A well-connected and conserved nucleoplasmic helicase is required for production of box C/D and H/ACA snoRNAs and localization of snoRNP proteins.

Biogenesis of small nucleolar RNA-protein complexes (snoRNPs) consists of synthesis of the snoRNA and protein components, snoRNP assembly, and localization to the nucleolus. Recently, two nucleoplasmic proteins from mice were observed to bind to a model box C/D snoRNA in vitro, suggesting that they function at an early stage in snoRNP biogenesis. Both proteins have been described in other contexts. The proteins, called p50 and p55 in the snoRNA binding study, are highly conserved and related to each other. Both have Walker A and B motifs characteristic of ATP- and GTP-binding and nucleoside triphosphate-hydrolyzing domains, and the mammalian orthologs have DNA helicase activity in vitro. Here, we report that the Saccharomyces cerevisiae ortholog of p50 (Rvb2, Tih2p, and other names) is required for production of C/D snoRNAs in vivo and, surprisingly, H/ACA snoRNAs as well. Point mutations in the Walker A and B motifs cause temperature-sensitive or lethal growth phenotypes and severe defects in snoRNA accumulation. Notably, depletion of p50 (called Rvb2 in this study) also impairs localization of C/D and H/ACA core snoRNP proteins Nop1p and Gar1p, suggesting a defect(s) in snoRNP assembly or trafficking to the nucleolus. Findings from other studies link Rvb2 orthologs with chromatin remodeling and transcription. Taken together, the present results indicate that Rvb2 is involved in an early stage of snoRNP biogenesis and may play a role in coupling snoRNA synthesis with snoRNP assembly and localization.

An unexpected, conserved element of the U3 snoRNA is required for Mpp10p association.

The U3 small nucleolar ribonucleoprotein (snoRNP) is composed of a small nucleolar RNA (snoRNA) and at least 10 proteins. The U3 snoRNA base pairs with the pre-rRNA to carry out the A0, A1, and A2 processing reactions that lead to the release of the 18S rRNA from the nascent pre-rRNA transcript. The yeast U3 snoRNA can be divided into a short 5' domain (nt 1-39) and a larger 3' domain (73 to the 3' end) separated by a stretch of nucleotides called the hinge region (nt 40-72). The sequences required for pre-rRNA base pairing are found in the 5' domain and hinge region whereas the 3' domain is largely covered with proteins. Mpp10p, one of the protein components unique to the U3 snoRNP, plays a role in processing at the A1 and A2 sites. Because of its critical role in U3 snoRNP function, we determined which sequences in the U3 snoRNA are required for Mpp10p association. Unlike fibrillarin and all the previous U3 snoRNP components studied in this manner, sequences in the 3' domain are not sufficient for Mpp10p association. Instead, a conserved sequence element in the U3 snoRNA hinge region is required, placing Mpp10p near the 5' domain that carries out the pre-rRNA base-pairing interactions in the functional center of the U3 snoRNP.

Probing RNA in vivo with methylation guide small nucleolar RNAs.

Most box C/D small nucleolar RNAs (snoRNAs) direct the formation of 2'-O-methylated nucleotides in ribosomal RNA and, apparently, other RNAs present in the nucleolar complex. Sites to be modified are selected by a long (>10-nt) antisense guide sequence in the snoRNA and a distance measurement from a box D or D' element that follows the snoRNA guide sequence. Modification of the substrate occurs in the region of complementarity, at a position five nucleotides upstream from box D/D'. Methylation can be targeted to novel sites by expressing a snoRNA with a new guide sequence. In some cases methylation impairs the growth rate of the cell, indicating that a functionally important nucleotide has been altered. With a view to harnessing snoRNA-directed methylation for functional mapping, we have developed a method for constructing libraries of snoRNA genes that, in principle, can introduce methylation point mutations into any rRNA segment of interest. The strategy and procedures are described here, and preliminary results are presented that show the feasibility of using this technology to probe a region of the yeast large subunit rRNA that includes the core of the peptidyltransferase center.

Point mutations in yeast CBF5 can abolish in vivo pseudouridylation of rRNA.

In budding yeast (Saccharomyces cerevisiae), the majority of box H/ACA small nucleolar RNPs (snoRNPs) have been shown to direct site-specific pseudouridylation of rRNA. Among the known protein components of H/ACA snoRNPs, the essential nucleolar protein Cbf5p is the most likely pseudouridine (Psi) synthase. Cbf5p has considerable sequence similarity to Escherichia coli TruBp, a known Psi synthase, and shares the "KP" and "XLD" conserved sequence motifs found in the catalytic domains of three distinct families of known and putative Psi synthases. To gain additional evidence on the role of Cbf5p in rRNA biosynthesis, we have used in vitro mutagenesis techniques to introduce various alanine substitutions into the putative Psi synthase domain of Cbf5p. Yeast strains expressing these mutated cbf5 genes in a cbf5Delta null background are viable at 25 degrees C but display pronounced cold- and heat-sensitive growth phenotypes. Most of the mutants contain reduced levels of Psi in rRNA at extreme temperatures. Substitution of alanine for an aspartic acid residue in the conserved XLD motif of Cbf5p (mutant cbf5D95A) abolishes in vivo pseudouridylation of rRNA. Some of the mutants are temperature sensitive both for growth and for formation of Psi in the rRNA. In most cases, the impaired growth phenotypes are not relieved by transcription of the rRNA from a polymerase II-driven promoter, indicating the absence of polymerase I-related transcriptional defects. There is little or no abnormal accumulation of pre-rRNAs in these mutants, although preferential inhibition of 18S rRNA synthesis is seen in mutant cbf5D95A, which lacks Psi in rRNA. A subset of mutations in the Psi synthase domain impairs association of the altered Cbf5p proteins with selected box H/ACA snoRNAs, suggesting that the functional catalytic domain is essential for that interaction. Our results provide additional evidence that Cbf5p is the Psi synthase component of box H/ACA snoRNPs and suggest that the pseudouridylation of rRNA, although not absolutely required for cell survival, is essential for the formation of fully functional ribosomes.

A small nucleolar RNA:ribozyme hybrid cleaves a nucleolar RNA target in vivo with near-perfect efficiency.

A hammerhead ribozyme has been localized to the yeast nucleolus by using the U3 small nucleolar RNA as a carrier. The hybrid small nucleolar RNA:ribozyme, designated a "snorbozyme," is metabolically stable and cleaves a target U3 RNA with nearly 100% efficiency in vivo. This is the most efficient in vivo cleavage reported for a trans-acting ribozyme. A key advantage of the model substrate featured is that a stable, trimmed cleavage product accumulates. This property allows accurate kinetic measurements of authentic cleavage in vivo. The system offers new avenues for developing effective ribozymes for research and therapeutic applications.

A comprehensive database for the small nucleolar RNAs from Saccharomyces cerevisiae.

Small nucleolar RNAs (snoRNAs) are involved in cleavage of rRNA, modification of rRNA nucleotides and, perhaps, other aspects of ribosome biogenesis in eukaryotic cells. Scores of snoRNAs have been discovered in recent years from various eukaryotes, and the total number is predicted to be up to 200 different snoRNA species per individual organism. We have created a comprehensive database for snoRNAs from the yeast Saccharomyces cerevisiae which allows easy access to detailed information about each species known (almost 70 snoRNAs are featured). The database consists of three major parts: (i) a utilities section; (ii) a master table; and (iii) a collection of tables for the individual snoRNAs. The utilities section provides an introduction to the database. The master table lists all known S. cerevisiae snoRNAs and their major properties. Information in the individual tables includes: alternate names, size, family classification, genomic organization, sequences (with major features identified), GenBank accession numbers, occurrence of homologues, gene disruption phenotypes, functional properties and associated RNAs and proteins. All information is accompanied with appropriate literature references. The database is available on the World Wide Web (http://www.bio.umass. edu/biochem/rna-sequence/Yeast_snoRNA_Database/snoRNA_ DataBase.html), and should be useful for a wide range of snoRNA studies.

Effect of local sequence inversions on the crystalline antiparallel beta-sheet lamellar structures of periodic polypeptides: implications for chain-folding.

The crystal structure and texture of the monodisperse periodic polypeptide [(AG)3EG(GA)3EG]10 (poly(+/-AG)3EG: A=alanine, G=glycine, E=glutamic acid) were analyzed by X-ray diffraction, Fourier transform infrared spectroscopy, and electron microscopy. Structure determination was aided by comparison with the recently described structure for the related periodic polypeptide [(AG)3EG]36 by Krejchi et al. (Macromolecules 1997;30:5012). Texture-oriented samples of poly(+/-AG)3EG were obtained by crystallization of the polymer from aqueous formic acid solution. The evidence supports an antiparallel (ap) beta-sheet protein structure and the X-ray diffraction signals index on an orthorhombic unit cell with parameters: a=0.950 nm (hydrogen-bond direction), b=1.052 nm (apbeta-sheet stacking direction), c=6.95 nm (chain direction). The absence of the (010) diffraction signal, a prominent signal in the poly(AG)3EG diffraction pattern, implies that the apbeta-sheets are 'apolar', i.e. both surfaces are equally populated with alanyl methyl groups. Selective line broadening of wide-angle diffraction signals with l not equal to 0 gives an estimated crystal size of approximately/= 4 nm in the chain direction. This observation, coupled with the appearance of low-angle particle interference peaks, indicates a crystal thickness considerably less than the chain length and suggests an adjacent-re-entry chain-folded lamellar structure incorporating the apbeta-sheet architecture. The polypeptide folds through gamma-turns, in-phase with the pseudo-octapeptide repeat; the glutamic acid residues occur on the lamellar surfaces. These results and those from the crystalline lamellae of poly(AG)3EG suggest that beta-turns are not compatible with these repetitively stacked apbeta-sheet structures. This implies that intersheet interactions of alanyl methyl groups and glycyl alpha-protons are not sufficiently strong to dictate the folding geometry in these structures.

Intronic snoRNA biosynthesis in Saccharomyces cerevisiae depends on the lariat-debranching enzyme: intron length effects and activity of a precursor snoRNA.

The eukaryotic small nucleolar RNAs (snoRNAs) are involved in processing of pre-rRNA and modification of rRNA nucleotides. Some snoRNAs are derived from mono- or polycistronic transcription units, whereas others are encoded in introns of protein genes. The present study addresses the role of the RNA lariat-debranching enzyme (Dbr1p) in the synthesis and function of intronic snoRNAs in the yeast Saccharomyces cerevisiae. Intronic snoRNA production was determined to depend on Dbr1p. Accumulation of mature intronic snoRNAs is reduced in a dbr1 mutant; instead, intronic snoRNAs are "trapped" within host intron lariats. Interestingly, the extent of intronic snoRNA accumulation in the form of lariats in dbr1 cells varied among different intronic snoRNAs. Intronic snoRNAs encoded within shorter introns, such as U24 and snR38, accumulate more unprocessed lariat precursors than those encoded within longer introns, e.g., U18 and snR39. This correlation was corroborated by experiments conducted with model intron:U24 snoRNA constructs. These results support a splicing-dependent exonucleolytic pathway for the biosynthesis of intronic snoRNAs. Curiously, U24 in a lariat may be functional in directing methylation of ribosomal RNA.

The snoRNA box C/D motif directs nucleolar targeting and also couples snoRNA synthesis and localization.

Most small nucleolar RNAs (snoRNAs) fall into two families, known as the box C/D and box H/ACA snoRNAs. The various box elements are essential for snoRNA production and for snoRNA-directed modification of rRNA nucleotides. In the case of the box C/D snoRNAs, boxes C and D and an adjoining stem form a vital structure, known as the box C/D motif. Here, we examined expression of natural and artificial box C/D snoRNAs in yeast and mammalian cells, to assess the role of the box C/D motif in snoRNA localization. The results demonstrate that the motif is necessary and sufficient for nucleolar targeting, both in yeast and mammals. Moreover, in mammalian cells, RNA is targeted to coiled bodies as well. Thus, the box C/D motif is the first intranuclear RNA trafficking signal identified for an RNA family. Remarkably, it also couples snoRNA localization with synthesis and, most likely, function. The distribution of snoRNA precursors in mammalian cells suggests that this coupling is provided by a specific protein(s) which binds the box C/D motif during or rapidly after snoRNA transcription. The conserved nature of the box C/D motif indicates that its role in coupling production and localization of snoRNAs is of ancient evolutionary origin.

Functional mapping of the U3 small nucleolar RNA from the yeast Saccharomyces cerevisiae.

The U3 small nucleolar RNA participates in early events of eukaryotic pre-rRNA cleavage and is essential for formation of 18S rRNA. Using an in vivo system, we have developed a functional map of the U3 small nucleolar RNA from Saccharomyces cerevisiae. The test strain features a galactose-dependent U3 gene in the chromosome and a plasmid-encoded allele with a unique hybridization tag. Effects of mutations on U3 production were analyzed by evaluating RNA levels in cells grown on galactose medium, and effects on U3 function were assessed by growing cells on glucose medium. The major findings are as follows: (i) boxes C' and D and flanking helices are critical for U3 accumulation; (ii) boxes B and C are not essential for U3 production but are important for function, most likely due to binding of a trans-acting factor(s); (iii) the 5' portion of U3 is required for function but not stability; and, (iv) strikingly, the nonconserved hairpins 2, 3, and 4, which account for 50% of the molecule, are not required for accumulation or function.

Smectic ordering in solutions and films of a rod-like polymer owing to monodispersity of chain length.

Solutions and melts of stiff ('rod-like') macromolecules often exhibit nematic liquid crystalline phases characterized by orientational, but not positional, molecular order. Smectic phases, in which macromolecular rods are organized into layers roughly perpendicular to the direction of molecular orientation, are rare, owing at least in part to the polydisperse nature (distribution of chain lengths) of polymers prepared by conventional polymerization processes. Bacterial methods for polypeptide synthesis, in which artificial genes encoding the polymer are expressed in bacterial vectors, offer the opportunity to make macromolecules with very well defined chain lengths. Here we show that a monodisperse derivative of poly(gamma-benzyl alpha,L-glutamate) prepared in this way shows smectic ordering in solution and in films. This result suggests that methods for preparing monodisperse polymers might provide access to new smectic phases with layer spacings that are susceptible to precise control on the scale of tens of nanometres.

The rRNA-processing function of the yeast U14 small nucleolar RNA can be rescued by a conserved RNA helicase-like protein.

The phylogenetically conserved U14 small nucleolar RNA is required for processing of rRNA, and this function involves base pairing with conserved complementary sequences in 18S RNA. With a view to identifying other important U14 interactions, a stem-loop domain required for activity of Saccharomyces cerevisiae U14 RNAs (the Y domain) was first subjected to detailed mutational analysis. The mapping results showed that most nucleotides of the Y domain can be replaced without affecting function, except for loop nucleotides conserved among five different yeast species. Defective variants were then used to identify both intragenic and extragenic suppressor mutations. All of the intragenic mutations mapped within six nucleotides of the primary mutation, suggesting that suppression involves a change in conformation and that the loop element is involved in an essential intermolecular interaction rather than intramolecular base pairing. A high-copy extragenic suppressor gene, designated DBP4 (DEAD box protein 4), encodes an essential, putative RNA helicase of the DEAD-DEXH box family. Suppression by DBP4 (initially CA4 [T.-H. Chang, J. Arenas, and J. Abelson, Proc. Natl. Acad. Sci. USA 87:1571-1575, 1990]) restores the level of 18S rRNA and is specific for the Y domain but is not allele specific. DBP4 is predicted to function either in assembly of the U14 small nucleolar RNP or, more likely, in its interaction with other components of the rRNA processing apparatus. Mediating the interaction of U14 with precursor 18S RNA is an especially attractive possibility.

Effects of amino acid side-chain volume on chain packing in genetically engineered periodic polypeptides.

The fidelity of bacterial protein synthesis allows the production of architecturally well-defined polymeric materials through precise control of chain length, sequence, stereochemistry, and interchain interactions. In the present paper, we examine the relation between amino acid residue volume and crystalline unit cell dimensions, in a set of periodic protein polymers of repeating unit sequence -(AlaGly)3-X-Gly-, where X is Asn, Phe, Ser, Val, or Tyr. The proteins were overexpressed in Escherichia coli, purified by simple procedures based on acid/ethanol precipitation or insolubility in aqueous sodium dodecyl sulfate, and processed to form oriented crystalline mats by precipitation from formic acid under mechanical shear. X-ray diffraction analyses revealed that the basic structures of the -(AlaGly)3-X-Gly- polymers are identical to that previously reported for [(AlaGly)3-GluGly]36, [Krejchi, M.T., Atkins, E.D.T., Waddon, A.J., Fournier, M.J., Mason, T.L., and Tirrell, D.A. (1994) Science 265, 1427-1432], with the oligoalanylglycine segments forming antiparallel beta-sheets and the substituted amino acids occurring within three-residue folds at the lamellar surfaces. The X-ray diffraction signals for each member of the family index on an orthorhombic unit cell; the a-axis (hydrogen bond direction) and c-axis (chain direction) spacings remain invariant but the b-axis (sheet stacking direction) spacing increases with increasing volume of the substituted amino acid. The results obtained from a variant with alternating Glu and Lys substitution at the X position, together with the results previously reported for poly(L-alanylglycine) [Panitch, A., Matsuki, K., Cantor, E.J., Cooper, S.J., Atkins, E.D.T., Fournier, M.J., Mason, T.L., and Tirrell, D.A. (1997) Macromolecules 30, 42-49] are included for comparison. The average intersheet stacking distance (b/2) increases linearly with the volume of the amino acid inserted at position X. Because the chain-folded lamellar architecture adopted by these periodic polypeptides accommodates a wide range of residues differing in charge, steric bulk, and hydrophobicity, these results illustrate a new approach to the engineering of intermolecular interactions in polymeric solids.

Small nucleolar RNAs direct site-specific synthesis of pseudouridine in ribosomal RNA.

Ten ACA yeast small nucleolar RNAs (snoRNAs) were shown to be required for site-specific synthesis of pseudouridine psi in ribosomal RNA. A common secondary folding motif for the snoRNAs and rRNA target segments predicts that site selection involves: (1) base pairing of the snoRNA with complementary rRNA elements flanking the site of modification, and (2) identification of a uridine located at a near-constant distance from the snoRNA ACA box. The model is supported by mutations showing that: (1) reducing the complementarity between the snoRNA and rRNA disrupts psi formation, and (2) altering the distance between the ACA box and target uridine causes an adjacent uridine to be modified. This discovery implies that most snoRNAs function in targeting nucleotide modification in rRNA: ribose methylation for the box C/D snoRNAs and psi formation for the ACA snoRNAs.

Synthesis of functional eukaryotic ribosomal RNAs in trans: development of a novel in vivo rDNA system for dissecting ribosome biogenesis.

Active 18S and 25S ribosomal RNAs were produced in trans in yeast, from plasmids containing RNA polymerase II transcription signals and rDNA fragments with unique hybridization tags. Analyses were carried out in cells with temperature-sensitive RNA polymerase I. Functional rRNAs were derived from separate 18S and 5.8/25S rRNA coding units, however, active 25S rRNA could be produced only by cotranscription with 5.8S rRNA. The results demonstrate that the polycistronic organization of the large rDNA operon is not required for successful processing of rRNA or assembly of functional ribosomes. The split operon system should facilitate future efforts to dissect eukaryotic ribosome biogenesis.

SnoRNAs as tools for RNA cleavage and modification.

Eukaryotic small nucleolar RNAs (snoRNAs) influence rRNA synthesis at two stages: nucleolytic processing and selection of nucleotides to be ribose methylated (Nm) or converted to pseudouridine (psi). The two modification functions and some processing activities involve direct base pairing of snoRNA with rRNA. In addition to rRNA-targeting sequences, snoRNA function depends on the presence of conserved box elements involved in snoRNA synthesis and localization. The present investigation is directed at using snoRNAs as tools for two purposes: 1) introducing nucleotide modifications into novel sites in rRNA and other snoRNAs, and: 2) targeting nucleolar RNAs for destruction using snoRNA:ribozyme chimers ('snorbozymes'). Early results demonstrate that snoRNAs can be used for both applications.

Differential clearance of glycoforms of IgG in normal and autoimmune-prone mice.

In order to understand better the origins of the elevated levels of the glycoform of IgG that lacks galactose on both arms of the oligosaccharide chain (G0%) located in the Fc, which occurs in man and mouse with age, and in particular in autoimmune disease, we investigated the clearance of two glycosylated forms of IgG2a and IgG1 in normal (BALB/c) and autoimmune-prone (MRL/1pr, MRL/+, and non-obese diabetic (NOD)) mice. To investigate the possibility of different rates of catabolism, enzymatically generated glycoforms of monomeric IgG1 and IgG2a (fully glycosylated or G0%), were iodinated and injected into the tail vein of the mice. We found that the G0% IgG2a remained in circulation significantly longer than the fully glycosylated variants, in all of the mouse strains tested. In contrast, the two forms of IgG1 had similar kinetics in all the autoimmune-prone mice, whereas in BALB/c, there was a longer half-life (t1/2) for G0% IgG1. These data suggest that there may be differences in the ability of the IgG glycoforms to bind to the Fc gamma receptors, in particular Fc gamma RI. The clearance rates were found to vary among the strains studied, with MRL/1pr having the fastest catabolic rates for all glycoforms and IgG subclasses tested. This appeared to be due to the presence of circulating IgG and IgM rheumatoid factors (RF). There were significantly increased frequencies and titres for both IgM and IgG RF in MRL/1pr mice compared with the other strains. In contrast, interferon-gamma, known to induce the Fc gamma RI, was found to be similar in the sera, in all of the strains of mice examined. These results suggest that RF probably play an important biological function in the MRL/1pr mice and aid in the clearance of circulating IgG. Our study shows that the state of glycosylation of IgG affects the t1/2 in vivo, and that by removing the terminal sugars (sialic acid and galactose), the antibody (IgG2a) will remain in circulation significantly longer. These observations may thus provide a partial explanation for the increase in relative percentage of this glycoform that occurs with age.

The RNA world of the nucleolus: two major families of small RNAs defined by different box elements with related functions.

We have discovered that all known yeast and vertebrate small nucleolar RNAs (snoRNAs), except for the MRP/7-2 RNA, fall into two major classes. One class is defined by conserved boxes C and D and the other by a novel element: a consensus ACA triplet positioned 3 nt before the 3' end of the RNA. A role for the ACA box is snoRNA stability has been established by mutational analysis of a yeast ACA snoRNA (snR 11). Full function of the box depends on the integrity of an adjacent upstream stem. All members of the yeast ACA family are associated with the GAR1 protein. Binding of this or another common small nucleolar ribonucleoprotein particle protein is predicted to be a critical entry point to snoRNA posttranscriptional life, including precise formation of the snoRNA 3' end.

An essential domain in Saccharomyces cerevisiae U14 snoRNA is absent in vertebrates, but conserved in other yeasts.

U14 is a small nucleolar RNA (snoRNA) required for early cleavages of eukaryotic precursor rRNA. The U14 RNA from Saccharomyces cerevisiae is distinguished from its vertebrate homologues by the presence of a stem-loop domain that is essential for function. This element, known as the Y-domain, is located in the U14 sequence between two universal sequences that base pair with 18S rRNA. Sequence data obtained for the U14 homologues from four additional phylogenetically distinct yeasts showed the Y-domain is not unique to S.cerevisiae. Comparison of the five Y-domain sequences revealed a common stem-loop structure with a conserved loop sequence that includes eight invariant nucleotides. Conservation of these features suggests that the Y-domain is a recognition signal for an essential interaction. Several plant U14 RNAs were found to contain similar structures, though with an unrelated consensus sequence in the loop portion. The U14 gene from the most distantly related yeast, Schizosaccharomyces pombe, was found to be active in S.cerevisiae, showing that Y-domain function is conserved and that U14 function can be provided by variants in which the essential elements are embedded in dissimilar flanking sequences. This last result suggests that U14 function may be determined solely by the essential elements.

Expression and purification of a spider silk protein: a new strategy for producing repetitive proteins.

Synthetic genes were constructed based on the known sequence of the spider dragline silk protein MaSp 2. The genes had 8, 16, or 32 contiguous units of the consensus repeat sequence of the protein. These artificial genes were constructed using a strategy involving compatible but nonregenerable restriction sites, which allowed construction of very large inserts in a precisely controlled manner. This strategy should have general utility in the controlled construction of repetitive proteins composed of identical or different repeat units. The protein from the 16-unit repeat was produced in Escherichia coli at levels up to 10 mg/g wet wt of cells although yields of 1-2 mg/g were more typical. The protein was easily purified with high recovery using an affinity column. The purified protein had the predicted amino acid composition and N-terminal sequence after cleavage of a leader sequence. The methodology described will allow production of sufficient quantities of protein for basic structure/function studies including production of synthetic fibers.