The RNA‐binding protein Hfq is important for ribosome biogenesis and affects translation fidelity

Ribosome biogenesis is a complex process involving multiple factors. Here, we show that the widely conserved RNA chaperone Hfq, which can regulate sRNA‐mRNA basepairing, plays a critical role in rRNA processing and ribosome assembly in Escherichia coli. Hfq binds the 17S rRNA precursor and facilitates its correct processing and folding to mature 16S rRNA. Hfq assists ribosome assembly and associates with pre‐30S particles but not with mature 30S subunits. Inactivation of Hfq strikingly decreases the pool of mature 70S ribosomes. The reduction in ribosome levels depends on residues located in the distal face of Hfq but not on residues found in the proximal and rim surfaces which govern interactions with the sRNAs. Our results indicate that Hfq‐mediated regulation of ribosomes is independent of its function as sRNA‐regulator. Furthermore, we observed that inactivation of Hfq compromises translation efficiency and fidelity, both features of aberrantly assembled ribosomes. Our work expands the functions of the Sm‐like protein Hfq beyond its function in small RNA‐mediated regulation and unveils a novel role of Hfq as crucial in ribosome biogenesis and translation.     

The 60S associated ribosome biogenesis factor LSG1‐2 is required for 40S maturation in Arabidopsis thaliana

Ribosome biogenesis involves a large ensemble of trans‐acting factors, which catalyse rRNA processing, ribosomal protein association and ribosomal subunit assembly. The circularly permuted GTPase Lsg1 is such a ribosome biogenesis factor, which is involved in maturation of the pre‐60S ribosomal subunit in yeast. We identified two orthologues of Lsg1 in Arabidopsis thaliana. Both proteins differ in their C‐terminus, which is highly charged in atLSG1‐2 but missing in atLSG1‐1. This C‐terminus of atLSG1‐2 contains a functional nuclear localization signal in a part of the protein that also targets atLSG1‐2 to the nucleolus. Furthermore, only atLSG1‐2 is physically associated with ribosomes suggesting its function in ribosome biogenesis. Homozygous T‐DNA insertion lines are viable for both LSG1 orthologues. In plants lacking atLSG1‐2 18S rRNA precursors accumulate and a 20S pre‐rRNA is detected, while the amount of pre‐rRNAs that lead to the 25S and 5.8S rRNA is not changed. Thus, our results suggest that pre‐60S subunit maturation is important for the final steps of pre‐40S maturation in plants. In addition, the lsg1‐2 mutants show severe developmental defects, including triple cotyledons and upward curled leaves, which link ribosome biogenesis to early plant and leaf development.     

Rice SNF2 family helicase ENL1 is essential for syncytial endosperm development

The endosperm of cereal grains represents the most important source of human nutrition. In addition, the endosperm provides many investigatory opportunities for biologists because of the unique processes that occur during its ontogeny, including syncytial development at early stages. Rice endospermless 1 (enl1) develops seeds lacking an endosperm but carrying a functional embryo. The enl1 endosperm produces strikingly enlarged amoeboid nuclei. These abnormal nuclei result from a malfunction in mitotic chromosomal segregation during syncytial endosperm development. The molecular identification of the causal gene revealed that ENL1 encodes an SNF2 helicase family protein that is orthologous to human Plk1‐Interacting Checkpoint Helicase (PICH), which has been implicated in the resolution of persistent DNA catenation during anaphase. ENL1‐Venus (enhanced yellow fluorescent protein (YFP)) localizes to the cytoplasm during interphase but moves to the chromosome arms during mitosis. ENL1‐Venus is also detected on a thread‐like structure that connects separating sister chromosomes. These observations indicate the functional conservation between PICH and ENL1 and confirm the proposed role of PICH. Although ENL1 dysfunction also affects karyokinesis in the root meristem, enl1 plants can grow in a field and set seeds, indicating that its indispensability is tissue‐dependent. Notably, despite the wide conservation of ENL1/PICH among eukaryotes, the loss of function of the ENL1 ortholog in Arabidopsis (CHR24) has only marginal effects on endosperm nuclei and results in normal plant development. Our results suggest that ENL1 is endowed with an indispensable role to secure the extremely rapid nuclear cycle during syncytial endosperm development in rice.     

FtsHi1/ARC1 is an essential gene in Arabidopsis that links chloroplast biogenesis and division

The Arabidopsis arc1 (accumulation and replication of chloroplasts 1) mutant has pale seedlings and smaller, more numerous chloroplasts than the wild type. Previous work has suggested that arc1 affects the timing of chloroplast division but does not function directly in the division process. We isolated ARC1 by map‐based cloning and discovered it encodes FtsHi1 (At4g23940), one of several FtsHi proteins in Arabidopsis. These poorly studied proteins resemble FtsH metalloproteases important for organelle biogenesis and protein quality control but are presumed to be proteolytically inactive. FtsHi1 bears a predicted chloroplast transit peptide and localizes to the chloroplast envelope membrane. Phenotypic studies showed that arc1 (hereafter ftsHi1‐1), which bears a missense mutation, is a weak allele of FtsHi1 that disrupts thylakoid development and reduces de‐etiolation efficiency in seedlings, suggesting that FtsHi1 is important for chloroplast biogenesis. Consistent with this finding, transgenic plants suppressed for accumulation of an FtsHi1 fusion protein were often variegated. A strong T‐DNA insertion allele, ftsHi1‐2, caused embryo‐lethality, indicating that FtsHi1 is an essential gene product. A wild‐type FtsHi1 transgene rescued both the chloroplast division and pale phenotypes of ftsHi1‐1 and the embryo‐lethal phenotype of ftsHi1‐2. FtsHi1 overexpression produced a subtle increase in chloroplast size and decrease in chloroplast number in wild‐type plants while suppression led to increased numbers of small chloroplasts, providing new evidence that FtsHi1 negatively influences chloroplast division. Taken together, our analyses reveal that FtsHi1 functions in an essential, envelope‐associated process that may couple plastid development with division.     

Enhanced NOLC1 promotes cell senescence and represses hepatocellular carcinoma cell proliferation by disturbing the organization of nucleolus

The nucleolus is a key organelle that is responsible for the synthesis of rRNA and assembly of ribosomal subunits, which is also the center of metabolic control because of the critical role of ribosomes in protein synthesis. Perturbations of rRNA biogenesis are closely related to cell senescence and tumor progression; however, the underlying molecular mechanisms are not well understood. Here, we report that cellular senescence‐inhibited gene (CSIG) knockdown up‐regulated NOLC1 by stabilizing the 5′UTR of NOLC1 mRNA, and elevated NOLC1 induced the retention of NOG1 in the nucleolus, which is responsible for rRNA processing. Besides, the expression of NOLC1 was negatively correlated with CSIG in the aged mouse tissue and replicative senescent 2BS cells, and the down‐regulation of NOLC1 could rescue CSIG knockdown‐induced 2BS senescence. Additionally, NOLC1 expression was decreased in human hepatocellular carcinoma (HCC) tissue, and the ectopic expression of NOLC1 repressed the proliferation of HCC cells and tumor growth in a HCC xenograft model.     

Mutations in the nucleolar proteins Tma23 and Nop6 suppress the malfunction of the Nep1 protein

The nucleolar Saccharomyces cerevisiae protein Nep1 was previously shown to bind to a specific site of the 18S rRNA and to be involved in assembly of Rps19p into pre-40S ribosome subunits. Here we report on the identification of tma23 and nop6 mutations as recessive suppressors of a nep1(ts) mutant allele and the nep1 deletion as well. Green fluorescent protein fusions localized Tma23p and Nop6p within the nucleolus, indicating their function in ribosome biogenesis. The high lysine content of both proteins and an RNA binding motif in the Nop6p amino acid sequence suggest RNA-binding functions for both factors. Surprisingly, in contrast to Nep1p, Tma23p and Nop6p seem to be specific for fungi as no homologues could be found in higher eukaryotes. In contrast to most other ribosome biogenesis factors, Tma23p and Nop6p are nonessential in S. cerevisiae. Interestingly, the tma23 mutants showed a considerably increased resistance against the aminoglycoside G418, probably due to a structural change in the 40S ribosomal subunit, which could be the result of incorrectly folded 18S rRNA gene, missing rRNA modifications or the lack of a ribosomal protein.     

AtBUD13 affects pre‐mRNA splicing and is essential for embryo development in Arabidopsis

Pre‐mRNA splicing is an important step for gene expression regulation. Yeast Bud13p (bud‐site selection protein 13) regulates the budding pattern and pre‐mRNA splicing in yeast cells; however, no Bud13p homologs have been identified in plants. Here, we isolated two mutants that carry T‐DNA insertions at the At1g31870 locus and shows early embryo lethality and seed abortion. At1g31870 encodes an Arabidopsis homolog of yeast Bud13p, AtBUD13. Although AtBUD13 homologs are widely distributed in eukaryotic organisms, phylogenetic analysis revealed that their protein domain organization is more complex in multicellular species. AtBUD13 is expressed throughout plant development including embryogenesis and AtBUD13 proteins is localized in the nucleus in Arabidopsis. RNA‐seq analysis revealed that AtBUD13 mutation predominantly results in the intron retention, especially for shorter introns (≤100 bases). Within this group of genes, we identified 52 genes involved in embryogenesis, out of which 22 are involved in nucleic acid metabolism. Our results demonstrate that AtBUD13 plays critical roles in early embryo development by effecting pre‐mRNA splicing.     

Methionine metabolism is essential for SIRT1‐regulated mouse embryonic stem cell maintenance and embryonic development

Methionine metabolism is critical for epigenetic maintenance, redox homeostasis, and animal development. However, the regulation of methionine metabolism remains unclear. Here, we provide evidence that SIRT1, the most conserved mammalian NAD⁺‐dependent protein deacetylase, is critically involved in modulating methionine metabolism, thereby impacting maintenance of mouse embryonic stem cells (mESCs) and subsequent embryogenesis. We demonstrate that SIRT1‐deficient mESCs are hypersensitive to methionine restriction/depletion‐induced differentiation and apoptosis, primarily due to a reduced conversion of methionine to S‐adenosylmethionine. This reduction markedly decreases methylation levels of histones, resulting in dramatic alterations in gene expression profiles. Mechanistically, we discover that the enzyme converting methionine to S‐adenosylmethionine in mESCs, methionine adenosyltransferase 2a (MAT2a), is under control of Myc and SIRT1. Consistently, SIRT1 KO embryos display reduced Mat2a expression and histone methylation and are sensitive to maternal methionine restriction‐induced lethality, whereas maternal methionine supplementation increases the survival of SIRT1 KO newborn mice. Our findings uncover a novel regulatory mechanism for methionine metabolism and highlight the importance of methionine metabolism in SIRT1‐mediated mESC maintenance and embryonic development.     

DGCR8 is essential for tumor progression following PTEN loss in the prostate

In human prostate cancer, the microRNA biogenesis machinery increases with prostate cancer progression. Here, we show that deletion of the Dgcr8 gene, a critical component of this complex, inhibits tumor progression in a Pten‐knockout mouse model of prostate cancer. Early stages of tumor development were unaffected, but progression to advanced prostatic intraepithelial neoplasia was severely inhibited. Dgcr8 loss blocked Pten null‐induced expansion of the basal‐like, but not luminal, cellular compartment. Furthermore, while late‐stage Pten knockout tumors exhibit decreased senescence‐associated beta‐galactosidase activity and increased proliferation, the simultaneous deletion of Dgcr8 blocked these changes resulting in levels similar to wild type. Sequencing of small RNAs in isolated epithelial cells uncovered numerous miRNA changes associated with PTEN loss. Consistent with a Pten–Dgcr8 association, analysis of a large cohort of human prostate tumors shows a strong correlation between Akt activation and increased Dgcr8 mRNA levels. Together, these findings uncover a critical role for microRNAs in enhancing proliferation and enabling the expansion of the basal cell compartment associated with tumor progression following Pten loss.     

The DC1‐domain protein VACUOLELESS GAMETOPHYTES is essential for development of female and male gametophytes in Arabidopsis

In this work we identified VACUOLELESS GAMETOPHYTES (VLG) as a DC1 domain‐containing protein present in the endomembrane system and essential for development of both female and male gametophytes. VLG was originally annotated as a gene coding for a protein of unknown function containing DC1 domains. DC1 domains are cysteine‐ and histidine‐rich zinc finger domains found exclusively in the plant kingdom that have been named on the basis of similarity with the C1 domain present in protein kinase C (PKC). In Arabidopsis, both male and female gametophytes are characterized by the formation of a large vacuole early in development; this is absent in vlg mutant plants. As a consequence, development is arrested in embryo sacs and pollen grains at the first mitotic division. VLG is specifically located in multivesicular bodies or pre‐vacuolar compartments, and our results suggest that vesicular fusion is affected in the mutants, disrupting vacuole formation. Supporting this idea, AtPVA12 – a member of the SNARE vesicle‐associated protein family and previously related to a sterol‐binding protein, was identified as a VLG interactor. A role for VLG is proposed mediating vesicular fusion in plants as part of the sterol trafficking machinery required for vacuole biogenesis in plants.     

The deubiquitinase USP36 promotes snoRNP group SUMOylation and is essential for ribosome biogenesis

SUMOylation plays a crucial role in regulating diverse cellular processes including ribosome biogenesis. Proteomic analyses and experimental evidence showed that a number of nucleolar proteins involved in ribosome biogenesis are modified by SUMO. However, how these proteins are SUMOylated in cells is less understood. Here, we report that USP36, a nucleolar deubiquitinating enzyme (DUB), promotes nucleolar SUMOylation. Overexpression of USP36 enhances nucleolar SUMOylation, whereas its knockdown or genetic deletion reduces the levels of SUMOylation. USP36 interacts with SUMO2 and Ubc9 and directly mediates SUMOylation in cells and in vitro. We show that USP36 promotes the SUMOylation of the small nucleolar ribonucleoprotein (snoRNP) components Nop58 and Nhp2 in cells and in vitro and their binding to snoRNAs. It also promotes the SUMOylation of snoRNP components Nop56 and DKC1. Functionally, we show that knockdown of USP36 markedly impairs rRNA processing and translation. Thus, USP36 promotes snoRNP group SUMOylation and is critical for ribosome biogenesis and protein translation.     

Ceramide inhibits development and cytokinesis and induces apoptosis in preimplantation bovine embryos

Ceramide is a second messenger induced by various cellular insults that plays a regulatory role in apoptosis. The objective of the present study was to determine whether ceramide signaling can occur in the preimplantation embryo by testing (1) effects of ceramide on development, cytokinesis, and apoptosis and (2) whether heat shock, which can induce apoptosis in embryos, causes activation of neutral or acidic sphingomyelinases responsible for generation of ceramide. Treatment of embryos > or =16 cells collected at Day 5 after insemination with 50 microM C(2)-ceramide increased caspase-9 activity and the proportion of blastomeres undergoing apoptosis but did not increase caspase-8 activity. Induction of apoptosis was more extensive when culture with ceramide was for 24 hr than for 9 hr. Ceramide also reduced the proportion of embryos that developed to the blastocyst stage when exposure was for 24 hr. At the two-cell stage, a period in development when apoptosis responses are blocked, culture of embryos with ceramide did not increase caspase-9 activity or the proportion of blastomeres that were apoptotic. However, culture with ceramide for 24 hr reduced cell proliferation and caused an increase in multinucleated cells because of inhibition of cytokinesis. Exposure of Day 5 embryos to a heat shock of 41 degrees C for 15 hr increased neutral sphingomyelinase activity but did not change acid sphingomyelinase activity. In conclusion, ceramide can regulate embryo development and apoptosis in a time and stage-of-development dependent manner and ceramide generation can be activated by cellular insult. Thus, the ceramide signaling pathway is present in the preimplantation embryo.