The Ribosome Assembly Factor LSG1 Interacts with Vesicle-Associated Membrane Protein-Associated Proteins (VAPs).

LSG1 is a conserved GTPase involved in ribosome assembly. It is required for the eviction of the nuclear export adapter NMD3 from the pre-60S subunit in the cytoplasm. In human cells, LSG1 has also been shown to interact with vesicle-associated membrane protein-associated proteins (VAPs) that are found primarily on the endoplasmic reticulum.

The Ribosome Assembly Factor Reh1 is Released from the Polypeptide Exit Tunnel in the Pioneer Round of Translation.

Assembly of functional ribosomal subunits and successfully delivering them to the translating pool is a prerequisite for protein synthesis and cell growth. In the ribosome assembly factor Reh1 binds to pre-60S subunits at a late stage during their cytoplasmic maturation. Previous work shows that the C-terminus of Reh1 inserts into the polypeptide exit tunnel (PET) of the pre-60S subunit.

A single 2'-O-methylation of ribosomal RNA gates assembly of a functional ribosome.

RNA modifications are widespread in biology and abundant in ribosomal RNA. However, the importance of these modifications is not well understood. We show that methylation of a single nucleotide, in the catalytic center of the large subunit, gates ribosome assembly.

In vitro characterization of Dhr1 from Saccharomyces cerevisiae.

The RNA helicase Dhr1 from S. cerevisiae is an essential enzyme required for the assembly of the cytosolic small ribosomal subunit (SSU). A critical feature of the SSU is the central pseudoknot, an RNA fold that organizes the overall architecture of the subunit and connects all four domains of the 18S ribosomal RNA (rRNA).

Release of the ribosome biogenesis factor Bud23 from small subunit precursors in yeast.

The two subunits of the eukaryotic ribosome are produced through quasi-independent pathways involving the hierarchical actions of numerous -acting biogenesis factors and the incorporation of ribosomal proteins. The factors work together to shape the nascent subunits through a series of intermediate states into their functional architectures. One of the earliest intermediates of the small subunit (SSU or 40S) is the SSU processome which is subsequently transformed into the pre-40S intermediate.

Genetics animates structure: leveraging genetic interactions to study the dynamics of ribosome biogenesis.

The assembly of eukaryotic ribosomes follows an assembly line-like pathway in which numerous trans-acting biogenesis factors act on discrete pre-ribosomal intermediates to progressively shape the nascent subunits into their final functional architecture. Recent advances in cryo-electron microscopy have led to high-resolution structures of many pre-ribosomal intermediates; however, these static snapshots do not capture the dynamic transitions between these intermediates. To this end, molecular genetics can be leveraged to reveal how the biogenesis factors drive these dynamic transitions.

Bud23 promotes the final disassembly of the small subunit Processome in Saccharomyces cerevisiae.

The first metastable assembly intermediate of the eukaryotic ribosomal small subunit (SSU) is the SSU Processome, a large complex of RNA and protein factors that is thought to represent an early checkpoint in the assembly pathway. Transition of the SSU Processome towards continued maturation requires the removal of the U3 snoRNA and biogenesis factors as well as ribosomal RNA processing. While the factors that drive these events are largely known, how they do so is not.

The L1 stalk is required for efficient export of nascent large ribosomal subunits in yeast.

The ribosomal protein Rpl1 (uL1 in universal nomenclature) is essential in yeast and constitutes part of the L1 stalk which interacts with E site ligands on the ribosome. Structural studies of nascent pre-60S complexes in yeast have shown that a domain of the Crm1-dependent nuclear export adapter Nmd3, binds in the E site and interacts with Rpl1, inducing closure of the L1 stalk. Based on this observation, we decided to reinvestigate the role of the L1 stalk in nuclear export of pre-60S subunits despite previous work showing that Rpl1-deficient ribosomes are exported from the nucleus and engage in translation.

Tsr4 Is a Cytoplasmic Chaperone for the Ribosomal Protein Rps2 in Saccharomyces cerevisiae.

Eukaryotic ribosome biogenesis requires the action of approximately 200 -acting factors and the incorporation of 79 ribosomal proteins (RPs). The delivery of RPs to preribosomes is a major challenge for the cell because RPs are often highly basic and contain intrinsically disordered regions prone to nonspecific interactions and aggregation. To counteract this, eukaryotes developed dedicated chaperones for certain RPs that promote their solubility and expression, often by binding eukaryote-specific extensions of the RPs.

Tightly-orchestrated rearrangements govern catalytic center assembly of the ribosome.

The catalytic activity of the ribosome is mediated by RNA, yet proteins are essential for the function of the peptidyl transferase center (PTC). In eukaryotes, final assembly of the PTC occurs in the cytoplasm by insertion of the ribosomal protein Rpl10 (uL16). We determine structures of six intermediates in late nuclear and cytoplasmic maturation of the large subunit that reveal a tightly-choreographed sequence of protein and RNA rearrangements controlling the insertion of Rpl10.

Utp14 interaction with the small subunit processome.

The SSU processome (sometimes referred to as 90S) is an early stable intermediate in the small ribosomal subunit biogenesis pathway of eukaryotes. Progression of the SSU processome to a pre-40S particle requires a large-scale compaction of the RNA and release of many biogenesis factors. The U3 snoRNA is a primary component of the SSU processome and hybridizes to the rRNA at multiple locations to organize the structure of the SSU processome.

The T-cell leukemia related rpl10-R98S mutant traps the 60S export adapter Nmd3 in the ribosomal P site in yeast.

Mutations in the ribosomal protein Rpl10 (uL16) can be drivers of T-cell acute lymphoblastic leukemia (T-ALL). We previously showed that these T-ALL mutations disrupt late cytoplasmic maturation of the 60S ribosomal subunit, blocking the release of the trans-acting factors Nmd3 and Tif6 in S. cerevisiae.

Nmd3 is a structural mimic of eIF5A, and activates the cpGTPase Lsg1 during 60S ribosome biogenesis.

During ribosome biogenesis in eukaryotes, nascent subunits are exported to the cytoplasm in a functionally inactive state. 60S subunits are activated through a series of cytoplasmic maturation events. The last known events in the cytoplasm are the release of Tif6 by Efl1 and Sdo1 and the release of the export adapter, Nmd3, by the GTPase Lsg1.

Bcp1 Is the Nuclear Chaperone of Rpl23 in Saccharomyces cerevisiae.

Eukaryotic ribosomes are composed of rRNAs and ribosomal proteins. Ribosomal proteins are translated in the cytoplasm and imported into the nucleus for assembly with the rRNAs. It has been shown that chaperones or karyopherins responsible for import can maintain the stability of ribosomal proteins by neutralizing unfavorable positive charges and thus facilitate their transports.

XRN1 Is a Species-Specific Virus Restriction Factor in Yeasts.

In eukaryotes, the degradation of cellular mRNAs is accomplished by Xrn1 and the cytoplasmic exosome. Because viral RNAs often lack canonical caps or poly-A tails, they can also be vulnerable to degradation by these host exonucleases. Yeast lack sophisticated mechanisms of innate and adaptive immunity, but do use RNA degradation as an antiviral defense mechanism.

Utp14 Recruits and Activates the RNA Helicase Dhr1 To Undock U3 snoRNA from the Preribosome.

In eukaryotic ribosome biogenesis, U3 snoRNA base pairs with the pre-rRNA to promote its processing. However, U3 must be removed to allow folding of the central pseudoknot, a key feature of the small subunit. Previously, we showed that the DEAH/RHA RNA helicase Dhr1 dislodges U3 from the pre-rRNA.

Understanding Biases in Ribosome Profiling Experiments Reveals Signatures of Translation Dynamics in Yeast.

Ribosome profiling produces snapshots of the locations of actively translating ribosomes on messenger RNAs. These snapshots can be used to make inferences about translation dynamics. Recent ribosome profiling studies in yeast, however, have reached contradictory conclusions regarding the average translation rate of each codon.

The DEAH-box helicase Dhr1 dissociates U3 from the pre-rRNA to promote formation of the central pseudoknot.

In eukaryotes, the highly conserved U3 small nucleolar RNA (snoRNA) base-pairs to multiple sites in the pre-ribosomal RNA (pre-rRNA) to promote early cleavage and folding events. Binding of the U3 box A region to the pre-rRNA is mutually exclusive with folding of the central pseudoknot (CPK), a universally conserved rRNA structure of the small ribosomal subunit essential for protein synthesis. Here, we report that the DEAH-box helicase Dhr1 (Ecm16) is responsible for displacing U3.

Genetic analysis of the ribosome biogenesis factor Ltv1 of Saccharomyces cerevisiae.

Ribosome biogenesis has been studied extensively in the yeast Saccharomyces cerevisiae. Yeast Ltv1 is a conserved 40S-associated biogenesis factor that has been proposed to function in small subunit nuclear export. Here we show that Ltv1 has a canonical leucine-rich nuclear export signal (NES) at its extreme C terminus that is both necessary for Crm1 interaction and Ltv1 export.

Physical and functional interaction between the methyltransferase Bud23 and the essential DEAH-box RNA helicase Ecm16.

The small ribosomal subunit assembles cotranscriptionally on the nascent primary transcript. Cleavage at site A2 liberates the pre-40S subunit. We previously identified Bud23 as a conserved eukaryotic methyltransferase that is required for efficient cleavage at A2.

Bypass of the pre-60S ribosomal quality control as a pathway to oncogenesis.

Ribosomopathies are a class of diseases caused by mutations that affect the biosynthesis and/or functionality of the ribosome. Although they initially present as hypoproliferative disorders, such as anemia, patients have elevated risk of hyperproliferative disease (cancer) by midlife. Here, this paradox is explored using the rpL10-R98S (uL16-R98S) mutant yeast model of the most commonly identified ribosomal mutation in acute lymphoblastic T-cell leukemia.

Ribosomes: lifting the nuclear export ban.

A recent study shows that nuclear export of the large ribosomal subunit is regulated by a GTPase that blocks recruitment of the nuclear export factor Nmd3 until remodeling of the pre-ribosome by the AAA-ATPase Rea1 (Midasin).