FTO controls reversible m6Am RNA methylation during snRNA biogenesis.

Small nuclear RNAs (snRNAs) are core spliceosome components and mediate pre-mRNA splicing. Here we show that snRNAs contain a regulated and reversible nucleotide modification causing them to exist as two different methyl isoforms, m1 and m2, reflecting the methylation state of the adenosine adjacent to the snRNA cap. We find that snRNA biogenesis involves the formation of an initial m1 isoform with a single-methylated adenosine (2'-O-methyladenosine, Am), which is then converted to a dimethylated m2 isoform (N6,2'-O-dimethyladenosine, m6Am). The relative m1 and m2 isoform levels are determined by the RNA demethylase FTO, which selectively demethylates the m2 isoform. We show FTO is inhibited by the oncometabolite D-2-hydroxyglutarate, resulting in increased m2-snRNA levels. Furthermore, cells that exhibit high m2-snRNA levels show altered patterns of alternative splicing. Together, these data reveal that FTO controls a previously unknown central step of snRNA processing involving reversible methylation, and suggest that epitranscriptomic information in snRNA may influence mRNA splicing.

Architectural arrangement of the small nuclear RNA (snRNA)-activating protein complex 190 subunit (SNAP190) on U1 snRNA gene promoter DNA.

Myb repeats ∼52 amino acid residues in length were first characterized in the oncogenic Myb transcription factor, which contains three tandem Myb repeats in its DNA-binding domain. Proteins of this family normally contain either one, two, or three tandem Myb repeats that are involved in protein-DNA interactions. The small nuclear RNA (snRNA)-activating protein complex (SNAPc) is a heterotrimeric transcription factor that is required for expression of small nuclear RNA genes. This complex binds to an essential promoter element, the proximal sequence element, centered ∼50 base pairs upstream of the transcription start site of snRNA genes. SNAP190, the largest subunit of SNAPc, uncharacteristically contains 4.5 tandem Myb repeats. Little is known about the arrangement of the Myb repeats in the SNAPc-DNA complex, and it has not been clear whether all 4.5 Myb repeats contact the DNA. By using a site-specific protein-DNA photo-cross-linking assay, we have now mapped specific nucleotides where each of the Myb repeats of Drosophila melanogaster SNAP190 interacts with a U1 snRNA gene proximal sequence element. The results reveal the topological arrangement of the 4.5 SNAP190 Myb repeats relative to the DNA and to each other when SNAP190 is bound to a U1 promoter as a subunit of SNAPc.

Regulation of human RNA polymerase III transcription by DNMT1 and DNMT3a DNA methyltransferases.

The human small nuclear RNA (snRNA) and small cytoplasmic RNA (scRNA) gene families encode diverse non-coding RNAs that influence cellular growth and division. Many snRNA and scRNA genes are related via their compact and yet powerful promoters that support RNA polymerase III transcription. We have utilized the human U6 snRNA gene family to examine the mechanism for regulated transcription of these potent transcription units. Analysis of nine U6 family members showed enriched CpG density within the promoters of actively transcribed loci relative to inert genes, implying a relationship between gene potency and DNA methylation. Indeed, both pharmacological inhibition of DNA methyltransferase (DNMT) activity and the forced diminution of DNMT-1, DNMT-3a, and DNMT-3b by siRNA targeting resulted in increased U6 levels in asynchronously growing MCF7 adenocarcinoma cells. In vitro transcription assays further showed that template methylation impedes U6 transcription by RNA polymerase III. Both DNMT-1 and DNMT-3a were detected at the U6-1 locus by chromatin immunoprecipitation directly linking these factors to RNA polymerase III regulation. Despite this association, the endogenous U6-1 locus was not substantially methylated in actively growing cells. However, both DNMT occupancy and low frequency methylation were correlated with increased Retinoblastoma tumor suppressor (RB) expression, suggesting that the RB status can influence specific epigenetic marks.

Subunit stoichiometry of the Drosophila melanogaster small nuclear RNA activating protein complex (SNAPc).

Small nuclear RNA activating protein complex (SNAPc) is a multi-subunit transcription factor required for expression of small nuclear RNA genes. This protein binds to a promoter element located approximately 40-65 bp upstream of the transcription start site. In Drosophila melanogaster, DmSNAPc contains three distinct polypeptide subunits: DmSNAP190, DmSNAP50, and DmSNAP43. The subunit stoichiometry in SNAPc complexed with DNA has not been examined. Therefore, the ability of differently tagged but otherwise identical subunits to associate with each other into the same protein-DNA complex was assayed by antibody super-shift analysis. The results reveal that DmSNAPc contains only a single copy of each of the three subunits.

Down-regulation of histone H2B by DNA-dependent protein kinase in response to DNA damage through modulation of octamer transcription factor 1.

Cells respond to double-stranded DNA breaks (DSBs) by pausing cell cycle progression to allow the repair machinery to restore genomic integrity. DNA-dependent protein kinase (DNA-PK), comprising a large catalytic subunit (DNA-PK(cs)) and the Ku antigen regulatory subunit (Ku70/Ku80), is activated in response to DSBs and is required for DNA repair through the nonhomologous end-joining pathway. Here we provide evidence that DNA-PK participates in altering specific gene expression in response to DNA damage by modulating the stability and transcriptional regulatory potential of the essential transcription factor octamer transcription factor 1 (Oct-1). Histone H2B and U2 RNA, whose expression are highly dependent on Oct-1, were strongly decreased in response to ionizing radiation in a DNA-PK-dependent manner, and Oct-1-dependent reporter gene transcription was repressed. Furthermore, Oct-1 phosphorylation in response to ionizing radiation increased in a DNA-PK-dependent manner. Paradoxically, down-regulation of transactivation correlated with the rapid DNA-PK-dependent stabilization of Oct-1. Stabilization of Oct-1 was dependent on the NH(2)-terminal region of Oct-1, which contains a transcriptional activation domain and which was phosphorylated by DNA-PK in vitro. These results suggest a mechanism for the regulation of Oct-1 in response to DNA damage through specific phosphorylation within the NH(2)-terminal transcriptional regulatory domain.

Target discrimination by RNA-binding proteins: role of the ancillary protein U2A' and a critical leucine residue in differentiating the RNA-binding specificity of spliceosomal proteins U1A and U2B".

The spliceosomal proteins U1A and U2B" each use a homologous RRM domain to bind specifically to their respective snRNA targets, U1hpll and U2hpIV, two stem-loops that are similar yet distinct in sequence. Previous studies have shown that binding of U2B" to U2hpIV is facilitated by the ancillary protein U2A', whereas specific binding of U1A to U1hpll requires no cofactor. Here we report that U2A' enables U2B" to distinguish the loop sequence of U2hpIV from that of U1hpll but plays no role in stem sequence discrimination. Although U2A' can also promote heterospecific binding of U1A to U2hpIV, a much higher concentration of the ancillary protein is required due to the approximately 500-fold greater affinity of U2A' for U2B". Additional experiments have identified a single leucine residue in U1A(Leu-44) that is critical for the intrinsic specificity of this protein for the loop sequence of U1 hpll in preference to that of U2hpIV. Our data suggest that most of the difference in RNA-binding specificity between U1A and U2B" can be accounted for by this leucine residue and by the contribution of the ancillary protein U2A' to the specificity of U2B".

Clusters of multiple different small nucleolar RNA genes in plants are expressed as and processed from polycistronic pre-snoRNAs.

Small nucleolar RNAs (snoRNAs) are involved in many aspects of rRNA processing and maturation. In animals and yeast, a large number of snoRNAs are encoded within introns of protein-coding genes. These introns contain only single snoRNA genes and their processing involves exonucleolytic release of the snoRNA from debranched intron lariats. In contrast, some U14 genes in plants are found in small clusters and are expressed polycistronically. An examination of U14 flanking sequences in maize has identified four additional snoRNA genes which are closely linked to the U14 genes. The presence of seven and five snoRNA genes respectively on 2.05 and 0.97 kb maize genomic fragments further emphasizes the novel organization of plant snoRNA genes as clusters of multiple different genes encoding both box C/D and box H/ACA snoRNAs. The plant snoRNA gene clusters are transcribed as a polycistronic pre-snoRNA transcript from an upstream promoter. The lack of exon sequences between the genes suggests that processing of polycistronic pre-snoRNAs involves endonucleolytic activity. Consistent with this, U14 snoRNAs can be processed from both non-intronic and intronic transcripts in tobacco protoplasts such that processing is splicing independent.

A suboptimal 5' splice site is a cis-acting determinant of nuclear export of polyomavirus late mRNAs.

Mouse polyomavirus has been used as a model system to study nucleocytoplasmic transport of mRNA. Three late mRNAs encoding the viral capsid proteins are generated by alternative splicing from common pre-mRNA molecules. mRNAs encoding the virion protein VP2 (mVP2) harbor an unused 5' splice site, and more than half of them remain fully unspliced yet are able to enter the cytoplasm for translation. Examination of the intracellular distribution of late viral mRNAs revealed, however, that mVP2 molecules are exported less efficiently than are mVP1 and mVP3, in which the 5' splice site has been removed by splicing. Point mutations and deletion analyses demonstrated that the efficiency of mVP2 export is inversely correlated with the strength of the 5' splice site and that unused 3' splice sites present in the mRNA have little or no effect on export. These results suggest that the unused 5' splice site is a key player in mVP2 export. Interestingly, mRNAs carrying large deletions but retaining the 5' splice site exhibited a wild-type mVP2 export phenotype, suggesting that there are no other constitutive cis-acting sequences involved in mVP2 export. RNA stability measurements confirmed that the subcellular distribution differences between these mRNAs were not due to differential half-lives between the two cellular compartments. We therefore conclude that the nuclear export of mVP2 is strongly influenced by a suboptimal 5' splice site. Furthermore, results comparing spliced and unspliced forms of mVP2 molecules indicated that the process of splicing does not enhance nuclear export. Since mVP2 and some of its mutant forms can accumulate in the cytoplasm in the absence of splicing, we propose that splicing is not a prerequisite for mRNA export in the polyomavirus system; rather, removal of splicing machinery from mRNAs may be required. The possibility that export of other viral mRNAs can be influenced by suboptimal splicing signals is also discussed.

Effect of anthracycline antitumor antibiotics (adriamycin and nogalamycin) and cycloheximide on the biosynthesis and processing of major UsnRNAs.

In the present study, anthracycline antitumor antibiotics (e.g. adriamycin and nogalamycin), the potent RNA synthesis inhibitors and cycloheximide, the protein synthesis inhibitor, have been used to understand the events of biosynthesis and processing of major UsnRNAs (U1-U6). The anthracyclines inhibit the UsnRNAs biosynthesis (in terms of labelling) differentially in a dose dependent manner. The inhibitory effect of adriamycin and nogalamycin reached plateau at a concentration of 2.5 micrograms/ 10(6) cells/ml and 0.1 microgram/10(6) cells/ml respectively and indicates that nogalamycin is more inhibitory than adriamycin. The inhibition of the UsnRNAs synthesis (in terms of labelling) became maximum within 30 min of incubation and remained unaltered even after 2 h. Thus, it shows that the anthracyclines preferentially inhibit the initiation of the UsnRNA genes' transcription as it has been seen in cases of other large RNAs' synthesis by some other laboratories. The higher inhibitory effect of the anthracyclines on the biosynthesis of U5 and U6 compared to other UsnRNAs indicates the presence of more binding sites on the U5 and U6 snRNA genes. In presence of the anthracyclines, there was high retention of cytoplasmic major pre-UsnRNAs/ UsnRNAs which indicates that the elongation of the UsnRNA synthesis is probably impaired along with initiation; because for the proper processing of the pre-UsnRNAs, formation of the correct secondary structure of that pre-UsnRNA is necessary. Cycloheximide showed some differential effect on the pol II transcribed UsnRNAs (U1-U5) biosynthesis (in terms of labelling) however it has no effect on the pol III transcribed U6 snRNA. It implies that in the pol II transcribed UsnRNAs, some transacting labile factors, either activator or inhibitor, are involved. Whereas, the processing of the UsnRNAs (either pol II or pol III transcribed) was affected more or less in a similar fashion in presence of cycloheximide, indicating the involvement of some transacting labile factors in this event.

Transcriptional pulse-chase analysis reveals a role for a novel snRNP-associated protein in the manufacture of spliceosomal snRNPs.

Vertebrate spliceosomal snRNAs associate with a conserved set of proteins, the Sm proteins, via a conserved RNA sequence, the Sm site. Assembly of this complex is required for the accumulation of stable snRNPs, hypermethylation of the 5' cap structure and nuclear import of the resultant particles. The function of individual core snRNP proteins is poorly understood, in part because of the difficulty of selectively inactivating individual polypeptides in vivo. Using a transcriptional pulse-chase method we have defined for the first time the steps of snRNP biogenesis in Saccharomyces cerevisiae. We describe a novel component of spliceosomal snRNPs, Brr1, which is distinct in sequence from Sm core proteins and yet which shares many of their properties, as well as a genetic interaction with the yeast homolog of Sm D1 core protein. Through a kinetic analysis of snRNP formation in wild-type and brr1 mutant cells we demonstrate specific defects in a subset of steps in the brr1 mutant: newly synthesized snRNAs are destabilized and 3'-end processing is slowed, whereas the cap hypermethylation reaction is unaffected. Notably, the stability of mature particles, as measured by promoter shut-off experiments, is normal in the absence of the Brr1 snRNP protein.

U1 small nuclear RNA chimeric ribozymes with substrate specificity for the Rev pre-mRNA of human immunodeficiency virus.

The in vivo effectiveness of ribozymes strongly depends on the correct choice of the vector molecule. High levels of expression, stability, active conformation, and correct cellular localization are the most important features for a ribozyme vector. We have exploited the utilization of the U1 small nuclear RNA (snRNA) as a vector for specifically targeting a ribozyme into the nucleus. The Rev pre-mRNA of human immunodeficiency virus type 1 was chosen as target for testing the activity of the Ul-ribozyme. The catalytic core of the hammerhead motif, plus the recognition sequences, substituted the stem-loop III of the U1 snRNA. The resulting construct displays efficient cleavage activity in vitro. In addition, in the in vivo system of Xenopus laevis oocytes, the Ul-chimeric ribozyme accumulates in large amounts in the nucleus and produces a considerable reduction of Rev pre-mRNA levels. The Rev-specific ribozyme was also inserted in a derivative of the Ul snRNA mutated in the region of pairing with the 5' splice site, such as to match it with the suboptimal splice junction of the Rev precursor. This construct shows more efficient reduction of Rev pre-mRNA in vivo than the wild-type U1 vector.

The SNAP45 subunit of the small nuclear RNA (snRNA) activating protein complex is required for RNA polymerase II and III snRNA gene transcription and interacts with the TATA box binding protein.

The RNA polymerase II and III small nuclear RNA (snRNA) promoters contain a common basal promoter element, the proximal sequence element (PSE). The PSE binds a multisubunit complex we refer to as the snRNA activating protein complex (SNAPc). At least four polypeptides are visible in purified SNAPc preparations, which migrate with apparent molecular masses of 43, 45, 50, and 190 kDa on SDS/polyacrylamide gels. In addition, purified preparations of SNAPc contain variable amounts of TATA box binding protein (TBP). An important question is whether the PSEs of RNA polymerase II and III snRNA promoters recruit the exact same SNAP complex or slightly different versions of SNAPc, differing, for example, by the presence or absence of a subunit. To address this question, we are isolating cDNAs encoding different subunits of SNAPc. We have previously isolated the cDNA encoding the 43-kDa subunit SNAP43. We now report the isolation of the cDNA that encodes the p45 polypeptide. Antibodies directed against p45 retard the mobility of the SNAPc-PSE complex in an electrophoretic mobility shift assay, indicating that p45 is indeed part of SNAPc. We therefore refer to this protein as SNAP45. SNAP45 is exceptionally proline-rich, interacts strongly with TBP, and, like SNAP43, is required for both RNA polymerase II and III transcription of snRNA genes.

Inefficient spliceosome assembly and abnormal branch site selection in splicing of an HIV-1 transcript in vitro.

Continuous replication of human immunodeficiency virus type I (HIV-1) requires balanced expression of spliced and nonspliced mRNAs in the cytoplasm. This process is regulated post-transcriptionally by the viral-encoded Rev protein. An important prerequisite for Rev responsiveness is the presence of weak splice sites in the viral mRNA. We have investigated the splicing of the second intron of the HIV-1 Tat/Rev transcript in vitro and show that the 3'-splice site region is responsible for the inefficient splicing of the HIV-1 transcript. In contrast, the HIV-1 5'-splice site is highly functional in combination with a heterologous 3'-splice site. Incubation of the HIV-1 transcript in nuclear extract leads to a rapid accumulation of 50 S nonproductive pre-spliceosome complexes. These complexes contain mainly U1 and U2 small nuclear ribonucleoproteins and are formed independently of the presence of the downstream 3'-splice site. The HIV-1 transcripts, which do proceed through the first splicing step, utilize primarily a uridine as the branch acceptor nucleotide. Sequence comparison with other HIV-1 introns suggests that nucleotides other than adenosines are commonly used as branch points in these viruses.

Structure and function of a human transcription factor TFIIIB subunit that is evolutionarily conserved and contains both TFIIB- and high-mobility-group protein 2-related domains.

Transcription factor TFIIIB plays a central role in transcription initiation by RNA polymerase III on genes encoding tRNA, 5S rRNA, and other small structural RNAs. We report the purification of a human TFIIIB-derived complex containing only the TATA-binding polypeptide (TBP) and a 90-kDa subunit (TFIIIB90) and the isolation of a cDNA clone encoding the 90-kDa subunit. The N-terminal half of TFIIIB90 exhibits sequence similarity to the yeast TFIIIB70 (BRF) and the class II transcription factor TFIIB and interacts weakly with TBP. The C-terminal half of TFIIIB90 contains a high-mobility-group protein 2 (HMG2)-related domain and interacts strongly with TBP. Recombinant TFIIIB90 plus recombinant human TBP substitute for human TFIIIB in a complementation assay for transcription of 5S, tRNA, and VA1 RNA genes, and both the TFIIB-related domain and the HMG2-related domain are required for this activity. TFIIIB90 is also required for transcription of human 7SK and U6 RNA genes by RNA polymerase III, but apparently within a complex distinct from the TBP/TFIIIB90 complex.

Regulation of angiogenic growth factor expression by hypoxia, transition metals, and chelating agents.

Recent work has indicated that oxygen-sensing mechanism(s) resembling those controlling erythropoietin production operate in many non-erythropoietin-producing cells. To pursue the implication that such a system might control other genes, we studied oxygen-regulated expression of mRNAs for vascular endothelial growth factor, platelet-derived growth factor (PDGF) A and B chains, placental growth factor (PLGF), and transforming growth factor in four different cell lines and compared the characteristics with those of erythropoietin regulation. Oxygen-regulated expression was demonstrated for each gene in at least one cell type. However, the response to hypoxia (1% oxygen) varied markedly, ranging from a 13-fold increase (PDGF-B in Hep G2 cells) to a 2-fold decrease (PLGF in the trophoblastic line BeWo). For each gene/cell combination, both the magnitude and direction of the response to hypoxia were mimicked by exposure to cobaltous ions or two different iron-chelating agents, desferrioxamine and hydroxypyridinones. These similarities with established characteristics of erythropoietin regulation indicate that a similar mechanism of oxygen sensing is operating on a variety of vascular growth factors, and they suggest that chelatable iron is closely involved in the mechanism.

Proximal sequence element-binding transcription factor (PTF) is a multisubunit complex required for transcription of both RNA polymerase II- and RNA polymerase III-dependent small nuclear RNA genes.

The proximal sequence element (PSE), found in both RNA polymerase II (Pol II)- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes, is specifically bound by the PSE-binding transcription factor (PTF). We have purified PTF to near homogeneity from HeLa cell extracts by using a combination of conventional and affinity chromatographic methods. Purified PTF is composed of four polypeptides with apparent molecular masses of 180, 55, 45, and 44 kDa. A combination of preparative electrophoretic mobility shift and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses has conclusively identified these four polypeptides as subunits of human PTF, while UV cross-linking experiments demonstrate that the largest subunit of PTF is in close contact with the PSE. The purified PTF activates transcription from promoters of both Pol II- and Pol III-transcribed snRNA genes in a PSE-dependent manner. In addition, we have investigated factor requirements in transcription of Pol III-dependent snRNA genes. We show that in extracts that have been depleted of TATA-binding protein (TBP) and associated factors, recombinant TBP restores transcription from U6 and 7SK promoters but not from the VAI promoter, whereas the highly purified TBP-TBP-associated factor complex TFIIIB restores transcription from the VAI but not the U6 or 7SK promoter. Furthermore, by complementation of heat-treated extracts lacking TFIIIC activity, we show that TFIIIC1 is required for transcription of both the 7SK and VAI genes, whereas TFIIIC2 is required only for transcription of the VAI gene. From these observations, we conclude (i) that PTF and TFIIIC2 function as gene-specific as gene-specific factors for PSE-and B-box-containing Pol III genes, respectively, (ii) that the form of TBP used by class III genes with upstream promoter elements differs from the from used by class III genes with internal promoters, and (iii) that TFIIIC1 is required for both internal and external Pol III promoters.

Morphology, immunophenotype, and distribution of latently and/or productively Epstein-Barr virus-infected cells in acute infectious mononucleosis: implications for the interindividual infection route of Epstein-Barr virus.

The present study was undertaken to unequivocally demonstrate the morphology, immunophenotype, and localization of Epstein Barr virus (EBV)-infected cells as well as the type of infection (latent versus productive) in tonsils of acute infectious mononucleosis. Paraffin sections from nine cases with clinical, serologic, and morphologic evidence of EBV infection were analyzed for the detection of small transcripts, designated EBER1 & 2, and BHLF1 by in situ hybridization (ISH) using nonisotopically labeled probes. ISH was combined with immunohistology, employing a broad panel of antibodies against B-, T-, epithelial-, macrophage-, and follicular dendritic cell (FDC)-antigens. All EBER-positive cells could be identified as lymphocytes, as they did not exhibit any morphologic or immunologic characteristics of epithelial cells, macrophages, or FDCs. A preferential accumulation of EBER-positive cells was noted around crypts, within surface squamous epithelium, and in the surroundings of necrosis. The majority of these lymphocytes could be shown to be B cells, which morphologically included Reed-Sternberg (RS)-like cells, immunoblasts, medium-sized lymphoid cells, as well as cells with plasmacytoid differentiation. In all cases, a varying number of EBER-positive T cells could be identified. ISH for BHLF1-RNA detection showed that almost all cases contained single positive small lymphoid cells, indicating a transition from latent to productive infection cycle. Such cells could also be detected within the crypt epithelium reaching up to its surface. Additional screening of 123 oropharyngeal mucosa samples from patients without evidence of acute EBV-infection, using the polymerase chain reaction for EBV-DNA detection combined with EBER- and BHLF1-ISH showed single latently infected lymphocytes in only one case. Our data imply that infected lymphocytes and not epithelial cells are, in fact, the reservoir for EBV infection, and that these are the cells that participate in the interindividual virus transfer.

Isolation and characterization of the tandemly repeated U6 genes from the sea urchin Strongylocentrotus purpuratus.

The tandemly repeated U6 genes were isolated from the sea urchin Strongylocentrotus purpuratus. Each 1.8 kb repeat unit contains a single U6 RNA sequence. There are no sequence similarities between the U6 promoter and other sea urchin snRNA genes, other than a long polypyrimidine tract 3' of the U6 sequence.

In vivo generation of highly abundant sequence-specific oligonucleotides for antisense and triplex gene regulation.

Antisense and triplex oligonucleotides continue to demonstrate potential as mediators of gene-specific repression of protein synthesis. However, inefficient and heterogeneous cellular uptake, intracellular sequestration, and rapid intracellular and extracellular degradation represent obstacles to their eventual clinical utility. Efficient cellular delivery of targeted ribozymes can present similar problems. In this report we describe a system for circumventing these obstacles and producing large quantities of short, sequence-specific RNA oligonucleotides for use in these gene regulation strategies. The oligonucleotides are generated from a vector containing promoter, capping, and termination sequences from the human small nuclear U6 gene, surrounding a synthetic sequence incorporating the oligonucleotide of interest. In vivo, these oligonucleotides are produced constitutively and without cell type specificity in levels up to 5 x 10(6) copies per cell, reach steady-state levels of expression within 9 hours post-transfection, and are still readily detectable 7 days post-transfection. In addition, these oligonucleotides are retained in the nucleus, obtain a 5' gamma-monomethyl phosphate cap, and have an intracellular half-life of approximately one hour. This expression vector provides a novel and efficient method of intracellular delivery of antisense or triplex RNA oligonucleotides (and/or ribozymes) for gene regulation, as well as a cost-effective means of comparing the biological activity arising from a variety of different potential oligonucleotide sequences.

m3G cap hypermethylation of U1 small nuclear ribonucleoprotein (snRNP) in vitro: evidence that the U1 small nuclear RNA-(guanosine-N2)-methyltransferase is a non-snRNP cytoplasmic protein that requires a binding site on the Sm core domain.

The RNA components of small nuclear ribonucleoproteins (U snRNPs) possess a characteristic 5'-terminal trimethylguanosine cap structure (m3G cap). This cap is an important component of the nuclear localization signal of U snRNPs. It arises by hypermethylation of a cotranscriptionally added m7G cap. Here we describe an in vitro assay for the hypermethylation, which employs U snRNP particles reconstituted in vitro from purified components and subsequent analysis by m3G cap-specific immunoprecipitation. Complementation studies in vitro revealed that both cytosol and S-adenosylmethionine are required for the hypermethylation of an m7G-capped U1 snRNP reconstituted in vitro, indicating that the U1 snRNA-(guanosine-N2)-methyltransferase is a trans-active non-snRNP protein. Chemical modification revealed one cytoplasmic component required for hypermethylation and one located on the snRNP: these components have different patterns of sensitivity to modification by N-ethylmaleimide and iodoacetic acid (IAA). In the presence of cytosol and S-adenosylmethionine, an intact Sm core domain is a necessary and sufficient substrate for cap hypermethylation. These data, together with our observation that isolated native U1 snRNPs but not naked U1 RNA inhibit the trimethylation of in vitro-reconstituted U1 snRNP, indicate that the Sm core binds the methyltransferase specifically. Moreover, isolated native U2 snRNP also inhibits trimethylation of U1 snRNP, suggesting that other Sm-class U snRNPs might share the same methyltransferase. IAA modification of m7G-capped U1 snRNPs inhibited hypermethylation when they were microinjected into Xenopus oocytes and consequently also inhibited nuclear import. In contrast, modification with IAA of m3G-capped U1 snRNPs reconstituted in vitro did not interfere with their nuclear transport in oocytes. These data suggest that m3G cap formation and nuclear transport of U1 snRNPs are mediated by distinct factors, which require distinct binding sites on the Sm core of U1 snRNP.