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 Synergistic defects in pre-rRNA processing from mutations in the U3-specific protein Rrp9 and U3 snoRNA

2020 ◽  
Vol 48 (7) ◽  
pp. 3848-3868 ◽  
Author(s):  
Guillaume Clerget ◽  
Valérie Bourguignon-Igel ◽  
Nathalie Marmier-Gourrier ◽  
Nicolas Rolland ◽  
Ludivine Wacheul ◽  
...  
Keyword(s):  
Protein Interaction Network ◽  
18S Rrna ◽  
Direct Interaction ◽  
Interaction Network ◽  
Specific Protein ◽  
Rrna Processing ◽  
Protein Protein Interaction ◽  
U3 Snorna ◽  
Ssu Processome ◽  
Protein Protein Interaction Network

Abstract U3 snoRNA and the associated Rrp9/U3-55K protein are essential for 18S rRNA production by the SSU-processome complex. U3 and Rrp9 are required for early pre-rRNA cleavages at sites A0, A1 and A2, but the mechanism remains unclear. Substitution of Arg 289 in Rrp9 to Ala (R289A) specifically reduced cleavage at sites A1 and A2. Surprisingly, R289 is located on the surface of the Rrp9 β-propeller structure opposite to U3 snoRNA. To understand this, we first characterized the protein-protein interaction network of Rrp9 within the SSU-processome. This identified a direct interaction between the Rrp9 β-propeller domain and Rrp36, the strength of which was reduced by the R289A substitution, implicating this interaction in the observed processing phenotype. The Rrp9 R289A mutation also showed strong synergistic negative interactions with mutations in U3 that destabilize the U3/pre-rRNA base-pair interactions or reduce the length of their linking segments. We propose that the Rrp9 β-propeller and U3/pre-rRNA binding cooperate in the structure or stability of the SSU-processome. Additionally, our analysis of U3 variants gave insights into the function of individual segments of the 5′-terminal 72-nt sequence of U3. We interpret these data in the light of recently reported SSU-processome structures.

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.

The N-terminal domain of Sxl protein disrupts Sxl autoregulation in females and promotes female-specific splicing of tra in males

Development ◽  
1999 ◽  
Vol 126 (13) ◽  
pp. 2841-2853 ◽  
Author(s):  
G. Deshpande ◽  
G. Calhoun ◽  
P.D. Schedl
Keyword(s):  
Fusion Protein ◽  
Rna Binding ◽  
Chimeric Protein ◽  
Dominant Negative ◽  
Terminal Domain ◽  
Binding Domains ◽  
Transcriptional Regulatory ◽  
Dominant Negative Activity ◽  
Female Specific

Sex determination in Drosophila depends upon the post-transcriptional regulatory activities of the Sex-lethal (Sxl) gene. Sxl maintains the female determined state and activates female differentiation pathways by directing the female-specific splicing of Sxl and tra pre-mRNAs. While there is compelling evidence that Sxl proteins regulate splicing by directly binding to target RNAs, previous studies indicate that the two Sxl RNA-binding domains are not in themselves sufficient for biological activity and that an intact N-terminal domain is also critical for splicing function. To further investigate the functions of the Sxl N terminus, we ectopically expressed a chimeric protein consisting of the N-terminal 99 amino acids fused to ss-galactosidase. The Nss-gal fusion protein behaves like a dominant negative, interfering with the Sxl autoregulatory feedback loop and killing females. This dominant negative activity can be attributed to the recruitment of the fusion protein into the large Sxl:Snf splicing complexes that are found in vivo and the consequent disruption of these complexes. In addition to the dominant negative activity, the Nss-gal fusion protein has a novel gain-of-function activity in males: it promotes the female-specific processing of tra pre-mRNAs. This novel activity is discussed in light of the blockage model for the tra splicing regulation.

scholarly journals In vivo reconstitution finds multivalent RNA-RNA interactions as drivers of mesh-like condensates

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Weirui Ma ◽  
Gang Zheng ◽  
Wei Xie ◽  
Christine Mayr
Keyword(s):  
Surface Area ◽  
Protein Trafficking ◽  
Rna Binding ◽  
Interaction Network ◽  
Large Surface Area ◽  
High Propensity ◽  
Membrane Protein Trafficking ◽  
Protein Components

Liquid-like condensates have been thought to be sphere-like. Recently, various condensates with filamentous morphology have been observed in cells. One such condensate is the TIS granule network that shares a large surface area with the rough endoplasmic reticulum and is important for membrane protein trafficking. It has been unclear how condensates with mesh-like shapes, but dynamic protein components are formed. In vitro and in vivo reconstitution experiments revealed that the minimal components are a multivalent RNA-binding protein that concentrates RNAs that are able to form extensive intermolecular mRNA-mRNA interactions. mRNAs with large unstructured regions have a high propensity to form a pervasive intermolecular interaction network that acts as condensate skeleton. The underlying RNA matrix prevents full fusion of spherical liquid-like condensates, thus driving the formation of irregularly shaped membraneless organelles. The resulting large surface area may promote interactions at the condensate surface and at the interface with other organelles.

scholarly journals Separable roles in vivo for the two RNA binding domains of a Drosophila A1-hnRNP homolog

RNA ◽  
1998 ◽  
Vol 4 (12) ◽  
pp. 1585-1598 ◽  
Author(s):  
KAI ZU ◽  
MARTHA L. SIKES ◽  
ANN L. BEYER
Keyword(s):  
Rna Binding ◽  
Binding Domains ◽  
Rna Binding Domains

scholarly journals Organizational analysis of elav gene and functional analysis of ELAV protein of Drosophila melanogaster and Drosophila virilis.

1991 ◽  
Vol 11 (6) ◽  
pp. 2994-3000 ◽  
Author(s):  
K M Yao ◽  
K White
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rna Binding ◽  
Terminal Region ◽  
Drosophila Virilis ◽  
Functional Domain ◽  
Amino Terminal ◽  
Binding Domains ◽  
Rna Binding Domains

Drosophila virilis genomic DNA corresponding to the D. melanogaster embryonic lethal abnormal visual system (elav) locus was cloned. DNA sequence analysis of a 3.8-kb genomic piece allowed identification of (i) an open reading frame (ORF) with striking homology to the previously identified D. melanogaster ORF and (ii) conserved sequence elements of possible regulatory relevance within and flanking the second intron. Conceptual translation of the D. virilis ORF predicts a 519-amino-acid-long ribonucleoprotein consensus sequence-type protein. Similar to D. melanogaster ELAV protein, it contains three tandem RNA-binding domains and an alanine/glutamine-rich amino-terminal region. The sequence throughout the RNA-binding domains, comprising the carboxy-terminal 346 amino acids, shows an extraordinary 100% identity at the amino acid level, indicating a strong structural constraint for this functional domain. The amino-terminal region is 36 amino acids longer in D. virilis, and the conservation is 66%. In in vivo functional tests, the D. virilis ORF was indistinguishable from the D. melanogaster ORF. Furthermore, a D. melanogaster ORF encoding an ELAV protein with a 40-amino-acid deletion within the alanine/glutamine-rich region was also able to supply elav function in vivo. Thus, the divergence of the amino-terminal region of the ELAV protein reflects lowered functional constraint rather than species-specific functional specification.

scholarly journals Synthesis and Assembly of the Box C+D Small Nucleolar RNPs

2000 ◽  
Vol 20 (8) ◽  
pp. 2650-2659 ◽  
Author(s):  
Denis L. J. Lafontaine ◽  
David Tollervey
Keyword(s):  
18S Rrna ◽  
Protein A ◽  
Small Nucleolar Rnas ◽  
Rrna Processing ◽  
Bona Fide ◽  
Polycistronic Transcripts ◽  
Nucleolar Rnas

ABSTRACT Two core small nucleolar RNP (snoRNP) proteins, Nop1p (fibrillarin in vertebrates) and Nop58p (also known as Nop5p) have previously been reported to be specifically associated with the box C+D class of small nucleolar RNAs (snoRNAs). Here we report that Nop56p, a protein related in sequence to Nop58p, is a bona fide box C+D snoRNP component; all tested box C+D snoRNAs were coprecipitated with protein A-tagged Nop56p. Analysis of in vivo snoRNP assembly indicated that Nop56p was stably associated with the snoRNAs only in the presence of Nop1p. In contrast, Nop58p and Nop1p associate independently with the snoRNAs. Genetic depletion of Nop56p resulted in inhibition of early pre-rRNA processing events at sites A0, A1, and A2 and mild depletion of 18S rRNA. However, Nop56p depletion did not lead to codepletion of the box C+D snoRNAs. This is in contrast to Nop58p, which was required for the accumulation of all tested box C+D snoRNAs. Unexpectedly, we found that Nop1p was specifically required for the synthesis and accumulation of box C+D snoRNAs processed from pre-mRNA introns and polycistronic transcripts.

scholarly journals The Splicing Factor U2AF Small Subunit Is Functionally Conserved between Fission Yeast and Humans

2004 ◽  
Vol 24 (10) ◽  
pp. 4229-4240 ◽  
Author(s):  
Christopher J. Webb ◽  
Jo Ann Wise
Keyword(s):  
Rna Binding ◽  
Mutational Analysis ◽  
Large Subunit ◽  
Time Frame ◽  
Small Subunit ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Binding Domains ◽  
Site Recognition

ABSTRACT The small subunit of U2AF, which functions in 3′ splice site recognition, is more highly conserved than its heterodimeric partner yet is less thoroughly investigated. Remarkably, we find that the small subunit of Schizosaccharomyces pombe U2AF (U2AFSM) can be replaced in vivo by its human counterpart, demonstrating that the conservation extends to function. Precursor mRNAs accumulate in S. pombe following U2AFSM depletion in a time frame consistent with a role in splicing. A comprehensive mutational analysis reveals that all three conserved domains are required for viability. Notably, however, a tryptophan in the pseudo-RNA recognition motif implicated in a key contact with the large subunit by crystallographic data is dispensable whereas amino acids implicated in RNA recognition are critical. Mutagenesis of the two zinc-binding domains demonstrates that they are neither equivalent nor redundant. Finally, two- and three-hybrid analyses indicate that mutations with effects on large-subunit interactions are rare whereas virtually all alleles tested diminished RNA binding by the heterodimer. In addition to demonstrating extraordinary conservation of U2AF small-subunit function, these results provide new insights into the roles of individual domains and residues.

scholarly journals Yeast 18S rRNA Dimethylase Dim1p: a Quality Control Mechanism in Ribosome Synthesis?

1998 ◽  
Vol 18 (4) ◽  
pp. 2360-2370 ◽  
Author(s):  
Denis L. J. Lafontaine ◽  
Thomas Preiss ◽  
David Tollervey
Keyword(s):  
18S Rrna ◽  
Small Subunit ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Rrna Processing ◽  
Small Subunit Rrna ◽  
Enzymatic Function ◽  
Processing Steps

ABSTRACT One of the few rRNA modifications conserved between bacteria and eukaryotes is the base dimethylation present at the 3′ end of the small subunit rRNA. In the yeast Saccharomyces cerevisiae, this modification is carried out by Dim1p. We previously reported that genetic depletion of Dim1p not only blocked this modification but also strongly inhibited the pre-rRNA processing steps that lead to the synthesis of 18S rRNA. This prevented the formation of mature but unmodified 18S rRNA. The processing steps inhibited were nucleolar, and consistent with this, Dim1p was shown to localize mostly to this cellular compartment. dim1-2 was isolated from a library of conditionally lethal alleles of DIM1. In dim1-2strains, pre-rRNA processing was not affected at the permissive temperature for growth, but dimethylation was blocked, leading to strong accumulation of nondimethylated 18S rRNA. This demonstrates that the enzymatic function of Dim1p in dimethylation can be separated from its involvement in pre-rRNA processing. The growth rate ofdim1-2 strains was not affected, showing the dimethylation to be dispensable in vivo. Extracts of dim1-2 strains, however, were incompetent for translation in vitro. This suggests that dimethylation is required under the suboptimal in vitro conditions but only fine-tunes ribosomal function in vivo. Unexpectedly, when transcription of pre-rRNA was driven by a polymerase II PGKpromoter, its processing became insensitive to temperature-sensitive mutations in DIM1 or to depletion of Dim1p. This observation, which demonstrates that Dim1p is not directly required for pre-rRNA processing reactions, is consistent with the inhibition of pre-rRNA processing by an active repression system in the absence of Dim1p.

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.

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