lack of TMG caps per se) underlies the cs phenotype.     

Characterization of a short isoform of human Tgs1 hypermethylase associating with small nucleolar ribonucleoprotein core proteins and produced by limited proteolytic processing

Tgs1 is the hypermethylase responsible for m(3)G cap formation of U small nuclear RNAs (U snRNAs) and small nucleolar RNAs (snoRNAs). In vertebrates, hypermethylation of snRNAs occurs in the cytoplasm, whereas this process takes place in the nucleus for snoRNAs. Accordingly, the hypermethylase is found in both compartments with a diffuse localization in the cytoplasm and a concentration in Cajal bodies in the nucleoplasm. In this study, we report that the Tgs1 hypermethylase exists as two species, a full-length cytoplasmic isoform and a shorter nuclear isoform of 65-70 kDa. The short isoform exhibits methyltransferase activity and associates with components of box C/D and H/ACA snoRNPs, pointing to a role of this isoform in hypermethylation of snoRNAs. We also show that production of the short Tgs1 isoform is inhibited by MG132, suggesting that it results from proteasomal limited processing of the full-length Tgs1 protein. Together, our results suggest that proteasome maturation constitutes a mechanism regulating Tgs1 function by generating Tgs1 species with different substrate specificities, subcellular localizations, and functions.     

Biochemical and genetic analysis of RNA cap guanine-N2 methyltransferases from Giardia lamblia and Schizosaccharomyces pombe

RNA cap guanine-N2 methyltransferases such as Schizosaccharomyces pombe Tgs1 and Giardia lamblia Tgs2 catalyze methylation of the exocyclic N2 amine of 7-methylguanosine. Here we performed a mutational analysis of Giardia Tgs2, entailing an alanine scan of 17 residues within the minimal active domain. Alanine substitutions at Phe18, Thr40, Asp76, Asn103 and Asp140 reduced methyltransferase specific activity to <3% of wild-type Tgs2, thereby defining these residues as essential. Alanines at Pro142, Tyr148 and Pro185 reduced activity to 7–12% of wild-type. Structure–activity relationships at Phe18, Thr40, Asp76, Asn103, Asp140 and Tyr148, and at three other essential residues defined previously (Asp68, Glu91 and Trp143) were gleaned by testing the effects of 18 conservative substitutions. Our results engender a provisional map of the Tgs2 active site, which we discuss in light of crystal structures of related methyltransferases. A genetic analysis of S. pombe Tgs1 showed that it is nonessential. An S. pombe tgs1Δ strain grows normally, notwithstanding the absence of 2,2,7-trimethylguanosine caps on its U1, U2, U4 and U5 snRNAs. However, we find that S. pombe requires cap guanine-N7 methylation catalyzed by the enzyme Pcm1. Deletion of the pcm1⁺ gene was lethal, as were missense mutations in the Pcm1 active site. Thus, whereas m⁷G caps are essential in both S. pombe and S. cerevisiae, m^2,2,7G caps are not.     

Cap analog substrates reveal three clades of cap guanine-N2 methyltransferases with distinct methyl acceptor specificities

The Tgs proteins are structurally homologous AdoMet-dependent eukaryal enzymes that methylate the N2 atom of 7-methyl guanosine nucleotides. They have an imputed role in the synthesis of the 2,2,7-trimethylguanosine (TMG) RNA cap. Here we exploit a collection of cap-like substrates to probe the repertoire of three exemplary Tgs enzymes, from mammalian, protozoan, and viral sources, respectively. We find that human Tgs (hTgs1) is a bona fide TMG synthase adept at two separable transmethylation steps: (1) conversion of m⁷G to m^2,7G, and (2) conversion of m^2,7G to m^2,2,7G. hTgs1 is unable to methylate G or m²G, signifying that both steps require an m⁷G cap. hTgs1 utilizes a broad range of m⁷G nucleotides, including mono-, di-, tri-, and tetraphosphate derivatives as well as cap dinucleotides with triphosphate or tetraphosphate bridges. In contrast, Giardia lamblia Tgs (GlaTgs2) exemplifies a different clade of guanine-N2 methyltransferase that synthesizes only a dimethylguanosine (DMG) cap structure and cannot per se convert DMG to TMG under any conditions tested. Methylation of benzyl⁷G and ethyl⁷G nucleotides by hTgs1 and GlaTgs2 underscored the importance of guanine N7 alkylation in providing a key π-cation interaction in the methyl acceptor site. Mimivirus Tgs (MimiTgs) shares with the Giardia homolog the ability to catalyze only a single round of methyl addition at guanine-N2, but is distinguished by its capacity for guanine-N2 methylation in the absence of prior N7 methylation. The relaxed cap specificity of MimiTgs is revealed at alkaline pH. Our findings highlight both stark and subtle differences in acceptor specificity and reaction outcomes among Tgs family members.     

Fibrillarin and Nop56 interact before being co-assembled in box C/D snoRNPs

Small nucleolar RNAs play crucial roles in ribosome biogenesis. They guide folding, site-specific nucleotide modifications and participate in cleavage of precursor ribosomal RNAs. To better understand how the biogenesis of the box C/D small nucleolar RNPs (snoRNPs) occur in a cellular context, we used a new approach based on the possibility of relocalizing a given nuclear complex by adding an affinity tag for B23 to one component of this complex. We selectively delocalized each core box C/D protein, namely 15.5kD, Nop56, Nop58 and fibrillarin, and analyzed the effect of such changes on other components of the box C/D snoRNPs. We show that modifying the localization and the mobility of core box C/D proteins impairs their association with box C/D snoRNPs. In addition, we demonstrate that fibrillarin and Nop56 directly interact in vivo. This interaction, indispensable for the association of both proteins with the box C/D snoRNPs, does not involve the glycine- and arginine-rich domain or the RNA-binding domain but the alpha-helix domain of fibrillarin. In addition, no RNA seems required to maintain fibrillarin-Nop56 interaction.     

Nucleolar trafficking of nucleostemin family proteins: common versus protein-specific mechanisms

The nucleolus has begun to emerge as a subnuclear organelle capable of modulating the activities of nuclear proteins in a dynamic and cell type-dependent manner. It remains unclear whether one can extrapolate a rule that predicts the nucleolar localization of multiple proteins based on protein sequence. Here, we address this issue by determining the shared and unique mechanisms that regulate the static and dynamic distributions of a family of nucleolar GTP-binding proteins, consisting of nucleostemin (NS), guanine nucleotide binding protein-like 3 (GNL3L), and Ngp1. The nucleolar residence of GNL3L is short and primarily controlled by its basic-coiled-coil domain, whereas the nucleolar residence of NS and Ngp1 is long and requires the basic and the GTP-binding domains, the latter of which functions as a retention signal. All three proteins contain a nucleoplasmic localization signal (NpLS) that prevents their nucleolar accumulation. Unlike that of the basic domain, the activity of NpLS is dynamically controlled by the GTP-binding domain. The nucleolar retention and the NpLS-regulating functions of the G domain involve specific residues that cannot be predicted by overall protein homology. This work reveals common and protein-specific mechanisms underlying the nucleolar movement of NS family proteins.     

Structural basis for m7G-cap hypermethylation of small nuclear,small nucleolar and telomerase RNA by the dimethyltransferase TGS1

The 5′-cap of spliceosomal small nuclear RNAs, some small nucleolar RNAs and of telomerase RNA was found to be hypermethylated in vivo. The Trimethylguanosine Synthase 1 (TGS1) mediates this conversion of the 7-methylguanosine-cap to the 2,2,7-trimethylguanosine (m₃G)-cap during maturation of the RNPs. For mammalian UsnRNAs the generated m^2,2,7G-cap is one part of a bipartite import signal mediating the transport of the UsnRNP-core complex into the nucleus. In order to understand the structural organization of human TGS1 as well as substrate binding and recognition we solved the crystal structure of the active TGS1 methyltransferase domain containing both, the minimal substrate m⁷GTP and the reaction product S-adenosyl-l-homocysteine (AdoHcy). The methyltransferase of human TGS1 harbors the canonical class 1 methyltransferase fold as well as an unique N-terminal, α-helical domain of 40 amino acids, which is essential for m⁷G-cap binding and catalysis. The crystal structure of the substrate bound methyltransferase domain as well as mutagenesis studies provide insight into the catalytic mechanism of TGS1.     

Nucleophosmin mutations alter its nucleolar localization by impairing G-quadruplex binding at ribosomal DNA

Nucleophosmin (NPM1) is an abundant nucleolar protein implicated in ribosome maturation and export, centrosome duplication and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia. Mutations at the C-terminal domain led to variant proteins that aberrantly and stably translocate to the cytoplasm. We have previously shown that NPM1 C-terminal domain binds with high affinity G-quadruplex DNA. Here, we investigate the structural determinants of NPM1 nucleolar localization. We show that NPM1 interacts with several G-quadruplex regions found in ribosomal DNA, both in vitro and in vivo. Furthermore, the most common leukemic NPM1 variant completely loses this activity. This is the consequence of G-quadruplex–binding domain destabilization, as mutations aimed at refolding the leukemic variant also result in rescuing the G-quadruplex–binding activity and nucleolar localization. Finally, we show that treatment of cells with a G-quadruplex selective ligand results in wild-type NPM1 dislocation from nucleoli into nucleoplasm. In conclusion, this work establishes a direct correlation between NPM1 G-quadruplex binding at rDNA and its nucleolar localization, which is impaired in the acute myeloid leukemia-associated protein variants.     

The extreme N-terminal domain of a hordeivirus TGB1 movement protein mediates its localization to the nucleolus and interaction with fibrillarin

The hordeiviral movement protein encoded by the first gene of the triple gene block (TGBp1) of Poa semilatent virus (PSLV), interacts with viral genomic RNAs to form RNP particles which are considered to be a form of viral genome capable of cell-to-cell and long-distance transport in infected plants. The PSLV TGBp1 contains a C-terminal NTPase/helicase domain (HELD) and an N-terminal extension region consisting of two structurally and functionally distinct domains: an extreme N-terminal domain (NTD) and an internal domain (ID). This study demonstrates that transient expression of TGBp1 fused to GFP in Nicotiana benthamiana leaves results in faint but obvious fluorescence in the nucleolus in addition to cytosolic distribution. Mutagenesis of the basic amino acids inside the NTD clusters A ¹¹⁶KSKRKKKNKK¹²⁵ and B ¹⁷⁵KKATKKESKKQTK¹⁸⁷ reveals that these clusters are indispensable for nuclear and nucleolar targeting of PSLV TGBp1 and may contain nuclear and nucleolar localization signals or their elements. The PSLV TGBp1 is able to bind to fibrillarin, the major nucleolar protein (AtFib2 from Arabidopsis thaliana) in vitro. This protein–protein interaction occurs between the glycine-arginine-rich (GAR) domain of fibrillarin and the first 82 amino acid residues of TGBp1. The interaction of TGBp1 with fibrillarin is also visualized in vivo by bimolecular fluorescence complementation (BiFC) during co-expression of TGBp1 or its deletion mutants, and fibrillarin as fusions to different halves of YFP in N. benthamiana plants. The sites responsible for nuclear/nucleolar localization and fibrillarin binding, have been located within the intrinsically disordered TGBp1 NTD. These data could suggest that specific functions of hordeivirus TGBp1 may depend on its interaction with nucleolar components.     

A novel lysine-rich domain and GTP binding motifs regulate the nucleolar retention of human guanine nucleotide binding protein, GNL3L

A variety of G-proteins and GTPases are known to be involved in nucleolar function. We describe here a new evolutionarily conserved putative human GTPase, guanine nucleotide binding protein-like 3-like (GNL3L). Genes encoding proteins related to GNL3L are present in bacteria and yeast to metazoa and suggests its critical role in development. Conserved domain search analysis revealed that the GNL3L contains a circularly permuted G-motif described by a G5-G4-G1-G2-G3 pattern similar to the HSR1/MMR1 GTP-binding protein subfamily. Highly conserved and critical residues were identified from a three-dimensional structural model obtained for GNL3L using the crystal structure of an Ylqf GTPase from Bacillus subtilis. We demonstrate here that GNL3L is transported into the nucleolus by a novel lysine-rich nucleolar localization signal (NoLS) residing within 1-50 amino acid residues. NoLS identified here is necessary and sufficient to target the heterologous proteins to the nucleolus. We show for the first time that the lysine-rich targeting signal interacts with the nuclear transport receptor, importin-beta and transports GNL3L into the nucleolus. Interestingly, depletion of intracellular GTP blocks GNL3L accumulation into the nucleolar compartment. Furthermore, mutations within the G-domains alter the GTP binding ability of GNL3L and abrogate wild-type nucleolar retention even in the presence of functional NoLS, suggesting that the efficient nucleolar retention of GNL3L involves activities of both basic NoLS and GTP-binding domains. Collectively, these data suggest that GNL3L is composed of distinct modules, each of which plays a specific role in molecular interactions for its nucleolar retention and subsequent function(s) within the nucleolus.     

RNA:(guanine-N2) methyltransferases RsmC/RsmD and their homologs revisited – bioinformatic analysis and prediction of the active site based on the uncharacterized Mj0882 protein structure

Background  

Escherichia coli guanine-N2 (m²G) methyltransferases (MTases) RsmC and RsmD modify nucleosides G1207 and G966 of 16S rRNA. They possess a common MTase domain in the C-terminus and a variable region in the N-terminus. Their C-terminal domain is related to the YbiN family of hypothetical MTases, but nothing is known about the structure or function of the N-terminal domain.     

Snurportin1, an m3G-cap-specific nuclear import receptor with a novel domain structure.

The nuclear import of the spliceosomal snRNPs U1, U2, U4 and U5, is dependent on the presence of a complex nuclear localization signal (NLS). The latter is composed of the 5'-2,2,7-terminal trimethylguanosine (m3G) cap structure of the U snRNA and the Sm core domain. Here, we describe the isolation and cDNA cloning of a 45 kDa protein, termed snurportin1, which interacts specifically with m3G-cap but not m7G-cap structures. Snurportin1 enhances the m3G-capdependent nuclear import of U snRNPs in both Xenopus laevis oocytes and digitonin-permeabilized HeLa cells, demonstrating that it functions as an snRNP-specific nuclear import receptor. Interestingly, solely the m3G-cap and not the Sm core NLS appears to be recognized by snurportin1, indicating that at least two distinct import receptors interact with the complex snRNP NLS. Snurportin1 represents a novel nuclear import receptor which contains an N-terminal importin beta binding (IBB) domain, essential for function, and a C-terminal m3G-cap-binding region with no structural similarity to the arm repeat domain of importin alpha.     

The Role of the Methyltransferase Domain of Bifunctional Restriction Enzyme RM.BpuSI in Cleavage Activity

Restriction enzyme (REase) RM.BpuSI can be described as a Type IIS/C/G REase for its cleavage site outside of the recognition sequence (Type IIS), bifunctional polypeptide possessing both methyltransferase (MTase) and endonuclease activities (Type IIC) and endonuclease activity stimulated by S-adenosyl-L-methionine (SAM) (Type IIG). The stimulatory effect of SAM on cleavage activity presents a major paradox: a co-factor of the MTase activity that renders the substrate unsusceptible to cleavage enhances the cleavage activity. Here we show that the RM.BpuSI MTase activity modifies both cleavage substrate and product only when they are unmethylated. The MTase activity is, however, much lower than that of M1.BpuSI and is thought not to be the major MTase for host DNA protection. SAM and sinefungin (SIN) increase the V_max of the RM.BpuSI cleavage activity with a proportional change in K_m, suggesting the presence of an energetically more favorable pathway is taken. We further showed that RM.BpuSI undergoes substantial conformational changes in the presence of Ca²⁺, SIN, cleavage substrate and/or product. Distinct conformers are inferred as the pre-cleavage/cleavage state (in the presence of Ca²⁺, substrate or both) and MTase state (in the presence of SIN and substrate, SIN and product or product alone). Interestingly, RM.BpuSI adopts a unique conformation when only SIN is present. This SIN-bound state is inferred as a branch point for cleavage and MTase activity and an intermediate to an energetically favorable pathway for cleavage, probably through increasing the binding affinity of the substrate to the enzyme under cleavage conditions. Mutation of a SAM-binding residue resulted in altered conformational changes in the presence of substrate or Ca²⁺ and eliminated cleavage activity. The present study underscores the role of the MTase domain as facilitator of efficient cleavage activity for RM.BpuSI.     

Cooperative signals governing ARF-mdm2 interaction and nucleolar localization of the complex

The ARF tumor suppressor protein stabilizes p53 by antagonizing its negative regulator, Mdm2 (Hdm2 in humans). Both mouse p19(ARF) and human p14(ARF) bind to the central region of Mdm2 (residues 210 to 304), a segment that does not overlap with its N-terminal p53-binding domain, nuclear import or export signals, or C-terminal RING domain required for Mdm2 E3 ubiquitin ligase activity. The N-terminal 37 amino acids of mouse p19(ARF) are necessary and sufficient for binding to Mdm2, localization of Mdm2 to nucleoli, and p53-dependent cell cycle arrest. Although a nucleolar localization signal (NrLS) maps within a different segment (residues 82 to 101) of the human p14(ARF) protein, binding to Mdm2 and nucleolar import of ARF-Mdm2 complexes are both required for cell cycle arrest induced by either the mouse or human ARF proteins. Because many codons of mouse ARF mRNA are not recognized by the most abundant bacterial tRNAs, we synthesized ARF minigenes containing preferred bacterial codons. Using bacterially produced ARF polypeptides and chemically synthesized peptides conjugated to Sepharose, residues 1 to 14 and 26 to 37 of mouse p19(ARF) were found to interact independently and cooperatively with Mdm2, while residues 15 to 25 were dispensable for binding. Paradoxically, residues 26 to 37 of mouse p19(ARF) are also essential for ARF nucleolar localization in the absence of Mdm2. However, the mobilization of the p19(ARF)-Mdm2 complex into nucleoli also requires a cryptic NrLS within the Mdm2 C-terminal RING domain. The Mdm2 NrLS is unmasked upon ARF binding, and its deletion prevents import of the ARF-Mdm2 complex into nucleoli. Collectively, the results suggest that ARF binding to Mdm2 induces a conformational change that facilitates nucleolar import of the ARF-Mdm2 complex and p53-dependent cell cycle arrest. Hence, the ARF-Mdm2 interaction can be viewed as bidirectional, with each protein being capable of regulating the subnuclear localization of the other.     

Crystal structure of tRNA m1G9 methyltransferase Trm10: insight into the catalytic mechanism and recognition of tRNA substrate

Transfer RNA (tRNA) methylation is necessary for the proper biological function of tRNA. The N¹ methylation of guanine at Position 9 (m¹G9) of tRNA, which is widely identified in eukaryotes and archaea, was found to be catalyzed by the Trm10 family of methyltransferases (MTases). Here, we report the first crystal structures of the tRNA MTase spTrm10 from Schizosaccharomyces pombe in the presence and absence of its methyl donor product S-adenosyl-homocysteine (SAH) and its ortholog scTrm10 from Saccharomyces cerevisiae in complex with SAH. Our crystal structures indicated that the MTase domain (the catalytic domain) of the Trm10 family displays a typical SpoU-TrmD (SPOUT) fold. Furthermore, small angle X-ray scattering analysis reveals that Trm10 behaves as a monomer in solution, whereas other members of the SPOUT superfamily all function as homodimers. We also performed tRNA MTase assays and isothermal titration calorimetry experiments to investigate the catalytic mechanism of Trm10 in vitro. In combination with mutational analysis and electrophoretic mobility shift assays, our results provide insights into the substrate tRNA recognition mechanism of Trm10 family MTases.