scholarly journals Mrd1p Is Required for Release of Base-Paired U3 snoRNA within the Preribosomal Complex

2009 ◽  
Vol 29 (21) ◽  
pp. 5763-5774 ◽  
Author(s):  
Pär Lundkvist ◽  
Sara Jupiter ◽  
Åsa Segerstolpe ◽  
Yvonne N. Osheim ◽  
Ann L. Beyer ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Rna Binding ◽  
Protein Complexes ◽  
Fundamental Problem ◽  
Ribosomal Subunit ◽  
Binding Domains ◽  
U3 Snorna ◽  
Conformational Rearrangements ◽  
Key Events ◽  
Processing Factors

ABSTRACT In eukaryotes, ribosomes are made from precursor rRNA (pre-rRNA) and ribosomal proteins in a maturation process that requires a large number of snoRNPs and processing factors. A fundamental problem is how the coordinated and productive folding of the pre-rRNA and assembly of successive pre-rRNA-protein complexes is achieved cotranscriptionally. The conserved protein Mrd1p, which contains five RNA binding domains (RBDs), is essential for processing events leading to small ribosomal subunit synthesis. We show that full function of Mrd1p requires all five RBDs and that the RBDs are functionally distinct and needed during different steps in processing. Mrd1p mutations trap U3 snoRNA in pre-rRNP complexes both in base-paired and non-base-paired interactions. A single essential RBD, RBD5, is involved in both types of interactions, but its conserved RNP1 motif is not needed for releasing the base-paired interactions. RBD5 is also required for the late pre-rRNP compaction preceding A2 cleavage. Our results suggest that Mrd1p modulates successive conformational rearrangements within the pre-rRNP that influence snoRNA-pre-rRNA contacts and couple U3 snoRNA-pre-rRNA remodeling and late steps in pre-rRNP compaction that are essential for cleavage at A0 to A2. Mrd1p therefore coordinates key events in biosynthesis of small ribosome subunits.

scholarly journals Imp3p and Imp4p, Two Specific Components of the U3 Small Nucleolar Ribonucleoprotein That Are Essential for Pre-18S rRNA Processing

1999 ◽  
Vol 19 (8) ◽  
pp. 5441-5452 ◽  
Author(s):  
Sarah J. Lee ◽  
Susan J. Baserga
Keyword(s):  
Ribosomal Proteins ◽  
18S Rrna ◽  
Rna Binding ◽  
Specific Protein ◽  
Rrna Processing ◽  
Binding Domains ◽  
U3 Snorna ◽  
Genes Encoding ◽  
Protein Components

ABSTRACT The function of the U3 small nucleolar ribonucleoprotein (snoRNP) is central to the events surrounding pre-rRNA processing, as evidenced by the severe defects in cleavage of pre-18S rRNA precursors observed upon depletion of the U3 RNA and its unique protein components. Although the precise function of each component remains unclear, since U3 snoRNA levels remain unchanged upon genetic depletion of these proteins, it is likely that the proteins themselves have significant roles in the cleavage reactions. Here we report the identification of two previously undescribed protein components of the U3 snoRNP, representing the first snoRNP components identified by using the two-hybrid methodology. By screening for proteins that physically associate with the U3 snoRNP-specific protein, Mpp10p, we have identified Imp3p (22 kDa) and Imp4p (34 kDa) (named for interacting with Mpp10p). The genes encoding both proteins are essential in yeast. Genetic depletion reveals that both proteins are critical for U3 snoRNP function in pre-18S rRNA processing at the A0, A1, and A2 sites in the pre-rRNA. Both Imp proteins associate with Mpp10p in vivo, and both are complexed only with the U3 snoRNA. Conservation of RNA binding domains between Imp3p and the S4 family of ribosomal proteins suggests that it might associate with RNA directly. However, as with other U3 snoRNP-specific proteins, neither Imp3p nor Imp4p is required for maintenance of U3 snoRNA integrity. Imp3p and Imp4p are therefore novel protein components specific to the U3 snoRNP with critical roles in pre-rRNA cleavage events.

scholarly journals Ribosomal protein L7a binds RNA through two distinct RNA-binding domains

2004 ◽  
Vol 385 (1) ◽  
pp. 289-299 ◽  
Author(s):  
Giulia RUSSO ◽  
Monica CUCCURESE ◽  
Gianluca MONTI ◽  
Annapina RUSSO ◽  
Angela AMORESANO ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Rna Binding ◽  
Limited Proteolysis ◽  
Ribosomal Subunit ◽  
The Other ◽  
Protein Domain ◽  
Binding Motif ◽  
Nucleic Acid Binding ◽  
Binding Domains ◽  
Rna Binding Domains

The human ribosomal protein L7a is a component of the major ribosomal subunit. We previously identified three nuclear-localization-competent domains within L7a, and demonstrated that the domain defined by aa (amino acids) 52–100 is necessary, although not sufficient, to target the L7a protein to the nucleoli. We now demonstrate that L7a interacts in vitro with a presumably G-rich RNA structure, which has yet to be defined. We also demonstrate that the L7a protein contains two RNA-binding domains: one encompassing aa 52–100 (RNAB1) and the other encompassing aa 101–161 (RNAB2). RNAB1 does not contain any known nucleic-acid-binding motif, and may thus represent a new class of such motifs. On the other hand, a specific region of RNAB2 is highly conserved in several other protein components of the ribonucleoprotein complex. We have investigated the topology of the L7a–RNA complex using a recombinant form of the protein domain that encompasses residues 101–161 and a 30mer poly(G) oligonucleotide. Limited proteolysis and cross-linking experiments, and mass spectral analyses of the recombinant protein domain and its complex with poly(G) revealed the RNA-binding region.

scholarly journals Factors Affecting Nuclear Export of the 60S Ribosomal Subunit In Vivo

2000 ◽  
Vol 11 (11) ◽  
pp. 3777-3789 ◽  
Author(s):  
Tracy Stage-Zimmermann ◽  
Ute Schmidt ◽  
Pamela A. Silver
Keyword(s):  
Ribosomal Protein ◽  
Nuclear Export ◽  
Ribosomal Proteins ◽  
Protein Import ◽  
Fluorescent Protein ◽  
Ribosomal Subunit ◽  
Rrna Processing ◽  
Factors Affecting ◽  
Processing Factors

In Saccharomyces cerevisiae, the 60S ribosomal subunit assembles in the nucleolus and then is exported to the cytoplasm, where it joins the 40S subunit for translation. Export of the 60S subunit from the nucleus is known to be an energy-dependent and factor-mediated process, but very little is known about the specifics of its transport. To begin to address this problem, an assay was developed to follow the localization of the 60S ribosomal subunit inS. cerevisiae. Ribosomal protein L11b (Rpl11b), one of the ∼45 ribosomal proteins of the 60S subunit, was tagged at its carboxyl terminus with the green fluorescent protein (GFP) to enable visualization of the 60S subunit in living cells. A panel of mutant yeast strains was screened for their accumulation of Rpl11b–GFP in the nucleus as an indicator of their involvement in ribosome synthesis and/or transport. This panel included conditional alleles of several rRNA-processing factors, nucleoporins, general transport factors, and karyopherins. As predicted, conditional alleles of rRNA-processing factors that affect 60S ribosomal subunit assembly accumulated Rpl11b–GFP in the nucleus. In addition, several of the nucleoporin mutants as well as a few of the karyopherin and transport factor mutants also mislocalized Rpl11b–GFP. In particular, deletion of the previously uncharacterized karyopherin KAP120 caused accumulation of Rpl11b–GFP in the nucleus, whereas ribosomal protein import was not impaired. Together, these data further define the requirements for ribosomal subunit export and suggest a biological function for KAP120.

scholarly journals The Role of the Schizosaccharomyces pombe gar2 Protein in Nucleolar Structure and Function Depends on the Concerted Action of its Highly Charged N Terminus and its RNA-binding Domains

1998 ◽  
Vol 9 (8) ◽  
pp. 2011-2023 ◽  
Author(s):  
Hélène Sicard ◽  
Marlène Faubladier ◽  
Jacqueline Noaillac-Depeyre ◽  
Isabelle Léger-Silvestre ◽  
Nicole Gas ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
18S Rrna ◽  
Rna Binding ◽  
Point Mutations ◽  
Ribosomal Subunit ◽  
Structural Domain ◽  
Nucleolar Structure ◽  
Terminal Domain ◽  
Binding Domains ◽  
Rna Binding Domains

Nonribosomal nucleolar protein gar2 is required for 18S rRNA and 40S ribosomal subunit production in Schizosaccharomyces pombe. We have investigated the consequences of the absence of each structural domain of gar2 on cell growth, 18S rRNA production, and nucleolar structure. Deletion of gar2 RNA-binding domains (RBDs) causes stronger inhibition of growth and 18S rRNA accumulation than the absence of the whole protein, suggesting that other factors may be titrated by its remaining N-terminal basic/acidic serine-rich domain. These drastic functional defects correlate with striking nucleolar hypertrophy. Point mutations in the conserved RNP1 motifs of gar2 RBDs supposed to inhibit RNA–protein interactions are sufficient to induce severe nucleolar modifications but only in the presence of the N-terminal domain of the protein. Gar2 and its mutants also distribute differently in glycerol gradients: gar2 lacking its RBDs is found either free or assembled into significantly larger complexes than the wild-type protein. We propose that gar2 helps the assembly on rRNA of factors necessary for 40S subunit synthesis by providing a physical link between them. These factors may be recruited by the N-terminal domain of gar2 and may not be released if interaction of gar2 with rRNA is impaired.

scholarly journals Assembly of the Trypanosoma brucei 60S Ribosomal Subunit Nuclear Export Complex Requires Trypanosome-Specific Proteins P34 and P37

2008 ◽  
Vol 8 (1) ◽  
pp. 77-87 ◽  
Author(s):  
Kimberly Prohaska ◽  
Noreen Williams
Keyword(s):  
Trypanosoma Brucei ◽  
Nuclear Export ◽  
Ribosomal Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Ribosomal Subunit ◽  
Subcellular Fractionation ◽  
Cellular Protein ◽  
Ribosomal Biogenesis ◽  
Specific Proteins

ABSTRACT We previously identified two Trypanosoma brucei RNA binding proteins, P34 and P37, and determined that they are essential for proper ribosomal assembly in this organism. Loss of these proteins via RNA interference is lethal and causes a decrease in both 5S rRNA levels and formation of 80S ribosomes, concomitant with a decrease in total cellular protein synthesis. These data suggest that these proteins are involved at some point in the ribosomal biogenesis pathway. In the current study, we have performed subcellular fractionation in conjunction with immune capture experiments specific for 60S ribosomal proteins and accessory factors in order to determine when and where P34 and P37 are involved in the ribosomal biogenesis pathway. These studies demonstrate that P34 and P37 associate with the 60S ribosomal subunit at the stage of the nucleolar 90S particle and remain associated subsequent to nuclear export. In addition, P34 and P37 associate with conserved 60S ribosomal subunit nuclear export factors exportin 1 and Nmd3, suggesting that they are components of the 60S ribosomal subunit nuclear export complex in T. brucei. Most significantly, the pre-60S complex does not associate with exportin 1 or Nmd3 in the absence of P34 and P37. These results demonstrate that, although T. brucei 60S ribosomal subunits utilize a nuclear export complex similar to that described for other organisms, trypanosome-specific factors are essential to the process.

scholarly journals The Small-Subunit Processome Is a Ribosome Assembly Intermediate

2004 ◽  
Vol 3 (6) ◽  
pp. 1619-1626 ◽  
Author(s):  
Kara A. Bernstein ◽  
Jennifer E. G. Gallagher ◽  
Brianna M. Mitchell ◽  
Sander Granneman ◽  
Susan J. Baserga
Keyword(s):  
Ribosomal Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Ribosome Assembly ◽  
Bona Fide ◽  
Protein Components ◽  
Ssu Processome

ABSTRACT 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.

scholarly journals A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis

2002 ◽  
Vol 13 (10) ◽  
pp. 3683-3695 ◽  
Author(s):  
Petra Björk ◽  
Göran Baurén ◽  
ShaoBo Jin ◽  
Yong-Guang Tong ◽  
Thomas R. Bürglin ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Ribosomal Subunit ◽  
Binding Domain ◽  
Dependent Manner ◽  
Binding Domains ◽  
40S Ribosomal Subunit ◽  
Rna Binding Domain

Synthesis of the ribosomal subunits from pre-rRNA requires a large number of trans-acting proteins and small nucleolar ribonucleoprotein particles to execute base modifications, RNA cleavages, and structural rearrangements. We have characterized a novel protein, RNA-binding domain-1 (RBD-1), that is involved in ribosome biogenesis. This protein contains six consensus RNA-binding domains and is conserved as to sequence, domain organization, and cellular location from yeast to human. RBD-1 is essential in Caenorhabditis elegans. In the dipteran Chironomus tentans, RBD-1 (Ct-RBD-1) binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In cytoplasmic extracts, 20–30% of Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.

RNase R mutants elucidate the catalysis of structured RNA: RNA-binding domains select the RNAs targeted for degradation

2009 ◽  
Vol 423 (2) ◽  
pp. 291-301 ◽  
Author(s):  
Rute Gonçalves Matos ◽  
Ana Barbas ◽  
Cecília Maria Arraiano
Keyword(s):  
Rna Binding ◽  
Protein Complexes ◽  
Rna Degradation ◽  
Rnase R ◽  
Independent Manner ◽  
Binding Domains ◽  
Modular Domain ◽  
Rnase Ii ◽  
Rna Binding Domains ◽  
First Time

The RNase II superfamily is a ubiquitous family of exoribonucleases that are essential for RNA metabolism. RNase II and RNase R degrade RNA in the 3′→5′ direction in a processive and sequence-independent manner. However, although RNase R is capable of degrading highly structured RNAs, the RNase II activity is impaired by the presence of secondary structures. RNase II and RNase R share structural properties and have a similar modular domain organization. The eukaryotic RNase II homologue, Rrp44/Dis3, is the catalytic subunit of the exosome, one of the most important protein complexes involved in the maintenance of the correct levels of cellular RNAs. In the present study, we constructed truncated RNase II and RNase R proteins and point mutants and characterized them regarding their exoribonucleolytic activity and RNA-binding ability. We report that Asp280 is crucial for RNase R activity without affecting RNA binding. When Tyr324 was changed to alanine, the final product changed from 2 to 5 nt in length, showing that this residue is responsible for setting the end-product. We have shown that the RNB domain of RNase II has catalytic activity. The most striking result is that the RNase R RNB domain itself degrades double-stranded substrates even in the absence of a 3′-overhang. Moreover, we have demonstrated for the first time that the substrate recognition of RNase R depends on the RNA-binding domains that target the degradation of RNAs that are ‘tagged’ by a 3′-tail. These results can have important implications for the study of poly(A)-dependent RNA degradation mechanisms.

Structural organization of escherichia coli ribosomes as determined by immuno electron microscopy

1978 ◽  
Vol 36 (2) ◽  
pp. 166-167 ◽  
Author(s):  
G. Stöffler ◽  
R.W. Bald ◽  
J. Dieckhoff ◽  
H. Eckhard ◽  
R. Lührmann ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Ribosomal Proteins ◽  
Structural Organization ◽  
Three Dimensional ◽  
Ribosomal Subunit ◽  
Spatial Arrangement ◽  
Small Subunit ◽  
Antibody Binding ◽  
Immuno Electron Microscopy

A central step towards an understanding of the structure and function of the Escherichia coli ribosome, a large multicomponent assembly, is the elucidation of the spatial arrangement of its 54 proteins and its three rRNA molecules. The structural organization of ribosomal components has been investigated by a number of experimental approaches. Specific antibodies directed against each of the 54 ribosomal proteins of Escherichia coli have been performed to examine antibody-subunit complexes by electron microscopy. The position of the bound antibody, specific for a particular protein, can be determined; it indicates the location of the corresponding protein on the ribosomal surface.The three-dimensional distribution of each of the 21 small subunit proteins on the ribosomal surface has been determined by immuno electron microscopy: the 21 proteins have been found exposed with altogether 43 antibody binding sites. Each one of 12 proteins showed antibody binding at remote positions on the subunit surface, indicating highly extended conformations of the proteins concerned within the 30S ribosomal subunit; the remaining proteins are, however, not necessarily globular in shape (Fig. 1).

Protein-induced conformational changes in 16S rRNA during assembly of the Escherichia Coli 30S ribosomal subunit: A STEM study

1987 ◽  
Vol 45 ◽  
pp. 910-911
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
16S Rrna ◽  
Conformational Changes ◽  
Ribosomal Proteins ◽  
Structural Changes ◽  
Ribosomal Subunit ◽  
Compact Structure ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
30S Subunit

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

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