Biomedical subjects

Sander Granneman

Publications and source records attributed to Sander Granneman.

13 recordsLinked to original sources

PubMed2006

The nucleolar protein Esf2 interacts directly with the DExD/H box RNA helicase, Dbp8, to stimulate ATP hydrolysis.

While 18 putative RNA helicases are involved in ribosome biogenesis in Saccharomyces cerevisiae, their enzymatic properties have remained largely biochemically uncharacterized. To better understand their function, we examined the enzymatic properties of Dpb8, a DExD/H box protein previously shown to be required for the synthesis of the 18S rRNA. As expected for an RNA helicase, we demonstrate that recombinant Dbp8 has ATPase activity in vitro, and that this activity is dependent on an intact ATPase domain. Strikingly, we identify Esf2, a nucleolar putative RNA binding protein, as a binding partner for Dbp8, and show that it enhances Dbp8 ATPase activity by decreasing the K(M) for ATP. Thus, we have uncovered Esf2 as the first example of a protein co-factor that has a stimulatory effect on a nucleolar RNA helicase. We show that Esf2 can bind to pre-rRNAs and speculate that it may function to bring Dbp8 to the pre-rRNA, thereby both regulating its enzymatic activity and guiding Dbp8 to its site of action.

Adenosine Triphosphatases↗

PubMed2006

The PINc domain protein Utp24, a putative nuclease, is required for the early cleavage steps in 18S rRNA maturation.

Ribosome biogenesis is a complex process that requires >150 transacting factors, many of which form macromolecular assemblies as big and complex as the ribosome itself. One of those complexes, the SSU processome, is required for pre-18S rRNA maturation. Although many of its components have been identified, the endonucleases that cleave the pre-18S rRNA have remained mysterious. Here we examine the role of four previously uncharacterized PINc domain proteins, which are predicted to function as nucleases, in yeast ribosome biogenesis. We also included Utp23, a protein homologous to the PINc domain protein Utp24, in our analysis. Our results demonstrate that Utp23 and Utp24 are essential nucleolar proteins and previously undescribed components of the SSU processome. In that sense, both Utp23 and Utp24 are required for the first three cleavage steps in 18S rRNA maturation. In addition, single-point mutations in the conserved putative active site of Utp24 but not Utp23 abrogate its function in ribosome biogenesis. Our results suggest that Utp24 might be the elusive endonuclease that cleaves the pre-rRNA at sites A(1) and/or A(2.).

Base Sequence↗

PubMed2006

Comprehensive mutational analysis of yeast DEXD/H box RNA helicases required for small ribosomal subunit synthesis.

The 17 putative RNA helicases required for pre-rRNA processing are predicted to play a crucial role in ribosome biogenesis by driving structural rearrangements within preribosomes. To better understand the function of these proteins, we have generated a battery of mutations in five putative RNA helicases involved in 18S rRNA synthesis and analyzed their effects on cell growth and pre-rRNA processing. Our results define functionally important residues within conserved motifs and demonstrate that lethal mutations in predicted ATP binding-hydrolysis motifs often confer a dominant negative phenotype in vivo when overexpressed in a wild-type background. We show that dominant negative mutants delay processing of the 35S pre-rRNA and cause accumulation of pre-rRNA species that normally have low steady-state levels. Our combined results establish that not all conserved domains function identically in each protein, suggesting that the RNA helicases may have distinct biochemical properties and diverse roles in ribosome biogenesis.

Alleles↗

PubMed2006

Comprehensive mutational analysis of yeast DEXD/H box RNA helicases involved in large ribosomal subunit biogenesis.

DEXD/H box putative RNA helicases are required for pre-rRNA processing in Saccharomyces cerevisiae, although their exact roles and substrates are unknown. To characterize the significance of the conserved motifs for helicase function, a series of five mutations were created in each of the eight essential RNA helicases (Has1, Dbp6, Dbp10, Mak5, Mtr4, Drs1, Spb4, and Dbp9) involved in 60S ribosomal subunit biogenesis. Each mutant helicase was screened for the ability to confer dominant negative growth defects and for functional complementation. Different mutations showed different degrees of growth inhibition among the helicases, suggesting that the conserved regions do not function identically in vivo. Mutations in motif I and motif II (the DEXD/H box) often conferred dominant negative growth defects, indicating that these mutations do not interfere with substrate binding. In addition, mutations in the putative unwinding domains (motif III) demonstrated that conserved amino acids are often not essential for function. Northern analysis of steady-state RNA from strains expressing mutant helicases showed that the dominant negative mutations also altered pre-rRNA processing. Coimmunoprecipitation experiments indicated that some RNA helicases associated with each other. In addition, we found that yeasts disrupted in expression of the two nonessential RNA helicases, Dbp3 and Dbp7, grew worse than when either one alone was disrupted.

Alleles↗

PubMed2005

Crosstalk in gene expression: coupling and co-regulation of rDNA transcription, pre-ribosome assembly and pre-rRNA processing.

Ribosomes, the large RNPs that translate mRNA into protein in the cytoplasm of eukaryotic cells, are synthesized in a subcompartment of the nucleus, the nucleolus. There, transcription by Pol I yields a pre-rRNA which is modified, cleaved and assembled with ribosomal proteins to make functional ribosomes. Previously, rRNA transcription and pre-rRNA cleavage in eukaryotes were considered to be separable steps in gene expression. However, recent findings suggest that these two steps in gene expression can be concurrent and are co-regulated. Unexpectedly, optimal rDNA transcription requires the presence of a defined subset of components of the pre-rRNA processing machinery.

Animals↗

PubMed2005

The putative NTPase Fap7 mediates cytoplasmic 20S pre-rRNA processing through a direct interaction with Rps14.

One of the proteins identified as being involved in ribosome biogenesis by high-throughput studies, a putative P-loop-type kinase termed Fap7 (YDL166c), was shown to be required for the conversion of 20S pre-rRNA to 18S rRNA. However, the mechanism underlying this function has remained unclear. Here we demonstrate that Fap7 is strictly required for cleavage of the 20S pre-rRNA at site D in the cytoplasm. Genetic depletion of Fap7 causes accumulation of only the 20S pre-rRNA, which could be detected not only in 43S preribosomes but also in 80S-sized complexes. Fap7 is not a structural component of 43S preribosomes but likely transiently interacts with them by directly binding to Rps14, a ribosomal protein that is found near the 3' end of the 18S rRNA. Consistent with an NTPase activity, conserved residues predicted to be required for nucleoside triphosphate (NTP) hydrolysis are essential for Fap7 function in vivo. We propose that Fap7 mediates cleavage of the 20S pre-rRNA at site D by directly interacting with Rps14 and speculate that it is an enzyme that functions as an NTP-dependent molecular switch in 18S rRNA maturation.

Adenylate Kinase↗

PubMed2004

RNA polymerase I transcription and pre-rRNA processing are linked by specific SSU processome components.

Sequential events in macromolecular biosynthesis are often elegantly coordinated. The small ribosomal subunit (SSU) processome is a large ribonucleoprotein (RNP) required for processing of precursors to the small subunit RNA, the 18S, of the ribosome. We have found that a subcomplex of SSU processome proteins, the t-Utps, is also required for optimal rRNA transcription in vivo in the yeast Saccharomyces cerevisiae. The t-Utps are ribosomal chromatin (r-chromatin)-associated, and they exist in a complex in the absence of the U3 snoRNA. Transcription is required neither for the formation of the subcomplex nor for its r-chromatin association. The t-Utps are associated with the pre-18S rRNAs independent of the presence of the U3 snoRNA. This association may thus represent an early step in the formation of the SSU processome. Our results indicate that rRNA transcription and pre-rRNA processing are coordinated via specific components of the SSU processome.

Blotting, Western↗

PubMed2004

Ribosome biogenesis: of knobs and RNA processing.

The synthesis of ribosomes in eukaryotes involves processing of pre-ribosomal RNA (pre-rRNA) and sequential assembly of a large number of ribosomal proteins on the rRNAs. Although we have gained tremendous insights into the processing of pre-rRNA intermediates in the last three decades, little was known about the dynamic nature of ribosome biogenesis. Only recently the development of efficient affinity-purification procedures and mass-spectrometry techniques has allowed the isolation of large pre-ribosomal complexes, which led to the identification of several ribosome assembly intermediates and a large number of novel ribosome assembly factors. In this mini-review, we summarize some of the discoveries that have been made in the field of ribosome biogenesis in the past 30 years and highlight some key aspects about what remains to be learned.

Macromolecular Substances↗

PubMed2004

The small-subunit processome is a ribosome assembly intermediate.

The small-subunit (SSU) processome is a large ribonucleoprotein required for the biogenesis of the 18S rRNA and likely corresponds to the terminal knobs visualized by electron microscopy on the 5' end of nascent rRNAs. The original purification of the SSU processome of Saccharomyces cerevisiae resulted in the identification of 28 proteins. Here, we characterize 12 additional protein components, including five small-ribosomal-subunit proteins (Rps4, Rps6, Rps7, Rps9, and Rps14) that had previously been copurified. Our multiple criteria for including a component as a bona fide SSU processome component included coimmunoprecipitation with Mpp10 (an SSU processome component), the U3 snoRNA, and the anticipated pre-rRNAs. Importantly, the association of specific ribosomal proteins with the SSU processome suggests that the SSU processome has roles in both pre-rRNA processing and ribosome assembly. These ribosomal proteins may be analogous to the primary or secondary RNA binding proteins first described in bacterial in vitro ribosome assembly maps. In addition to the ribosomal proteins and based on the same experimental approach, we found seven other proteins (Utp18, Noc4, Utp20, Utp21, Utp22, Emg1, and Krr1) to be bona fide SSU processome proteins.

Blotting, Northern↗

PubMed2004

Role of pre-rRNA base pairing and 80S complex formation in subnucleolar localization of the U3 snoRNP.

In the nucleolus the U3 snoRNA is recruited to the 80S pre-rRNA processing complex in the dense fibrillar component (DFC). The U3 snoRNA is found throughout the nucleolus and has been proposed to move with the preribosomes to the granular component (GC). In contrast, the localization of other RNAs, such as the U8 snoRNA, is restricted to the DFC. Here we show that the incorporation of the U3 snoRNA into the 80S processing complex is not dependent on pre-rRNA base pairing sequences but requires the B/C motif, a U3-specific protein-binding element. We also show that the binding of Mpp10 to the 80S U3 complex is dependent on sequences within the U3 snoRNA that base pair with the pre-rRNA adjacent to the initial cleavage site. Furthermore, mutations that inhibit 80S complex formation and/or the association of Mpp10 result in retention of the U3 snoRNA in the DFC. From this we propose that the GC localization of the U3 snoRNA is a direct result of its active involvement in the initial steps of ribosome biogenesis.

Cell Nucleolus↗

PubMed2003

Probing the yeast proteome for RNA-processing factors.

A method has been developed to identify proteins required for the biogenesis of non-coding RNA in yeast, using a microarray to screen for aberrant patterns of RNA processing in mutant strains, and new proteins involved in the processing of ribosomal and non-coding RNAs have been found.

Fungal Proteins↗

PubMed2003

The human Imp3 and Imp4 proteins form a ternary complex with hMpp10, which only interacts with the U3 snoRNA in 60-80S ribonucleoprotein complexes.

Ribosome biogenesis requires a vast number of trans-acting factors many of which are required for the chemical modification and processing of the pre-rRNA component. The U3 snoRNP complex is required for the early cleavage steps in pre-rRNA processing. We have cloned cDNAs encoding the human and mouse homologs of the yeast U3 snoRNP-associated proteins Imp3 and Imp4. Both human proteins localize to nucleoli and interact with the U3 snoRNA. The results of complementation experiments show that, in contrast to mouse Imp4, mouse Imp3 can partially alleviate the growth defect of the corresponding yeast null strain, indicating that the role of Imp3 in pre-rRNA processing is evolutionarily conserved. The results of density gradient centrifugation experiments show that, in contrast to hU3-55K, the human Imp3 and Imp4 proteins predominantly interact with the U3 snoRNA in 60-80S ribonucleoprotein complexes. In addition, we have found that hImp3, hImp4 and hMpp10 can form a stable hetero-trimeric complex in vitro, which is generated by direct interactions of both hImp3 and hImp4 with hMpp10. The analysis of hImp3 and hImp4 mutants indicated that their binding to hMpp10 correlates with their nucleolar accumulation, strongly suggesting that the formation of the ternary complex of hImp3, hImp4 and hMpp10 is required for their association with nucleolar components.

Amino Acid Sequence↗

PubMed2002

The hU3-55K protein requires 15.5K binding to the box B/C motif as well as flanking RNA elements for its association with the U3 small nucleolar RNA in Vitro.

The 15.5K protein directly binds to the 5' stem-loop of the U4 small nuclear RNA, the small nucleolar (sno) RNA box C/D motif, and the U3 snoRNA-specific box B/C motif. The box B/C motif has also been shown to be essential for the association of the U3 small nucleolar ribonucleoprotein-specific protein hU3-55K. We therefore set out to determine how 15.5K and hU3-55K recognize the box B/C motif. By using an in vitro assembly assay, we show that hU3-55K effectively binds a sub-fragment of the U3 snoRNA surrounding the B/C motif that we have named the U3BC RNA. The association of hU3-55K with the U3BC RNA is dependent on the binding of 15.5K to the box B/C motif. The association of hU3-55K with the U3BC RNA was found to be also dependent on a conserved RNA structure that flanks the box B/C motif. Furthermore, we show that hU3-55K, a WD 40 repeat containing protein, directly cross-links to the U3BC RNA. Our data support a new structural model of the box B/C region of the U3 snoRNA in which the box B/C motif is base-paired to form a structure highly similar to that of both the U4 5' stem-loop and the box C/D motif.

Amino Acid Sequence↗