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 m2,2,7G-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

The divergent eukaryote Trichomonas vaginalis has an m7G cap methyltransferase capable of a single N2 methylation

Eukaryotic RNAs typically contain 5′ cap structures that have been primarily studied in yeast and metazoa. The only known RNA cap structure in unicellular protists is the unusual Cap4 on Trypanosoma brucei mRNAs. We have found that T. vaginalis mRNAs are protected by a 5′ cap structure, however, contrary to that typical for eukaryotes, T. vaginalis spliceosomal snRNAs lack a cap and may contain 5′ monophophates. The distinctive 2,2,7-trimethylguanosine (TMG) cap structure usually found on snRNAs and snoRNAs is produced by hypermethylation of an m7G cap catalyzed by the enzyme trimethylguanosine synthase (Tgs). Here, we biochemically characterize the single T. vaginalis Tgs (TvTgs) encoded in its genome and demonstrate that TvTgs exhibits substrate specificity and amino acid requirements typical of an RNA cap-specific, m7G-dependent N2 methyltransferase. However, recombinant TvTgs is capable of catalysing only a single round of N2 methylation forming a 2,7-dimethylguanosine cap (DMG) as observed previously for Giardia lamblia. In contrast, recombinant Entamoeba histolytica and Trypanosoma brucei Tgs are capable of catalysing the formation of a TMG cap. These data suggest the presence of RNAs with a distinctive 5′ DMG cap in Trichomonas and Giardia lineages that are absent in other protist lineages

Isoforms of U1-70k control subunit dynamics in the human spliceosomal U1 snRNP

Most human protein-encoding genes contain multiple exons that are spliced together, frequently in alternative arrangements, by the spliceosome. It is established that U1 snRNP is an essential component of the spliceosome, in human consisting of RNA and ten proteins, several of which are post- translationally modified and exist as multiple isoforms. Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle. Using mass spectrometry we investigate the composition and dynamics of the native human U1 snRNP and compare native and recombinant complexes to isolate the effects of various subunits and isoforms on the overall stability. Our data reveal differential incorporation of four protein isoforms and dynamic interactions of subunits U1-A, U1-C and Sm-B/B’. Results also show that unstructured post- ranslationally modified C-terminal tails are responsible for the dynamics of Sm-B/B’ and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo. These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.This work was funded by: BBSRC (OVM), BBSRC and EPSRC (HH and NM), EU Prospects (HH), European Science Foundation (NM), the Royal Society (CVR), and fellowship from JSPS and HFSP (YM and DAPK respectively)

Structure–function analysis and genetic interactions of the yeast branchpoint binding protein Msl5

Saccharomyces cerevisiae Msl5 (branchpoint binding protein) orchestrates spliceosome assembly by binding the branchpoint sequence 5′-UACUAAC and establishing cross intron-bridging interactions with other components of the splicing machinery. Reciprocal tandem affinity purifications verify that Msl5 exists in vivo as a heterodimer with Mud2 and that the Msl5–Mud2 complex is associated with the U1 snRNP. By gauging the ability of mutants of Msl5 to complement msl5Δ, we find that the Mud2-binding (amino acids 35–54) and putative Prp40-binding (PPxY100) elements of the Msl5 N-terminal domain are inessential, as are the C-terminal proline-rich domain (amino acids 382–476) and two zinc-binding CxxCxxxxHxxxxC motifs (amino acids 273–286 and 299–312). A subset of conserved branchpoint RNA-binding amino acids in the central KH-QUA2 domain (amino acids 146–269) are essential pairwise (Ile198–Arg190; Leu256–Leu259) or in trios (Leu169–Arg172–Leu176), whereas other pairs of RNA-binding residues are dispensable. We used our collection of viable Msl5 mutants to interrogate synthetic genetic interactions, in cis between the inessential structural elements of the Msl5 polypeptide and in trans between Msl5 and yeast splicing factors (Mud2, Nam8 and Tgs1) that are optional for vegetative growth. The results suggest a network of important but functionally buffered protein–protein and protein–RNA interactions between the Mud2–Msl5 complex at the branchpoint and the U1 snRNP at the 5′ splice site

A synthetic snRNA m3G-CAP enhances nuclear delivery of exogenous proteins and nucleic acids

Accessing the nucleus through the surrounding membrane poses one of the major obstacles for therapeutic molecules large enough to be excluded due to nuclear pore size limits. In some therapeutic applications the large size of some nucleic acids, like plasmid DNA, hampers their access to the nuclear compartment. However, also for small oligonucleotides, achieving higher nuclear concentrations could be of great benefit. We report on the synthesis and possible applications of a natural RNA 5′-end nuclear localization signal composed of a 2,2,7-trimethylguanosine cap (m3G-CAP). The cap is found in the small nuclear RNAs that are constitutive part of the small nuclear ribonucleoprotein complexes involved in nuclear splicing. We demonstrate the use of the m3G signal as an adaptor that can be attached to different oligonucleotides, thereby conferring nuclear targeting capabilities with capacity to transport large-size cargo molecules. The synthetic capping of oligos interfering with splicing may have immediate clinical applications

Mapping the binding site of snurportin 1 on native U1 snRNP by cross-linking and mass spectrometry

Mass spectrometry allows the elucidation of molecular details of the interaction domains of the individual components in macromolecular complexes subsequent to cross-linking of the individual components. Here, we applied chemical and UV cross-linking combined with tandem mass-spectrometric analysis to identify contact sites of the nuclear import adaptor snurportin 1 to the small ribonucleoprotein particle U1 snRNP in addition to the known interaction of m3G cap and snurportin 1. We were able to define previously unknown sites of protein–protein and protein–RNA interactions on the molecular level within U1 snRNP. We show that snurportin 1 interacts with its central m3G-cap-binding domain with Sm proteins and with its extreme C-terminus with stem-loop III of U1 snRNA. The crosslinking data support the idea of a larger interaction area between snurportin 1 and U snRNPs and the contact sites identified prove useful for modeling the spatial arrangement of snurportin 1 domains when bound to U1 snRNP. Moreover, this suggests a functional nuclear import complex that assembles around the m3G cap and the Sm proteins only when the Sm proteins are bound and arranged in the proper orientation to the cognate Sm site in U snRNA

Mapping targets for small nucleolar RNAs in yeast

Background: Recent analyses implicate changes in the expression of the box C/D class of small nucleolar RNAs (snoRNAs) in several human diseases. Methods: Here we report the identification of potential novel RNA targets for box C/D snoRNAs in budding yeast, using the approach of UV crosslinking and sequencing of hybrids (CLASH) with the snoRNP proteins Nop1, Nop56 and Nop58. We also developed a bioinformatics approach to filter snoRNA-target interactions for bona fide methylation guide interactions. Results: We recovered 241,420 hybrids, out of which 190,597 were classed as reproducible, high energy hybrids. As expected, the majority of snoRNA interactions were with the ribosomal RNAs (rRNAs). Following filtering, 117,047 reproducible hybrids included 51 of the 55 reported rRNA methylation sites. The majority of interactions at methylation sites were predicted to guide methylation. However, competing, potentially regulatory, binding was also identified. In marked contrast, following CLASH performed with the RNA helicase Mtr4 only 7% of snoRNA-rRNA interactions recovered were predicted to guide methylation. We propose that Mtr4 functions in dissociating inappropriate snoRNA-target interactions. Numerous snoRNA-snoRNA interactions were recovered, indicating potential cross regulation. The snoRNAs snR4 and snR45 were recently implicated in site-directed rRNA acetylation, and hybrids were identified adjacent to the acetylation sites. We also identified 1,368 reproducible snoRNA-mRNA interactions, representing 448 sites of interaction involving 39 snoRNAs and 382 mRNAs. Depletion of the snoRNAs U3, U14 or snR4 each altered the levels of numerous mRNAs. Targets identified by CLASH were over-represented among these species, but causality has yet to be established. Conclusions: Systematic mapping of snoRNA-target binding provides a catalogue of high-confidence binding sites and indicates numerous potential regulatory interactions

A role for the Cajal-body-associated SUMO isopeptidase USPL1 in snRNA transcription mediated by RNA polymerase II

Cajal bodies are nuclear structures that are involved in biogenesis of snRNPs and snoRNPs, maintenance of telomeres and processing of histone mRNA. Recently, the SUMO isopeptidase USPL1 was identified as a component of Cajal bodies that is essential for cellular growth and Cajal body integrity. However, a cellular function for USPL1 is so far unknown. Here, we use RNAi-mediated knockdown in human cells in combination with biochemical and fluorescence microscopy approaches to investigate the function of USPL1 and its link to Cajal bodies. We demonstrate that levels of snRNAs transcribed by RNA polymerase (RNAP) II are reduced upon knockdown of USPL1 and that downstream processes such as snRNP assembly and pre-mRNA splicing are compromised. Importantly, we find that USPL1 associates directly with U snRNA loci and that it interacts and colocalises with components of the Little Elongation Complex, which is involved in RNAPII-mediated snRNA transcription. Thus, our data indicate that USPL1 plays a key role in RNAPII-mediated snRNA transcription

WRAP53 Is Essential for Cajal Body Formation and for Targeting the Survival of Motor Neuron Complex to Cajal Bodies

The WRAP53 protein regulates the formation and maintenance of Cajal bodies (nuclear sub-organelles), as well as directs the recruitment of nuclear factors to Cajal bodies

Identification of Gemin5 as a Novel 7-Methylguanosine Cap-Binding Protein

A unique attribute of RNA molecules synthesized by RNA polymerase II is the presence of a 7-methylguanosine (m(7)G) cap structure added co-transcriptionally to the 5' end. Through its association with trans-acting effector proteins, the m(7)G cap participates in multiple aspects of RNA metabolism including localization, translation and decay. However, at present relatively few eukaryotic proteins have been identified as factors capable of direct association with m(7)G.Employing an unbiased proteomic approach, we identified gemin5, a component of the survival of motor neuron (SMN) complex, as a factor capable of direct and specific interaction with the m(7)G cap. Gemin5 was readily purified by cap-affinity chromatography in contrast to other SMN complex proteins. Investigating the underlying basis for this observation, we found that purified gemin5 associates with m(7)G-linked sepharose in the absence of detectable eIF4E, and specifically crosslinks to radiolabeled cap structure after UV irradiation. Deletion analysis revealed that an intact set of WD repeat domains located in the N-terminal half of gemin5 are required for cap-binding. Moreover, using structural modeling and site-directed mutagenesis, we identified two proximal aromatic residues located within the WD repeat region that significantly impact m(7)G association.This study rigorously identifies gemin5 as a novel cap-binding protein and describes an unprecedented role for WD repeat domains in m(7)G recognition. The findings presented here will facilitate understanding of gemin5's role in the metabolism of non-coding snRNAs and perhaps other RNA pol II transcripts