Las1L Is a Nucleolar Protein Required for Cell Proliferation and Ribosome Biogenesis

Abstract

Ribosome biogenesis is a highly regulated process ensuring that cell growth (increase in biomass) is coordinated with cell proliferation. The formation of eukaryotic ribosomes is a multistep process initiated by the transcription and processing of rRNA in the nucleolus. Concomitant with this, several preribosomal particles, which transiently associate with numerous nonribosomal factors before mature 60S and 40S subunits are formed and exported in the cytoplasm, are generated. Here we identify Las1L as a previously uncharacterized nucleolar protein required for ribosome biogenesis. Depletion of Las1L causes inhibition of cell proliferation characterized by a G 1 arrest dependent on the tumor suppressor p53. Moreover, we demonstrate that Las1L is crucial for ribosome biogenesis and that depletion of Las1L leads to inhibition of rRNA processing and failure to synthesize the mature 28S rRNA. Taken together, our data demonstrate that Las1L is essential for cell proliferation and biogenesis of the 60S ribosomal subunit.Cell growth (increase in cell mass) and cell division must be coordinated during proliferation for cells to remain at a constant size (13). Biogenesis of eukaryotic ribosomes is a finely tuned process ensuring that the protein synthesis capacity of a cell meets the demands of growth associated with proliferation (31). Ribosome biogenesis is initiated by the transcription of rRNA by RNA polymerase I (Pol I) (28S, 18S, and 5.8S rRNA) in the nucleolus and by RNA polymerase III (5S rRNA) in the nucleoplasm. After undergoing cotranscriptional site-specific modification, the 47S pre-rRNA transcript is submitted to a series of endonucleolytic cleavages and exonucleolytic digestion steps to remove internal and external transcribed spacer (ITS and ETS) regions and generate the mature 18S, 28S, and 5.8S rRNAs (21,26,27,36). Mature rRNAs are then assembled with ribosomal proteins to form the 40S (18S rRNA) and 60S (28S, 5.8S, and 5S rRNA) ribosomal particles. These steps involve a large assortment of factors in addition to the RNA and protein components of the ribosome itself. More than 200 additional accessory proteins and noncoding small RNAs engage in complex reactions of processing, assembly, and nuclear export to generate functional cytoplasmic 40S and 60S subunits (36). Although a large collection of evolutionarily conserved proteins has been implicated in processing and assembly of ribonucleoproteins, many details of these pathways and the precise function of several of these factors remain unresolved (28, 54).Perturbation of rRNA transcription or rRNA processing or deletion of ribosomal proteins has been shown to induce nucleolar stress and stabilization of the tumor suppressor p53 (4, 9-11, 16, 23, 25, 32, 58, 61, 62). It was previously demonstrated that, following nucleolar stress, several ribosomal proteins can bind to HDM2 (an E3 ubiquitin ligase) and inhibit its ability to ubiquitinate and target p53 for degradation, leading to accumulation of p53 and subsequent G 1 cell cycle arrest. This suggests t...

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

“…Recent analyses established that human Las1L is required for large subunit rRNA synthesis (7). To precisely characterize the involvement of Las1L in ITS2 processing, we extracted total RNA from HeLa cells treated for 72 h with siRNAs targeting the LAS1L…”

“…Consistently, it was recently shown to be required for large ribosome subunit synthesis (7). Las1L depletion, like other nucleolar stresses (reviewed in reference 4), induces a tumor surveillance pathway resulting in p53 stabilization, G 1 arrest, and ultimately cell death (7). Yeast Las1 has known functions in cell morphogenesis (surface growth) and cell division (13).…”

Congenital lethal motor neuron disease with a novel defect in ribosome biogenesis

“…16 Loss of LAS1 in S cerevisiae interrupts the production of the large, 60S ribosomal subunit by disruption of the processing of the 47S pre-rRNA at the second intervening sequence (ITS2), which separates the 5.8S and 28S rRNA sequences. 30,32 Processing of the 28S rRNA is dependent on the five friends of methylated Chtop (5FMC) complex, which includes LAS1L, SENP3, WDR18, TEX10, and PELP1. Collectively, this complex coordinates the rate of formation of the large ribosomal subunit and controls its distribution from the nucleolus.…”

“…Significantly elevated levels of apoptosis were noted in the morphant embryos (figure 2, G and H), consistent with previous reports that LAS1L is necessary for cellcycle progression. 30 To demonstrate sufficiency of human LAS1L RNA to rescue the phenotype induced by spliceblocking of exon 10 in the zebrafish and to demonstrate pathogenicity of the p.S477N substitution, we performed rescue experiments. First, we injected fulllength human LAS1L RNA into zebrafish embryos after first injecting the splice-blocking morpholino to eliminate the native protein.…”

“…Recent studies have demonstrated an important role for LAS1L in ribosomal biogenesis. 10,30 Ribosomal biogenesis is a complex process coordinating production of 4 ribosomal RNAs from 2 different RNA templates, 80 core proteins, and numerous associated proteins (figure 4). 16 Loss of LAS1 in S cerevisiae interrupts the production of the large, 60S ribosomal subunit by disruption of the processing of the 47S pre-rRNA at the second intervening sequence (ITS2), which separates the 5.8S and 28S rRNA sequences.…”