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H A Raué

Publications and source records attributed to H A Raué.

At least 19 recordsLinked to original sources
PubMed2006

Analysis of sequence and structural features that identify the B/C motif of U3 small nucleolar RNA as the recognition site for the Snu13p-Rrp9p protein pair.

The eukaryal Snu13p/15.5K protein binds K-turn motifs in U4 snRNA and snoRNAs. Two Snu13p/15.5K molecules bind the nucleolar U3 snoRNA required for the early steps of preribosomal processing. Binding of one molecule on the C'/D motif allows association of proteins Nop1p, Nop56p, and Nop58p, whereas binding of the second molecule on the B/C motif allows Rrp9p recruitment. To understand how the Snu13p-Rrp9p pair recognizes the B/C motif, we first improved the identification of RNA determinants required for Snu13p binding by experiments using the systematic evolution of ligands by exponential enrichment. This demonstrated the importance of a U.U pair stacked on the sheared pairs and revealed a direct link between Snu13p affinity and the stability of helices I and II. Sequence and structure requirements for efficient association of Rrp9p on the B/C motif were studied in yeast cells by expression of variant U3 snoRNAs and immunoselection assays. A G-C pair in stem II, a G residue at position 1 in the bulge, and a short stem I were found to be required. The data identify the in vivo function of most of the conserved residues of the U3 snoRNA B/C motif. They bring important information to understand how different K-turn motifs can recruit different sets of proteins after Snu13p association.

Base Pairing↗
PubMed2001

All three functional domains of the large ribosomal subunit protein L25 are required for both early and late pre-rRNA processing steps in Saccharomyces cerevisiae.

Mutational analysis has shown that the integrity of the region in domain III of 25S rRNA that is involved in binding of ribosomal protein L25 is essential for the production of mature 25S rRNA in the yeast Saccharomyces cerevisiae. However, even structural alterations that do not noticeably affect recognition by L25, as measured by an in vitro assay, strongly reduced 25S rRNA formation by inhibiting the removal of ITS2 from the 27S(B) precursor. In order to analyze the role of L25 in yeast pre-rRNA processing further we studied the effect of genetic depletion of the protein or mutation of each of its three previously identified functional domains, involved in nuclear import (N-terminal), RNA binding (central) and 60S subunit assembly (C-terminal), respectively. Depletion of L25 or mutating its (pre-)rRNA-binding domain blocked conversion of the 27S(B) precursor to 5.8S/25S rRNA, confirming that assembly of L25 is essential for ITS2 processing. However, mutations in either the N- or the C-terminal domain of L25, which only marginally affect its ability to bind to (pre-)rRNA, also resulted in defective ITS2 processing. Furthermore, in all cases there was a notable reduction in the efficiency of processing at the early cleavage sites A0, A1 and A2. We conclude that the assembly of L25 is necessary but not sufficient for removal of ITS2, as well as for fully efficient cleavage at the early sites. Additional elements located in the N- as well as C-terminal domains of L25 are required for both aspects of pre-rRNA processing.

Binding Sites↗
PubMed2001

Identification of cis-acting elements involved in 3'-end formation of Saccharomyces cerevisiae 18S rRNA.

In yeast, the 3' end of mature 18S rRNA is generated by endonucleolytic cleavage of the 20S precursor at site D. Available data indicate that the major cis-acting elements required for this processing step are located in relatively close proximity to the cleavage site. To identify these elements, we have studied the effect of mutations in the mature 18S and ITS1 sequences neighboring site D on pre-rRNA processing in vivo. Using clustered point mutations, we found that alterations in the sequence spanning site D from position -5 in 18S rRNA to +6 in ITS1 reduced the efficiency of processing at this site to different extents as demonstrated by the lower level of the mature 18S rRNA and the increase in 20S pre-rRNA in cells expressing only mutant rDNA units. More detailed analysis revealed an important role for the residue located 2 nt upstream from site D (position -2), whereas sequence changes at position -1, +1, and +2 relative to site D had no effect. The data further demonstrate that the proposed base pairing between the 3' end of 18S rRNA and the 5' end of ITS1 is not important for efficient and accurate processing at site D, nor for the formation of functional 40S ribosomal subunits. These results were confirmed by analyzing the accumulation of the D-A2 fragment derived from the mutant 20S pre-rRNA in cells that lack the Xrn1p exonuclease responsible for its degradation. The latter results also showed that the accuracy of cleavage was affected by altering the spacer sequence directly downstream of site D but not by mutations in the 18S rRNA sequence preceding this site.

Base Sequence↗
PubMed2000

Domain III of Saccharomyces cerevisiae 25 S ribosomal RNA: its role in binding of ribosomal protein L25 and 60 S subunit formation.

Domain III of Saccharomyces cerevisiae 25 S rRNA contains the recognition site for the primary rRNA-binding ribosomal protein L25, which belongs to the functionally conserved EL23/L25 family of ribosomal proteins. The EL23/L25 binding region is very complex, consisting of several irregular helices held together by long-distance secondary and tertiary interactions. Moreover, it contains the eukaryote-specific V9 (D7a) expansion segment. Functional characterisation of the structural elements of this site by a detailed in vitro and in vivo mutational analysis indicates the presence of two separate regions that are directly involved in L25 binding. In particular, mutation of either of two conserved nucleotides in the loop of helix 49 significantly reduces in vitro L25 binding, thus strongly supporting their role as attachment sites for the r-protein. Two other helices appear to be primarily required for the correct folding of the binding site. Mutations that abolish in vitro binding of L25 block accumulation of 25 S rRNA in vivo because they stall pre-rRNA processing at the level of its immediate precursor, the 27 S(B) pre-rRNA. Surprisingly, several mutations that do not significantly affect L25 binding in vitro cause the same lethal defect in 27 S(B) pre-rRNA processing. Deletion of the V9 expansion segment also leads to under-accumulation of mature 25 S rRNA and a twofold reduction in growth rate. We conclude that an intact domain III, including the V9 expansion segment, is essential for normal processing and assembly of 25 S rRNA.

Base Sequence↗
PubMed2000

Yeast Rrp9p is an evolutionarily conserved U3 snoRNP protein essential for early pre-rRNA processing cleavages and requires box C for its association.

Pre-rRNA processing in eukaryotic cells requires participation of several snoRNPs. These include the highly conserved and abundant U3 snoRNP, which is essential for synthesis of 18S rRNA. Here we report the characterization of Rrp9p, a novel yeast U3 protein, identified via its homology to the human U3-55k protein. Epitope-tagged Rrp9p specifically precipitates U3 snoRNA, but Rrp9p is not required for the stable accumulation of this snoRNA. Genetic depletion of Rrp9p inhibits the early cleavages of the primary pre-rRNA transcript at A0, A1, and A2 and, consequently, production of 18S, but not 25S and 5.8S, rRNA. The hU3-55k protein can partially complement a yeast rrp9 null mutant, indicating that the function of this protein has been conserved. Immunoprecipitation of extracts from cells that coexpress epitope-tagged Rrp9p and various mutant forms of U3 snoRNA limits the region required for association of Rrp9p to the U3-specific box B/C motif. Box C is essential, whereas box B plays a supportive role.

Amino Acid Sequence↗
PubMed2000

The final step in the formation of 25S rRNA in Saccharomyces cerevisiae is performed by 5'-->3' exonucleases.

The final stage in the formation of the two large subunit rRNA species in Saccharomyces cerevisiae is the removal of internal transcribed spacer 2 (ITS2) from the 27SB precursors. This removal is initiated by endonucleolytic cleavage approximately midway in ITS2. The resulting 7S pre-rRNA, which is easily detectable, is then converted into 5.8S rRNA by the concerted action of a number of 3'-->5' exonucleases, many of which are part of the exosome. So far the complementary precursor to 25S rRNA resulting from the initial cleavage in ITS2 has not been detected and the manner of its conversion into the mature species is unknown. Using various yeast strains that carry different combinations of wild-type and mutant alleles of the major 5'-->3' exonucleases Rat1p and Xrn1p, we now demonstrate the existence of a short-lived 25.5S pre-rRNA whose 5' end is located closely downstream of the previously mapped 3' end of 7S pre-rRNA. The 25.5S pre-rRNA is converted into mature 25S rRNA by rapid exonucleolytic trimming, predominantly carried out by Rat1p. In the absence of Rat1p, however, the removal of the ITS2 sequences from 25.5S pre-rRNA can also be performed by Xrn1p, albeit somewhat less efficiently.

Base Sequence↗
PubMed1999

Nuclear and nucleolar localization of Saccharomyces cerevisiae ribosomal proteins S22 and S25.

Nuclear import usually relies on the presence of nuclear localization sequences (NLSs). NLSs are recognized by NLS receptors (importins), which target their substrates to the nuclear pore. We identified the NLSs of the yeast ribosomal proteins S22 and S25 and studied the former by mutational analysis. Furthermore, in S25 the nucleolar targeting information was found to overlap with its NLS. Comparison with previously published data on yeast ribosomal protein NLSs and computer analysis indicates the existence of a novel type of ribosomal protein-specific NLS that differs from the classical Chelsky and bipartite NLSs. The existence of such a ribosomal protein-specific NLS is in accordance with the recent identification of ribosomal protein-specific importins.

Amino Acid Sequence↗
PubMed1999

The roles of Rrp5p in the synthesis of yeast 18S and 5.8S rRNA can be functionally and physically separated.

The yeast nucleolar protein Rrp5p is the only known trans-acting factor that is essential for the synthesis of both 18S rRNA and the major, short form of 5.8S (5.8Ss) rRNA, which were thought to be produced in two independent sets of pre-rRNA processing reactions. To identify domains within Rrp5p required for either processing pathway, we have analyzed a set of eight deletion mutants that together cover the entire RRP5 sequence. Surprisingly, only one of the deletions is lethal, indicating that regions encompassing about 80% of the protein can be removed individually without disrupting its essential biological function. Biochemical analysis clearly demonstrated the presence of two distinct functional domains. Removal of each of three contiguous segments from the N-terminal half specifically inhibits the formation of 5.8Ss rRNA, whereas deleting part of the C-terminal region of the protein only blocks the production of 18S rRNA. The latter phenotype is also caused by a temperature-sensitive mutation within the same C-terminal region. The two functional regions identified by the mutational analysis appear to be correlated with the structural domains detected by computer analysis. They can even be physically separated, as demonstrated by the fact that full Rrp5p activity can be supplied by two contiguous protein fragments expressed in trans.

Cell Survival↗
PubMed1997

Variable region V1 of Saccharomyces cerevisiae 18S rRNA participates in biogenesis and function of the small ribosomal subunit.

The role of helix 6, which forms the major portion of the most 5'-located expansion segment of Saccharomyces cerevisiae 18S rRNA, was studied by in vivo mutational analysis. Mutations that increased the size of the helical part and/or the loop, even to a relatively small extent, abolished 18S rRNA formation almost completely. Concomitantly, 35S pre-rRNA and an abnormal 23S precursor species accumulated. rDNA units containing these mutations did not support cell growth. A deletion removing helix 6 almost completely, on the other hand, had a much less severe effect on the formation of 18S rRNA, and cells expressing only the mutant rRNA remained able to grow, albeit at a much reduced rate. Disruption of the apical A.U base pair by a single point mutation did not cause a noticeable reduction in the level of 18S rRNA but did result in a twofold lower growth rate of the cells. This effect could not be reversed by introduction of a second point mutation that restores base pairing. We conclude that both the primary and the secondary structure of helix 6 play an important role in the formation and the biological function of the 40S subunit.

Base Sequence↗
PubMed1997

Variable regions V13 and V3 of Saccharomyces cerevisiae contain structural features essential for normal biogenesis and stability of 5.8S and 25S rRNA.

The homologous ribosomal RNA species of all organisms can be folded into a common "core" secondary structure. In addition, eukaryotic rRNAs contain a large number of segments, located at fixed positions, that are highly variable in size and sequence from one organism to another. We have investigated the role of the two largest of these variable regions in Saccharomyces cerevisiae 25S rRNA, V13, and V3, by mutational analysis in a yeast strain that can be rendered completely dependent on the synthesis of mutant (pre-)rRNA. We found that approximately half of variable region V13 can be deleted without any phenotypic effect. The remaining portion, however, contains multiple structural features whose disturbance causes serious growth defects or lethality. Accumulation of 25S rRNA is strongly reduced by these mutations, at least in part because they inhibit processing of ITS2. Removal of even a relatively small portion of V3 also strongly reduces the cellular growth rate and larger deletions are lethal. Interestingly, some of the deletions in V3 cause accumulation of 27S(A) pre-rRNA and, moreover, appear to interfere with the close coupling between the processing cleavages at sites A3 and B1(S). These results demonstrate that both variable regions play an important role in 60S subunit formation.

Base Sequence↗
PubMed1996

Rat RL23a ribosomal protein efficiently competes with its Saccharomyces cerevisiae L25 homologue for assembly into 60 S subunits.

The large subunit protein RL23a from rat liver ribosomes shows 62% sequence identity with the primary rRNA-binding ribosomal protein L25 from Saccharomyces cerevisiae. In vitro binding studies indicated that both r-proteins are able to recognise the L25 binding site on yeast 25 S rRNA and its structural homologue on mammalian 28 S rRNA with equal efficiency. To determine whether the two r-proteins are also functionally equivalent in vivo, a single plasmid-borne copy of either the wild-type L25 gene or the RL23a cDNA, driven by the L25 promoter, was introduced into a yeast strain in which the chromosomal L25 gene is under control of the glucose-repressible GALI-10 promoter. No difference in growth rate could be detected between the two types of transformants when cultured on glucose-based medium. In cells that co-express epitope-tagged versions of L25 and RL23a from single-copy genes, approximately 35% of the 60 S subunits contained the heterologous protein as determined by Western analysis. This value could be increased to 55% by overexpressing RL23a using a multi-copy plasmid. These data demonstrate that rat RL23a can act as a highly efficient substitute for its yeast counterpart in the assembly of functional yeast ribosomes even in the presence of the endogenous L25 protein.

Amino Acid Sequence↗
PubMed1996

Overexpression of binding protein and disruption of the PMR1 gene synergistically stimulate secretion of bovine prochymosin but not plant thaumatin in yeast.

When the heterologous proteins thaumatin and bovine prochymosin are produced in yeast cells as a fusion with the yeast invertase secretory signal peptide, less than 2% of the product is secreted in a biologically active form into the medium. The remainder accumulates intracellularly in a misfolded conformation. We investigated whether this poor secretion can be improved by overexpression of binding protein (BiP) one of the major chaperones in eukaryotic cells. Indeed, a tenfold increase in the level of binding protein, as a result of the introduction of extra copies of the kar2 gene into yeast cells containing a single, integrated copy of the invertase/prochymosin fusion gene, caused more than a 20-fold increase in the amount of extracellular prochymosin. By additional disruption of the PMR1 gene of these cells we were able to obtain secretion of virtually all of the prochymosin produced. Export of thaumatin, on the other hand, was not significantly stimulated by binding protein overexpression.

Animals↗
PubMed1995

Evolutionarily conserved structural elements are critical for processing of Internal Transcribed Spacer 2 from Saccharomyces cerevisiae precursor ribosomal RNA.

Structural features of Internal Transcribed Spacer 2 (ITS2) important for the correct and efficient removal of this spacer from Saccharomyces cerevisiae pre-rRNA were identified by in vivo mutational analysis based upon phylogenetic comparison with its counterparts from four different yeast species. Compatibility between ITS2 structure and the S. cerevisiae processing machinery was found to have been maintained over only a short evolutionary distance, in contrast to the situation for ITS1. Nevertheless, cis-acting elements required for correct and efficient processing are confined predominantly to those regions of the spacer that show the highest degree of evolutionary conservation. Mutation or deletion of each of these regions severely reduced production of mature 26 S, but not 17 S rRNA, mainly by impeding processing of the 29 SB precursor. In some cases, however, conversion of 29SA into 29 SB pre-rRNA also appeared to be affected. Deletion of non-conserved segments, on the other hand, caused little or no disturbance in processing. Surprisingly, some combinations of such individually neutral deletions had a severe negative effect on the removal of ITS2, suggesting a requirement for a higher-order structure of ITS2. Finally, even structural alterations of ITS2 that did not noticeably affect processing, significantly reduced the growth rate of cells that exclusively express the mutant rDNA units. We take this as further evidence for a direct role of ITS2 in the formation of fully functional 60 S ribosomal subunits.

Base Sequence↗
PubMed1995

Development and application of an in vivo system to study yeast ribosomal RNA biogenesis and function.

We have developed a system for mutational analysis of Saccharomyces cerevisiae ribosomal RNA in vivo in which yeast cells can be made completely dependent on mutant rRNA and ribosomes by a simple switch in carbon source. The system is based on a yeast strain defective in RNA polymerase I (Pol I) transcription [Nogi et al. (1991) Proc. Natl. Acad. Sci. USA 88, 3962-3966]. This normally inviable strain was rescued by integration of multiple copies of the complete 37S pre-rRNA operon under control of the inducible, Pol II-transcribed GAL7 promoter into the rDNA repeat on chromosome XII. The resulting YJV100 strain can only grow on medium containing galactose as the carbon source. A second, episomal vector was constructed in which the rDNA unit was placed under control of the constitutive PGK1 promoter. YJV100 cells transformed with this vector are now also able to grow on glucose-based medium making the cells completely dependent on plasmid-encoded rRNA. We show that the Pol II-transcribed pre-rRNA is processed and assembled similarly to authentic Pol I-synthesised pre-rRNA, making this 'in vivo Pol II system' suitable for the detailed analysis of rRNA mutations, even highly deleterious ones, affecting ribosome biogenesis or function. A clear demonstration of this is our finding that an insertion into variable region V8 in 17S rRNA, previously judged to be neutral with respect to processing of 17S rRNA, its assembly into 40S subunits and the polysomal distribution of these subunits [Musters et al. (1989), Mol. Cell. Biol. 9, 551-559], is in fact a lethal mutation.

Base Sequence↗
PubMed1995

The pathway to maturity: processing of ribosomal RNA in Saccharomyces cerevisiae.

The 17-18S, 5.8S, and 25-28S rRNA species of eukaryotic cells are transcribed by RNA polymerase I into a single precursor molecule, from which external and internal spacer sequences are subsequently removed in an order series of nucleolytic reactions. Whereas the order of the cleavage reactions has long been established, only recently has significant progress been made in detailing the cis-acting elements and the trans-acting factors involved in this process. The use of recently developed systems for in vivo mutational analysis of yeast rDNA has greatly enhanced our knowledge of cis-acting structural features within the pre-rRNA, which are critical for correct and efficient removal of the spacer sequences. The same systems also allow a link to be forged between trans-acting processing factors and these cis-acting elements. In this review the newly obtained information will be summarized, focused predominantly on pre-rRNA processing in the yeast Saccharomyces cerevisiae.

Fungal Proteins↗
PubMed1994

Mutational analysis of the C-terminal region of Saccharomyces cerevisiae ribosomal protein L25 in vitro and in vivo demonstrates the presence of two distinct functional elements.

A previous analysis of yeast ribosomal protein L25 implicated an evolutionarily conserved motif of seven amino acids near the C terminus (positions 120 to 126) in specific binding of the protein to domain III of 26 S rRNA. We analyzed the effect of various point mutations in this amino acid sequence on the capacity of the protein to interact in vitro with its binding site on the rRNA. Most of the mutations tested, including some conservative replacements, strongly reduced or abolished rRNA binding, further supporting a pivotal role for the motif in the specific interaction between L25 and 26 S rRNA. We have also determined the ability of the various mutant L25 species to complement in vivo for the absence of wild-type protein in cells that conditionally express the chromosomal L25 gene. Surprisingly, up to a fivefold reduction in the in vitro binding capacity of L25 is tolerated without affecting the ability of the mutant protein to support (virtually) wild-type rates of 60 S subunit formation and cell growth. Mutations that completely abolish recognition of 26 S rRNA, however, block the formation of 60 S particles, demonstrating that binding of L25 to this rRNA is an essential step in the assembly of the large ribosomal subunit. Using the same combination of approaches we identified an element, located between positions 133 and 139, that is indispensable for the ability of L25 to support a normal rate of 60 S subunit formation, but plays a relatively minor role in determining the rRNA-binding capacity of the protein. In particular, the presence of a hydrophobic amino acid at position 135 was found to be highly important. These results indicate that the element in question is crucial for a step in the assembly of the 60 S subunit subsequent to association of L25 with 26 S rRNA.

Amino Acid Sequence↗
PubMed1994

Separate structural elements within internal transcribed spacer 1 of Saccharomyces cerevisiae precursor ribosomal RNA direct the formation of 17S and 26S rRNA.

Structural features of Internal Transcribed Spacer 1 (ITS1) that direct its removal from Saccharomyces cerevisiae pre-rRNA during processing were identified by an initial phylogenetic approach followed by in vivo mutational analysis of specific structural elements. We found that S. cerevisiae ITS1 can functionally be replaced by the corresponding regions from the yeasts Torulaspora delbrueckii, Kluyveromyces lactis and Hansenula wingei, indicating that structural elements required in cis for processing are evolutionarily conserved. Despite large differences in size, all ITS1 regions conform to the secondary structure proposed by Yeh et al. [Biochemistry 29 (1990) 5911-5918], showing five domains (I-V; 5'-->3') of which three harbour an evolutionarily highly conserved element. Removal of most of domain II, including its highly conserved element, did not affect processing. In contrast, highly conserved nucleotides directly downstream of processing site A2 in domain III play a major role in production of 17S, but not 26S rRNA. Domain IV and V are dispensable for 17S rRNA formation although an alternative, albeit inefficient, processing route to mature 17S rRNA may be mediated by a conserved region in domain IV. Each of these two domains is individually sufficient for efficient production of 26S rRNA, suggesting two independent processing pathways. We conclude that ITS1 is organized into two functionally and structurally distinct halves.

Base Sequence↗