ISG20L2, a Novel Vertebrate Nucleolar Exoribonuclease Involved in Ribosome Biogenesis

Couté, Yohann; Kindbeiter, Karine; Belin, Stéphane; Dieckmann, Régis; Duret, Laurent; Bezin, Laurent; Sanchez, Jean‐Charles; Diaz, Jean‐Jacques

doi:10.1074/mcp.m700510-mcp200

Cited by 44 publications

(59 citation statements)

References 76 publications

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“…The second type contains RPs of both the 40S and 60S subunits but with a higher ratio of 60S proteins, such as the complexes described for NCL, nucleophosmin, Par14, and NOP56 (26,(65)(66)(67)(68). The third type is highly enriched in 60S RPs and includes SBDS and ISG20L2 (69,70). DDX24 and NIP7 are present specifically together in the RPS19 and Par14 complexes (27), consistent with our observations, suggesting the importance of DDX24 polyubiquitylation in this association.…”

Section: L S S L a E E S N R T D E E E Q A I I S R V V L E A A S S supporting

confidence: 78%

MDM2 Mediates Nonproteolytic Polyubiquitylation of the DEAD-Box RNA Helicase DDX24

Yamauchi

Nishiyama

Moroishi

et al. 2014

Molecular and Cellular Biology

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MDM2 mediates the ubiquitylation and thereby triggers the proteasomal degradation of the tumor suppressor protein p53. However, genetic evidence suggests that MDM2 contributes to multiple regulatory networks independently of p53 degradation. We have now identified the DEAD-box RNA helicase DDX24 as a nucleolar protein that interacts with MDM2. DDX24 was found to bind to the central region of MDM2, resulting in the polyubiquitylation of DDX24 both in vitro and in vivo. Unexpectedly, however, the polyubiquitylation of DDX24 did not elicit its proteasomal degradation but rather promoted its association with preribosomal ribonucleoprotein (pre-rRNP) processing complexes that are required for the early steps of pre-rRNA processing. Consistently with these findings, depletion of DDX24 in cells impaired pre-rRNA processing and resulted both in abrogation of MDM2 function and in consequent p53 stabilization. Our results thus suggest an unexpected role of MDM2 in the nonproteolytic ubiquitylation of DDX24, which may contribute to the regulation of pre-rRNA processing. MDM2 belongs to the family of RING finger-type ubiquitin ligases (E3s) and functions as a pivotal negative regulator of the tumor suppressor protein p53 (1, 2). MDM2 inhibits p53 function by two distinct mechanisms: it abrogates the transactivation activity of p53 through direct binding to the NH 2 -terminal region of the protein (3, 4), and it mediates the polyubiquitylation of p53 and thereby targets it for degradation by the 26S proteasome (5-7). On the other hand, p53 binds to the promoter of the MDM2 gene and activates its expression (8, 9), thereby completing a negative-feedback loop that is responsible for strict regulation of p53 function in the absence of stress. Exposure of cells to stress, however, results in downregulation of both the abundance and the activity of MDM2 as well as consequent stabilization and subsequent activation of p53 (1, 2). The mechanisms by which p53 escapes from the inhibitory action of MDM2 differ among cell types and stress signals.The role of the nucleolus as a stress sensor has recently emerged. Many types of stress signal converge on steps of ribosome biogenesis and thereby activate p53 (10). A group of ribosomal proteins (RPs), including RPL5, RPL11, and RPL23, serve as transmitters of stress signaling. These proteins are released from the nucleolus in response to stress, bind to and inhibit the activity of MDM2 in the nucleoplasm (11), and eventually activate p53 (11-18).Inhibition of precursor rRNA (pre-rRNA) processing is a key mechanism for induction of nucleolar stress (19). In mammals, the ϳ200 genes encoding 47S pre-rRNA, which serves as a precursor for 18S, 5.8S, and 28S rRNAs, are organized in five tandem arrays on the short arms of acrocentric chromosomes. These genes are transcribed by RNA polymerase I (Pol I) in association with the Pol I-specific basal factors SL1 (also known as TAF1B) and UBF (20). The transcribed 47S pre-rRNA undergoes a series of endo-and exonucleolytic cleavages to remove the spacer ...

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Section: L S S L a E E S N R T D E E E Q A I I S R V V L E A A S S supporting

confidence: 78%

MDM2 Mediates Nonproteolytic Polyubiquitylation of the DEAD-Box RNA Helicase DDX24

Yamauchi

Nishiyama

Moroishi

et al. 2014

Molecular and Cellular Biology

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“…ISG20L2 is a 39-59 exoRNase that is involved in ribosome biogenesis (Coute et al, 2008), although any further biological function has yet to be determined. In contrast, ISG15 is a relatively wellcharacterized ubiquitin homologue.…”

Section: Discussionmentioning

confidence: 99%

Comparison of U2OS and Huh-7 cells for identifying host factors that affect hepatitis C virus RNA replication

Jones¹,

Domingues²,

Targett-Adams³

et al. 2010

Journal of General Virology

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Host cell factors are critical to all stages of the hepatitis C virus (HCV) life cycle. While many cellular proteins that regulate HCV genome synthesis have been identified, the mechanisms engaged in this process are incompletely understood. To identify novel cellular proteins involved in HCV RNA replication, we screened a library of small interfering RNAs (siRNAs) targeting 299 cellular factors, which principally function in RNA interactions. For the screen, a robust system was established using two cell lines (derived from Huh-7 and U2OS cells) that replicated tricistronic subgenomic replicons (SGRs). We found that the U2OS cell line gave lower levels of intracellular HCV RNA replication compared with Huh-7 cells and was more readily transfected by siRNAs. Consequently, increased gene silencing and greater effects on HCV replication were observed in the U2OS cell line. Thus, U2OS cells provided a suitable and more sensitive alternative to Huh-7 cells for siRNA studies on HCV RNA replication. From the screen, several cellular proteins that enhanced and suppressed HCV RNA replication were identified. One of the genes found to downregulate viral RNA synthesis, ISG15, is expressed in response to alpha interferon and may therefore partly contribute to the clearance of virus from infected individuals. A second gene that inhibited HCV RNA levels was the 59-39 exoRNase XRN1, which suggested a role for cellular RNA degradation pathways in modulating the abundance of viral genomes. Therefore, this study provides an important framework for future detailed analyses of these and other cellular proteins. INTRODUCTIONHepatitis C virus (HCV) possesses a single-stranded (ss), positive-sense RNA genome of approximately 9.6 kb . The RNA is translated to yield a polyprotein that is cleaved by cellular (signal peptidase and signal peptide peptidase) and virus-encoded proteases (the NS2-3 autoprotease and NS3-4A serine protease), thereby generating the mature HCV proteins. The nonstructural (NS) proteins NS3, NS4A, NS4B, NS5A and NS5B are located at punctate structures on the endoplasmic reticulum (ER) membrane, which represent viral replication complexes (RCs) (Egger et al., 2002;Gosert et al., 2003;Targett-Adams et al., 2008). Although the NS3-NS5B proteins are essential for HCV RNA synthesis, replication is dependent on host cell factors. A number of cellular proteins that participate in HCV RNA replication have been identified (see below for references). However, characterization of the complete complement of cellular factors required for HCV replication remains undefined.Small inhibitory RNA (siRNA) technology is a powerful approach to identify host cell factors that contribute to viral replication and as such, several groups have screened siRNA libraries targeting cellular genes. For example, screening with libraries that target protein kinases (Supekova et al., 2008), transporter proteins and transcription factors (Ng et al., 2007), previously documented HCV-interacting proteins (Randall et al., 2007) and proteins implicated...

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“…In addition to Las1L, Dom3Z, and Xrn2, our analysis of lowmolecular-weight RNAs (Fig. 10B) included targeting two conserved 3=-5= exoRNases, Eri1 and Isg20L2, with known or suspected functions in 5.8S rRNA 3=-end formation in mammals (1,9), the exosome subunit ExoSC10 (human Rrp6), and the ATPdependent 3=-5= RNA helicase Skiv2L2 (human Dob1/Mtr4). Skiv2L2 is a core exosome cofactor with known functions in 5.8S rRNA 3=-end formation in yeast and humans (10,39).…”

Section: Figmentioning

confidence: 99%

The Evolutionarily Conserved Protein LAS1 Is Required for Pre-rRNA Processing at Both Ends of ITS2

Schillewaert

Wacheul

Lhommé³

et al. 2012

Molecular and Cellular Biology

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Ribosome synthesis entails the formation of mature rRNAs from long precursor molecules, following a complex pre-rRNA processing pathway. Why the generation of mature rRNA ends is so complicated is unclear. Nor is it understood how pre-rRNA processing is coordinated at distant sites on pre-rRNA molecules. Here we characterized, in budding yeast and human cells, the evolutionarily conserved protein Las1. We found that, in both species, Las1 is required to process ITS2, which separates the 5.8S and 25S/28S rRNAs. In yeast, Las1 is required for pre-rRNA processing at both ends of ITS2. It is required for Rrp6-dependent formation of the 5.8S rRNA 3= end and for Rat1-dependent formation of the 25S rRNA 5= end. We further show that the Rat1-Rai1 5=-3= exoribonuclease (exoRNase) complex functionally connects processing at both ends of the 5.8S rRNA. We suggest that prerRNA processing is coordinated at both ends of 5.8S rRNA and both ends of ITS2, which are brought together by pre-rRNA folding, by an RNA processing complex. Consistently, we note the conspicuous presence of ϳ7-or 8-nucleotide extensions on both ends of 5.8S rRNA precursors and at the 5= end of pre-25S RNAs suggestive of a protected spacer fragment of similar length. Ribosomes are essential to all life forms. Ribogenesis is a major metabolic activity requiring the coordinated expression of the core RNA and protein components of the small and large subunits and also of a myriad of trans-acting protein and RNA factors, their maturation, packaging, and transport (reviewed in references 21, 29, and 42). Despite this great complexity, ribogenesis is an extremely robust process. Quality control and fail-safe mechanisms are available at all steps along the assembly pathway to ensure that a sufficient amount of functional ribosomes is provided at all times (reviewed in references 24 and 30).In eukaryotes, ribogenesis is initiated in the nucleolus. There, at the interface between the cortical side of fibrillar centers (FCs) and the surrounding dense fibrillar components (DFCs), RNA polymerase I is recruited and threaded along the ribosomal DNA (rDNA), producing pre-rRNA transcripts that extend into the DFC, where pre-rRNAs undergo cotranscriptional modification. In fast-growing budding yeast cells, up to 50 to 70% of pre-rRNA molecules are also subjected to cotranscriptional cleavage (3,23,28,36). Cotranscriptional cleavage occurs in internal transcribed spacer 1 (ITS1), separating the precursors destined to mature into the small and large ribosome subunit rRNAs, i.e., the 18S and 5.8S-25S rRNAs, respectively (28,36,47). This strategy, involving the cosynthesis of three of the four mature rRNAs from a single long transcript, contributes to coordinating the synthesis of the various components of the translational machinery. This strategy has been extensively conserved throughout evolution and predates the eukaryotes, since the Bacteria and Archaea also synthesize their rRNAs from polycistronic precursors (reviewed in reference 31).The synthesis of mature rRNAs relie...

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ISG20L2, a Novel Vertebrate Nucleolar Exoribonuclease Involved in Ribosome Biogenesis

Cited by 44 publications

References 76 publications

MDM2 Mediates Nonproteolytic Polyubiquitylation of the DEAD-Box RNA Helicase DDX24

MDM2 Mediates Nonproteolytic Polyubiquitylation of the DEAD-Box RNA Helicase DDX24

Comparison of U2OS and Huh-7 cells for identifying host factors that affect hepatitis C virus RNA replication

The Evolutionarily Conserved Protein LAS1 Is Required for Pre-rRNA Processing at Both Ends of ITS2

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