Proteomic Identification of Small-Subunit Ribosome Assembly Factors in Trypanosoma brucei.

The putative yeast GTPase Nug1, which is associated with several pre-60 S particles in the nucleolus and nucleoplasm, consists of an N-terminal domain, which is found only in eukaryotic orthologues, and middle and C-terminal domains that are conserved throughout eukaryotes, bacteria, and archaea. Here, we analyzed the role of the eukaryote-specific Nug1 N-domain (Nug1-N). We show that the essential Nug1-N is sufficient and necessary for nucle(ol)ar targeting and association with pre-60 S particles. Nug1-N exhibits RNA binding activity and is genetically linked in an allele-specific way to the pre-60 S factors Noc2, Noc3, and Dbp10. In contrast, the middle domain, which exhibits a circularly permuted GTPase fold and an intrinsic GTP hydrolysis activity in vitro, is not essential for cell growth. The conserved Nug1 C-domain, which has a yet uncharacterized fold, is also essential for ribosome biogenesis. Our findings suggest that Nug1 associates with pre-60 S subunits via its essential N-terminal RNA-binding domain and exerts a non-essential regulative role in pre-60 S subunit biogenesis via its central GTPase domain.

In humans and other holozoan organisms, the ribosomal protein eS30 is synthesized as a fusion protein with the ubiquitin-like protein FUBI. However, FUBI is not part of the mature 40S ribosomal subunit and cleaved off by an as-of-yet unidentified protease. How FUBI-eS30 processing is coordinated with 40S subunit maturation is unknown. To study the mechanism and importance of FUBI-eS30 processing, we expressed non-cleavable mutants in human cells, which affected late steps of cytoplasmic 40S maturation, including the maturation of 18S rRNA and recycling of late-acting ribosome biogenesis factors. Differential affinity purification of wild-type and non-cleavable FUBI-eS30 mutants identified the deubiquitinase USP36 as a candidate FUBI-eS30 processing enzyme. Depletion of USP36 by RNAi or CRISPRi indeed impaired FUBI-eS30 processing and moreover, purified USP36 cut FUBI-eS30 in vitro. Together, these data demonstrate the functional importance of FUBI-eS30 cleavage and identify USP36 as a novel protease involved in this process.

Decision letter: Processing of the ribosomal ubiquitin-like fusion protein FUBI-eS30/FAU is required for 40S maturation and depends on USP36

status: Published

Ribosomes are large macromolecular machineries composed of both protein and RNA constituents with a species-dependent molecular mass of at least ~3.3 MDa for the fully assembled eukaryotic 80S ribosome. Their catalytic activity is dependent on ribosomal RNA; therefore, ribosomes are bona fide ribozymes, and as such they mediate the final step of gene expression from DNA to RNA to protein by peptide bond formation between amino acids. Importantly, spatial separation of ribosome function and biogenesis into distinct cellular compartments allows for intricate regulatory mechanisms and rigorous quality control. Ribosome biogenesis occurs predominantly in the nucleolus and nucleus of the cell with final cytoplasmic maturation and quality control steps. Briefly, nucleolar ribosomal RNA together with ~200 trans-acting assembly factors co-transcriptionally forms the 40S and 60S pre-ribosomal subunits into which ~80 ribosomal proteins are incorporated in a hierarchical fashion. Recent studies, including this thesis, have identified a novel class of dedicated ribosome assembly chaperones, in addition to the ~200 trans-acting ribosome assembly factors, which facilitate ribosomal protein shuttling. Ribosomal proteins are generated in the cytoplasm, and with only few exceptions they all have to enter the nucleus for incorporation into the pre-ribosomal subunits. Assembly chaperones can bind and protect unassembled ribosomal proteins either co-translationally or following nuclear import and shuttle them in a timely fashion to their destination sites at the maturing pre-ribosomal subunits. The first chapter of this thesis describes the identification and characterization of a dedicated assembly chaperone for the large ribosomal subunit protein RpL4, termed Acl4. Interestingly, Acl4 and likely also other dedicated assembly chaperones not only interact with ribosomal proteins to avoid aggregation and to shield them from unfavorable interactions, but also protect their client proteins from cellular degradation by the ubiquitin-proteasome machinery. Ribosomes are built by assembling equimolar amounts of ribosomal proteins, which generates a challenge for the cell to ensure stoichiometric quantities of ribosomal proteins. Recent studies have demonstrated that stoichiometric levels of ribosomal proteins are established by cellular degradation of excess protein via ubiquitination of unassembled components. The second chapter of this thesis describes a conserved degradation pathway, which is dependent on the E3 ubiquitin ligase Tom1 to mark unprotected and unassembled ribosomal proteins and target them for degradation. Moreover, it is demonstrated in the third chapter for the first time how an assembly chaperone protects its client ribosomal protein from ubiquitination and proteasome-mediated degradation. High resolution structures of the Acl4•RpL4 complex as well as RpL4 in complex with the nuclear transport factor Kap104 visualize the molecular interactions of those proteins and uncover the molecular mechanism of protecting conserved Tom1-target sites within RpL4. Together, the reported results identify and characterize both a novel degradation pathway as well as a protection mechanism for ribosomal proteins and advance the understanding of the intricate regulation of ribosome biogenesis.

The nucleolus, the most prominent structure observed in the nucleus, is often called a “ribosome factory.” Cells spend an enormous fraction of their resources to achieve the mass-production of ribosomes required by rapid growth. On the other hand, ribosome biogenesis is also tightly controlled, and must be coordinated with other cellular processes. Ribosomal proteins and ribosome biogenesis factors are attractive candidates for this link. Recent results suggest that some of them have functions beyond ribosome biogenesis. Here we review recent progress on ribosome biogenesis factors, Ebp2 and Rrs1, in yeast Saccharomyces cerevisiae. In this organism, Ebp2 and Rrs1 are found in the nucleolus and at the nuclear periphery. At the nuclear envelope, these proteins interact with a membrane-spanning SUN domain protein, Mps3, and play roles in telomere clustering and silencing along with the silent information regulator Sir4. We propose that a protein complex consisting Ebp2, Rrs1 and Mps3 is involved in a wide range of activities at the nuclear envelope.

Unco-ordinate regulation of ribosomal RNA and ribosomal protein synthesis during L 6E 9 myoblast differentiation

Transcriptomic analysis of ribosome biogenesis and pre-rRNA processing during growth stress in Entamoeba histolytica

Insulin-like growth factor-I (IGF-I) signaling is strongly associated with cell growth and regulates the rate of synthesis of the rRNA precursor, the first and the key stage of ribosome biogenesis. In a screen for mediators of IGF-I signaling in cancer, we recently identified several ribosome-related proteins, including NEP1 (nucleolar essential protein 1) and WDR3 (WD repeat 3), whose homologues in yeast function in ribosome processing. The WDR3 gene and its locus on chromosome 1p12-13 have previously been linked with malignancy. Here we show that IGF-I induces expression of WDR3 in transformed cells. WDR3 depletion causes defects in ribosome biogenesis by affecting 18 S rRNA processing and also causes a transient down-regulation of precursor rRNA levels with moderate repression of RNA polymerase I activity. Suppression of WDR3 in cells expressing functional p53 reduced proliferation and arrested cells in the G(1) phase of the cell cycle. This was associated with activation of p53 and sequestration of MDM2 by ribosomal protein L11. Cells lacking functional p53 did not undergo cell cycle arrest upon suppression of WDR3. Overall, the data indicate that WDR3 has an essential function in 40 S ribosomal subunit synthesis and in ribosomal stress signaling to p53-mediated regulation of cell cycle progression in cancer cells.

[This corrects the article DOI: 10.1186/s13100-015-0053-5.].

BackgroundA large number of Saccharomyces cerevisiae cellular factors modulate the movement of the retrovirus-like transposon Ty1. Surprisingly, a significant number of chromosomal genes required for Ty1 transposition encode components of the translational machinery, including ribosomal proteins, ribosomal biogenesis factors, protein trafficking proteins and protein or RNA modification enzymes.ResultsTo assess the mechanistic connection between Ty1 mobility and the translation machinery, we have determined the effect of these mutations on ribosome biogenesis and Ty1 transcriptional and post-transcriptional regulation. Lack of genes encoding ribosomal proteins or ribosome assembly factors causes reduced accumulation of the ribosomal subunit with which they are associated. In addition, these mutations cause decreased Ty1 + 1 programmed translational frameshifting, and reduced Gag protein accumulation despite at least normal levels of Ty1 mRNA. Several ribosome subunit mutations increase the level of both an internally initiated Ty1 transcript and its encoded truncated Gag-p22 protein, which inhibits transposition.ConclusionsTogether, our results suggest that this large class of cellular genes modulate Ty1 transposition through multiple pathways. The effects are largely post-transcriptional acting at a variety of levels that may include translation initiation, protein stability and subcellular protein localization.Electronic supplementary materialThe online version of this article (doi:10.1186/s13100-015-0053-5) contains supplementary material, which is available to authorized users.

As part of the innate immune system, the important cellular pattern recognition receptor (PRR) RIG-I senses the presence of incoming viral particles by binding viral RNA, leading to the transcription of type I interferons (IFN). These cytokines are then secreted and bind to the type I IFN receptor (IFNAR), which induces expression of hundreds of interferon-stimulated gene products (ISGs) that generate an antiviral environment. Kaposi’s sarcoma-associated herpesvirus (KSHV) is a human oncovirus. To establish lifelong infection, KSHV has evolved sophisticated mechanisms to modulate the innate immune response of its host. We identified the poorly characterised KSHV ORF20 protein as a potent inhibitor of the RIG-I-mediated type I IFN response using a luciferase-based reporter screen with 85 open reading frames (ORFs). To better understand the role of ORF20, we used the unbiased approach of quantitative affinity purification coupled to mass spectrometry (q-AP-MS) to identify the cellular interaction partners of ORF20. We found that ORF20 interacted with a variety of ribosomal and nucleolar proteins. Immunofluorescence analysis showed their colocalisation in the nucleolus. Interestingly, distinct localisation patterns of ORF20 correlated with the dispersal of nucleolar and ribosomal proteins from the nucleolus. Furthermore, the antiviral ISG oligoadenylate synthetase-like protein (OASL) was identified as an interaction partner of ORF20 by q-AP-MS. We found that ORF20 specifically interacted with the OAS domain of OASL and that ORF20 and OASL colocalised in the nucleolus. OASL was shown to amplify the type I IFN activation by directly interacting with RIG-I. However, we found that ORF20 did not interfere with colocalisation or interaction of OASL and RIG-I. Surprisingly, we found that the inhibitory effect of ORF20 on RIG-I signalling was independent of OASL expression. By analysing the cellular interaction partners of OASL by q-AP-MS, we found that ORF20 and OASL shared a highly similar interactome. Like ORF20, OASL interacted and colocalised with numerous ribosomal and nucleolar proteins. As the nucleolus is the site of ribosome biogenesis and ORF20 and OASL interact with a number of ribosomal proteins, our data suggest that the cellular protein OASL may have a role for ribosome function, which might be manipulated by the KSHV protein ORF20 to promote KSHV infection.

Ribosome biogenesis and cell cycle are coordinated processes. Recent studies in mammalian cell lines have showed that ribosome biogenesis is linked to tumorgenesis, where mutation or depletion of ribosomal factors, leads cancer cell proliferation. The yeast Saccharomyces cerevisae is a useful model organism for understanding the connections between ribosome biogenesis and cell cycle control. Only a handful of studies have been done and these have mainly focused on different transacting factors involved in ribosome biogenesis. Here we are focusing on the role of ribosomal proteins. Our lab has found that depleting the ribosomal protein Rpl4 results in defects in both rRNA processing and cell cycle with accumulation of di‐ and tri‐ budded cells. This discovery has led our lab to investigate other ribosomal proteins also have implications in these two cellular pathways. Depletion of ribosomal proteins Rpl7A, Rpl18A, Rpl37, Rpl40A in S. cerevisae also resulted in yeast cells with di‐ and tri‐ buds, unlike the wild type cells, which only produce a single bud at a time. To fully understand which stage of cell cycle is defective, fluorescent activated cell sorter and microscopy analysis are in progress. We are currently characterizing defects in rRNA processing and ribosomal subunit assembly during depletion of these ribosomal proteins.

Ribosome biogenesis is a multi-step process that initiates within the nucleolus, terminates in the cytoplasm, and determines the rate of protein synthesis. Ribosome biogenesis is essential for maintaining liver function. In eukaryotes, it involves producing and assembling approximately 200 factors and 80 ribosomal proteins. Mutations in ribosome proteins, ribosomal RNA processing, and ribosome assembly factors in the liver can result in liver disease. Hepatitis C virus causes acute or chronic infection and liver disease, which can progress to liver cirrhosis, cancer, and death. This review provides an overview of the effects of ribosomal biogenesis, including ribosomal RNA, ribosomal proteins, and ribosome biogenesis factors, on liver regeneration, hepatitis C virus, nonalcoholic fatty liver disease, liver fibrosis, cirrhosis, and liver cancer. It lists drugs that exploit ribosome biogenesis to treat liver cancer. Targeting ribosome biogenesis shows promise as a therapeutic approach. A better understanding of this process will contribute to developing effective and targeted therapeutic strategies for ribosome biogenesis disorders.

In Drosophila tissue culture cells, the synthesis of ribosomal proteins was inhibited by a 1-h 37 degrees C heat shock. Ribosomal protein synthesis was repressed to a greater extent than that of most other proteins synthesized by these cells at 25 degrees C. After a 1-h heat shock, when the cells were returned to 25 degrees C, the ribosomal proteins were much slower than most other 25 degrees C proteins to return to pre-heat shock levels of synthesis. Relative to one another, all the ribosomal proteins were inhibited and later recovered to normal levels of synthesis at the same rate and to the same extent. Unlike the ribosomal proteins, the precursor to the large rRNAs was continually synthesized during heat shock, although at a slightly reduced level, but was not processed. It was rapidly degraded, with a half-life of approximately 16 min. Pre-heat shock levels of synthesis, stability, and correct processing were restored only when ribosomal protein synthesis returned to at least 50% of that seen in non-heat-shocked cells.