Iron-sulfur-protein biogenesis in eukaryotes

Iron-sulfur (Fe-S) clusters (ISCs) are versatile, ancient co-factors of proteins that are involved in electron transport, enzyme catalysis and regulation of gene expression. The synthesis of ISCs and their insertion into apoproteins involves the function of complex cellular machineries. In eukaryotes, the mitochondrial ISC-assembly machinery is involved in the maturation of all cellular iron-sulfur proteins. A mitochondrial export machinery and a recently discovered cytosolic assembly system specifically participate in the maturation of cytosolic and nuclear iron-sulfur proteins. Of the approximately 20 assembly components, more than ten are encoded by essential genes, which indicates that the process is indispensable for life. Mutations in two of the assembly components lead to neurological diseases. The essential character of Fe-S-protein biogenesis in eukaryotes and its importance for human disease identifies this evolutionary ancient process as one of the most important biosynthetic pathways of life.     

Low-molecular-weight RNA of eukaryotes: biogenesis, subcellular localization, functions

The experimental data on the organization and expression of the low-molecular-weight RNA genes on the subcellular localization, metabolism and evolution of these RNA are summarized. The processes in which they are involved are discussed. The biological role of the low-molecular-weight RNA is paid a special attention. A hypothesis concerning the involvement of these molecules in the mechanism of active transport of proteins from the cytoplasm into the nucleus of the cell is put forward.     

Ribosome biogenesis: of knobs and RNA processing

The synthesis of ribosomes in eukaryotes involves processing of pre-ribosomal RNA (pre-rRNA) and sequential assembly of a large number of ribosomal proteins on the rRNAs. Although we have gained tremendous insights into the processing of pre-rRNA intermediates in the last three decades, little was known about the dynamic nature of ribosome biogenesis. Only recently the development of efficient affinity-purification procedures and mass-spectrometry techniques has allowed the isolation of large pre-ribosomal complexes, which led to the identification of several ribosome assembly intermediates and a large number of novel ribosome assembly factors. In this mini-review, we summarize some of the discoveries that have been made in the field of ribosome biogenesis in the past 30 years and highlight some key aspects about what remains to be learned.     

Yeast and human RNA helicases involved in ribosome biogenesis: Current status and perspectives

Ribosome biogenesis is a fundamental process that is conserved in eukaryotes. Although spectacular progress has been made in understanding mammalian ribosome synthesis in recent years, by far, this process has still been best characterised in the yeast Saccharomyces cerevisiae. In yeast, besides the rRNAs, the ribosomal proteins and the 75 small nucleolar RNAs, more than 250 non-ribosomal proteins, generally referred to as trans-acting factors, are involved in ribosome biogenesis. These factors include nucleases, RNA modifying enzymes, ATPases, GTPases, kinases and RNA helicases. Altogether, they likely confer speed, accuracy and directionality to the ribosome synthesis process, however, the precise functions for most of them are still largely unknown. This review summarises our current knowledge on eukaryotic RNA helicases involved in ribosome biogenesis, particularly focusing on the most recent advances with respect to the molecular roles of these enzymes and their co-factors in yeast and human cells. This article is part of a Special Issue entitled: The Biology of RNA helicases—Modulation for life.     

Organelle biogenesis and intracellular lipid transport in eukaryotes.

The inter- and intramembrane transport of phospholipids, sphingolipids, and sterols involves the most fundamental processes of membrane biogenesis. Identification of the mechanisms involved in these lipid transport reactions has lagged significantly behind that for intermembrane protein traffic until recently. Application of methods that include fluorescently labeled and spin-labeled lipid analogs, new cellular fractionation techniques, topographically specific chemical modification techniques, the identification of organelle-specific metabolism, permeabilized cell methodology, and yeast molecular genetics has contributed to revealing a diverse biochemical array of transport processes for lipids. Compelling evidence now exists for ATP-dependent, ATP-independent, vesicle-dependent, and vesicle-independent transport processes that are lipid and membrane specific. ATP-dependent transport processes include the transbilayer movement of phosphatidylserine and phosphatidylethanolamine at the plasma membrane and the transport of phosphatidylserine from its site of synthesis to the mitochondria. ATP-independent processes include the transbilayer movement of virtually all lipids at the endoplasmic reticulum, the movement of phosphatidylserine between the inner and outer mitochondrial membranes, and the transfer of nascent phosphatidylcholine and phosphatidylethanolamine to the plasma membrane. The ATP-independent movement of lipids between organelles is believed to be due to the action of lipid transfer proteins, but this still remains to be proved. Vesicle-based transport mechanisms (which are also inherently ATP dependent) include the transport of nascent cholesterol, sphingomyelin, and glycosphingolipids from the Golgi apparatus to the plasma membrane and the recycling of sphingolipids and selected pools of phosphatidylcholine from the plasma membrane to the cell interior. The vesicles involved in cholesterol transport to the plasma membrane are different from those involved in bulk protein transport to the cell surface. The vesicles involved in recycling sphingomyelin to and from the cell surface are different from those involved in the assembly of newly synthesized sphingolipids into the plasma membrane. The preliminary characterization of these lipid translocation processes suggests divergent rather than unifying mechanisms for lipid transport in organelle assembly.     

Developmentally regulated, combinatorial RNA processing modulates AMPA receptor biogenesis

The subunit composition determines AMPA receptor (AMPA-R) function and trafficking. Mechanisms underlying channel assembly are thus central to the efficacy and plasticity of glutamatergic synapses. We previously showed that RNA editing at the Q/R site of the GluR2 subunit contributes to the assembly of AMPA-R heteromers by attenuating formation of GluR2 homotetramers. Here we report that this function of the Q/R site depends on subunit contacts between adjacent ligand binding domains (LBDs). Changes of LBD interface contacts alter GluR2 assembly properties, forward traffic, and expression at synapses. Interestingly, developmentally regulated RNA editing within the LBD (at the R/G site) produces analogous effects. Our data reveal that editing to glycine reduces the self-assembly competence of this critical subunit and slows GluR2 maturation in the endoplasmic reticulum (ER). Therefore, RNA editing sites, located at strategic subunit interfaces, shape AMPA-R assembly and trafficking in a developmentally regulated manner.     

Ribosomal RNA processing and an RNase R family member in chloroplasts of Arabidopsis

An Arabidopsis mutant rnr1, which has a defect in the basic genetic system in chloroplasts, was isolated using the screening of the high chlorophyll fluorescence phenotype. Whereas chlorophyll fluorescence and immunoblot studies showed the mutant had reduced activities of photosystems I and II, molecular characterization of the mutant suggested that a T-DNA insertion impaired the expression of a gene encoding a RNase R family member with a targeting signal to chloroplasts. Since RNase R family members have a 3–5 exoribonuclease activity, we examined the RNA profile in chloroplasts. In rnr1 the intercistronic cleavage between 23S and 4.5S rRNA was impaired, and a significant reduction in rRNA in chloroplasts was found, suggesting that RNR1 functions in the maturation of chloroplast rRNA. The present results suggest that defects in the genetic system in chloroplasts cause high chlorophyll fluorescence, pale green leaf, and marked reduction in the growth rate, whereas the levels of some chloroplast RNA were higher in rnr1 than in the wild-type.     

Ribosomal protein L33 is required for ribosome biogenesis, subunit joining, and repression of GCN4 translation

We identified a mutation in the 60S ribosomal protein L33A (rpl33a-G76R) that elicits derepression of GCN4 translation (Gcd- phenotype) by allowing scanning preinitiation complexes to bypass inhibitory upstream open reading frame 4 (uORF4) independently of prior uORF1 translation and reinitiation. At 37 degrees C, rpl33a-G76R confers defects in 60S biogenesis comparable to those produced by the deletion of RPL33A (DeltaA). At 28 degrees C, however, the 60S biogenesis defect is less severe in rpl33a-G76R than in DeltaA cells, yet rpl33a-G76R confers greater derepression of GCN4 and a larger reduction in general translation. Hence, it appears that rpl33a-G76R has a stronger effect on ribosomal-subunit joining than does a comparable reduction of wild-type 60S levels conferred by DeltaA. We suggest that rpl33a-G76R alters the 60S subunit in a way that impedes ribosomal-subunit joining and thereby allows 48S rRNA complexes to abort initiation at uORF4, resume scanning, and initiate downstream at GCN4. Because overexpressing tRNAiMet suppresses the Gcd- phenotype of rpl33a-G76R cells, dissociation of tRNAiMet from the 40S subunit may be responsible for abortive initiation at uORF4 in this mutant. We further demonstrate that rpl33a-G76R impairs the efficient processing of 35S and 27S pre-rRNAs and reduces the accumulation of all four mature rRNAs, indicating an important role for L33 in the biogenesis of both ribosomal subunits.     

Characterization and mutational analysis of yeast Dbp8p, a putative RNA helicase involved in ribosome biogenesis

RNA helicases of the DEAD box family are involved in almost all cellular processes involving RNA molecules. Here we describe functional characterization of the yeast RNA helicase Dbp8p (YHR169w). Our results show that Dbp8p is an essential nucleolar protein required for biogenesis of the small ribosomal subunit. In vivo depletion of Dbp8p resulted in a ribosomal subunit imbalance due to a deficit in 40S ribosomal subunits. Subsequent analyses of pre-rRNA processing by pulse–chase labeling, northern hybridization and primer extension revealed that the early steps of cleavage of the 35S precursor at sites A₁ and A₂ are inhibited and delayed at site A₀. Synthesis of 18S rRNA, the RNA moiety of the 40S subunit, is thereby blocked in the absence of Dbp8p. The involvement of Dbp8p as a bona fide RNA helicase in ribosome biogenesis is strongly supported by the loss of Dbp8p in vivo function obtained by site-directed mutagenesis of some conserved motifs carrying the enzymatic properties of the protein family.