Intersection of the Kap123p-mediated nuclear import and ribosome export pathways

Kap123p is a yeast beta-karyopherin that imports ribosomal proteins into the nucleus prior to their assembly into preribosomal particles. Surprisingly, Kap123p is not essential for growth, under normal conditions. To further explore the role of Kap123p in nucleocytoplasmic transport and ribosome biogenesis, we performed a synthetic fitness screen designed to identify genes that interact with KAP123. Through this analysis we have identified three other karyopherins, Pse1p/Kap121p, Sxm1p/Kap108p, and Nmd5p/Kap119p. We propose that, in the absence of Kap123p, these karyopherins are able to supplant Kap123p's role in import. In addition to the karyopherins, we identified Rai1p, a protein previously implicated in rRNA processing. Rai1p is also not essential, but deletion of the RAI1 gene is deleterious to cell growth and causes defects in rRNA processing, which leads to an imbalance of the 60S/40S ratio and the accumulation of halfmers, 40S subunits assembled on polysomes that are unable to form functional ribosomes. Rai1p localizes predominantly to the nucleus, where it physically interacts with Rat1p and pre-60S ribosomal subunits. Analysis of the rai1/kap123 double mutant strain suggests that the observed genetic interaction results from an inability to efficiently export pre-60S subunits from the nucleus, which arises from a combination of compromised Kap123p-mediated nuclear import of the essential 60S ribosomal subunit export factor, Nmd3p, and a DeltaRAI1-induced decrease in the overall biogenesis efficiency.     

Involvement of the specific nucleolar protein SURF6 in regulation of proliferation and ribosome biogenesis in mouse NIH/3T3 fibroblasts

The nucleolar proteins which link cell proliferation to ribosome biogenesis are regarded to be potentially oncogenic. Here, in order to examine the involvement of an evolutionary conserved nucleolar protein SURF6/Rrp14 in proliferation and ribosome biogenesis in mammalian cells, we established stably transfected mouse NIH/3T3 fibroblasts capable of conditional overexpression of the protein. Cell proliferation was monitored in real-time, and various cell cycle parameters were quantified based on flow cytometry, Br-dU-labeling and conventional microscopy data. We show that overexpression of SURF6 accelerates cell proliferation and promotes transition through all cell cycle phases. The most prominent SURF6 pro-proliferative effects include a significant reduction of the population doubling time, from 19.8 ± 0.7 to 16.2 ± 0.5 hours (t-test, p < 0.001), and of the length of cell division cycle, from 17.6 ± 0.6 to 14.0 ± 0.4 hours (t-test, p < 0.001). The later was due to the shortening of all cell cycle phases but the length of G1 period was reduced most, from 5.7 ± 0.4 to 3.8 ± 0.3 hours, or by ～30%, (t-test, p < 0.05). By Northern blots and qRT-PCR, we further showed that the acceleration of cell proliferation was concomitant with an accumulation of rRNA species along both ribosomal subunit maturation pathways. It is evident, therefore, that like the yeast homologue Rrp14, mammalian SURF6 is involved in various steps of rRNA processing during ribosome biogenesis. We concluded that SURF6 is a novel positive regulator of proliferation and G1/S transition in mammals, implicating that SURF6 is a potential oncogenic protein, which can be further studied as a putative target in anti-cancer therapy.     

Characterization of karyopherin cargoes reveals unique mechanisms of Kap121p-mediated nuclear import

In yeast there are at least 14 members of the beta-karyopherin protein family that govern the movement of a diverse set of cargoes between the nucleus and cytoplasm. Knowledge of the cargoes carried by each karyopherin and insight into the mechanisms of transport are fundamental to understanding constitutive and regulated transport and elucidating how they impact normal cellular functions. Here, we have focused on the identification of nuclear import cargoes for the essential yeast beta-karyopherin, Kap121p. Using an overlay blot assay and coimmunopurification studies, we have identified 30 putative Kap121p cargoes. Among these were Nop1p and Sof1p, two essential trans-acting protein factors required at the early stages of ribosome biogenesis. Characterization of the Kap121p-Nop1p and Kap121p-Sof1p interactions demonstrated that, in addition to lysine-rich nuclear localization signals (NLSs), Kap121p recognizes a unique class of signals distinguished by the abundance of arginine and glycine residues and consequently termed rg-NLSs. Kap104p is also known to recognize rg-NLSs, and here we show that it compensates for the loss of Kap121p function. Sof1p is also transported by Kap121p; however, its import can be mediated by a piggyback mechanism with Nop1p bridging the interaction between Sof1p and Kap121p. Together, our data elucidate additional levels of complexity in these nuclear transport pathways.     

Yeast Rrp14p is a nucleolar protein involved in both ribosome biogenesis and cell polarity

We previously cloned RRP14/YKL082c, whose product exhibits two-hybrid interaction with Ebp2p, a regulatory factor of assembly of 60S ribosomal subunits. Depletion of Rrp14p results in shortage of 60S ribosomal subunits and retardation of processing from 27S pre-rRNA to 25S rRNA. Furthermore, 35S pre-rRNA synthesis appears to decline in Rrp14p-depleted cells. Rrp14p interacts with regulatory factors of 60S subunit assembly and also with Utp11p and Faf1p, which are regulatory factors required for assembly of 40S ribosomal subunits. We propose that Rrp14p is involved in ribosome synthesis from the beginning of 35S pre-rRNA synthesis to assembly of the 60S ribosomal subunit. Disruption of RRP14 causes an extremely slow growth rate of the cell, a severe defect in ribosome synthesis, and a depolarized localization of cortical actin patches throughout the cell cycle. These results suggest that Rrp14p has dual functions in ribosome synthesis and polarized cell growth.     

NOP132 is required for proper nucleolus localization of DEAD-box RNA helicase DDX47

Previously, we described a novel nucleolar protein, NOP132, which interacts with the small GTP binding protein RRAG A. To elucidate the function of NOP132 in the nucleolus, we identified proteins that interact with NOP132 using mass spectrometric methods. NOP132 associated mainly with proteins involved in ribosome biogenesis and RNA metabolism, including the DEAD-box RNA helicase protein, DDX47, whose yeast homolog is Rrp3, which has roles in pre-rRNA processing. Immunoprecipitation of FLAG-tagged DDX47 co-precipitated rRNA precursors, as well as a number of proteins that are probably involved in ribosome biogenesis, implying that DDX47 plays a role in pre-rRNA processing. Introduction of NOP132 small interfering RNAs induced a ring-like localization of DDX47 in the nucleolus, suggesting that NOP132 is required for the appropriate localization of DDX47 within the nucleolus. We propose that NOP132 functions in the recruitment of pre-rRNA processing proteins, including DDX47, to the region where rRNA is transcribed within the nucleolus.     

Role of the yeast Rrp1 protein in the dynamics of pre-ribosome maturation

The Saccharomyces cerevisiae gene RRP1 encodes an essential, evolutionarily conserved protein necessary for biogenesis of 60S ribosomal subunits. Processing of 27S pre-ribosomal RNA to mature 25S rRNA is blocked and 60S subunits are deficient in the temperature-sensitive rrp1-1 mutant. We have used recent advances in proteomic analysis to examine in more detail the function of Rrp1p in ribosome biogenesis. We show that Rrp1p is a nucleolar protein associated with several distinct 66S pre-ribosomal particles. These pre-ribosomes contain ribosomal proteins plus at least 28 nonribosomal proteins necessary for production of 60S ribosomal subunits. Inactivation of Rrp1p inhibits processing of 27SA(3) to 27SB(S) pre-rRNA and of 27SB pre-rRNA to 7S plus 25.5S pre-rRNA. Thus, in the rrp1-1 mutant, 66S pre-ribosomal particles accumulate that contain 27SA(3) and 27SB(L) pre-ribosomal RNAs.     

Ribosome biogenesis is sensed at the Start cell cycle checkpoint

In the yeast Saccharomyces cerevisiae it has long been thought that cells must reach a critical cell size, called the "setpoint," in order to allow the Start cell cycle transition. Recent evidence suggests that this setpoint is lowered when ribosome biogenesis is slowed. Here we present evidence that yeast can sense ribosome biogenesis independently of mature ribosome levels and protein synthetic capacity. Our results suggest that ribosome biogenesis directly promotes passage through Start through Whi5, the yeast functional equivalent to the human tumor suppressor Rb. When ribosome biogenesis is inhibited, a Whi5-dependent mechanism inhibits passage through Start before significant decreases in both the number of ribosomes and in overall translation capacity of the cell become evident. This delay at Start in response to decreases in ribosome biogenesis occurs independently of Cln3, the major known Whi5 antagonist. Thus ribosome biogenesis may be sensed at multiple steps in Start regulation. Ribosome biogenesis may thus both delay Start by increasing the cell size setpoint and independently may promote Start by inactivating Whi5.     

Perturbation of RNA Polymerase I transcription machinery by ablation of HEATR1 triggers the RPL5/RPL11-MDM2-p53 ribosome biogenesis stress checkpoint pathway in human cells

Ribosome biogenesis is an energy consuming process which takes place mainly in the nucleolus. By producing ribosomes to fuel protein synthesis, it is tightly connected with cell growth and cell cycle control. Perturbation of ribosome biogenesis leads to the activation of p53 tumor suppressor protein promoting processes like cell cycle arrest, apoptosis or senescence. This ribosome biogenesis stress pathway activates p53 through sequestration of MDM2 by a subset of ribosomal proteins (RPs), thereby stabilizing p53. Here, we identify human HEATR1, as a nucleolar protein which positively regulates ribosomal RNA (rRNA) synthesis. Downregulation of HEATR1 resulted in cell cycle arrest in a manner dependent on p53. Moreover, depletion of HEATR1 also caused disruption of nucleolar structure and activated the ribosomal biogenesis stress pathway – RPL5 / RPL11 dependent stabilization and activation of p53. These findings reveal an important role for HEATR1 in ribosome biogenesis and further support the concept that perturbation of ribosome biosynthesis results in p53-dependent cell cycle checkpoint activation, with implications for human pathologies including cancer.     

Ribosomal 18 S RNA Processing by the IGF-I-responsive WDR3 Protein Is Integrated with p53 Function in Cancer Cell Proliferation

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 G₁ 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.     

RRP1B targets PP1 to mammalian cell nucleoli and is associated with Pre-60S ribosomal subunits

A pool of protein phosphatase 1 (PP1) accumulates within nucleoli and accounts for a large fraction of the serine/threonine protein phosphatase activity in this subnuclear structure. Using a combination of fluorescence imaging with quantitative proteomics, we mapped the subnuclear localization of the three mammalian PP1 isoforms stably expressed as GFP-fusions in live cells and identified RRP1B as a novel nucleolar targeting subunit that shows a specificity for PP1β and PP1γ. RRP1B, one of two mammalian orthologues of the yeast Rrp1p protein, shows an RNAse-dependent localization to the granular component of the nucleolus and distributes in a similar manner throughout the cell cycle to proteins involved in later steps of rRNA processing. Quantitative proteomic analysis of complexes containing both RRP1B and PP1γ revealed enrichment of an overlapping subset of large (60S) ribosomal subunit proteins and pre-60S nonribosomal proteins involved in mid-late processing. Targeting of PP1 to this complex by RRP1B in mammalian cells is likely to contribute to modulation of ribosome biogenesis by mechanisms involving reversible phosphorylation events, thus playing a role in the rapid transduction of cellular signals that call for regulation of ribosome production in response to cellular stress and/or changes in growth conditions.     

Rrp5p, a trans-acting factor in yeast ribosome biogenesis, is an RNA-binding protein with a pronounced preference for U-rich sequences

Rrp5p is a trans-acting factor important for biogenesis of both the 40S and 60S subunit of the Saccharomyces cerevisiae ribosome. The protein contains 12 tandemly repeated S1 RNA binding motifs in its N-terminal region, suggesting the ability to interact directly with the pre-rRNA. In vitro binding studies, using immunopurified Rrp5p and in vitro transcribed, 32P-UTP-labeled RNA fragments, revealed that Rrp5p is a general RNA-binding protein with a strong preference for single-stranded sequences rich in uridines. Co-immunoprecipitation studies in yeast cells expressing ProtA-tagged Rrp5p showed that the protein is still associated with pre-ribosomal particles containing 27SA2 pre-rRNA but not with particles containing the 27SB precursor. Thus, Rrp5p appears to dissociate from the 66S pre-ribosome upon or immediately after further processing of 27SA2 pre-rRNA, suggesting the presence of (an) important binding site(s) within the 3'-terminal portion of ITS1. The location of these possible binding site(s) was further delimited using rrp2-1 mutant cells, which accumulate the 5'-extended 5.8S pre-rRNA species. The results indicate that association of Rrp5p with the pre-ribosome is abolished upon removal of a 30-nt region downstream from site A2, which contains two short, single-stranded U stretches. Sequence comparison shows that only the most 5' of these two U-rich stretches is conserved among yeast species whose ITS1 can functionally replace the S. cerevisiae spacer. The implications for the role of Rrp5p in yeast ribosome biogenesis are discussed.     

Cell cycle regulated transport controlled by alterations in the nuclear pore complex

Eukaryotic cells have developed mechanisms for regulating the nuclear transport of macromolecules that control various cellular events including movement through defined stages of the cell cycle. In yeast cells, where the nuclear envelope remains intact throughout the cell cycle, these transport regulatory mechanisms must also function during mitosis. We have uncovered a mechanism for regulating transport that is controlled by M phase specific molecular rearrangements in the nuclear pore complex (NPC). These changes allow a transport inhibitory nucleoporin, Nup53p, to bind the karyopherin Kap121p specifically during mitosis, slowing its movement through the NPC and inducing cargo release. Yeast strains that possess defects in the function of Kap121p or the fidelity of the inhibitory pathway are delayed in mitosis. We propose that fluctuations in Kap121p transport mediated by the NPC contribute to controlling the subcellular distribution of molecules that direct progression through mitosis.     

Mammalian WDR12 is a novel member of the Pes1-Bop1 complex and is required for ribosome biogenesis and cell proliferation

Target genes of the protooncogene c-myc are implicated in cell cycle and growth control, yet the linkage of both is still unexplored. Here, we show that the products of the nucleolar target genes Pes1 and Bop1 form a stable complex with a novel member, WDR12 (PeBoW complex). Endogenous WDR12, a WD40 repeat protein, is crucial for processing of the 32S precursor ribosomal RNA (rRNA) and cell proliferation. Further, a conditionally expressed dominant-negative mutant of WDR12 also blocks rRNA processing and induces a reversible cell cycle arrest. Mutant WDR12 triggers accumulation of p53 in a p19ARF-independent manner in proliferating cells but not in quiescent cells. Interestingly, a potential homologous complex of Pes1-Bop1-WDR12 in yeast (Nop7p-Erb1p-Ytm1p) is involved in the control of ribosome biogenesis and S phase entry. In conclusion, the integrity of the PeBoW complex is required for ribosome biogenesis and cell proliferation in mammalian cells.